WO2016014992A2

WO2016014992A2 - Compositions for treatment, diagnosis and prognosis of diseases

Info

Publication number: WO2016014992A2
Application number: PCT/US2015/042084
Authority: WO
Inventors: Jenny L. PERSSON; Heather H. JOHNSON
Original assignee: Everest Biosciences, Inc.
Priority date: 2014-07-24
Filing date: 2015-07-24
Publication date: 2016-01-28
Also published as: WO2016014992A3

Abstract

Disclosed herein are novel pharmaceutically-useful compositions, which compositions may be useful in the treatment of diseases such as cancer. Also disclosed is the use of the compositions for disease diagnosis and prognosis, treatment selection and disease progression monitoring.

Description

COMPOSITIONS FOR TREATMENT, DIAGNOSIS AND PROGNOSIS OF

DISEASES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. Provisional Patent Application 62/028,783, filed July 24, 2014, the entire disclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to novel pharmaceutically-useful compositions, which compositions may be useful in the treatment of diseases such as cancer. The invention also relates to use of the compositions for disease diagnosis and prognosis, treatment selection, and disease progression monitoring.

BACKGROUND OF THE INVENTION

[0003] Phosphoinositides has long beene known to present in cellular membranes. The phosphoinositide family consists of seven derivatives of phosphatidylmositol (Ptdlns) that are formed through the phosphorylation of the 3-, 4- and 5-positions of the inositol ring. Despite their low abundance in the cell, phosphoinositides are important regulators of a large variety of cellular processes. The production of the different phosphoinositide species is spatially and temporally regulated through the actions of kinases, phosphatases and phospholipases, some of which can be localized in different subcellular compartments.

[0004] Key to determining the biological roles of phosphoinositides is understanding the enzymes involved in their metabolism. Although many such enzymes have now been identified, there is still much to learn about their cellular functions. Phosphatidylmositol 4-phosphate 5-kinases (PIP5Ks) are a group of kinases that catalyse the production of phosphatidylmositol (4,5)-bisphosphate [PtdIns(4,5)P2]. There are 3 PIP5Ks in human including PIP5K (also known as and hereafter is referered as PIP5K1A), ΡΙΡ5Κβ (also known as PIP5K1B) and PIP5Ky (also known as PIP5K1C).

[0005] PIP5Ks has been linked to diseases in humans. A report has linked the mutation of human ΡΙΡ5Κγ to a lethal congenital contractural syndrome type 3 (LCCS3) characterized by multiple joint contractures, micrognathia and anterior-horn atrophy in the spinal cord. The origin of this disease was traced back to a mutation (G757A) in the kinase domain of ΡΙΡ5Κγ that renders the protein unable to phosphorylate PtdIns(4)/^>. ΡΙΡ5Κγ is highly expressed in the brain and the symptoms are linked to major neurological defects. In neurons, PtdIns(4,5)P2 is important for different processes, including synaptic-vesicle endocytosis and neurite outgrowth. A lack of PtdIns(4,5)/^>2 might impinge on these processes, resulting in the neurological defects found in LCCS3. The activity of phosphatidylmositol 4-kinase (PI4K) and of PIP5K is increased in different hepatoma cell lines as compared with that in normal liver cells. Together with the fact that PtdIns(3,4,5)P3 levels are altered in many cancers because of mutations of the phosphatase and tensin homolog (PTEN) (which negatively regulates the levels of PtdIns(3,4,5)P3), it was suggested increased PtdIns(4,5)P2 levels (through changes in PIP5K activity) might also be important to sustain increased PtdIns(3,4,5).P3 production during cancer progression.

[0006] Cancer is a class of diseases that affects people world-wide. Generally, cells in a benign tumor retain their differentiated features and do not divide in a completely uncontrolled manner. A benign tumor is usually localized and non-metastatic.

[0007] In a malignant tumor, cells become undifferentiated, do not respond to the body's growth control signals, and multiply in an uncontrolled manner. Malignant tumors are generally divided into two categories: primary and secondary. Primary tumors arise directly from the tissue in which they are found. Secondary tumors may be originated from the primary tumors or may be originated elsewhere in the body, and are capable of spreading to distant sites (metastasizing) or metastasis. The common routes for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems or blood streams.

[0008] There is a need to develop new diagnosis and treatment for diseases such as cancer.

SUMMARY OF THE INVENTION

[0009] The present invention provides compounds and formulation thereof for treatment of diseases, such as cancer, spinal cord injur, and AIDS.

[0010] In one aspect, the present invention provides a method for treatment of a disease in a subject in which inhibition of PIP5K1A is desired and/or required, comprising: administering a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.

[0011] In another aspect, the present invention provides a method for treatment of a disease in a subject characterized by over activation of PIP5K1A and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.

[0012] In some embodiments, the inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 5% and 100% as determined by an in vitro or in vivo assay. In some embodiments, the the inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 40% and 80% as determined by an in vitro or in vivo assay.

[0013] In another aspect, the present invention provides a method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.

[0014] In some embodiments, the inhibitor of PIP5K1A is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity as determined by an in vitro or in vivo assay. In some embodiments, the inhibitor of PIP5K1A is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity as determined by an in vitro or in vivo assay.

[0015] In another aspect, the present invention provides a method for treatment of a disease in a subject characterized by over activation of PI3 Kinase and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.

[0016] In some embodiments, the inhibitor of PIP5K1A is capable of reducing production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) to between about 50 %> and 150%o of normal production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay. In some embodiments, the inhibitor of PIP5K1A is capable of reducing production level of phosphatidylinositol 3,4,5- bisphosphate (PtdIns(3,4,5)P2) to between about 80 %> and 120%o of normal production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal , as determined by an in vitro or in vivo assay.

[0017] In yet another aspect, the present invention provides a method for treatment of a disease in a subject characterized by over activation of phospholipase C (PLC) and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.

[0018] In some embodiments, the inhibitor of PIP5K1A is capable of reducing PLC activity to between about 50 % and 150% of normal PLC activity as determined by an in vitro or in vivo assay. In some embodiments, the inhibitor of PIP5K1A is capable of reducing PLC activity to between about 80 % and 120% of normal PLC activity as determined by an in vitro or in vivo assay.

[0019] In a further aspect, the present invention provides method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of modulator of PDK/Akt pathway, wherein the modulator is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity as determined by an in vitro or in vivo assay.

[0020] In some embodiments, the modulator of PI3K/Akt pathway is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity as determined by an in vitro or in vivo assay. [0021] In some embodiments, the disease is cancer, spinal cord injury, or AIDS. In some embodiments, the cancer is selected from the group consisting of: hemangioma, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas, pyogenic granulomas, leukemia, myelodysplastic syndromes (MDS), prostate cancer, breast cancer, skin cancer, bone cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, bladder, gall bladder, ovary, cervix, pancreas, rectum, parathyroid, thyroid, esophagus, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papilllary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, non-small cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

[0022] In some embodiments, the inhibitor of PIP5K1A, or modulator of PI3K/Akt pathway comprises a small molecule, an RNA, a peptide, or an antibody.

[0023] In another aspect, the present invention provides a method of diagnosing a subject suspect of having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to diagnosis of the disease, thereby diagnosing the subject with regard to the disease.

[0024] In some embodiments, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient diagnosed as having the disease.

[0025] In another aspect, the present invention provides a method for determining disease stage of a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to disease stage of the disease, thereby determining disease stage of the subject with regard to the disease.

[0026] In some embodiments, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having the disease of different disease stages.

[0027] In another aspect, the present invention provides a method for monitoring disease progression of a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to disease progression of the disease, thereby determining disease progression of the subject with regard to the disease.

[0028] In some embodiments, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having the disease of different disease progression.

[0029] In another aspect, the present invention provides a method for determining a prognosis for a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to prognosis of the disease, thereby determining prognosis of the subject with regard to the disease.

[0030] In some embodiments, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient determined as having the disease of different prognosis outcome.

[0031] In another aspect, the present invention provides a method of determining effect of treatment of a subject having a disease condition with a pharmaceutical composition, comprising: (a) providing a biological sample from a subject that has been subject to a treatment of a disease; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to treatment effect of the disease, thereby determining the treatment effect of the pharmaceutical composition. [0032] In another aspect, the present invention provides a method of determining whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIP5K1A, and/or an inhibitor of PI3K/Akt pathway, and/or an inhibitor of PLC pathway, comprising: (a) providing a biological sample from a subject t having a disease condition; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIPK1 A, and/or an inhibitor of PDK/Akt pathway, and/or an inhibitor of PLC pathway, thereby determining whether the subject should be treated with the pharmaceutical composition.

[0033] In some embodiments, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient that has been subjected to the treatment and achieved treatment effect.

[0034] In some embodiments, the one or more genes comprises a gene selected from the group consisting of: Akt, pAkt (phosphorylated Akt), Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21 , Cytokeratin fragments 21 -1, EGFR mutations, Estrogen receptor (ER)/progesterone receptor (PR), Androgen receptor (AR), Fibrin/fibrinogen, HER4, HER2/neu, Immunoglobulins, KIT, KRAS , mutation analysis, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), BRCA1 , ERCC1, TYMS, RRM1, TUBB3, STMN1 , MDR1 , GSTP1 , TOP2A, IGF1R, VEGFR1/VEGFR2, FGFR1 /FGFR2/FGFR3/FGFR4, PTEN, KIT, Cyclin Al , Cyclin Dl , GADD45, DDB2, VEGF, MMP-2, MMP-9, CDK1, Chkl , FAK, pFAK (phosphorylated FAK), PIK3CA , CYLD, Nflx, Impadl , DCAMKL-1, Casein kinase II alpha (Ck2a), Phosphotidic acid (PA), Racl , RhoA, Ajuba (LIMP), pRB (phosphorylated RB), ARF6 , and PLD2 .

[0035] In some embodiments, the expression levels are mRNA expression level or protein expression level, or both.

[0036] In another aspect, the present invention provides a method for reducing over activation of PI3K/Akt pathway in a subject, comprising administering the subject with an inhibitor of PIP5K1A, thereby reducing over activation of the PDK/Akt pathway in the subject. [0037] In another aspect, the present invention provides a method for reducing over activation of PI3K/Akt pathway in a cell, comprising contacting the cell with an inhibitor of PIP5K1A, thereby reducing over activation of the PI3K/Akt pathway in the cell.

[0038] In another aspect, the present invention provides a method for reducing over activation of PLC pathway in a subject, comprising administering the subject with an inhibitor of PIP5K1A, thereby reducing over activation of the PLC pathway in the subject.

[0039] In another aspect, the present invention provides a method for reducing over activation of PLC pathway in a cell, comprising contacting the cell with an inhibitor of PIP5K1A, thereby reducing over activation of the PLC pathway in the cell.

INCORPORATION BY REFERENCE

[0040] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0042] Figure 1 : depicts depletion of PIP5K1A by RNAi in prostate cancer PC-3 cells. PIP5K1A expression was completely blocked by RNAi treatment at 48 and 72 hrs.

[0043] Figure 2A-B: Figure 2 A and Figure 2B depict inhibition of Akt phosphorylation by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. Akt phosphorylation and activation were significantly inhibited by blocking PIP5K1 A expression with RNAi treatment.

[0044] Figure 3A-B: Figure 3A and Figure 3B depict inhibition of expression of Akt downstream effector cyclin Dl by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. Cyclin Dl expression was significantly inhibited by blocking PIP5K1 A expression with RNAi treatment.

[0045] Figure 4 depicts increased expression of Akt downstream negative effector p27 by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. P27 expression was significantly increased by blocking PIP5K1A expression with RNAi treatment.

[0046] Figure 5 depicts expression of PIP5K1A in prostate cancer patient tumor samples. PIP5K1A is over-expressed in prostate cancer patient tissues and its expression level correlates with cancer grade: very low in benign prostatic hyperplasia (BPH) patients, low in low grade prostate cancer patients and at the highest level in high grade metastatic prostate cancer patients.

[0047] Figure 6 depicts statistical analysis of PIP5K1A expression level in benign patients and malignant prostate cancer patients. There is a statistically significant difference between PIP5K1A levels in benign and malignant prostate cancer patients (P<0.001).

[0048] Figure 7A-C depicts correlation (FIG. 7C) of PIP5K1A expression (FIG. 7B) and Androgen Receptor (AR) expression (FIG. 7A) in prostate cancer patients. Both AR and PIP5K1A are over-expressed in malignant prostate cancer patients. There is a strong and statistically significant correlation between AR and PIP5K1A expression with correlation coefficient at 0.633 (P<0.01).

[0049] Figure 8 depicts expression of PIP5K1A in healthy and lung cancer patient samples. PIP5K1A is over-expressed in lung cancer patient tissues and its expression level correlates with cancer grade: very low in healthy subjects, low in non-invasive Lung Squamous Cell Carcinoma and very high in invasive Non-Small Cell Lung Cancer patients.

[0050] Figure 9A-G. Characterization of ΡΙΡ5Κ1α as a specific target for a novel anticancer compound ISA-201 1B. Figure 9A. The chemical structure of ISA-201 1B is presented. Figure 9B. Dose-dependent effect of ISA-201 1B on proliferation of PC-3 cells was assessed using the nonradioactive MTS proliferation assay. Mean absorbance for control and 10 μΜ ISA-201 IB treated cells were 0.53 and 0.31 respectively (difference = 0.22; 95% CI = 0.26 to 0.36; P = 0.003). Mean absorbance for 20 μΜ ISA-201 IB treated cells was 0.26 (difference = 0.27; 95% CI = 0.23 to 0.29; P < 0.001). Mean absorbance for 50 μΜ ISA-201 IB treated cells was 0.1 1 (difference = 0.42; 95% CI = 0.09 to 0.14; P < 0.001). Data means are from three independent experiments performed with upper 95% confidence intervals. Figure 9C. Difference in the effect of ISA-201 IB on proliferation between PC-3 cells and 22Rvl cells is shown. The effect of ISA-201 IB on PC3 cells at 10 μΜ (P=0.0006) and 50 μΜ (P=0.025) is significantly stronger than on 22Rvl cells. Data means are from three independent experiments. **P < 0.01 and *P < 0.05 are indicated. Figure 9D. Schematic illustration on high-throughput kinase profiling technology using KINOMEscan^® platform. Bound kinase levels in test compound and control wells are compared. The experiments were performed in triplicates. Figure 9E. The binding affinity of ISA-201 IB with ΡΙΡ5Κ1 α and a group of kinases was obtained from the assay indicated in Figure 9D is shown. Figure 9F. PC-3 cells were treated with DMSO as control, docetaxel (DTX) at 50 nM, ISA-201 IB and its analog 2011 A at 20 μΜ for 48 hours. Protein lysates from each treatment were subjected to immunoblot analysis. Antibody against ΡΙΡ5Κ1α was used as a probe. Data are the means of three independent experiments. *P < 0.05. Figure 9G. PC-3 cells were treated with DMSO as control, docetaxel (DTX) at 50 nM, ISA-2009, ISA-201 IB, and 2011A at 20 μΜ for 48 hours. Antibody against PKARl a was used as a probe for immunoblotting. [0051] Figure 10A-B. The effect of ISA-201 1B on growth of PC-3 tumor xenografts in vivo. Figure 10A. Growth of tumor xenografts treated with vehicle (Control), docetaxel (10 mg/kg), ISA- 201 IB (40 mg/kg), and docetaxel (10 mg/kg) in combination with ISA-2011B (40 mg/kg) every second day. Treatment started on day 0 and ended on day 20 (n = 6 mice per group). Mean tumor volumes and upper 95% confidence intervals are shown. **/><0.01. Figure 10B. Tumors from each group were collected and weighted at the end of experiment. On day 20, mean tumor mass of control group = 0.183 g, ISA-201 IB treated group = 0.027 g, difference = 0.156 g, 95% CI = 0.012 to 0.041 , P < 0.006, ISA-201 IB + docetaxel treated group = 0.024 g, difference = 0.159 g, 95% CI = 0.016 to 0.031, P < 0.001). Mean tumor mass in grams (g) and upper 95%o confidence intervals are shown. ** P<0.01.

[0052] Figure 11A-D. Evaluation of the clinical importance of ΡΙΡ5Κ1α and its link with PIP2 and AR in PCa patients. Figure 1 1A. Immunohistochemical analysis of a TMA containing BPH and paired PCa specimens from 48 PCa patients. Representative microphotographs are shown. Scale bars are indicated and are applied to all images in panel a. Figure 11B. Box-plot quantitative comparison between BPH and paired cancer specimens from 48 PCa patients are shown. The paired Wilcoxon's rank sum test analyses are shown for ΡΙΡ5Κ1α, PIP2, and AR. **P < 0.01. Figure 11C. Box-plots presenting expression of genes encoding ΡΙΡ5Κ1α, AKT2 and AR. Tumor specimens from distinct subgroups of patients with primary [n=181] and metastatic [n=37] PCa were assessed. **P< 0.01. Figure 11D. Kaplan-Meier survival analysis based on BCR-free showing the difference between patients with low or high expression of the regulators are shown. Differences in BCR-free survivals between two groups were calculated using the log-rank test.

[0053] Figure 12A-K. ΡΙΡ5Κ1 α overexpression promotes malignant phenotype in non-malignant epithelial PNTIA cells via AKT/AR/CDKl pathways. Figure 12A. Immunoblots show the expression of ΡΙΡ5Κ1α, phosphorylated AKT, cyclin Dl , and CDK1 in a panel of PCa cell lines including PNTIA, LNCaP, and PC-3. Figure 12B. The effect of ΡΙΡ5Κ1 α overexpression on the morphology of PNTIA cells. Representative immune-fluorescent images show the overexpression of ΡΙΡ5Κ1 α and its co -localization with β-Tubulin. DAPI was used to stain the nucleus of cells. The scale bar is indicated. Figure 12C. Representative images of the immunofluorescent cells overexpressing ΡΙΡ5Κ1α or control vector, stained with PIP2 antibody. Figure 12D. Immunoblots show the effect of ΡΙΡ5Κ1α overexpression on PNTIA cells. The mean expression of ΡΙΡ5Κ1 α in cells transfected with pLPS-EGFP control vector was 7.42 and the mean value in cells transfected with pLPS-PIP5Kla was 10.96 (difference = 3.54; 95% CI = 5.89 to 16.02; P = 0.047). The mean expression of AKT in cell transfected with pLPS-EGFP control vector was 3.78 and the mean value in cells transfected with pLPS-PIP5Kla was 29.02 (difference = 25.24; 95% CI = 21.67 to 36.37, P = 0.021). Data is presented as average of three independent experiments (±SD). *P <0.05. **P < 0.01. Figure 12E. Immunoblots show the effect of overexpression of ΡΙΡ5Κ1 α on cyclin Dl , cyclin El , and (Figure 12F) cyclin A2, CDKl , and cyclin Bl or (Figure 12G, H) phosphorylated FAK, Twist, VEGF and MMP9 in PNT1A cells. Figure 121. The number of invaded cells is indicated. Data is presented as an average of triplicates (±SD). *P= 0.014. Figure 12J. Immunoblots show the expression of AR in PNT1A cells overexpressing ΡΙΡ5Κ1 α or control vector. Figure 12K. Cytoplasmic (Cyt) and nuclear (Nuc) fractions were separated from PNT1A cells overexpressing ΡΙΡ5Κ1 α. Cells were subjected to immunoprecipitation (IP) assay as shown in the right panel. Antibody to CDKl was used to pull down the immunocomplexes, and antibody to IgG was used as a negative control. Antibodies against AR or CDKl were used for immunoblot analysis (IB). The cell lysates from cytoplasmic and nuclear fractions were used as "Input" controls as indicated in the left panel. Blotting of actin served as loading control, and antibody against lamin B was used as a control for the nuclear fraction.

[0054] Figure 13A-H. The effect of ISA-201 1B on ΡΙΡ5Κ1α and its downstream regulators in androgen-sensitive LNCaP cells. Figure 13 A. LNCaP cells were treated with docetaxel at 50 nM and ISA-201 1B (20 μΜ) and the lysates were subjected to immunoblot analysis. Antibodies against ΡΙΡ5Κ1α and β-Actin were used. Figure 13B. LNCaP cells were treated with DMSO as vehicle control (Control), etoposide (20 μΜ), ISA-201 1A (20 μΜ), ISA-201 1B (20 μΜ), tadalafil (20 μΜ), and docetaxel (DTX) at 50 nM. Quantification of the immunoblots of phosphorylated AKT in LNCaP cells treated with vehicle control or ISA-201 1B are shown. Data are representative of three independent experiments. *P < 0.05. Figure 13C. Representative immunofluorescent images show the expression and subcellular localization of phosphorylated AKT in LCaP cells treated with vehicle control or ISA-201 1B. Figure 13D. Immunoblots show the expression of phosphorylated CREB in LCaP cells treated with different agents as indicated. Figure 13E. Representative immunofluorescent images show the expression and subcellular localization of CDKl in LCaP cells treated with vehicle control or ISA-201 IB. Figure 13F. LNCaP cells were treated with docetaxel (50 nM), ISA-2009, ISA- 2011 A, and ISA-201 IB (20 μΜ). The lysates were subjected to immunoblot analysis using antibodies against P27 and SKP2 as probes. Figure 13G. Representative immunofluorescent images show the expression and subcellular localization of AR and PSA in LNCaP cells treated with vehicle control or ISA-201 IB. Figure 13H. Immunoblots show the expression of AR and PSA in LNCaP cells that were treated with docetaxel (50 nM), ISA-2009, ISA-201 1A, and ISA-201 IB (20 μΜ).

[0055] Figure 14A-J. ISA-201 1 inhibits tumor invasion through inhibition PIP5K la-mediated cancer cell pathways. Holomonitor M3 imaging system was applied to capture living PC-3 cells in culture during treatment. Figure 14A. Representative microphotographs of PC-3 cells treated with vehicle control, etoposide (50 μΜ), docetaxel (100 nM), and ISA-201 IB (50 μΜ) for 48 hours are shown. The scale bar is indicated. Figure 14B. Representative immunofluorescent images show the expression and subcellular localization of PIP3 in PC-3 cells treated with vehicle control or ISA- 201 IB. The scale bar is indicated. Figure 14C. PC-3 cells were treated with vehicle control (DMSO), 50 μΜ of etoposide, ISA-2011 A, ISA-201 IB, tadalafil, and docetaxel (DTX) at 100 nM for 48 hours. The protein lysates were subjected to immunoblots. Antibodies to phosphorylated AKT and total AKT were used as probes. Data is representative of three independent experiments. *P < 0.05. (Figure 14D and E) Immunoblots shows the expression of the key cell cycle proteins in PC-3 cells treated with different agents as indicated. Figure 14F. Cell cycle distribution of PC-3 cells treated with vehicle control or ISA-2011B. Figure 14G. Representative FACS plots show the apoptosis status of cells treated with DMSO as vehicle control, docetaxel (DTX) or ISA-2011 (left panel). Data is representative of three independent experiments. **P < 0.01 (right panel). Figure 14H. Adhesion assay of PC-3 cells treated with DMSO as vehicle control, etoposide, and ISA-201 1B. Plates were coated with fibronectin prior to seeding the cells. Mean number of adherent cells in control is 1.62 xl O⁴; mean adherent cells treated with ISA-201 1B is 0.63 xlO⁴ (difference = 0.99 xlO⁴; 95% CI = 0.53 xlO⁴ to 0.77 xl O⁴; P= 0.002). Data is representative of three independent experiments. Upper 95% confidence intervals are shown. **P < 0.01 (right panel). Figure 141. Invasion assays of PC-3 cells treated with DMSO as vehicle control or ISA-201 1B. Figure 14J. Immunoblots of phosphorylated FAK in PC-3 cells after 48 hour treatment with DMSO as vehicle control, docetaxel (DTX) at 500 nM, ISA-201 1A, and ISA-201 IB (20 μΜ' and 50μΜ^Λ).

[0056] Figure 15A-I. The effect of ΡΙΡ5Κ1α inhibition on AKT pathways in PC-3 cells. Figure 15A and B. ΡΙΡ5Κ1α was depleted by transfecting PC-3 cells with ΡΙΡ5Κ1α siRNA (KD) or scramble control (Ct). Immunoblots for ΡΙΡ5Κ1α and phosphorylated AKT in PC-3 cells that were transfected with ΡΙΡ5Κ1α siRNA (KD) or scramble control (Ct) are shown. Data is presented as average of three independent experiments (±SD). **P <0.01. Figure 15C and D. Immunoblots showing the effect of ΡΙΡ5Κ1α knockdown on cyclin Dl, cyclin A2, CDK1, and cyclin B l in PC-3 cells. Figure 15E. Morphology of PC-3 cells expressing siRNA to ΡΙΡ5Κ1α and PNT1A cells overexpressing ΡΙΡ5Κ1 α is shown. Antibody to alpha-tubulin was used for staining. The scale bar is indicated. Figure 15F. Immunoblots of phosphorylated FAK in PC-3 cells transfected with ΡΙΡ5Κ1 α siRNA or scramble control are shown. Data is presented as average of three independent experiments (±SD). Mean expression of phosphorylated FAK in control was 15.30, and in ΡΙΡ5Κ1α knockdown was 5.66 (difference = 9.58; 95% CI = 4.89 to 6.42; P < 0.001). Figure 15G. Immunoblots of P27, Twist, and MMP9 in PC-3 cells transfected with ΡΙΡ5Κ1α siRNA or scramble control are shown. Figure 15H. Immunoblots of c-PARP in PC-3 cells treated with vehicle control (DMSO) or ISA-201 IB (20 μΜ' and 50 μΜ^Λ), with or without ΡΙΡ5Κ1α or scramble siRNA co-transfection are shown. Figure 151. Subcutaneous xenografts were established by implanting PC-3 cells expressing control scramble siRNA or PIP5Kla-siRNA into nude mice. The tumors were measured on day 6 after implantation and were followed for a total of 20 days (n = 7 mice per group). Two groups of mice bearing PC3 cells that expressed control siRNA (Control) or PIP5Kl a-siRNA (KD-PIP5Kla) are indicated. Difference in tumor growth between the two groups is calculated and **P =0.008 is indicated. [0057] Figure 16. Schematic model shows the downstream signaling pathway of ΡΙΡ5Κ1α and the effect of ISA-2011B inhibition. The model depicts ΡΙΡ5Κ1α as the key kinase responsible for generation of PIP2. In turns, PIP2 serves as a precursor molecule required for PI3K activity and generation of PIP3. PIP3 is involved in the downstream activation of AKT, and AKT -related signaling pathways. ISA-2011B is able to enter into the cell, and inhibit ΡΙΡ5Κ1α. This leads to the reduced production of PIP2 and PIP3 and a subsequent inhibition of PI3K/AKT, but sustained P27 and down-regulation of CDK1 and other cell cycle regulators. Inhibition of AR signaling pathways is mediated through CDK1 in part via feedback loop.

[0058] Figure 17A-D. Effect of ISA-2011B in various aggressive cancer cell lines. HTC-1 16 (Figure 17A) and CaCo2 (Figure 17B) colon cancer cells, MDA-MB-231 breast cancer cells (Figure 17C), and U937 leukemic cells (Figure 17D) were purchased from American Type Culture Collection. The cells were treated with vehicle control (DMSO) or ISA-2011B dissolved in 100% DMSO. The treatments were carried out for 48 h, at 10 μΜ, 50 μΜ, and 100 μΜ of ISA-201 IB. The figure shows the viable cell count for every treatment group.

[0059] Figure 18. Gene alteration profiles in primary and metastatic patient samples. Gene alteration profiles for PIP5K1A, AKT2, AR, and PTEN in primary (n = 188) and metastatic (n = 37) patients. The proportion of alterations is indicated. The types of alterations, including amplification, mutation, mRNA up-regulation, mRNA down-regulation, and homozygous deletion, are indicated.

[0060] Figure 19. Correlation between level of PTEN mRNA expression and disease-free survival in PCa patients. Kaplan-Meier survival analysis based on biochemical recurrence-free (BCR-free) in PCa patients with high or low PTEN expression. PCa patients with a lower level of PTEN in their tumors (n = 21) had significantly poorer disease-free survival than those with a higher level (n = 175). The follow-up time is 160 months, as indicated. Differences in BCR-free survivals between two groups were calculated using the log-rank test, P < 0.001.

[0061] Figure 20. Effect of ΡΙΡ5Κ1α overexpression on AR in PNT1A cells. Representative immunofluorescent images show the expression and localization of AR in PNT1A cells overexpressing ΡΙΡ5Κ1α or control vector. The cells were stained with antibody to AR (green) and DAPI (blue). The merge of these two images is shown.

[0062] Figure 21. Interaction between CDK1 and AR in PNT1A cells. Cytoplasmic (Cyt) and nuclear (Nuc) fractions were separated from PNT1A cells expressing pPLPS Control vector and were subjected to immunoprecipitation (IP) assay as shown (Right). Antibody to CDK1 was used to pull down the immunocomplexes, and antibody to IgG was used as a negative control. Antibodies against AR or CDK1 were used for immunoblot analysis (IB) to detect AR or CDK1 in cytoplasmic or nuclear compartment. The cell lysates from cytoplasmic and nuclear fractions were used as "Input" controls, as indicated (Left). Blotting of actin served as loading control, and antibody against lamin B was used as a control for the nuclear fraction.

[0063] Figure 22. Effect of ISA-2011B in the androgen-dependent LNCaP cell line. LNCaP prostate cancer cells were purchased from American Type Culture Collection. The cells were treated with vehicle control (DMSO) or ISA-2011B dissolved in 100% DMSO. The treatments were carried out for 48 h, at 10 μΜ, 50 μΜ, and 100 μΜ of ISA-201 IB. The figure shows the viable cell count for every treatment group.

DETAILED DESCRIPTION OF THE INVENTION

[0064] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry and nucleic acid chemistry and hybridization are those well known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references, which are provided throughout this document. The nomenclature used herein and the laboratory procedures in analytical chemistry, and organic synthetic described below are those well known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

I. PIP5K1A Inhibitor for Treatment of Diseases

[0065] In one aspect, the present invention provides compositions and methods related to using inhibitors of PIP5K1A for treatment of diseases.

A. PIP5K1A

[0066] PIP5K1A catalyses the phosphorylation of phosphatidylmositol 4- phosphate (PtdIns4P) to form phosphatidylmositol 4,5-bisphosphate (PtdIns(4,5)P2). PtdIns(4,5)P2 is involved in a variety of cellular processes and is the substrate to form phosphatidylmositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), another second messenger. The majority of PtdIns(4,5)P2 is thought to be produced via type I phosphatidylmositol 4-phosphate 5-kinases (PIP5Ks) given the abundance of PtdIns4P. In addition, PIP5K1A can catalyse the phosphorylation of phosphatidylmositol 3,4- bisphosphate (PtdIns(3,4)P2) to form phosphatidylmositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). PIP5K1A has been known as one of the PIP5Ks that participates in a variety of cellular processes such as actin cytoskeleton organization, cell adhesion, migration and phagocytosis. PIP5K1A is required for membrane ruffling formation, actin organization and focal adhesion formation during directional cell migration by controlling integrin-induced translocation of RAC1 to the plasma membrane. PIP5K1A , together with PIP5K1C, is required for phagocytosis, but they regulate different types of actin remodeling at sequential steps. PIP5K1A promotes particle ingestion by activating WAS that induces Arp2/3 dependent actin polymerization at the nascent phagocytic cup. PIP5K1A, together with PIP5K1B, is required after stimulation of G-protein coupled receptors for stable platelet adhesion. PIP5K1A plays a role during calcium-induced keratinocyte differentiation. PIP5K1A is recruited to the plasma membrane by the E-cadherin/beta-catenin complex where it provides the substrate PtdIns(4,5)P2 for the production of PtdIns(3,4,5)P3, diacylglycerol and inositol 1,4,5-trisphosphate that mobilize internal calcium and drive keratinocyte differentiation. PIP5K1A, together with PIP5K1C, also have a role during embryogenesis. PIP5K1A is highly expressed in heart, placenta, skeletal muscle, kidney and pancreas. PIP5K1A is detected at lower levels in brain, lung and liver. Four isoforms of the human protein PIP5K1 A are produced by alternative splicing.

[0067] In one aspect, the present invention provides a method for treatment of a disease in a subject in which inhibition of PIP5K1A is desired and/or required. The method comprises administering a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5KlA.

[0068] Without being bound by any particular theory, it is noted that PIP5K1A is a regulator of multiple signal transduction pathways that involved in various diseases, such as cancer. Exemplary paths are the PDK/Akt pathway and the phospholipase C (PLC) pathway.

[0069] The expression level and/or activity in the cells of disease tissues may be normal or more likely, higher than normal (over-expressed). Nevertheless, as over activation of many signal transduction pathways, such as the PDK/Akt pathway and the PLC pathway, are involved in various diseases conditions, such as cancer, downregulation of PIP5K1A activity reduces the activity of these pathways. Downregulation of PI3K/Akt and/or PLC pathway activity in turn results in reversion or curing of disease conditions.

[0070] In certain disease conditions, such as cancer, PIP5K1A is over-expressed. For example, PIP5K1A is over-expressed in prostate cancer patient tissues. Moreover, its expression level correlates with cancer grade: very low in benign prostatic hyperplasia (BPH) patients, low in low grade prostate cancer patients and at the highest level in high grade metastatic prostate cancer patients. See Figure 5.

[0071] Statistical analysis of PIP5K1A expression level in benign patients and malignant prostate cancer patients showed that there is a statistically significant difference between PIP5K1A levels in benign and malignant prostate cancer patients (P<0.001). See Figure 6.

[0072] Both AR and PIP5K1A are over-expressed in malignant prostate cancer patients. There is a strong and statistically significant correlation between AR and PIP5K1 A expression with correlation coefficient at 0.633 (PO.01). See Figure 7A-C. [0073] PIP5K1A is also over-expressed in lung cancer patient tissues and its expression level correlates with cancer grade: very low in healthy subjects, low in non-invasive Lung Squamous Cell Carcinoma and very high in invasive Non-Small Cell Lung Cancer patients. See Figure 8.

[0074] As described in more details herein, the present invention provides various inhibitors of PIP5K1 A that can be used to reduce PIPKIA activity.

[0075] Reduction of PIP5K1A activity can be measured either as relative to the level of over- expression or relative to the level of normal expression, as determined by an in vitro or in vivo assay.

[0076] In some embodiments, PIP5K1A activity is reduced by about 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% in comparison to the over-expression level.

[0077] In some embodiments, the inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 5% and 100% as determined by an in vitro or in vivo assay.

[0078] In some embodiments, the inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 40% and 80% as determined by an in vitro or in vivo assay.

[0079] In some embodiments, inhibitor of PIP5K1A is capable of reducing PIP5K1A activity to 1 , 2, 3, 4, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,1 15, 120, 125, 130, 135, 140, 145, 150, 155, or 160 % of normal PIP5K1A activity as determined by an in vitro or in vivo assay.

[0080] In some embodiments, inhibitor of PIP5K1A is capable of reducing PIP5K1A activity to about 1 % and 150% of normal PIP5K1A activity of as determined by an in vitro or in vivo assay.

[0081] In some embodiments, inhibitor of PIP5K1A is capable of reducing PIP5K1A activity to between about 50 % and 150% of normal PIP5K1A activity of as determined by an in vitro or in vivo assay.

[0082] By "PIP5K1A activity" herein is meant the mRNA expression level, protein expression level, or kinase activity of PIPKIA. Measurement of mRNA expression level, protein expression level, or kinase level is carried out using methods known in the art and/or disclosed herein.

B. PIP5K1A Modulators

[0083] In another aspect, the present invention provides various PIP5K1A modulators obtained using the methods provided herein.

[0084] By "PIP5K1A" herein is meant phosphatidylinositol-4-phosphate 5-kinase, type I, alpha. The DNA and protein sequences of PIPKIA from various species are known in the art and can be obtained from various public domain sources, such as GeneBank. Preferably, the PIP5K1A used here is of a mammal, preferably of human, rat, or mice. PIP5K1A nucleic acids (e.g. cDNA or genomic DNA) is synthesized or cloned from various sources and used to generate transgenic cells or animals, or to generate PIP5K1 A polypeptide or a fragment thereof to be used in the assays provided herein.

[0085] By "PIP5K1A modulator" herein is meant a molecule that can affect the expression (mRNA level or protein level) and/or the activity (e.g., kinase activity) of PIP5K1A, as determined by an in vivo or in vitro assay. A modulator can either decrease (inhibitor) or enhance (activator) the expression and/or the activity of PIP5K1 A. Preferably, the modulator is an inhibitor.

[0086] The methods of the invention utilize PIP5K1A polypeptides, antibodies or nucleic acids which encode PIP5K1A polypeptides for identifying candidate bioactive agents which bind to PIP5K1A, which modulate the activity of PIP5K1A, or which alter the expression of PIP5K1A within cells.

[0087] By "candidate bioactive agent" herein is meant any molecule which binds to PIP5K1A, modulates the kinase activity of PIP5K1A, and/or alters the expression of PIP5K1A within cells. A molecule, as described herein, can be an oligopeptide, small organic molecule, polysaccharide, antibody or polynucleotide, etc. Generally a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.

[0088] Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons (D).

[0089] Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.

[0090] Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.

[0091] In some embodiments, the candidate bioactive agents are proteins. By "protein" herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For example, homo- phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes amino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.

[0092] In some embodiments, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of multicellular eukaryotic proteins may be made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of multicellular eukaryotic proteins, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.

[0093] In some embodiments, the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.

[0094] In some embodiments, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.

[0095] In some embodiments, the candidate bioactive agents are nucleic acids.

[0096] As described above generally for proteins, nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes may be used as is outlined above for proteins.

[0097] In some embodiments, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.

[0098] In some embodiments, short RNA (RNAi), anti-sense RNAs, microRNMs (miRNA) and DNAs can be used as therapeutic agents for blocking the expression of certain PIP5K1A genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane (Zamecnik et al., (1986), Proc. Natl. Acad. Sci. USA 83:4143-4146). The anti- sense oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups. In some embodiments, PIP5K1A anti-sense RNAs and DNAs can be used to prevent PIP5K1A gene transcription into mRNAs, to inhibit translation of PIP5K1A mRNAs into proteins, and to block activities of pre-existing PIP5K1A proteins.

[0099] As used herein, a multivalent cation indicator is a molecule that is readily permeable to a cell membrane or otherwise amenable to transport into a cell e.g., via liposomes, etc., and upon entering a cell, exhibits a fluorescence that is either enhanced or quenched upon contact with a multivalent cation. Examples of multivalent cation indicators useful in the invention are set out in Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals. 1 1th ed. Molecular Probes, Inc. Eugene, OR (2013); incorporated herein by reference in its entirety.

[0001] In some embodiments for binding assays, either PIP5K1A or the candidate bioactive agent is labelled with, for example, a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide means of detecting the binding of the candidate agent to PIP5K1A. The label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that can be detected. Alternatively, the label can be a labelled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalysed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labelled as described above, thereby, providing a detectable signal for the bound PIP5K1A. As known in the art, unbound labelled streptavidin is removed prior to analysis. Alternatively, PIP5K1A can be immobilized or covalently attached to a surface and contacted with a labelled candidate bioactive agent. Alternatively, a library of candidate bioactive agents can be immobilized or covalently attached to a biochip and contacted with a labelled PIP5K1A. Procedures which employ biochips are well known in the art.

[0101] In some embodiments, the kinase activity PIP5K1A is measured in intact cells, preferably PC-3 cells, which have high PIP5K1A activity.

[0102] In some embodiments for screening for candidate bioactive agents which modulate expression levels of PIP5K1A within cells, candidate agents can be used which wholly suppress the expression of PIP5K1A within cells, thereby altering the cellular phenotype. In a further preferred embodiment, candidate agents can be used which reduce the expression of PIP5K1A within cells, thereby altering the cellular phenotype. Examples of these candidate agents include antisense cDNAs and DNAs, regulatory binding proteins and/or nucleic acids, as well as any of the other candidate bioactive agents herein described which modulate transcription or translation of nucleic acids encoding PIP5K1A.

[0103] In some embodiments, the invention provides antibodies which specifically bind to unique epitopes on the human PIP5K1 A polypeptide, e.g. unique epitopes of the protein.

[0104] In another embodiment, the invention provides antibodies which specifically bind to unique epitopes on the mouse PIP5K1A polypeptide, e.g. unique epitopes of the protein.

[0105] The anti-PIP5Kl A polypeptide antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the PIP5K1A polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

[0106] The anti-PIP5KlA polypeptide antibodies may further comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. [0107] The immunizing agent will typically include the PIP5K1A polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which prevent the growth of HGPRT-deficient cells.

[0108] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Rockville, Md. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

[0109] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against a PIP5K1A polypeptide. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme- linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0110] After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

[0111] The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0112] The monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S. Pat. No. 4,816,567; Morrison et al., supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non- immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

[0113] The anti-PIP5KlA polypeptide antibodies may further comprise monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

[0114] The anti-PIP5KlA polypeptide antibodies may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fabi, F(abi)₂ or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non- human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. I n some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

[0115] Methods for humanizing non -human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0116] Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by the introducing of human immunoglobulin loci into transgenic animals, e.g. mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661 ,016, and in the following scientific publications: Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

[0117] The anti-PIP5KlA polypeptide antibodies may further comprise heteroconjugate antibodies. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Pat. No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0118] In a further embodiment, the anti-PIP5KlA polypeptide antibodies may have various utilities. For example, anti-PIP5KlA polypeptide antibodies may be used in diagnostic assays for PIP5K1A polypeptides, e.g., detecting its expression in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in the diagnostic assays can be labelled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as .sup.3H, .sup. l4C, .sup.32P, .sup.35S, or .sup.1251, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta- galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13 : 1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).

[0119] In some embodiments, the PIP5K1A inhibitor comprises a small interference RNA (siRNA) and modulates kinase expression through RNA interference (RNAi). The siRNA is designed and produced using methods known in the art.

[0120] Figure 1 depicts depletion of PIP5K1A by RNAi in prostate cancer PC-3 cells. PIP5K1A expression was completely blocked by RNAi treatment at 48 and 72 hrs.

[0121] Figures 2A and 2B depict inhibition of Akt phosphorylation by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. Akt phosphorylation and activation were significantly inhibited by blocking PIP5K1A expression with RNAi treatment.

[0122] Figure 3 A and 3B depict inhibition of expression of Akt downstream effector cyclin Dl by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. Cyclin Dl expression was significantly inhibited by blocking PIP5K1 A expression with RNAi treatment.

[0123] Figure 4 depicts increased expression of Akt downstream negative effector p27 by PIP5K1A RNAi treatment in prostate cancer PC-3 cells. P27 expression was significantly increased by blocking PIP5K1A expression with RNAi treatment. 3. PBK/Akt Pathway

[0124] In another aspect, the present invention provides a method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising: administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.

[0125] The PI3K/AKT/mTOR pathway is an intracellular signalling pathway important in cell proliferation, apoptosis and angiogenesis, and hence in cancers, e.g. breast cancer and non-small-cell lung cancer. PI3K activation activates AKT which activates mTOR. In many cancers this pathway is overactive thus increasing cancer cell proliferation, reducing cancer cell apoptosis, and promoting tumor angiogenesis and metastasis. Thus some experimental cancer drugs aim to inhibit the signalling pathway of PDK/AKT/mTOR. The PI3K pathway may be overactive because PTEN is faulty or deficient.

[0126] As disclosed herein, in fact, suppression of PIP5K1A using PIP5K1A inhibitor results in lower activity of Akt. See Example 2.

[0127] Akt, also known as Protein Kinase B (PKB), is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration. Akt has three family members, Aktl, Akt2 and Akt3.

[0128] Akt is associated with tumor cell survival, proliferation, and invasiveness. The activation of Akt is also one of the most frequent alterations observed in human cancer and tumor cells. Tumor cells that have constantly active Akt may depend on Akt for survival. A mosaic activating mutation (c. 49G→A, p.Glul7Lys) in AKT1 is associated with the Proteus Syndrome, which causes overgrowth of skin, connective tissue, brain and other tissues. Akt inhibitors may treat cancers such as neuroblastoma. In addition, miltefosine is approved for leishmaniasis and under investigation for other indications including HIV.

[0129] AKT activation is associated with many malignancies, however, and it has been observed a converse role for AKT and one of its downstream effector FOXOs in acute myeloid leukemia (AML). It was claimed that low levels of AKT activity associated with elevated levels of FOXOs are required to maintain the function and immature state of leukemia-initiating cells (LICs). FOXOs are active, implying reduced Akt activity, in about 40% of AML patient samples regardless of genetic subtype; and either activation of Akt or compound deletion of FOXO 1/3/4 reduced leukemic cell growth in a mouse model.

[0130] By "Akt activity" herein is meant the mRNA expression level, protein expression level, or kinase activity of Akt. Measurement of mRNA expression level, protein expression level, or kinase level is carried out using methods known in the art and/or disclosed herein. [0131] Reduction of Akt activity can be measured either as relative to the level of over-expression or relative to the level of normal expression, as determined by an in vitro or in vivo assay.

[0132] In some embodiments, Akt activity is reduced by about 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% in comparison to the over-expression level.

[0133] In some embodiments, inhibitor of PIP5K1A is capable of reducing Akt activity by between about 5% and 100% as determined by an in vitro or in vivo assay.

[0134] In some embodiments, inhibitor of PIP5K1A is capable of reducing Akt activity by between about 40% and 80% as determined by an in vitro or in vivo assay.

[0135] In some embodiments, inhibitor of PIP5K1A is capable of reducing Akt activity to 1 , 2, 3, 4, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10,1 15, 120, 125, 130, 135, 140, 145, 150, 155, or 160 % of normal Akt activity as determined by an in vitro or in vivo assay.

[0136] In some embodiments, inhibitor of PIP5K1A is capable of reducing Akt activity to about 1 % and 150% of normal Akt activity of as determined by an in vitro or in vivo assay.

[0137] In some embodiments, inhibitor of PIP5K1A is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity of as determined by an in vitro or in vivo assay.

[0138] In some embodiments, inhibitor of PIP5K1A is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity of as determined by an in vitro or in vivo assay.

[0139] In a further aspect, the present invention provides method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of modulator of PDK/Akt pathway, wherein the modulator is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity as determined by an in vitro or in vivo assay.

[0140] In some embodiments, the inhibitor of PIP5K1A is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity of as determined by an in vitro or in vivo assay.

[0141] In some embodiments, the modulator of PDK/Akt pathway comprises a small molecule, an RNA, a peptide, or an antibody.

[0142] In some embodiments, the modulator of PDK/Akt pathway comprises a PIP5K1A inhibitor as provided herein.

[0143] In some embodiments, the modulator of PDK/Akt is obtained using methods similar to the methods provided herein for obtaining PIP5klA modulator, as a person skilled in the art will appreciate. [0144] In a further aspect, the present invention provides a method for treatment of a disease in a subject characterized by over activation of PI3 Kinase and is in need of such treatment, comprising: administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.,

[0145] Phosphatidylinositide 3-kinases (PI 3-kinases or PBKs) are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. PBKs are a family of related intracellular signal transducer enzymes capable of phosphorylating the 3 position hydroxyl group of the inositol ring of phosphatidylmositol (Ptdlns). They are also known as phosphatidylinositol-3- kinases. Specifically, PI3K catalyses the phosphorylation of phosphatidylmositol 4,5-bisphosphate (PtdIns(4,5)P2) to form phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2). Phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) then activates Akt and a myriad of downstream effectors. This pathway, with oncogene PIK3CA and tumor suppressor PTEN (gene), is implicated in tumorigenesis of many cancers, and insensitivity of cancer tumors to insulin and IGF1 in calorie restriction.

[0146] Reduction of the enzymatic substrates of PI3K, such as phosphatidylmositol 4,5- bisphosphate (PtdIns(4,5)P2), can lead to reduction in production of its enzymatic products, such as phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2), thus can be used to reduce PI3K activity. Such reduction of PI3K activity can be measured by level of its enzymatic products, such as phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2), either as relative to the level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K is over-expressed or relative to the level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.

[0147] In general, PIP5K1A inhibitor may not directly inhibit PI3K activity, but rather inhibits production of PI3K enzymatic substrate phosphatidylmositol 4,5-bisphosphate (PtdIns(4,5)P2, so it cannot catalyse production of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2). As such, reduction of PI3K activity generally is measured by level of its enzymatic products, such as phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2), either as relative to the level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K is over-expressed or relative to the level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.

[0148] In some embodiments, production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) is reduced by about 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% in comparison to production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K is over-expressed, as determined by an in vitro or in vivo assay. [0149] In some embodiments, inhibitor of PIP5K1A is capable of reducing production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) to between about 1 , 2, 3, 4, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10,115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 % of normal production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.

[0150] In some embodiments, inhibitor of PIP5K1A is capable of reducing production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) to between about 1 % and 150% of normal production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.

[0151] In some embodiments, inhibitor of PIP5K1A is capable of reducing production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) to between about 50 %> and 150%o of normal production level of phosphatidylmositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.

4. PLC Pathway

[0152] In a further aspect, the present invention provides method for treatment of a disease in a subject characterized by over activation of phospholipase C (PLC) and is in need of such treatment, comprising: administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.

[0153] Phospholipase C (PLC) is a class of enzymes that cleave phospholipids just before the phosphate group. It is most commonly taken to be synonymous with the human forms of this enzyme, which play an important role in eukaryotic cell physiology, in particular signal transduction pathways. Thirteen types of mammalian phospholipase C are classified into six isotypes (β, γ, δ, ε, ζ, η) according to structure.

[0154] Reduction of PLC activity can be measured either as relative to the level of over-expression or relative to the level of normal expression, as determined by an in vitro or in vivo assay.

[0155] In some embodiments, PLC activity is reduced by about 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%o in comparison to the over-expression level.

[0156] In some embodiments, inhibitor of PIP5K1A is capable of reducing PLC activity by between about 5% and 100% as determined by an in vitro or in vivo assay.

[0157] In some embodiments, inhibitor of PIP5K1A is capable of reducing PLC activity by between about 40% and 80% as determined by an in vitro or in vivo assay.

[0158] In some embodiments, inhibitor of PIP5K1A is capable of reducing PLC activity to 1 , 2, 3, 4, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10,115, 120, 125, 130, 135, 140, 145, 150, 155, or 160 % of normal PLC activity of as determined by an in vitro or in vivo assay.

[0159] In some embodiments, inhibitor of PIP5K1A is capable of reducing PLC activity to about 1 % and 150% of normal PLC activity of as determined by an in vitro or in vivo assay.

[0160] In some embodiments, inhibitor of PIP5K1A is capable of reducing PLC activity to between about 50 % and 150% of normal PLC activity of as determined by an in vitro or in vivo assay.

[0161] By "PLC activity" herein is meant the mRNA expression level, protein expression level, or lipase activity of PLC. Measurement of mRNA expression level, protein expression level, or lipase level is carried out using methods known in the art and disclosed herein.

[0162] In some embodiments, inhibitor of PIP5K1A is capable of reducing PLC activity to between about 50 % and 150% of normal PLC activity of as determined by an in vitro or in vivo assay.

[0163] In some embodiments, inhibitor of PIP5K1A is capable of reducing PLC activity to between about 80 % and 120% of normal PLC activity of as determined by an in vitro or in vivo assay.

//. Diagnosis, Prognosis, Selection of Patients, and Biomarker

[0164] In one aspect, the present invention provides compositions and methods related to using PIP5K1A gene as a biomarker for diagnosis, prognosis, or monitoring of various diseases (preferably cancer) and selection of patients for treatment with certain diseases.

A. Diagnosis

[0165] In one aspect, the present invention provides a method of diagnosing a subject suspect of having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of one or more genes in the biological sample, wherein at least one of said one or more genes is PIP5K1A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to diagnosis of the disease, thereby diagnosing the subject with regard to the disease.

B. Biological sample

[0166] By "biological sample" herein is meant any biological sample suspected of containing a target gene and/or gene product to be detected (e.g. PIP5K1A), such as polynucleotides or polypeptides or fragments thereof, and may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support), protein (in solution or bound to a solid support such as for western blot analysis), peptide (in solution or bound to a solid support such as for western blot analysis), an extract from cells, blood, urine, marrow, body fluid, or a tissue, and the like.

[0167] Biological samples useful in the practice of the methods of the invention may be obtained from any mammal in which a diseases condition is suspected, confirmed, or developing. In one embodiment, the mammal is a human, and the human may be a candidate for a therapeutic for the treatment of a cancer, e.g. prostate cancer. The human candidate may be a patient currently being treated with, or considered for treatment with, a PIP5K1 A inhibitor, such as those provided herein. In another embodiment, the mammal is large animal, such as a horse or cow, while in other embodiments, the mammal is a small animal, such as a dog or cat, all of which are known to develop cancers, including lung carcinomas.

[0168] Any biological sample comprising cells (or extracts of cells) from a mammalian cancer is suitable for use in the methods of the invention. Circulating tumor cells may also be obtained from serum using tumor markers, cytokeratin protein markers or other methods of negative selection as described (see Ma et al., Anticancer Res. 23(1A): 49-62 (2003)). Serum and bone marrow samples may be particularly preferred for patients with leukemia. For cancers involving solid tumors, such as sarcomas and carcinomas, the biological sample may comprise cells obtained from a tumor biopsy, which maybe be obtained according to standard clinical techniques.

[0169] Circulating tumor cells ("CTCs") may be purified, for example, using the kits and reagents sold under the trademarks VITA-ASSAYS™, VITA-CAP™, and CELLSEARCH® (commercially available from Vitatex, LLC (a Johnson and Johnson corporation). Other methods for isolating CTCs are described (see, for example, PCT Publication No. WO/2002/020825, CristofaniUi et al., New Engl. J. of Med. 351 (8):781-791 (2004), and Adams et al., J. Amer. Chem. Soc. 130(27): 8633-8641 (July 2008)). In a particular embodiment, a CTC may be isolated and identified as having originated from the lung.

C . Detection of PIP5 K 1 A Polypeptide

[0170] In some embodiments, the PIP5K1A Polypeptide is detected by an immunoassay. An PIP5K1A protein or peptide is generated to produce antibodies (monoclonal or polyclonal) specific for PIP5K1A proteins. Such antibodies are then used in an assay to detect the presence of PIP5K1A.

[0171] PIP5K1A is generally detected using a PIP5K1A polypeptide-specific reagent. By "PIP5K1A polypeptide-specific reagent" herien is meant any reagent, biological or chemical, capable of specifically binding to, detecting and/or quantifying the presence/level of expressed PIP5K1A polypeptide in a biological sample. The term includes, but is not limited to, the preferred antibody and reagents discussed below, and equivalent reagents are within the scope of the present invention. [0172] Reagents suitable for use in practice of the methods of the invention include an PIP5K1A polypeptide-specific antibody.

[0173] Human PIP5K1A polypeptide-specific antibodies may also bind to highly homologous and equivalent epitopic peptide sequences in other mammalian species, for example murine or rabbit, and vice versa. Antibodies useful in practicing the methods of the invention include (a) monoclonal antibodies, (b) purified polyclonal antibodies that specifically bind to the target polypeptide (e.g. the PIP5K1A polypeptide), (c) antibodies as described in (a)-(b) above that bind equivalent and highly homologous epitopes or phosphorylation sites in other non-human species (e.g. mouse, rat), and (d) fragments of (a)-(c) above that bind to the antigen (or more preferably the epitope) bound by the exemplary antibodies disclosed herein

[0174] By "antibody" or "antibodies" herein is meant all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. The antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-1 1 (1989); Morrision et al., Proc. Nat'l. Acad. Sci. 81 : 6851 (1984); Neuberger et al., Nature 312: 604 (1984)). The antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No. 4,816,567 (Cabilly et al.). The antibodies may also be chemically constructed specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel et al.)

[0175] The invention is not limited to use of antibodies, but includes equivalent molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a fusion-protein or truncated-protein specific manner, to essentially the same epitope to which an PIP5K1A polypeptide-specific antibody in the methods of the invention binds. See, e.g., Neuberger et al., Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods of the invention further described below.

[0176] Polyclonal antibodies useful in practicing the methods of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing a desired fusion-protein specific epitope (e.g. the fusion junction of an Rspo fusion protein described herein), collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, and purifying polyclonal antibodies having the desired specificity, in accordance with known procedures. The antigen may be a synthetic peptide antigen comprising the desired epitopic sequence, selected and constructed in accordance with well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czernik, Methods In Enzymology, 201 : 264-283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21 -49 (1962)). Polyclonal antibodies produced as described herein may be screened and isolated as further described below.

[0177] Further still, U.S. Pat. No. 5,194,392, Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) that is a topological equivalent of the epitope (i.e., a "mimotope") that is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, this method involves detecting or determining a sequence of monomers that is a topographical equivalent of a ligand that is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Pat. No. 5,480,971 , Houghten et al. (1996) discloses linear Ci-C-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest. Thus, non-peptide analogs of the epitope-bearing peptides of the invention also can be made routinely by these methods.

[0178] Antibodies employed in the methods of the invention may be further characterized by, and validated for, use in a particular assay format, for example flow cytometry (FC), immunohistochemistry (IHC), and/or Immunocytochemistry (ICC). Antibodies may also be advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE), or labels such as quantum dots, for use in multi-parametric analyses along with other signal transduction (phospho-AKT, phospho- Erk 1/2) and/or cell marker (cytokeratin) antibodies.

D . Detection of PIP5 K 1 A Polynucleotide

[0179] PIP5K1A -specific reagents provided by the invention also include nucleic acid probes and primers suitable for detection of a PIP5K1A polynucleotide.

[0180] In some embodiments, the PIP5K1A gene is detected by PCR, such as regular PCR, Reverse Transcription PCR, Real-time PCR (Q-PCR) or digital PCR. A pair of primers is used to amplify the PIP5K1A gene. The primers are designed based on the target gene sequence to be amplified.

[0181] In some embodiments, the PIP5K1A gene is detected by other hybridization-based methods, such as microarray, branched DNA (QUANTIGENE^®), VIEWRNA^® or RNASCOPE^®.

E. Detection of PIP5K1A Overexpression

[0182] In another aspect, the present invention provides compositions and methods for detection of PIP5K1A overexpression. Overexpression of PIP5K1A may or may not co-exist with overexpression or activation of PI3K, Akt, or PLC.

[0183] PIP5K1A overexpression can be overexpression of either PIP5K1A mRNA or polypeptide, or both. The PIP5K1 A can be either wild-type or a variant of PIP5K1 A. [0184] PIP5K1A overexpression is determined relevant to a baseline expression level, which is obtained by measuring expression level of PIP5K1A (mRNA or polypeptide) in normal cells or a normal subject population (e.g., normal human population).

[0185] The expression level of PIP5K1A mRNA level is measured using methods known in the art, such as Northern blot, RT-PCR, RT-PCT combined with Real-time PCR, digital PCR, DNA array, high throughput sequencing, or in situ hybridization, and the like.

[0186] The expression level of PIP5K1A, either at mRNA level or protein level, is measured using methods known in the art, such as Western blot, protein array, immunohistology staining, and the like.

[0187] In general, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient diagnosed as having the disease.

F. Disease Monitoring, Disease Stage Determination, Prognosis

[0188] In one aspect, the present invention provides a method for monitoring disease progression of a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to disease progression of the disease, thereby determining disease progression of the subject with regard to the disease.

[0189] In some embodiments, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having the disease of different disease progression.

[0190] In anther aspect, the present invention provides a method for determining disease stage of a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of the one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of the one or more genes, wherein the reference expression levels correlate to disease stage of the disease, thereby determining disease stage of the subject with regard to the disease.

[0191] In some embodiments, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having said disease of different disease stages. [0192] As disclosed herein, PIP5kl A expression level can be used for determining disease stage of (e.g. cancer grade) as the level correlates to disease stage (e.g. low level in BPH, medium level in low grade prostate cancer, high level in high grade of cancer, highest level in metastatic cancer).

[0193] In another aspect, the present invention provides a method for determining a prognosis for a subject diagnosed as having a disease, comprising: (a) providing a biological sample from a subject; (b) measuring expression levels for one or more genes and/or product of one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of one or more genes, wherein the reference expression levels correlate to prognosis of the disease, thereby determining prognosis of the subject with regard to the disease.

[0194] In general, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient diagnosed as having the disease of different progression stages.

F. Determining Effect of Treatment

[0195] In another aspect, the present invention provides a method of determining effect of treatment of a subject having a disease condition with a pharmaceutical composition, comprising: (a) providing a biological sample from a subject that has been subject to a treatment of a disease; (b) measuring expression levels for one or more genes and/or product of one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of one or more genes, wherein the reference expression levels correlate to treatment effect of the disease, thereby determining the treatment effect of the pharmaceutical composition.

[0196] In general, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient that has been subjected to said treatment and achieved treatment effect.

G. Treatment Selection

[0197] In another aspect, the present invention provides a method of determining whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIP5K1A, and/or an inhibitor of PI3K/Akt pathway, and/or an inhibitor of PLC pathway, comprising: (a) providing a biological sample from a subject t having a disease condition; (b) measuring expression levels for one or more genes and/or product of one or more genes in the biological sample, wherein at least one of the one or more genes is PIP5K1 A gene; and (c) comparing the measured expression levels to reference expression levels for the one or more genes and/or product of one or more genes, wherein the reference expression levels correlate to whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIPK1 A, and/or an inhibitor of PDK/Akt pathway, and/or an inhibitor of PLC pathway, thereby determining whether the subject should be treated with the pharmaceutical composition.

[0198] In general, the reference expression levels are determined using a sample from a normal subject and/or a sample from a patient that has been subjected to said treatment and achieved treatment effect.

EL Additional Biomarker Genes

[0199] In the methods provided in, one or more genes is combined with PIP5KA as a biomarker, and such gene is selected from the group consisting of: Akt, pAkt (phosphorylated Akt), Alpha- fetoprotein (AFP), Beta-2 -microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21, Cytokeratin fragments 21-1 , EGFR mutations, Estrogen receptor (ER)/progesterone receptor (PR), Androgen receptor (AR), Fibrin/fibrinogen, HER4, HER2/neu, Immunoglobulins, KIT, KRAS , mutation analysis, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), BRCAl, ERCCl , TYMS, RRM1 , TUBB3, STMN1 , MDR1, GSTP1 , TOP2A, IGF 1R, VEGFR1/VEGFR2, FGFR1/FGFR2/FGFR3/FGFR4, PTEN, KIT, Cyclin Al , Cyclin Dl, GADD45, DDB2, VEGF, MMP-2, MMP-9, CDK1 , Chkl , FAK, pFAK (phosphorylated FAK), PIK3CA , CYLD, Nflx, Impadl, DCAMKL-1, Casein kinase II alpha (Ck2a), Phosphotidic acid (PA), Racl , RhoA, Ajuba (LIMP), pRB (phosphorylated RB), ARF6, FOXOs and PLD2 .

L Drug Screening

[0002] In another aspect, the present invention provides methods for reducing PI3K/Akt over activation using an inhibitor of PIP5KA. The methods can be used in screening for new inhibitor of PIP5K1A.

[0201] In one aspect, the present invention provides a method for reducing over activation of PI3K/Akt in a subject, comprising administering the subject with an inhibitor of PIP5K1A, thereby reducing over activation of the PI3K/Akt in the subject.

[0202] In another aspect, the present invention provides a method for reducing over activation of PI3K/Akt in a cell, comprising contacting the cell with an inhibitor of PIP5K1A, thereby reducing over activation of the PDK/Akt in the cell.

Medical and Pharmaceutical Uses

[0203] Compounds disclosed herein include small molecules, biologies (RNA, DNA, RNA/DNA hybrid, antibody, peptide and polymer). [0204] Compounds of the invention are indicated as pharmaceuticals. According to a further aspect of the invention, there is provided a compound of the invention, as hereinbefore (but without any provisos, where applicable), for use as a pharmaceutical. There is also provided a synthetic form of a compound of the invention (but without any provisos, where applicable), for use as a pharmaceutical.

[0205] For the avoidance of doubt, although compounds of the invention may possess pharmacological activity as such, certain pharmaceutically-acceptable (e.g. "protected") derivatives of compounds of the invention may exist or be prepared which may not possess such activity, but may be administered, including bot not limited to parenterally or orally and thereafter be metabolized in the body to form compounds of the invention. Such compounds (which may possess some pharmacological activity, provided that such activity is appreciably lower than that of the "active" compounds to which they are metabolized) may therefore be described as "prodrugs" of compounds of the invention.

[0206] By "prodrug of a compound of the invention", we include compounds that form a compound of the invention, in an experimentally-detectable amount, within a predetermined time (e.g. about 1 hour), following an administration, including but not limited to oral or parenteral administration. All prodrugs of the compounds of the invention are included within the scope of the invention.

[0207] Furthermore, certain compounds of the invention may possess no or minimal pharmacological activity as such, but may be administered, including but not limited to parenterally or orally, and thereafter be metabolized in the body to form compounds of the invention that possess pharmacological activity as such. Such compounds (which also includes compounds that may possess some pharmacological activity, but that activity is appreciably lower than that of the "active" compounds of the invention to which they are metabolized), may also be described as "prodrugs".

[0208] Thus, the compounds of the invention are useful because they possess pharmacological activity, and/or are metabolized in the body following an administration, including but not limited to oral or parenteral administration, to form compounds which possess pharmacological activity.

[0209] Compounds of the invention (as hereinbefore defined but without the proviso(s)) may be useful in the treatment of a cancer. By "cancer", we mean any disease that arises from an uncontrolled growth of cells (e.g. uncontrolled division), invasion (e.g. direct growth into adjacent tissue) or metastasis. By "uncontrolled growth", we include an increase in the number and/or size of cancer cells (also referred to herein as "proliferation"). By "metastasis" we mean the movement or migration (e.g. invasiveness) of cancer cells from a primary tumor site in the body of a subject to one or more other areas within the subject's body (where the cells can then form secondary tumors). Thus, in one embodiment the invention provides compounds and methods for inhibiting, in whole or in part, the formation of secondary tumors in a subject with cancer.

[0210] Advantageously, the compounds of the invention may be capable of inhibiting the proliferation and/or metastasis of cancer cells selectively.

[0211] By "selectively" we mean that the compounds of the invention may inhibit the proliferation and/or metastasis of cancer cells to a greater extent than it modulates the function (e.g. proliferation) of non-cancer cells. Preferably, the compounds of the invention inhibit the proliferation and/or metastasis of cancer cells only.

[0212] Compounds of the invention may be suitable for use in the treatment of various diseases, such as cancer and spinal cord injury.

[0213] Compounds of the invention may be suitable for use in the treatment of any cancer type, including all tumors (non-solid and solid tumors). The cancer type may include (but is not limited to) benign tumors (such as hemangiomas, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas and pyogenic granulomas) and malignant tumors (such as leukemia, myelodysplastic syndromes (MDS), prostate cancer, breast cancer, skin cancer, bone cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, bladder, gall bladder, ovary, cervix, pancreas, rectum, parathyroid, thyroid, esophagus, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papilllary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, non-small cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, epidermoid carcinomas, and other carcinomas and sarcomas). Others that may be mentioned include cancers of the testis, genitourinary tract, glioblastoma, neuroblastoma, keratoacanthoma, epidermoid carcinoma, large cell carcinoma, follicular carcinoma, undifferentiated carcinoma, papilliary carcinoma, seminona, myeloid disorders, lymphoid disorders, hairy cells, buccal cavity and pharynx (oral), lip, tongue, mouth, small intestine, colon-rectum, large intestine, rectum, brain and central nervous system, Hodgkin's and leukaemia; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocitic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkett's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; and other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xeroderma pigmentosum, keratoxanthoma, thyroid follicular cancer and Kaposi's sarcoma).

[0214] In particular, compounds of the invention may possess potent inhibitory activity on the growth and invasion of hormone-refractory and aggressive human prostate tumors (for example, as may be shown in xenograft mouse models). It is particularly preferred therefore that the compounds of the invention may be useful in the treatment of aggressive cancers such as prostate cancer.

[0215] Compounds of the invention may reduce the rate of cell proliferation when tested in an assay using a PC-3 cancer cell line (e.g. obtained from ATCC). The compounds may thus possess a beneficial inhibitory effect on the ability of tumors of this type, and of cancers generally, to survive. The PC-3 cancer cell line has several properties that represent the hormone-independent and invasive prostate cancer. For instance, PC-3 cells lack functional androgen receptor (AR) signalling, grow rapidly in culture medium and can form large and very aggressive tumors when implanted into nude mice. Hence, the biological tests described hereinafter (e.g. PC-3 xenograft mouse models) provide a sound predication of the utility of the compounds tested by mimicking the gradual dissemination of prostate carcinoma cells in vivo.

[0216] Further, it has been reported that even the widely used cancer drugs such as AVASTIN™ or docetaxel alone or in combination had low inhibitory effect on PC-3 cells (Petrylak DP: Future directions in the treatment of androgen-independent prostate cancer. Urology 65: 8-12, 200538.2007; and Hung H: Bevacizumab plus 5-fluorouracil induce growth suppression in the CWR-22 and CWR- 22R prostate cancer xenografts. Molecular cancer therapeutics 6: 2149-2157, 2007).

[0217] The compounds of the invention may target multiple cellular pathways that are associated with tumor growth, apoptosis, angiogenesis and metastasis. The compounds of the invention may also affect other cancer pathways, such as cell cycle regulation and inflammation.

[0218] The compounds of the invention may therefore be VEGF (vascular endothelial growth factor) inhibitors, e.g. they may inhibit the expression of VEGF and/or VEGF receptors including but not limited to VEGF and/or VEGF receptor 2 (as may be shown in a test described herein). This may occur selectively, or, may be one of a plurality of the mechanisms by which the compounds of the invention act to treat cancer. The VEGF signalling pathway is known to be linked to tumor vascularisation and invasion, and hence compounds of the invention may possess anti-cancer effects by inhibiting angiogenesis (and may therefore be classed as anti-angiogenesis agents). In another embodiment of the invention therefore, the compounds of the invention may be useful in the treatment of a disease in which the inhibition of angiogenesis (and/or VEGF) is desired and/or required.

[0219] The term "inhibition" may refer to any measurable reduction and/or prevention, which in the context of angiogenesis refers to the reduction and/or prevention of angiogenesis (e.g. the expression of VEGF receptors including but not limited to VEGF and VEGF receptor 2). The inhibitory activity may be measured by comparing the angiogenesis inhibition in a sample containing a compound of the invention and (a) VEGF receptor(a), such as VEGF and/or VEGF receptor 2, with an equivalent sample of in the absence of a compound of the invention. The measurable change may be objective (e.g. measurable by some test or marker, for example in an in vitro or in vivo assay or test, such as one described hereinafter, or otherwise another suitable assay or test known to those skilled in the art) or subjective (e.g. the subject gives an indication of or feels an effect).

[0220] Compounds of the invention are indicated both in the therapeutic and/or prophylactic treatment of the above-mentioned conditions.

[0221] According to a further aspect of the present invention, there is provided a method of treatment of a disease (e.g. cancer or another disease as mentioned herein) which may be associated with, or affected by, angiogenesis (and/or VEGF; e.g. an inhibition of the expression of VEGF and/or VEGF receptor 2) is desired and/or required, which method comprises administration of a therapeutically effective amount of a compound of the invention but without the proviso(s), as hereinbefore defined, to a patient suffering from, or susceptible to, such a condition.

[0222] "Patients" include mammalian (including human) patients. Hence, the method of treatment discussed above may include the treatment of a human or animal body.

[0223] The term "effective amount" refers to an amount of a compound, which confers a therapeutic effect on the treated patient. The effect may be objective (e.g. measurable by some test or marker) or subjective (e.g. the subject gives an indication of or feels an effect).

[0224] Compounds of the invention may be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, sublingually, by any other parenteral route or via inhalation, in a pharmaceutically acceptable dosage form.

[0225] Compounds of the invention may be administered alone, but are preferably administered by way of known pharmaceutical formulations, including tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration, and the like. The type of pharmaceutical formulation may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.

[0226] Such formulations may be prepared in accordance with standard and/or accepted pharmaceutical practice. Otherwise, the preparation of suitable formulations may be achieved non- inventively by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.

[0227] According to a further aspect of the invention there is thus provided a pharmaceutical formulation including a compound of the invention, as hereinbefore defined but without the proviso(s), in admixture with a pharmaceutically acceptable adjuvant, diluent and/or carrier.

[0228] Depending on e.g. potency and physical characteristics of the compound of the invention (i.e. active ingredient), pharmaceutical formulations that may be mentioned include those in which the active ingredient is present in at least 1% (or at least 10%, at least 30% or at least 50%) by weight. That is, the ratio of active ingredient to the other components (i.e. the addition of adjuvant, diluent and carrier) of the pharmaceutical composition is at least 1 :99 (or at least 10:90, at least 30:70 or at least 50:50) by weight.

[0229] The amount of compound of the invention in the formulation will depend on the severity of the condition, and on the patient, to be treated, as well as the compound(s) which is/are employed, but may be determined non-inventively by the skilled person.

[0230] The invention further provides a process for the preparation of a pharmaceutical formulation, as hereinbefore defined, which process comprises bringing into association a compound of the invention, as hereinbefore defined, or a pharmaceutically acceptable ester, amide, solvate or salt thereof with a pharmaceutically-acceptable adjuvant, diluent or carrier.

[0231] Compounds of the invention may also be combined with other therapeutic agents that may be useful in the treatment of a cancer and/or a proliferative disease (e.g. another VEGF inhibitor as described herein). Compounds of the invention may also be combined with other therapies.

[0232] According to a further aspect of the invention, there is provided a combination product comprising:

(A) a compound of the invention, as hereinbefore defined but without the proviso(s); and

(B) one or more therapeutic agent(s) that is useful in the treatment of cancer and/or a proliferative disease,

wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically- acceptable adjuvant, diluent or carrier. [0233] Such combination products provide for the administration of a compound of the invention in conjunction with the other therapeutic agent, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises a compound of the invention, and at least one comprises the other therapeutic agent, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including a compound of the invention and the other therapeutic agent).

[0234] Thus, there is further provided:

(1) a pharmaceutical formulation including a compound of the invention, as hereinbefore defined but without the proviso(s), one or more therapeutic agent(s) that is useful in the treatment of cancer and/or a proliferative disease, and a pharmaceutically-acceptable adjuvant, diluent or carrier; and

(2) a kit of parts comprising components:

(a) a pharmaceutical formulation including a compound of the invention, as hereinbefore defined but without the proviso(s), in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier; and

(b) a pharmaceutical formulation including one or more therapeutic agent(s) that is useful in the treatment of cancer and/or a proliferative disease in admixture with a pharmaceutically- acceptable adjuvant, diluent or carrier,

which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.

[0235] The invention further provides a process for the preparation of a combination product as hereinbefore defined but without the proviso(s), which process comprises bringing into association a compound of the invention, as hereinbefore defined, or a pharmaceutically acceptable ester, amide, solvate or salt thereof with the other therapeutic agent (or agents) that is (or are) useful in the treatment of cancer and/or a proliferative disease, and at least one pharmaceutically-acceptable adjuvant, diluent or carrier.

[0236] By "bringing into association", we mean that the two components are rendered suitable for administration in conjunction with each other.

[0237] Thus, in relation to the process for the preparation of a kit of parts as hereinbefore defined, by bringing the two components "into association with" each other, we include that the two components of the kit of parts may be:

(i) provided as separate formulations (i.e. independently of one another), which are subsequently brought together for use in conjunction with each other in combination therapy; or (ii) packaged and presented together as separate components of a "combination pack" for use in conjunction with each other in combination therapy.

[0238] Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of the invention may be administered at varying therapeutically effective doses to a patient in need thereof. However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

[0239] Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 10 g (e.g. 1000 mg) per day of a compound of the invention. For example, the dose range may be between 1 mg/kg and 1000 mg/kg (e.g. between 10 mg/kg and 500 mg/kg, preferably between about 20 mg/kg and 200 mg/kg) such as the does ranges employed in the mouse models described hereinafter.

[0240] In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

[0241] Compounds of the invention may have the advantage that they target multiple pathways involving tumor growth, apoptosis, angiogenesis and metastases. The compounds of the invention may also be effective angiogenesis inhibitors (and/or VEGF inhibitors), i.e. they may (for example, selectively, or as one mode of action) inhibit angiogenesis (and/or VEGF; e.g. they may inhibit the expression of VEGF and VEGF receptor 2).

[0242] Compounds of the invention may also have the advantage that they may be more efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer side effects than, be more easily absorbed than, and/or have a better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance) than, and/or have other useful pharmacological, physical, or chemical properties over, compounds known in the prior art, whether for use in the above-stated indications or otherwise.

[0243] For instance, compounds of the invention may be well tolerated by the patient, i.e. show less or no side effects (e.g. weight loss or other toxic side effects) for example as compared to other therapeutic agents. This may be the case even at high concentrations/doses of the compounds of the invention. The compounds of the invention may also display good potency (e.g. better potency than other therapeutic agents) at a relatively or comparatively lower dose. Hence the compound of the invention may have a large therapeutic window.

[0244] The compounds of the invention may have advantages over other known chemotherapeutic agents (e.g. AVASTIN™, docetaxel and/or etoposide), which includes better potency and better safety profile (i.e. reduced side effects). Such comparative advantages may be shown in biological tests such as those described hereinafter.

[0245] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

EXAMPLES

Example 1

[0246] Akt Activity Assay

[0247] Akt activity is assessed by obtaining cells/tissue^ood/urine/body fluid sample from a subject with a disease, and to perform an in vitro Akt activity assay, which utilizes an Akt-specific antibody to immunoprecipitate Akt in the lysate obtained from collected cells/tissue/blood/urine/body fluid sample. Akt activity is determined in a kinase reaction using recombinant GSK-3a as substrate. Phosphorylation of the GSK-3a is analyzed by Western blot analysis using the phospho-GSK-3a specific antibody. Akt activity in the subject with a disease is measured by the level of phospho- GSK-3a.

Example 2

A. Materials and Methods

[0248] Patient samples and data collection

[0249] This study was approved by the ethics committee of Lund University, Sweden and the Helsinki Declaration of Human Rights was strictly observed. The tissue microarray PRC961 contained samples from patient cases including prostate hyperplasia (N=12) and localized prostate cancer (N=48) (Pantomics Inc.). The tumor samples did not contain significant amount of normal tissues. Tissue microarray MTU951 contained different types of cancer specimens from 40 patients (Pantomics, Inc) was used. The cancer types included Adrenocortical carcinoma, bladder cancer, bone cancer, brain cancer, esophagus cancer, stomach cancer, colon cancer, liver cancer, pancreatic cancer, ovarian cancer, thyroid cancer, testicular cancer.

[0250] Immunohistochemistry

[0251] Deparaffinization of paraffin embedded tissue samples (7 μηι) on tissue microarray slides was performed, and was followed by antigen retrieval in which slides were boiled in 0.01 M citrate buffer, pH 6.0, for 10 min. The staining procedure was performed using a semiautomatic staining machine (Ventana ES, Ventana Inc., Tucson, AZ). The specimens were viewed and the microphotographs were taken under 40 X magnification with a Nikon 800 microscope. The specimens were evaluated. The staining intensity was scored as 0 (negative), 1 (weakly positive), 2 (moderately positive) and 3 (strong or very strongly positive).

[0252] Sources of antibodies for Western Blotting

[0253] The following antibodies were used: polyclonal antibodies against PIP5K1A and phoshorylated Akt and Cyclin Dl (Cell Signalling Technology, Danvers, MA), P27 (Santa Cruz Technology, Santa Cruz) and β-Actin (MP Biomedicals, Illkirch France). Secondary antibodies were HRP-conjugated anti-mouse IgG and anti-rabbit IgG (GE Healthcare).

[0254] siRNA silencing of PIP5K1A in PC-3 cells

[0255] For siRNA experiments, 2 xlO⁶ human prostate cancer PC-3 cells were transfected with 10 nM siRNA non-targeting (Ctrl) or oligos of siRNA to PIP5K1A (Invitrogen, Grand Island, NY) and cultured for 24, 48 or 72 hours. Transfections were performed with Microporator MP-100 (Digital- Bio Technology, Seoul, Korea) according to the manufacturer's instructions.

[0256] Two oligos of siRNAs to PIP5K1A were used to silence PIP5K1A. The siRNAs were designed to target exon 14 of PIP5K1A gene to specifically silence PIP5K1A. The first oligo duplex contains sense and anti-sense strands with the sequences of GCG UUC ACC UUG GUC GUC CUG AUG U (SEQ ID NO:l) and ACA UCA GGA CGA CCA AGG UGA ACG C (SEQ ID NO:2). The second oligo duplex contains sense and anti-sense strands with the sequences of CCU UCC GCU ACU UCC GGG AGC UAU U (SEQ ID NO:3) and AAU AGC UCC CGG AAG UAG CGG AAG G (SEQ ID NO:4). The annealed oligos were produced and mixed by Invitrogen.

[0257] Immunoblot analysis

[0258] The cells were harvested and lysed in ice-cold RIPA buffer (120 mM NaCl, 50 mM Tris- HC1 pH 7.6, 50 mM NaF, O.lmM Na3V04, 1% NP40, 1 mM phenylmethylsulfonyl fluoride (PMSF)) (Sigma, St. Louis, MD) and 15 % protease inhibitor cocktail Complete Mini (Roche, Basel, Switzerland). 20μg of the protein was separated with 12% SDS-PAGE gels and transferred onto nitrocellulose membranes. Signals were visualized using the Enhanced ChemiLuminescence detection system (Millipore Corp Sweden, Solna, Sweden) and documented with an Alphalmager CCD system. Densitometric quantification of immunoblots was performed by the ImageJ Image Analysis Software (NIH, Baltimore, MD) and represented as fold change relative to control and was normalized with actin staining.

[0259] Statistical analysis of gene expression profiles

[0260] Two-sample t tests were used to identify the genes that are differentially expressed between metastasis and primary tumors with a significance level of P< 0.05. Tukey-test, ANOVA, Mann- Whitney test and Spearman rank correlation test were performed with the Statistical Package for the Social Sciences software (SPSS, version 17; Chicago). P-value <0.05 was considered as significant.

B. Results

[0261] Inhibition of PIP5K1A Down-regulates Akt Activation

[0262] To study the role of PIP5K1A in regulating PI3 Kinase/ Akt pathway, PIP5K1A expression was silenced by RNAi-mediated knockdown in PC-3 cells. PIP5K1A expression was completely inhibited after 48 and 72 hours of treatment with siRNA to PIP5K1A (Figure 1). Knockdown of PIP5K1A led to significantly lower level of phosphorylated Akt (Figure 2A-B). Akt phosphorylation is a critical step in activation of PI3 Kinase/ Akt pathway, so the result demonstrates the role of PIP5K1A in activation of PI3 Kinase/ Akt pathway.

[0263] Inhibition of PIP5K1A Down-regulates Akt Downstream Effector Cyclin Dl

[0264] Cyclin Dl is a known down-stream effector of the PI3 Kinase/ Akt pathway. Inhibition of PIP5K1 A also led to the decreased level of cyclin Dl expression (Figure 3A-B).

[0265] Inhibition of PIP5K1A Up-regulates Akt Downstream Negative Effector p27

[0266] P27 is a known down-stream negative effector of the PI3 Kinase/ Akt pathway. Inhibition of PIP5K1A led to increased expression of P27 (Figure 4). These results supports the role of PIP5K1 A in regulating PI3 Kinase/ Akt pathway.

[0267] Expression of PIP5K1A Protein in Prostate Hyperplasia Tissues and Prostate Cancer Specimens

[0268] To investigate the clinical relevance of PIP5K1A pathway in the development and progression of cancers, protein expression level of PIP5K1A in clinical specimens obtained from patients with prostate hyperplasia tissues (BPH) and malignant prostate cancers was evaluated. Prostate cancer specimens had significantly higher level of PIP5K1A expression than that of the BPH prostate tissues (p<0.001) (Figure 5 and 6). More importantly, PIP5K1A expression level correlates with cancer grade: very low in benign prostatic hyperplasia (BPH) patients, low in low grade prostate cancer patients and at the highest level in high grade metastatic prostate cancer patients (Figure 5). [0269] Expression of PIP5K1A Correlates with AR Expression in Prostate Hyperplasia Tissues and Prostate Cancer Specimens

[0270] Similarly, expression of androgen receptor (AR) was significantly higher in cancer specimens as compared with BPH tissues (p<0.01) (Figure 7A). Spearman rank correlation statistical analysis showed that level of PIP5K1A expression positively correlated with AR expression in prostate cancer specimens (p<0.01) (Figure 7B).

[0271] Expression of PIP5K1A Protein in Healthy and Lung Cancer Specimens

[0272] Expression of PIP5K1A was evaluated in normal lung tissues, and non-invasive lung sequamous cell carcinoma and invasve non-small cell lung cancer specimens. PIP5K1A is over- expressed in lung cancer patient tissues as compared to normal lung tissue. More importantly, its expression level correlates with cancer grade and invasiveness: very low in normal lung tissues, low in non-invasive lung sequamous cell carcinoma specimens and very high in invasive non-small cell lung cancer specimens (Figure 8).

Example 3

[0273] Method to Assess Kinase Inhibition Activity

[0274] Kinase Inhibition Assay

[0275] Protein kinases are phosphoryl transferases that transfer the γ-phosphate of ATP to conserved serine, threonine, or tyrosine residues on specific substrate proteins. Kinase activity assay involves the quantification of this phosphoryl transfer by detection of the production of the phosphorylated product or the change in the ratio of ATP to ADP. The radioisotope filtration binding assay is used to measure kinase inhibition activity of a putative kinase inhibitor. The reactions are performed using radioisotope labeled γ-ΑΤΡ. The incorporation of this radiolabeled phosphate into the kinase substrate is then assayed after a series of binding and washing steps to remove unincorporated radioisotope. This allows for the detection of kinase phosphoryl transfer activity, which is directly proportional to the amount of phosphorylated substrate. The kinase activity in the presence and absence of the putative kinase inhibitor is measured to determine and quantify the kinase inhibition activity.

Example 4

[0276] Method to Identify PIP5K1 A Inhibitors

[0277] Kinase Inhibitor Screening Methods

[0278] For high-throughput screening of kinase inhibitors, many convenient and automation methods to assess kinase activity can be used with detection methods using radioisotope, fluorescence emission, chemiluminescence emission, production of phosphorylated substrates or the binding of potential chemical inhibitors to the target kinase. The high-throughput screening methods include radiometric based filtration binding assay, radiometric based scintillation proximity assay (SPA), fluorescence Intensity assay (FI), fluorescence polarization assay (FP), fluorescence resonance energy transfer assay (FRET), time-resolved fluorescence assay (TRF), time-resolved fluorescence resonance energy transfer assay (TR-FRET), ELISA-based assay, luminescence detection assay, mobility shift assay and ligand-kinase binding assay.

[0279] For example, a library of chemical compounds that are potential PIP5K1A inhibitors can be tested in a high-throughput PIP5K1A inhibition screening to identify compounds that can inhibit PIP5K1A in the μΜ range. Further tests on the putative inhibitors can be performed to identify compounds with kinase inhibition activity in nM concentration range. These molecules will also be tested in membrane preparations and only compounds capable of inhibiting PIP5K1A activity in its natural environment as well as in the original high throughput assay will be identified as PIP5K1A inhibitors. In addition, kinase inhibition selectivity can be tested to identify specific PIP5K1A inhibitors. The identified inhibitors can be further tested in vivo such as in xenograft animal model studies.

Example 5

[0280] [0003] Nitrogen-containing heterocyclic compounds are important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4- phosphate 5-kinase alpha (PIP5Ka) is a lipid kinase, similar to phosphatidylinositol 3-kinase (PI3K). However, the role of ΡΙΡ5Κ1α in oncogenic processes and the development of inhibitors which selectively target ΡΙΡ5Κ1α have not been reported. In the present study, we report that overexpression of ΡΙΡ5Κ1α is associated with poor prognosis in prostate cancer, and correlates with an elevated level of the androgen receptor (AR). Overexpression of ΡΙΡ5Κ1α in PNT1A non-malignant cells results in an increased AKT activity, and an increased survival as well as invasive malignant phenotype, while siRNA-mediated knockdown of ΡΙΡ5Κ1 α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a novel role for ΡΙΡ5Κ1α as a druggable target for our newly developed compound ISA-201 1B using a high-throughput KINOMEscan^® platform. ISA-201 1B is discovered during our synthetic studies of C-l indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-201 1B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting ΡΙΡ5Κ1α associated PI3K/AKT and the downstream survival, proliferation and invasion pathways. Further, siRNA-mediated knockdown of ΡΙΡ5Κ1α exerts similar effects on PC3 cells as ISA-201 1B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, ΡΙΡ5Κ1α has a high potential as a drug target, and compound ISA-201 1B is interesting for further development of targeted cancer therapy. [0281] Prostate cancer is the most common malignancy, and the third leading cancer-related cause of death among men of western world. Treatment options at advanced stages of the disease are scarce, and better therapies are in urgent need. In our study, we show that the clinically-relevant lipid kinase ΡΙΡ5Κ1α plays an important role in cancer cell invasion and survival by regulating the PI3K/AKT/AR pathways. Elevated level of ΡΙΡ5Κ1α contributes to cancer cell proliferation, survival and invasion. In this context we introduce a newly developed compound ISA-201 IB with promising anticancer effects by inhibiting the ΡΙΡ5Κ1α associated AKT pathways. Conclusively, we propose that ΡΙΡ5Κ1α may be used as a potential therapeutic target for treatment of advanced prostate cancer.

[0282] The mainstay of cancer treatment largely consists of nonspecific cytotoxic agents with severe side-effects in treated cancer patients (1). It is thus essential to discover novel drugs and their targets to more selectively eradicate cancer cells. Within the pharmaceutically important heterocyclic compounds, the 1 ,2,3,4-tetrahydroquinoline and 1,2,3,4-tetrahydroisoquinoline ring systems are common structural motifs found in several biologically active compounds (2-6). The discovery and development of a novel class of 1,2,3,4-tetrahydroisoquinoline derivatives as selective anticancer drugs represents new challenges in discovery and development of anticancer drugs.

[0283] The phosphoinositide family of lipids consists of several derivatives of phosphatidylinositols (Ptdlns) that are formed through series of phosphorylation by enzymes termed phosphatidylinositol-phosphate kinases (PIPKs) (7). Among the PIPKs, PI3 -kinases (PI3K) are the best characterized enzymes that commonly are targets for anticancer drugs (8). Less is known about PI-4-phosphate 5 kinases (PIP5K), enzymes that are upstream of PI3K (9, 10). The PIP5K family of lipid kinases consists of the three isozymes α, β, and γ (11 -13). ΡΙΡ5Κ1α is located in the chromosomal region lq21.3 (14), the product of which is predominantly responsible for the synthesis of PtdIns-4,5-P₂ (PIP2), a substrate used by PI3K to produce PtdIns-3,4,5-P₃ (PIP3) (15). PIP3 in turn activates the AKT family of serine/threonine kinases (16, 17). The aberrant activation of AKT is one of the most frequently observed alterations in human cancer cells, and elevated level of phosphorylated AKT at Ser-473 site (pSer-473) is associated with mutations in PIK3CA (pi 10a), a PI3K subunit or in phosphatase and tensin homologue (PTEN) gene in metastatic cancers (18, 19). In prostate cancer (PCa), PI3K/AKT has been reported to cross-activate androgen receptor (AR)- mediated signaling to promote progression of castration resistant PCa (20-24). AR signaling has also been used as targets for designing drugs to treat lethal metastatic PCa (24-28).

[0284] Given the fact that ΡΙΡ5Κ1α produces PIP2, which is required for the activation of PI3K/AKT, we anticipate that ΡΙΡ5Κ1 α may play an important role in cancer progression. It is of importance to investigate whether ΡΙΡ5Κ1α may be used as potential target for developing effective novel anticancer drugs. It is known that ΡΙΡ5Κ1α is expressed at low level in lipid tissues and is dispensable during organ development, as deletion of ΡΙΡ5Κ1α does not result in lethal defects in mice but causes impaired spermatogenesis in males (29, 30). A recent reported study shows that ΡΙΡ5Κ1α is highly expressed in the human MDA-MB-231 breast cancer cell line, suggesting that overexpression of ΡΙΡ5Κ1 α is associated with malignant diseases (31).

[0285] In this study we present our discovery of a diketopiperazine fused C-1 indol-3-yl substituted tetrahydroisoquinoline, termed ISA-201 IB, as a novel anticancer drug that effectively inhibits growth of PCa tumor in vivo, and invasion of PCa cells in vitro. We unravel a novel role of ΡΙΡ5Κ1α as a target for ISA-201 IB, and as a central factor that regulates PI3K/AKT and AR signaling pathways, which are involved in regulation of cell proliferation, survival, and invasion.

[0286] RESULTS

[0287] Discovery of ISA-2011B

[0288] ISA-201 IB, a diketopiperazine fused C-1 indol-3-yl substituted 1 ,2,3,4- tetrahydroisoquinoline derivative, was discovered as a result of our development of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolies via a Pictet-Spengler approach (32) (Figure 9A), and was found to have a potent inhibitory effect on proliferation in various types of aggressive cancer cell lines (Figure 17). The dose-dependent effect of ISA-201 IB on proliferation of PC-3 cells was determined by MTS assay. The proliferation rate of PC-3 cells after treatment with ISA-201 IB at 10, 20 and 50 μΜ was significantly reduced to 58.77%, 48.65% and 21.62%> of vehicle-treated controls respectively. Mean absorbance for control cells and cells that were treated with 10 μΜ ISA-201 IB were 0.53 and 0.31 respectively (difference = 0.22; 95% CI = 0.26 to 0.36; P = 0.003) (Figure 9B). Further, the effect of ISA-201 IB is significantly stronger on PC-3 cells with PTEN mutation at 10 μΜ ( =0.0006) and 50 μΜ ( =0.025) than on 22Rvl cells which contain intact PTEN gene (Figure 9C).

[0289] One of the most compelling applications of comprehensive high-throughput kinase profiling is the screening of the compound across a panel of kinases to identify the binding targets (33). We screened ISA-201 IB across 460 kinases covering more than 80% of the human catalytic protein kinome (Figure 9D). ISA-201 IB exhibited the highest binding affinity to ΡΙΡ5Κ1 α, and to MARK1 and MARK4 as well (Figure 9E). Since ΡΙΡ5Κ1 α is linked to PI3K/AKT pathways, it was selected for further validation. ISA-201 IB treatment inhibited ΡΙΡ5Κ1 α expression by 78.6% in PC-3 cells, as determined by immunoblot analysis (Figure 9F). As measured by densitometric quantification of the blots from three independent experiments using Image J program, the mean value of ΡΙΡ5Κ1α expression was 9.43 in control cells compared with 2.01 in ISA-201 IB treated cells (95% CI = 0.31 to 4.34; P = 0.013) (Figure 9F). ISA-201 1A, an analog of ISA-201 IB, also reduced the level of ΡΙΡ5Κ1α, but treatment with a taxane-based drug (docetaxel) did not markedly inhibit ΡΙΡ5Κ1α expression (Figure 9F). However, ISA-201 IB did not inhibit PKARla, a key receptor of cyclic AMP/PKA, which also activates AKT (34) (Figure 9G). Collectively these results indicate that ISA-201 IB inhibits ΡΙΡ5Κ1 α, but not proteins that are unrelated to ΡΙΡ5Κ1α. [0290] ISA-2011 effectively inhibited tumor growth in xenograft mouse models

[0291] The most significant pre-clinical extension of this work is to determine the therapeutic benefit of ISA-201 IB in vivo. We examined the effect of ISA-201 IB on growth of invasive human prostate tumor grafted into mice. When the mean tumor volume reached 50 mm³, treatment of xenograft mice with vehicle control, ISA-201 IB, docetaxel and combination of ISA-201 IB and docetaxel was initiated, and lasted for 20 days. Growth of tumors in mice treated with ISA-201 IB regressed compared with tumors in control (Figure 10A, mean volume of vehicle control = 500.00 mm³, ISA-201 IB treated = 42.00 mm³, difference = 458.00 mm³, 95% CI = 7.70 to 76.30; P < 0.001 , two-sided t test). Similarly, this was also the case for tumors treated with ISA-201 IB and docetaxel in a combination (Figure 10A, mean volume of ISA-201 IB + docetaxel treated group= 36.00 mm³, 95% CI = 30.40 to 41.60 P < 0.001, two-sided t test). Although tumors treated with docetaxel also regressed (Figure 10A), but adverse effect of docetaxel on mice was pronounced. In contrast, no adverse effect such as weight loss was observed in mice treated with ISA-201 IB and docetaxel in combination. This suggests that ISA-201 IB interacts with docetaxel to reduce its off-target effects with unknown mechanisms. On day 20, tumor mass of the treatment groups were significantly smaller compared to that of control group (Figure 10B, as compared to control, for ISA-201 IB treated, P < 0.006; for ISA-201 IB + docetaxel, P < 0.001).

[0292] Effect of ISA-2011B on tumor growth is associated with its effect on ΡΙΡ5Κ1α, which is highly expressed in human PCa

[0293] The most important step during drug development is to characterize the associations between target and disease, and to explore the potential role of the new targets. Having identified significant anticancer effect of ISA-201 IB in PCa growth in vivo, we went on to study the clinical importance of ΡΙΡ5Κ1 α in PCa patient samples. A tissue microarray (TMA) that contained biopsies of benign prostate hyperplasia (BPH) and paired cancer tissues from 48 PCa patients were immunostained with antibodies against ΡΙΡ5Κ1 α, PIP2 and AR (Figure 11A). ΡΙΡ5Κ1α, PIP2, and AR expression was significantly higher in PCa tissues when compared to BPH epithelium, as determined by Wilcoxon's signed rank test (P< 0.001) (Figure 11B). There was a significantly positive correlation between ΡΙΡ5Κ1α and PIP2 (r² = 0.477, P = 0.01), and between ΡΙΡ5Κ1 α and AR (r² = 0.640, P = 0.01) as determined by Spearman-rank correlation test.

[0294] We next examined genes encoding ΡΙΡ5Κ1α, AKT1 , AKT2, PTEN and AR in primary (n=181) and metastatic (n=37) PCa tissues by using a public database (35). We observed alterations in genes encoding ΡΙΡ5Κ1 α, AKT2, PTEN and AR in subsets of PCa patients (Figure 18). Mann Whitney test revealed that expression of ΡΙΡ5Κ1 α, AKT2 and AR was significantly higher in metastatic lesions compared to primary PCa (p < 0.001) (Figure 1 1C). Gene amplification in ΡΙΡ5Κ1 α was observed in 2.8% (n = 5 out of 181) of primary cancer, and 16.2% (n = 6 out of 37) of metastatic PCa lesions (p = 0.001). The follow-up time from diagnosis to disease recurrence known as biochemical recurrence (BCR) ranged from 1 to 168 months (Figure 11D). A subset of patients with higher mRNA expression due to gene amplifications in ΡΙΡ5Κ1α, AKT2 and AR, or low mRNA expression due to PTEN mutation suffered poorer BCR-free survival (for ΡΙΡ5Κ1 α, <0.001 ; for AKT2, P = 0.01 ; for AR, <0.001) (Figure 11D and Figure 19). Thus, overexpression of ΡΙΡ5Κ1α, AKT2 and AR or down-regulation of PTEN is associated with poor prognosis of PCa patients.

[0295] Overexpression of PIP5Kla increased expression of cell proliferation markers and invasiveness

[0296] We further characterized the consequence and molecular mechanisms of ΡΙΡ5Κ1 α overexpression in PCa cells. Expression of ΡΙΡ5Κ1 α, pSer-473 AKT, and the key cell cycle regulators: cyclin Dl and cyclin-dependent kinase 1 (CDK1) was detected in non-malignant cells at relative lower level compared with the malignant PCa cells (Figure 12A). We introduced overexpression of ΡΙΡ5Κ1 α by transfecting non-malignant PNT1A cells with pLPS-PIP5Kla-EGFP vector or pLPS-EGFP control. As determined by immuno staining of cells with antibodies against ΡΙΡ5Κ1α, β-Tubulin, and PIP2, we observed that overexpression of ΡΙΡ5Κ1α led to an enhanced β- Tubulin and PIP2 expression in PNT1A cells (Figure 12B, C). Overexpression of ΡΙΡ5Κ1α led to an increase in ΡΙΡ5Κ1α protein expression by 47.61% as compared to control (P = 0.047) (Figure 12D). This was coincident with the effect of ΡΙΡ5Κ1α overexpression on AKT activation. Expression of pSer-473 AKT increased by 666.81% in PNT1A cells transfected with pLPS-PIP5Kl a as compared to controls (Figure 4D, control transfected mean= 3.78; PIP5K1 a transfected mean = 29.02, difference = 25.24; 95%> CI = 21.67 to 36.37, P = 0.021). Thus, ΡΙΡ5Κ1α overexpression significantly increased activity of AKT. It is known that AKT, once activated, will go on to regulate multiple factors that control cell cycle proliferation, survival, invasion and metastasis via its kinase activity (36). Expression of the G0/G1 and S/G2/M cyclins and CDKs including cyclin Dl, cyclin El, cyclin A2, CDK1 and cyclin B l was increased in PNT1A cells that overexpressed ΡΙΡ5Κ1 α, compared to controls (Figure 12E, F). ΡΙΡ5Κ1α overexpression also resulted in an induction of series of key factors, downstream or upstream of AKT. These included phosphorylation of FAK, a focal adhesion molecule; Twist 1, a pro-invasion regulator (Figure 12G), and VEGF and MMP9, the factors that mediate angiogenesis (Figure 12H). We further assessed whether PNT1A cells overexpressing ΡΙΡ5Κ1α may have gained malignant invasive feature by performing invasion assay. PNT1A cells overexpressing ΡΙΡ5Κ1α gained ability to invade through the extracellular matrix-coated membrane with an invasive capacity of 412% higher than the controls (P = 0.014) (Figure 121). Interestingly, overexpression of ΡΙΡ5Κ1α greatly increased expression of AR as determined by immunoblot analysis (Figure 12 J) and immunostaining (Figure 20). Immunoprecipitation assays further revealed that AR formed protein-protein complexes with CDK1 in the nuclear compartment of PNT1A cells expressing control vector or ΡΙΡ5Κ1 α vector (Figure 21 ; Figure 12K). Thus, ΡΙΡ5Κ1α is able to activate PI3K/AKT pathway and enhance cross-interactions of AKT with AR, probably through CDK1.

[0297] ISA-2011B inhibits the elevated survival, proliferation and invasion signaling

[0298] As demonstrated in Figure 1 1A, ΡΙΡ5Κ1α is highly expressed in primary and metastatic PCa, and in androgen-dependent malignant LNCaP cells. We next tested whether inhibition of ΡΙΡ5Κ1α by ISA-201 1B might lead to successful inhibition of deregulated PI3K/AKT and AR pathways. Treatment of LNCaP cells with ISA-201 1B and its analogs ISA-201 1A reduced ΡΙΡ5Κ1 α expression (Figure 13 A), and inhibited proliferation of LNCaP cells (Figure 22). ISA-2011B significantly inhibited pSer-473 AKT by 75.55% in compared to control (Figure 5B, mean pSer-473 AKT expression in control treated = 13.43, mean expression in ISA-201 IB treated = 10.15, difference = 3.28; 95% CI = 3.34 to 16.95; P = 0.026). While expression of pSer-473 AKT was not affected by etoposide, docetaxel, and tadalafil, an approved drug for treatment of BPH, which exhibits slight similarity in the chemical structure as compared to ISA-201 IB (Figure 13b). The expression of pSer- 473 AKT was barely detectable in ISA-201 IB treated cells which displayed characteristics of apoptotic bodies and fragmented nucleus (Figure 13C). In contrast, expression of phosphorylated CREB, a downstream factor of PKA pathway remained unchanged in cells treated with ISA-201 IB, ISA-201 1A, etoposide, tadalafil and docetaxel (Figure 13D). ISA-201 IB treatment led to a down- regulation of CDK1 (Figure 13E). Interestingly, ISA-201 IB treatment inhibited SKP2, a key ubiquitin enzyme that mediates P27 degradation, and ISA-201 IB increased expression of P27, an inhibitor that is required to block cell proliferation by inhibiting cyclins and CDKs (Figure 13F). ISA-201 IB treatment also led to a remarkable inhibition of AR and PSA expression (Figure 13G, H). However, docetaxel, ISA-2009 and ISA-201 1 A had no effect on AR and PSA expression (Figure 13H). This data suggest that ISA-201 IB mediated inhibitory effect on PCa proliferation is associated with its ability to inhibit ΡΙΡ5Κ1 α, which transduce the inhibitory effect to its downstream signaling pathways including AKT, AR, and cell cycle.

[0299] ISA-2011B target PIP5Kla/AKT/AR pathways to inhibit tumor cell growth and induce apoptosis in aggressive PCa cells

[0300] We further tested whether inhibition of ΡΙΡ5Κ1α by ISA-201 IB might lead to successful inhibition in cancer cell invasion in PC-3 cells. A live cell imaging system, Holomonitor M3 imaging analysis was used to monitor the effect of ISA-201 IB, etoposide and docetaxel on morphological changes in living cells in real-time for up to 48 hours. ISA-201 IB treatment led to a reduction in cell size, and changes in morphology, which was also achieved by docetaxel treatment (Figure 14A). PI3K activates AKT signaling pathways through PIP2 and PIP3 (17). PIP3 expression was greatly reduced in viable cells and was barely detectable in cells with fragmented nucleus after ISA-201 IB treatment (Figure 14B). Similar to what was observed in LNCaP cells that were treated with ISA-201 IB, the decreased ΡΙΡ5Κ1α was coincident with a reduced expression of phosphorylated AKT in PC-3 cells treated with ISA-201 1B. The mean pSer-473 AKT expression in control treated cells was 18.44 compared with 5.49 in ISA-2011B treated cells with a decrease down to 29.77% (difference = 12.95, 95% CI = 2.84 to 8.14; P = 0.001) (Figure 14C). Similar to what was observed in LNCaP cells, etoposide, tadalafil and docetaxel did not exhibit inhibitory effect on AKT activity in PC-3 cells (Figure 14C). Expression of the key cell cycle regulators including cyclin Dl , cyclin El, cyclin A2, CDK1 , phosphorylated CDK1 , and cyclin B l was remarkably inhibited by ISA-201 1B (Figure 6D and E). ISA-2011B treatment led to an accumulation of cells at G2/M phase of cell cycle, a characteristics of cells that are unable to undergo proliferation (mean G2/M phase cells in control group is 14.87% and in treated group is 19.83% (difference = 4.96%; 95% CI = 15.01% to 20.79%; P = 0.047) (Figure 14F). In contrast to docetaxel, ISA-2011B treatment led to an increased rate of apoptosis with constant rate of unspecific necrosis in PC-3 cells as compared to controls (P < 0.001) (Figure 14G). As the cell-cell attachment is essential for cancer cells to expand and invade, we tested whether ISA-201 1B may prevent adherence and attachment of PC-3 cells. PC-3 cells treated with ISA-201 IB or solvent were subjected to adhesion assay (Figure 14H). The rate of adherence in cells treated with ISA-201 IB was only 38.82% compared to that of control (P= 0.002) (Figure 14H). We further assessed the effect of ISA-201 IB on invasiveness of PC-3 cells using invasion assay. ISA- 201 IB inhibited invasiveness of PC-3 cells down to 55.96% of control (Figure 141). Mean absorbance in control was 0.121 ; mean absorbance in ISA-201 IB treated was 0.068 (Figure 61, difference = 0.053; 95% CI = 0.066 to 0.070; P < 0.001). Expression of phosphorylated FAK, a factor that promotes invasion was almost diminished in PC-3 cells treated with ISA-201 IB and ISA-201 1 A at dose-dependent fashion (Figure 13 J). However, docetaxel did not show pronounced effect on FAK phosphorylation (Figure 14J). Taken together, ISA-201 IB inhibits proliferation, survival, and invasion in androgen-insensitive PCa cells. Moreover, this effect may be mediated through ΡΙΡ5Κ1 α and its downstream PI3K/AKT.

[0301] The effect of depletion of ΡΙΡ5Κ1α in PC-3 cells

[0302] We next asked whether inhibition of ΡΙΡ5Κ1α via siRNA-mediated knockdown may achieve the same effect as that of ISA-201 IB treatment in PC-3 cells. As such, we silenced ΡΙΡ5Κ1 α by transfecting PC-3 cells with ΡΙΡ5Κ1α or scramble siRNA. Immunoblot analysis confirmed that expression of ΡΙΡ5Κ1 α was reduced to 34.45% in cells transfected with ΡΙΡ5Κ1 α siRNA compared to control. Mean ΡΙΡ5Κ1α expression in control was 13.49; mean value in ΡΙΡ5Κ1α knockdown was 4.65 (Figure 15A, difference = 8.84; 95% CI = 3.86 to 5.43, i^><0.001). Knockdown of ΡΙΡ5Κ1α resulted in a significant decrease in expression of pSer-473 AKT down to 57.88%o. Mean pSer-473 AKT in control was 29.46 and 17.05 in ΡΙΡ5Κ1α knockdown cells (Figure 15B, difference = 12.41 ; 95% CI = 7.80 to 26.31 , =0.007). Thus, the effect of inhibition of ΡΙΡ5Κ1 α via siRNA on AKT activation was equivalent to what was achieved by ISA-201 IB treatment. The expression of cyclin Dl, cyclin A2, CDK1 and cyclin Bl was decreased in PC-3 cells transfected with ΡΙΡ5Κ1α siRNA when compared to controls (Figure 15C, D). The morphology of PC-3 cells expressing siPIP5Kl a as determined by β-Tubulin staining exhibited remarkable differences when compared to PC-3 cells expressing control siRNA, but was similar to control PNT1A cells (Figure 15E). Conversely, the morphology of PNT1A cells overexpressing ΡΙΡ5Κ1α resembled aggressive PC-3 control cells (Figure 15E). The activation of FAK was reported to be linked to a switch towards aggressive phenotypes. Expression of phosphorylated FAK in PC-3 cells expressing siPIP5Kla decreased to 36.98% (P < 0.001). Depletion of ΡΙΡ5Κ1α led to an increase in P27 expression, and a decrease in twist 1 and MMP9 (Figure 15G). An apoptotic marker c-PARP is used to detect early apoptotic process. Immunoblot analysis for c-PARP expression was performed using lysates from PC-3 cells transfected with control siRNA or siPIP5Kl a, which were treated with ISA-201 IB or vehicle (Figure 15H). Two distinct isoforms of c-PARP were detected in PC-3 cells that were either treated with ISA- 201 IB at 50 μΜ alone, expressed siPIP5Kla alone, or expressed siPIP5Kl a and were treated with ISA-201 IB in a combination. This suggests that downregulation of ΡΙΡ5Κ1α through treatment or knockdown induced apoptosis (Figure 15H). We examined the effect of inhibition of ΡΙΡ5Κ1α on tumor growth by implanting PC-3 cells expressing control siRNA or siPIP5Kla into nude mice. PC-3 cells expressing control siRNA formed tumors, with the mean tumor volume of 148.5 mm³ on day 20. In contrast, PC-3 cells transfected with siPIP5Kl a barely gave rise to tumors, with the mean tumor volume of 4.9 mm³ on day 20 (Figure 151, mean volume of control siRNA tumors = 148.5 mm³; mean volume of siPIP5Kl a = 4.9 mm³ (only one mouse had tumor, difference = 143.6 mm³; 95% CI = -4.7 to 14.4; P = 0.008, two-sided t test). Taken together, the targeted inhibition of ΡΙΡ5Κ1 α and ISA- 201 IB treatment showed similar inhibitory effect on tumor growth in xenograft mice.

[0303] DISCUSSION

[0304] In this study, we present our discovery of a novel anti-cancer drug candidate, ISA-201 IB and the role of ΡΙΡ5Κ1 α, a target of ISA-201 IB in PCa progression. ISA-201 IB has a unique structure consisting of a diketopiperazine fused, methylenedioxy protected, 1 ,2,3,4- tetrahydroisoquinoline core with an electron rich trans-substituent at position 1. We show that ISA- 201 IB inhibits tumor growth by inhibiting the expression and activity of ΡΙΡ5Κ1α thereby affecting the downstream PI3K/AKT, AR and cell cycle pathways (Figure 16). We for the first time show that overexpression of ΡΙΡ5Κ1α in non-malignant PNT1A cells induces the invasive capacity of these cells, and increases expression of VEGF, phosphorylated FAK, Twist, and MMP9, the key factors that promote cancer cell proliferation and invasion. Conversely, inhibition of ΡΙΡ5Κ1α in PC-3 cells via siRNA-mediated knockdown or ISA-201 IB treatment reduces invasiveness, induces apoptosis and inhibits tumor growth in xenograft mice. ΡΙΡ5Κ1α, and its associated PIP2, and PIP3 are important lipids for membrane structure and actin polymerization, thus elevated levels of these lipids may lead to malignant transformation and progression of cancer cells into a more invasive phenotype. Our present study suggests that ΡΙΡ5Κ1 α has a great potential to be used as a drug target for treatment of PCa.

[0305] AKT can be activated by phosphorylation through two upstream pathways: through PI3K/PIP3, or through cAMP/PKA (36). In the present study, we show that ISA-201 IB inhibits AKT activity through ΡΙΡ5Κ1α/ΡΙ3Κ/ΡΙΡ3. In contrast, ISA-201 IB treatment has no effect on PKA receptor and the key protein phosphorylated CREB in cAMP/PKA pathway in LNCaP or PC-3 cells. Further, the effect of ISA-201 IB is significantly stronger on PC-3 cells with PTEN mutation than on 22Rvl cells which contain intact PTEN gene. 22Rvl cells, though less sensitive to ISA-201 IB, also displayed reduced proliferation after ISA-201 IB treatment. It is known that 22Rvl cells also have relatively high levels of pAKT and AR (37). Given that amplification of AKT2 or mutations in PIK3CA, in addition to PTEN mutation, are all responsible for activation of PI3K pathway (15, 38, 39), our data suggest that ISA-201 IB exerts its on-target effect on PCa cells by targeting pathways related to AKT/AR, rather than toxic effect.

[0306] The mechanism underlying the interactions between AR and PI3K/AKT still remains poorly understood. In the present study, we uncover several unrecognized inter-links among ΡΙΡ5Κ1α, PI3K/AKT, AR and CDKl in PCa. Previous studies show that the PI3K/AKT and AR pathways negatively regulate each other during castration resistance (38, 39). Our proposed model suggests that ISA-201 IB inhibits ΡΙΡ5Κ1α, which leads to a subsequent inhibition in PI3K/AKT levels, and sustained P27 and down-regulation of CDKl and other cell cycle regulators. Down- regulation of CDKl may lead to an inhibition in AR signaling pathways. This model is supported by our data that CDKl and AR form protein-protein complexes predominantly in the nuclear compartment of cells. The complexes of CDK1-AR are persistent in PNT1A cells overexpressing ΡΙΡ5Κ1α. In agreement with our findings, a previous reported study show that CDKl phosphorylates AR and thereby activates AR activity during progression of castration resistant PCa (40). This indicates that ISA-201 IB targets CDKl associated pathways that regulate AR activity.

[0307] One of the most significant pre-clinical extensions of this work is to determine the therapeutic benefit of ISA-201 IB in PCa growth. Treatment of xenograft mice with ISA-201 IB results in tumor regression. Similarly, PC-3 cells in which ΡΙΡ5Κ1 α is inhibited through knockdown only formed a small tumor in one out of seven mice, in contrast to control PC-3 cells which formed large tumors in all seven xenograft mice. These in vivo data are in agreement with the data obtained in cell line studies in which ISA-201 IB treatment inhibits tumor growth by inhibiting levels of ΡΙΡ5Κ1α and pAKT S473. However, due to the small size of tumors at the end of experiment, it is difficult to measure the levels of pAKT S473 in vivo.

[0308] In the present study, we also observed that treatment with ISA201 IB in combination with docetaxel completely blocked the progression of invasive prostate cancer locally, and profoundly inhibited tumor growth. Docetaxel inhibits mitosis and induces apoptosis in cancer cells as well as normal proliferating cells and is highly toxic. We found that ISA-2011B together with docetaxel showed less toxic effects in mice compared with docetaxel alone. Since the interaction between two drugs often results in changes in metabolic pathways and binding of drugs to cellular membranes, our data suggest that the interaction between ISA-2011B and docetaxel via unknown mechanisms may lead to a reduced off-target effects in mice bearing tumors.

[0309] Taken together, our novel findings in our present study provide valuable information on the novel targets and anticancer drugs, which hold a great potential for further development of advanced prostate cancer treatment. It will be interesting to further systematically investigate the on-target effect of ISA-2011B treatment on pAKT S473 in vivo by using several well-designed in vivo mouse models in our future studies.

[0310] MATERIALS AND METHODS

[0311] KINOMEscan®

[0312] The interaction of ISA-2011B with 442 kinases covering more than 80% of the human catalytic protein kinome was tested using KINOMEscan assay. Screening "hits" are identified by measuring the amount of kinase captured in test versus control samples by using quantitative RT- PCR.

[0313] Tissue Specimens, Tissue Microarrays, cDNA Microarrays, and CGH Arrays

[0314] Tissue microarrays containing BPH and PCa tissues from 48 patients were purchased from Pantomics Inc. (Richmond, CA). mRNA expression data of ΡΙΡ5Κ1α, AKT2, PTEN and AR was extracted from the dataset in the cBioPortal database (35). The study was approved by the Ethics Committee, Lund University and the Helsinki Declaration of Human Rights was strictly observed.

[0315] MTS Proliferation Assay

[0316] The effects of ISA-201 IB, docetaxel, Etoposide and Tadalafil on PCa cell lines and various types of cancer cell lines were determined using the nonradioactive MTS proliferation assay (Promega Biotech) according to manufacturer's protocol. Viability was determined by measuring the absorbance at 490 nm wavelength, on Infinite® M200 multimode microplate reader (Tecan Sunrise™).

[0317] Mouse Models of Human Xenograft Tumors

[0318] Four sets of treatment experiments in mouse models bearing human PC-3 tumors were performed. All animal experiments met the requirement of Lund University Animal Care Regulation. For treatment experiments, BALB/c nude mice aged 8-12 weeks were used in the experiments. 4 ^χ 10⁶ tumor cells were implanted into the mice (6 mice/group). Tumor xenografts were treated with vehicle (control), docetaxel (10 mg/kg), ISA-2011B (40 mg/kg), and docetaxel (10 mg/kg) in combination with ISA-201 IB (40 mg/kg) every second day. For ΡΙΡ5Κ1α knockdown experiments, 2 x 10⁶ PC-3 cells transfected with control or ΡΙΡ5Κ1α siRNA were implanted into the NRMI nude mice (7 mice/group).

[0319] Plasmids, Stable Transfection, and siRNA Knockdown Assay

[0320] For transfection, pLPS-3'EGFP vector containing full-length human ΡΙΡ5Κ1α cDNA as "PIP5K1 a-EGFP" or control empty vector were used. Cells overexpressing PIP5Kla- pLPS-3'EGFP or pLPS-3'EGFP vector were selected by culturing cells in medium containing G418 antibiotics (400 g/ml) (Sigma Aldrich, Stockholm).

[0321] FACS-Based Cell Cycle Analysis and Apoptosis Assay

[0322] Cell cycle analysis of PCa cells after treatment with ISA-201 IB was performed. To measure apoptosis, cells were collected after treatment for 48 hours and were Subsequent stained with FITC-conjugated Annexin V and 7-AAD according to the manufacturers' Protocol (BD Biosciences, San Jose, CA, USA).

[0323] Invasion Assay

[0324] The Boyden trans-well chambers were used for invasion assay according to the manufacturer's protocol (Merck KGaA, Darmstadt, Germany).

[0325] Statistical Analysis

[0326] All outcome variables are presented are representative of at least three independent experiments. All statistical testes were two-sided, and P values less than 0.05 were considered to be statistically significant. Statistical software SPSS, version 21 (Chicago) was used.

[0327] KINOMEscan. The interaction of ISA-201 IB with 442 kinases covering more than 80% of the human catalytic protein kinome was tested using the KINOMEscan assay. We used competition binding assays by interrogating compounds against human protein kinome. The binding of ISA- 201 IB to the kinase active site directly (sterically) or indirectly (allosterically) was determined. We initially screened each compound against the panel at a single concentration (10 μΜ) to identify candidate kinase targets and determined a quantitative dissociation constant (Kd) for each interaction, as previously described (1). Schematic illustration on high-throughput kinase profiling technology using KINOMEscan platform was shown. Escherichia coli or mammalian cell-expressed kinases labeled with DNA tags for quantitative PCR readout are equilibrated together with test compound (ISA-201 IB) or DMSO as control. Briefly, known active site binding ligands are immobilized on each well of the microplates. After equilibration the wells are washed to remove unbound kinases. Screening "hits" are identified by measuring the amount of kinase captured in test vs. control samples by using quantitative RT-PCR. Bound kinase levels in test compound and control wells are compared. In a similar manner, dissociation constants (Kd) for test compound-kinase interactions are determined by measuring the amount of kinase capture.

[0328] Tissue Specimens, Tissue Microarrays, cDNA Microarrays, and CGH Arrays.

Paraffin- embedded sections containing representative tissue cores of prostate cancer (PCa) and corresponding benign prostate samples from 48 patients were purchased from Pantomics Inc. mRNA expression data of ΡΙΡ5Κ1 α, AKT2, phosphatase and tensin homolog (PTEN), and AR were extracted from the dataset in the cBioPortal database. The data were obtained by performing the Affymetrix U133A array platform using human normal tissues from primary PCa (n = 181) and metastatic PCa lesions (n = 37), as previously described (2). The study was approved by the Ethics Committee, Lund University, and the Helsinki Declaration of Human Rights was strictly observed. Tumor specimens from distinct subgroups of patients with primary (n = 181) and metastatic (n = 37) PCa were assessed. The Mann- Whitney test was performed to compare expression levels in metastatic cancers with those observed in nonmetastatic primary tumor tissues; **P < 0.01.

[0329] Immunohistochemistry Analysis. Immunohistochemistry on tumor tissue arrays was performed as previously described (3). The staining procedure was performed using a semiautomatic staining machine (Ventana ES; Ventana Inc.). The sections were viewed with an Olympus BX51 microscope at magnification of ^χ20 or x40. The specimens were evaluated by four different scientists; one of them is a specialist in pathology. The staining intensity was scored as 0 (negative), 1 (weakly positive or positive), 2 (moderate positive), or 3 (strongly or very strongly positive) using an arbitrary semiquantitative scale. The slides were scanned, and microphotographs were taken using the scanner (ScanscopeCS; Aperio).

[0330] Cell Culturing and Treatments. PC-3, 22Rvl , and PNT1A cells were purchased from American Type Culture Collection. Treatments with ISA-201 1B dissolved in DMSO and various types of drugs, including docetaxel, etoposide (Sigma-Aldrich), and tadalafil, was performed. A 0.5% DMSO concentration was used in in vitro experiments. Cells (0.2 ^χ 106 cells/mL) were seeded and allowed to attach to the plates by growing in 10% FBS phenol red-free RPMI-1640 medium for 24 h and then were treated with drugs alone or in combination for 24, 48, and 72 h. ISA-201 IB, ISA-2009, ISA-201 1A (ΙμΜ, 5 μΜ, 10 μΜ, 50 μΜ, and 100 μΜ), etoposide, tadalafil (20-50 μΜ), or docetaxel (25 nM) were used.

[0331] MTS Proliferation Assay. The effects of ISA-201 IB, docetaxel, etoposide, and tadalafil on PCa cell lines and various types of cancer cell lines were determined using the nonradioactive MTS proliferation assay (Promega Biotech) according to the manufacturer's protocol. Cancer cells (5 x 103 cells per well) were incubated for 24 h in a 96-well plastic plate that contained complete growth medium to allow attachment. The medium was then replaced with fresh medium containing ISA- 201 IB or the anticancer drugs as mentioned above at different concentrations and cultured for 24, 48, or 72 h. MTS reagent (20 μί) was then added, and cells were cultured for another 4 h. Viability was determined by measuring the absorbance at 490-nm wavelength, on an Infinite M200 multimode microplate reader (Tecan Sunrise).

[0332] Mouse Models of Human Xenograft Tumors. Four sets of treatment experiments in mouse models bearing human PC-3 tumors were performed. All animal experiments met the requirement of Lund University Animal Care Regulation. For ΡΙΡ5Κ1α knockdown experiments, 2 ^χ 106 PC-3 cells transfected with control or ΡΙΡ5Κ1α siRNA were implanted into NRMI nude mice (seven mice per group). For treatment experiments, BALB/c nude mice aged 8-12 wk were used in the experiments. Subcutaneous xenografts were established with PC-3 cells. Tumor cells (4 x 106) were implanted into the mice, and tumors were grown to 50 mm3 on average. Xenograft mice with tumors were randomized and were treated with different agents (n = 6 mice per group). Upon tumor initiation, aliquots of appropriate numbers of viable cells were suspended in 100 sterile vehicle solution and implanted s.c. into the flank. At the end of experiments, mice were killed under anesthesia using lethal i.p. injections. Tumor diameters were measured using calipers, and volumes were calculated using the formula a x b2/2, where a and b represent the larger and smaller diameters, respectively. When the mean tumor volume reached 50 mm3, treatment of xenograft mice with vehicle control, ISA-2011B, docetaxel, and combination of ISA-2011B and docetaxel was initiated and lasted for 20 d. Tumor xenografts were treated with vehicle (control), docetaxel (10 mg/kg), ISA- 201 IB (40 mg/kg), and docetaxel (10 mg/kg) in combination with ISA-2011B (40 mg/kg) every second day.

[0333] Plasmids, Stable Transfection, and siRNA Knockdown Assay. For transient transfection, pLPS-3'EGFP vector containing full-length human ΡΙΡ5Κ1 α cDNA as "PIP5K1 a- EGFP" or control empty vector were used. The vectors were purchased from Harvard Medical School. Transient transfection was performed using Lipofectamine 2000 transfection reagent (Life Technologies) according to the manufacturer's instructions. For stable transfection, PNT1A cells were transfected with Lipofectamine 2000 transfection reagent. Vectors (2-5 μg) were used in transfection experiments. Cells overexpressing ΡΙΡ5Κ1 α- pLPS-3'EGFP or pLPS-3'EGFP vector were selected by culturing cells in medium containing G418 antibiotics (400 g/mL) ( Sigma- Aldrich). Cells were maintained in medium with G418 antibiotics for a total of 2 wk. SiRNAs against ΡΙΡ5Κ1 α or siRNA negative control duplex were purchased from Life Technologies. SiRNAs (50 nM) were transfected into 1 x 105 PCa cells using Transfection Reagent TransIT-TKO according to the manufacturer's protocol (Minis Bio, LCC). After introduction of respective siRNA complexes into PCa cells, cells were then collected at 24, 48, and 72 h after transfection.

[0334] Antibodies. Primary antibodies against ΡΙΡ5Κ1 α, β-actin, pAkt, p27, cyclin Dl , cyclin E, cyclin B, cyclin-dependent kinase 1 (CDK1), pCDKl(Tyrl4-15), and PKA RI-α/β at 1 :500 dilutions (Cell Signaling Technology); PKA RIi at 1 : 1 ,000 (BD Biosciences Transduction Laboratories); androgen receptor (AR) at 1 :250; the p-CREB-1 at Ser-133 sites at 1 :500; P27, cyclin A2 (Santa Cruz Biotechnology Inc.), prostate-specific antigen (PSA) at 1 :500 (DAKO); and MMP-9 (Abeam) and anti β-actin at 1 : 10,000 (MP Biochemicals) were used. Secondary antibodies were HRP-conjugated anti- mouse IgG and anti-rabbit IgG (GE Healthcare).

[0335] FACS-Based Cell Cycle Analysis and Apoptosis Assay. Cell cycle analysis of PCa cells after treatment with ISA-201 1B was performed. Cells were harvested after treatment. Cells (1 ^x 106) were washed with PBS and fixed in 70% ice-cold ethanol at -20 °C overnight. Fixed cells were centrifuged at 400 x g and washed with PBS. The cells were stained with 50 g/mL propidium iodide (PI) (Sigma-Aldrich), 0.1 % Triton X-100 (Sigma-Aldrich), and 100 g/mL RNase-A (AppliChem) for 40 min at room temperature in the dark, and the ΡΙ-elicited fluorescence of individual cells was measured using flow cytometry (FACS Calibur; Becton Dickinson). To measure apoptosis, cells were collected after treatment for 48 h and were subsequently stained with FITC-conjugated Annexin V and 7-AAD according to the manufacturer's protocol (BD Biosciences). The cells were then subsequently subjected to flow cytometry analysis on a FACSCalibur (Becton Dickinson). The data were assessed using FCS Express software (DeNovo Software).

[0336] Subcellular Fractionation and Immunoprecipitation. Subcellular fractionation was prepared as in previously published work (4). Cell pellets were resuspended in ice-cold nuclei isolation buffer [10 mM HEPES (pH 7.9), 1.5 mM MgC12, 10 mM KC1, 0.5 mM DTT, 1 % Triton X- 100, 15% PI Complete Mini, and 1 mM PMSF], incubated on ice for 10 min, and then centrifuged. The supernatant containing the cytoplasmic fraction was collected, and the pellet was resuspended in ice-cold RIPA buffer [120 mM NaCl, 50 mM Tris HCl (pH 7.6), 50 mM NaF, 0.1 mM Na3V04, 1 % Nonidet P-40, and 1 mM PMSF; Sigma] and 15% protease inhibitor mixture Complete Mini (Roche), followed by incubation on ice for 20 min. After centrifugation the supernatant containing the final nuclear fraction was collected. The nuclear and cytoplasmic fractions were subjected to immunoblot analysis. The subcellular fractionation was controlled by detection of β-tubulin and Lamin B in the cytoplasmic and nuclear fractions, respectively. Cytoplasmic (Cyt) and nuclear (Nuc) fractions were separated from PNT1A cells overexpressing ΡΙΡ5Κ1α or control vector and were subjected to immunoprecipitation assay. Antibody to CDK1 was used to pull down the immunocomplexes, and antibody to IgG was used as a negative control. CDK1 antibody and antibody to IgG (negative control) (BD Biosciences) were incubated with 500 x,g of freshly prepared protein lysates and 30 of G-Sepharose beads (GE Healthcare) for 3 h at 4 °C. After incubation the samples were washed in RIPA buffer and prepared for immunoblot analysis. The cell lysates from cytoplasmic and nuclear fractions were used as "Input" controls. Blotting of actin served as loading control, and antibody against lamin B was used as a control for the nuclear fraction.

[0337] Immunoblot Analysis. The cells were harvested and lysed in ice-cold RIPA buffer. The protein (20-30 x,g) was separated with 12% SDS/PAGE gels and transferred onto nitrocellulose membranes. Signals were visualized using the Enhanced ChemiLuminescence detection system (Pierce) and documented with an Alphalmager CCD system. Densitometric quantification of immunoblots was performed by ImageJ Image Analysis Software (National Institutes of Health) and represented as fold change relative to control and was normalized with actin band.

[0338] Immunofluorescence and Morphological Analysis. PCa cells were grown on the glass coverslips in phenol red-free RPMI-1640 medium containing 10% FBS for 24 h and were then treated with ISA-201 IB or indicated drugs for 24 h. Cells were fixed with 4% paraformaldehyde in PBS. For blocking background staining from nonspecific interactions, Image-iT FX signal enhancer (Molecular Probes) was used. Primary antibodies against ΡΙΡ5Κ1α, PIP2, Phosphor-473 AKT, β-tubulin, AR, and PSA were used. The secondary antibodies, including rabbit anti-donkey conjugated to Rhodamine (Chemicon/Millipore International Inc.) or anti-goat conjugated to FITC antibodies at 1 :200 and goat anti-rabbit Alexa Fluor 488 at 1 :500 (Invitrogen, Stockholm, Sweden), were used. The counterstain 4',6-diamidino-2-phenylindole (SERVA Electrophoresis GmbH) was used to visualize cell nuclei. The slides were detected under an Olympus AX70 fluorescent microscope (Nikon DS-U1). The software ACT2U was used (ACT2U version. 1.5). Alternatively, the images were accessed, and photomicrographs were taken at x lO magnification using the HoloMonitor M3 (Phase Holographic Imaging AB).

[0339] Invasion Assay. For invasion assay using PNT1A cells, cells stably expressing control vector or PIP5Kla vector were cultured in RPMI-1640 medium containing 10% FBS for 5 d and followed by culturing in serum-free medium for an additional 2 d before being subjected to invasion assays. PNT1A cells in RPMI-1640 medium containing 10% FBS were also subjected to invasion assay. The effect of ISA-201 IB (50 μΜ) on the invasiveness of PC-3 cells was evaluated with Boyden transwell chambers (Merck KGaA) according to the manufacturer's protocol. In brief, 5 ^x 104 PC-3 cells in 300 of serum-free medium supplemented with 50 μΜ ISA-201 IB were seeded into the upper chamber of the system.

[0340] The lower well was filled with 10% FBS containing Ham's F-12 medium as chemoattractant and the same reagent treatment as the upper chamber. After 48 h of incubation, the noninvading cells in the upper chamber were wiped off, and the invasive cells on the lower membrane were stained for 15 min at room temperature and then dissolved in 10% acetic acid. The absorbance of the stained cells was measured on an ELISA plate reader. The effects of ΡΙΡ5Κ1α overexpression on the invasion capacity of PNT1A cells were evaluated using QCM High Sensitivity Noncross- linked Collagen invasion assay (Merck Millipore) as per the manufacturer's protocol. In brief, control and PIP5Kla-overexpressing PNTA cells were starved in serum- free growth medium for 96 h before seeding. The invasion assay was initiated by seeding 1.25 x 105 cells into respective inserts, in a total of 250 of serum-free growth medium (upper chamber). At the same time, 500 of complete growth medium supplemented with 10% FBS was added into the lower chamber. The cells were cultured for 48 h to allow migration through the collagen layer. After incubation the cells were stained, and the invaded cell number was determined by manual counting. Adhesion Assay. A 96-well plate was coated overnight at 4 °C with Fibronectin (AMS Biotechnology) diluted in Dulbecco's PBS at a final concentration 50 g/mL in a volume of 100 μ As negative controls, wells were incubated with Dulbecco's PBS only. After coating, the wells were washed with 1 * PBS three times, and 2% BSA in l x PBS was added to the wells and incubated for 1 h at 37 °C. The wells were then washed with l x PBS, and subsequently 1 x 105 cells that were pretreated with agents or vehicle control were added to the wells and incubated at 37 °C, 5% C02 for 6 h. The wells were then washed, and the adherent cells were collected and counted.

[0341] Statistical Analysis. Possible pairwise correlations between the groups were analyzed using the Spearman rank correlation test. The statistical significance of differences between groups was calculated by using paired the Wilcoxon rank sum test and Student's t test or the Kruskal-Wallis and Mann- Whitney tests (comparison of means), and the Fisher exact test was used for comparison of incidence, as indicated. For measurement of tumor growth, two-way repeated analysis of variance (ANOVA) was performed using the ANOVA test. Distributions of biochemical recurrence (BCR)- free survival were estimated using Kaplan-Meier survival analysis. Differences between survival curves were calculated using the log-rank test. All outcome variables presented are representative of at least three independent experiments. All statistical testes were two-sided, and P values <0.05 were considered to be statistically significant. The statistical software SPSS, version 21 was used.

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[0343] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein the terms "about" and "approximately" means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0344] The terms "a," "an," "the" and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0345] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0346] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0347] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term "consisting of excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0348] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.

[0349] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

1. A method for treatment of a disease in a subject in which inhibition of PIP5K1 A is desired and/or required, comprising:

administering a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.

2. A method for treatment of a disease in a subject characterized by over activation of PIP5K1 A and is in need of such treatment, comprising:

administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of PIP5K1A.

3. The method of claim 1 or 2, wherein said inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 5% and 100% as determined by an in vitro or in vivo assay.

4. The method of claim 3, wherein said inhibitor of PIP5K1A is capable of reducing PIP5K1A activity by between about 40% and 80% as determined by an in vitro or in vivo assay.

5. A method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising:

6. The method of claim 5, wherein said inhibitor of PIP5K1A is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity of as determined by an in vitro or in vivo assay.

7. The method of claim 6, wherein said inhibitor of PIP5K1A is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity of as determined by an in vitro or in vivo assay.

8. A method for treatment of a disease in a subject characterized by over activation of PI3 Kinase and is in need of such treatment, comprising:

9. The method of claim 8, wherein said inhibitor of PIP5K1 A is capable of reducing production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) to between about 50 %> and 150%o of normal production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal, as determined by an in vitro or in vivo assay.

10. The method of claim 9, wherein said inhibitor of PIP5K1A is capable of reducing production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) between about 80 % and 120% of normal production level of phosphatidylinositol 3,4,5-bisphosphate (PtdIns(3,4,5)P2) when PI3K expression level is normal , as determined by an in vitro or in vivo assay.

11. A method for treatment of a disease in a subject characterized by over activation of phospholipase C (PLC) and is in need of such treatment, comprising:

12. The method of claim 11, wherein said inhibitor of PIP5K1 A is capable of reducing PLC activity to between about 50 % and 150% of normal PLC activity of as determined by an in vitro or in vivo assay.

13. The method of claim 12, wherein said inhibitor of PIP5K1A is capable of reducing PLC activity to between about 80 % and 120% of normal PLC activity of as determined by an in vitro or in vivo assay.

14. A method for treatment of a disease in a subject characterized by over activation of Akt and is in need of such treatment, comprising:

administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of modulator of PI3K/Akt pathway, wherein said modulator is capable of reducing Akt activity to between about 50 % and 150% of normal Akt activity of as determined by an in vitro or in vivo assay.

15. The method of claim 14, wherein said modulator of PDK/Akt pathway is capable of reducing Akt activity to between about 80 % and 120% of normal Akt activity of as determined by an in vitro or in vivo assay.

16. The method of any of claims 1 - 15, wherein said disease is cancer, spinal cord injury, or AIDS.

17. The method of claim 16, wherein said cancer is selected from the group consisting of :

hemangioma, hepatocellular adenoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas, pyogenic granulomas, leukemia, myelodysplastic syndromes (MDS), prostate cancer, breast cancer, skin cancer, bone cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, bladder, gall bladder, ovary, cervix, pancreas, rectum, parathyroid, thyroid, esophagus, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papilllary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, non-small cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy- cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

18. The method of any one of claims 1 to 17, wherein said inhibitor of PIP5K1 A, or modulator of PI3K/Akt pathway comprises a small molecule, an RNA, a peptide, or an antibody.

19. A method of diagnosing a subject suspect of having a disease, comprising:

(a) providing a biological sample from a subject;

(b) measuring expression levels for one or more genes and/or product of said one or more genes in said biological sample, wherein at least one of said one or more genes is PIP5K1 A gene; and

(c) comparing said measured expression levels to reference expression levels for said one or more genes and/or product of said one or more genes, wherein said reference expression levels correlate to diagnosis of said disease,

thereby diagnosing said subject with regard to said disease.

20. The method of claim 19, wherein said reference expression levels are determined using a sample from a normal subject and/or a sample from a patient diagnosed as having said disease.

21. A method for determining disease stage of a subject diagnosed as having a disease, comprising:

(a) providing a biological sample from a subject;

(b) measuring expression levels for one or more genes and/or product of said one or more genes in said biological sample, wherein at least one of said one or more genes is PIP5K1A gene; and (c) comparing said measured expression levels to reference expression levels for said one or more genes and/or product of said one or more genes, wherein said reference expression levels correlate to disease stage of said disease,

thereby determining disease stage of said subject with regard to said disease.

22. The method of claim 21, wherein said reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having said disease of different disease stages.

23. A method for monitoring disease progression of a subject diagnosed as having a disease, comprising:

(a) providing a biological sample from a subject;

(c) comparing said measured expression levels to reference expression levels for said one or more genes and/or product of said one or more genes, wherein said reference expression levels correlate to disease progression of said disease,

thereby determining disease progression of said subject with regard to said disease.

24. The method of claim 23, wherein said reference expression levels are determined using a sample from a normal subject and/or a sample from a patient has been diagnosed as having said disease of different disease progression.

25. A method for determining a prognosis for a subject diagnosed as having a disease, comprising:

(a) providing a biological sample from a subject;

(c) comparing said measured expression levels to reference expression levels for said one or more genes and/or product of said one or more genes, wherein said reference expression levels correlate to prognosis of said disease,

thereby determining prognosis of said subject with regard to said disease.

26. The method of claim 25, wherein said reference expression levels are determined using a sample from a normal subject and/or a sample from a patient determined as having said disease of different prognosis outcome.

27. A method of determining effect of treatment of a subject having a disease condition with a pharmaceutical composition, comprising:

(a) providing a biological sample from a subject that has been subject to a treatment of a disease with a pharmaceutical composition ;

(c) comparing said measured expression levels to reference expression levels for said one or more genes and/or product of said one or more genes, wherein said reference expression levels correlate to treatment effect of said disease,

thereby determining the treatment effect of said pharmaceutical composition.

28. A method of determining whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIP5K1A, and/or an inhibitor of PI3K/Akt pathway, and/or an inhibitor of PLC pathway, comprising:

(a) providing a biological sample from a subject t having a disease condition;

(c) comparing said measured expression levels to reference expression levels for said one or more genes and/or product of said one or more genes, wherein said reference expression levels correlate to whether a subject having a disease condition should be treated with a pharmaceutical composition comprising an inhibitor of PIPKIA, and/or an inhibitor of PI3K/Akt pathway, and/or an inhibitor of PLC pathway,

thereby determining whether said subject should be treated with said pharmaceutical composition.

29. The method of claim 27 or 28, wherein said reference expression levels are determined using a sample from a normal subject and/or a sample from a patient that has been subjected to said treatment and achieved treatment effect.

30. The method of claim 28, wherein said inhibitor of PIP5K1A, or inhibitor of PDK/Akt pathway, or inhibitor of PLC pathway, comprises a small molecule, an RNA, a peptide, or an antibody.

31. The method of any of claims 19 - 30, wherein said disease is cancer, spinal cord injury, or AIDS.

32. The method of claim 31, wherein said cancer is selected from the group consisting of :

hemangioma, hepatocellular adnoma, cavernous haemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regenerative hyperplasia, trachomas, pyogenic granulomas, leukemia, myelodysplastic syndromes (MDS), prostate cancer, breast cancer, skin cancer, bone cancer, liver cancer, lung cancer, brain cancer, cancer of the larynx, bladder, gall bladder, ovary, cervix, pancreas, rectum, parathyroid, thyroid, esophagus, adrenal, neural tissue, head and neck, colon, stomach, bronchi, kidneys, basal cell carcinoma, squamous cell carcinoma of both ulcerating and papilllary type, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma, veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lung tumor, non-small cell lung tumor, gallstones, islet cell tumor, primary brain tumor, acute and chronic lymphocytic and granulocytic tumors, hairy- cell tumor, adenoma, hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuronms, intestinal ganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomater tumor, cervical dysplasia and in situ carcinoma, neuroblastoma, retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skin lesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenic and other sarcoma, malignant hypercalcemia, renal cell tumor, polycythemia vera, adenocarcinoma, glioblastoma multiforma, leukemias, lymphomas, malignant melanomas, epidermoid carcinomas, and other carcinomas and sarcomas.

33. The method of any one of claims 19 to 32, wherein said one or more genes comprises a gene selected from the group consisting of: Akt, pAkt (phosphorylated Akt), Alpha-fetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion gene, BRAF mutation V600E, CA15-3/CA27.29, CA19-9, CA-125, Carcinoembryonic antigen (CEA), CD20, Chromogranin A (CgA), Chromosomes 3, 7, 17, and 9p21, Cytokeratin fragments 21 - 1 , EGFR mutations, Estrogen receptor (ER)/progesterone receptor (PR), Androgen receptor (AR), Fibrin/fibrinogen, HER4, HER2/neu, Immunoglobulins, KIT, KRAS , mutation analysis, Lactate dehydrogenase, Nuclear matrix protein 22, Prostate-specific antigen (PSA), Urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), BRCA1 , ERCC1, TYMS, RRM1, TUBB3, STMN1 , MDR1, GSTP1 , TOP2A, IGF 1R, VEGFR 1 /VEGFR2 , FGFR1/FGFR2/FGFR3/FGFR4, PTEN, KIT, Cyclin Al, Cyclin Dl, GADD45, DDB2, VEGF, MMP-2, MMP-9, CDK1, Chkl, FAK, pFAK (phosphorylated FAK), PIK3CA , CYLD, Nflx, Impadl, DCAMKL-1 , Casein kinase II alpha (Ck2a), Phosphotidic acid (PA), Racl , RhoA, Ajuba (LIMP), pRB (phosphorylated RB), ARF6 , and PLD2 .

34. The method of any one of claims 19 - 33, wherein said expression levels are mRNA expression level or protein expression level, or both.

35. A method for reducing over activation of PI3K/Akt pathway in a subject, comprising: a administering said subject with an inhibitor of PIP5K1A, thereby reducing over activation of said PDK/Akt pathway in said subject.

36. A method for reducing over activation of PI3K/Akt pathway in a cell, comprising:

contacting said cell with an inhibitor of PIP5K1A, thereby reducing over activation of said

PI3K/Akt pathway in said cell.

37. A method for reducing over activation of PLC pathway in a subject, comprising:

administering said subject with an inhibitor of PIP5K1A, thereby reducing over activation of said PLC pathway in said subject.

38. A method for reducing over activation of PLC pathway in a cell, comprising:

PLC pathway in said cell.