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WO2008110491A2 - Chemotherapy of neoplastic diseases using combinations of rapamycin and compounds modulating mtor pathway alone or in combination with heat - Google Patents

Chemotherapy of neoplastic diseases using combinations of rapamycin and compounds modulating mtor pathway alone or in combination with heat Download PDF

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WO2008110491A2
WO2008110491A2 PCT/EP2008/052682 EP2008052682W WO2008110491A2 WO 2008110491 A2 WO2008110491 A2 WO 2008110491A2 EP 2008052682 W EP2008052682 W EP 2008052682W WO 2008110491 A2 WO2008110491 A2 WO 2008110491A2
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rapamycin
mtor
compound
cells
metformin
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PCT/EP2008/052682
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WO2008110491A3 (en
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Christoph Moroni
Don Benjamin
Marco Colombi
Klaus-Dieter Molle
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University Of Basel
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Rapamycin is known to inhibit T-cell proliferation at nanomolar concentrations, and its derivatives are more recently also in experimental clinical use as potential anti-neoplastic agents. However, it is observed that several cell types are not sensitive to treatment. Biomarkers which predict responsiveness of cancer cells to rapamycin or its derivatives are needed.
  • Metformin a drug used for the treatment of type Il diabetes, applied over prolonged times to diabetic patients appears to reduce the risk of cancer [J. M. M. Evans et al., BMJ
  • metformin inhibits mTOR-dependent translation initiation via the mTOR pathway components AMPK (adenosyl monophosphate activated kinase) and its target Tsc2 (tubreous sclerosis complex 2), and suggest that this may be a mechanism how metformin inhibits cancer cells. Growth inhibition reported is only partial. More than 40% of cells survive after treatment, indicating that under those conditions metformin alone is not adequate for cancer treatment.
  • AMPK adenosyl monophosphate activated kinase
  • Tsc2 tubreous sclerosis complex 2
  • US patent application 2007/10280918 describes a pharmaceutical composition for treating cancer comprising a combination of a PP2A methylating agent and an active principle selected from a large number of enzyme inhibitor compounds also naming rapamycin and metformin.
  • thermotherapy is an experimental mode of therapy of promising, yet limited success, where the underlying cellular determinants for sensitivity are not known.
  • the invention relates to compounds either blocking or activating the mTOR pathway for use in the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, in combination with rapamycin or a rapamycin- like compound and/or in combination with metformin or a metformin-like compound and/or in combination with thermal energy, and optionally in combination with further suitable inhibitor compounds.
  • mTOR pathway activating compounds sensitize cells to rapamycin and exert therapeutic synergism with rapamycin.
  • Compounds blocking the pathway are therapeutic on their own and additionally synergize with rapamycin and with mTOR pathway activators.
  • Compounds blocking or activating the mTOR pathway are, for example, compounds blocking or downregulating Tsc2, pten, Tsc1 , raptor, rictor, or blocking or downregulating other components of the mTOR complexes 1 and 2, or blocking or downregulating other components of the mTOR pathway including downstream elements of the mTOR pathway.
  • Particular examples of such compounds are anti-sense RNA, siRNA or shRNA downregulating Tsd , Tsc2 or pten.
  • Rapamycin or a rapamycin-like compound is a further example of a compound blocking the mTOR pathway.
  • Metformin exerts a powerful synergistic effect with rapamycin on some but not other cells.
  • the invention therefore, in particular, relates to compounds blocking or activating the mTOR pathway such as rapamycin or a rapamycin-like compound in combination with metformin or a metformin-like compound, and to other compounds blocking or activating the mTOR pathway in combination with rapamycin or a rapamycin-like compounds and/or in combination with metformin or a metformin-like compound for use in the treatment of the mentioned diseases.
  • the invention further relates to the use of a compound blocking or activating the mTOR pathway for the manufacture of a medicament for the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, in combination with rapamycin or a rapamycin-like compound and/or in combination with metformin or a metformin-like compound and/or in combination with thermal energy, and optionally in combination with an inhibitor of the MAPK, Wnt, Jak-Stat, Pl-3k, JUNK or hedgehog pathway and/or a DNA damaging agent, 2'-deoxy-glucose, an inducer of hypoxia, or a pyrogenic compound.
  • a compound blocking or activating the mTOR pathway for the manufacture of a medicament for the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, in combination with rapamycin or a rapamycin-like compound and/or in combination with metformin or a metformin-
  • the invention further relates to pharmaceutical preparations comprising a compound blocking or activating the mTOR pathway and one or several of the mentioned further inhibitors.
  • the invention further relates to a method of treating neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, comprising administering a therapeutically effective amount of a compound blocking or activating the mTOR pathway in combination with rapamycin or a rapamycin-like compound and/or in combination with thermotherapy and/or in combination with metformin or a metformin-like compound, and optionally in combination with an inhibitor of the MAPK, Wnt, Jak-Stat, Pl- 3K, JUNK or hedgehog pathway and/or a DNA damaging agent, 2'-deoxy-glucose, an inducer of hypoxia, or a pyrogenic compound.
  • the invention relates to a method of treating the mentioned diseases with compounds blocking or activating the mTOR pathway, such as rapamycin or a rapamycin-like compound or metformin or a metformin-like compound in combination with thermotherapy.
  • the invention further relates to a method for screening for a compound blocking the mTOR pathway using known IL-3 dependent cells that are insensitive to mTOR pathway blockade exerted by rapamycin, and in parallel known IL-3 independent mutant cells that are sensitive to rapamycin due to their addiction to the mTOR pathway.
  • Such mutants can be obtained by frameshift mutagen (ICR191 ) treatment of IL-3 dependent cells followed by selection of independent cells via IL-3 removal [K. F. Kiser et al., Oncogene 25:6595-6603 (2006)]. Suitable cells are identified by establishing rapamycin sensitivity of growth.
  • the invention further relates to a method to predict sensitivity of a tumor to the combination of mTOR pathway inhibition combined with application of thermal energy, based on the fact that cells with mutations in Pten display elevated levels of the gluconeogenic enzyme fructose-1 ,6-bisphosphatase 2 (FBP2) and are heat sensitive due to an energy burning "futile cycle".
  • FBP2 gluconeogenic enzyme fructose-1 ,6-bisphosphatase 2
  • Figure 1 Growth inhibition by rapamycin and rescue by IL-3
  • X-axis nanomolar concentration of rapamycin
  • Y-axis % of cell growth. Indicated cell lines were grown with indicated concentrations of rapamycin (X-axis in nM) for 48 h in presence or absence of IL-3 (as indicated), and growth (Y-axis in %) was determined.
  • Fig. 1A Rapamycin inhibits the mutant line D2c, but not its non-mutagenized precursor
  • cell hybrid 20xD2c is rapamycin insensitive, indicating that drug sensitivity is recessive and was acquired by a loss-of- function mutation restorable by cell fusion. All cells are tested in presence of IL-3.
  • Fig. 1 B Five additional IL-3 independent mutant lines, all derived from 15V4, are likewise sensitive to rapamycin. Drug inhibition is rescued in all cases by the presence of IL-3 during growth (dashed lines).
  • Fig. 1 C Clone D2c is different from all clones, in that it is not rescued by the addition of IL-3.
  • Figure 2 Growth of cells transformed by mTOR are sensitive to rapamycin.
  • X-axis nanomolar concentration of rapamycin;
  • Y-axis % of cell growth.
  • the sensitivity to rapamycin was determined following 48 h incubation with indicated concentrations of rapamycin. The fact that cells die by this mTOR antagonist proves that 15V4 can become addicted to mTOR-dependent growth by activation of the mTOR pathway.
  • Figure 3 Transformation of 15V4 to IL-3 independent growth by siRNA targeting PTEN or TSC2.
  • Fig. 3A 15V4 cells were infected with lentiviruses encoding small hairpin RNA (shRNA) targeting PTEN (shPTEN), TSC2 (shTSC2), or a non-targeting control sequence (shCTRL) and selected for puromycin resistance for 3 days. Then, IL-3 was removed and the number of viable cells determined at the indicated times (days without IL-3: 0, 1 , 2, 3, 4, 7, 13, 18, 23). Results indicate that downregulation of both PTEN and TSC2 is sufficient to transform cells to IL-3 independent growth.
  • shRNA small hairpin RNA
  • shPTEN PTEN
  • TSC2 TSC2
  • shCTRL non-targeting control sequence
  • Fig. 3B X-axis: nanomolar concentration of rapamycin; Y-axis: % of cell growth.
  • the upper panel shows sensitivity of shPTEN cells described in 3A to rapamycin treatment.
  • the lower panel shows sensitivity of shTSC2 cells described in 3A to rapamycin treatment. Drug inhibition is rescued in shPTEN and shTSC2 cells by the presence of IL-3 during growth.
  • the sensitivity to rapamycin was determined following 48 h incubation with indicated concentrations of rapamycin.
  • the fact that cells die by this mTOR antagonist proves that 15V4 can become addicted to mTOR-dependent growth by activation of the mTOR pathway.
  • X-axis time in hours; Y-axis: cells per milliliter.
  • Precursor cells 15V4 (upper panel), mutants 6.5 (middle panel) and D2c (lower panel) were infected with lentiviruses carrying shRNA targeting raptor (shRap), a component of mTORCI , or rictor (shRic), a component of mTORC2, or non-targeting (shCon). After selection with puromycin for 2 days, IL-3 was removed (-IL-3) or not (+IL-3) and the number of viable cells determined over time.
  • mTORCI is vital for growth of D2c, and mTORC2 for 6.5.
  • Figure 5 Metformin inhibits growth of 6.5, but not of normal precursor cells 15V4 X-axis: millimolar concentration of metformin; Y-axis: % of cell growth (note the logarithmic scale). Cells were incubated at the indicated nM concentrations of rapamycin (R) and metformin, alone or in combination, and growth was determined after 48 h.
  • Panel A precursor 15V4 cells are insensitive even to the combination of drugs.
  • Panels B, C D2c is sensitive to rapamycin, but hardly to metformin. No change in presence of IL-3.
  • Panels D, E rapamycin sensitive 6.5 cells are sensitive to metformin alone, with a very strong synergistic effect when combined with rapamycin. Drug inhibition is rescued in 6.5 cells by the presence of IL-3 during growth.
  • Figure 6 Identification of marker gene FBP2 by Affymetrix DNA microchip analysis. RNA expression from triplicate cultures are analyzed. All cells including mutants were grown in the presence of IL-3 before RNA isolation. Indicated are the number of transcripts which show a change factor of at least two-fold.
  • A Comparison of mutants 6.5, 6.8 and H1 .2 with precursor 15V4. Numbers outside the circles indicate the number of transcripts up- or downregulated in the respective mutant compared to precursor line 15V4. Arrows pointing up: upregulated transcripts; arrows pointing down: downregulated transcripts. For example, in clone 6.5, 180 transcripts are upregulated, and 193 transcripts are downregulated. Note that 12 transcripts are upregulated and 24 transcripts are downregulated in all three mutants.
  • B Comparison of mutants 6.5, 6.8 and H1 .2 with their backfusion hybrids 6.5 x 20, 6.8 x 20 and H1.2 x 20. Arrows pointing up: upregulated transcripts in mutant versus hybrid; arrows pointing down: downregulated transcripts in mutant versus hybrid. Note that 69 transcripts are elevated in all mutants versus their hybrids, while 32 transcripts are downregulated in all hybrids.
  • D Relative expression level of FBP2 expression in precursor cells, mutants (with and without rapamycin) and hybrids.
  • Y-axis arbitrary units of expression.
  • Figure 7 Analysis of FBP2 expression by real-time quantitative PCR
  • X-axis indicated cell lines.
  • Y-axis relative number of transcripts with 15V4 set as 1 (exponential units).
  • the invention relates to compounds modulating the mTOR pathway either as activators thus sensitizing cells to rapamycin (rapamycin sensitizers) or as inhibitors thus acting in synergy with rapamycin, for use as a medicament in combination with rapamycin or a rapamycin-like compound and/or in combination with metformin or a metformin-like compound and/or in combination with thermal energy, for the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated.
  • rapamycin sensitizers rapamycin sensitizers
  • inhibitors thus acting in synergy with rapamycin
  • the invention further relates to the application of thermal energy together with the aforesaid inhibitors of the mTOR pathway, based on the observation that mTOR pathway activation, particularly due to Pten mutation, renders tumor cells sensitive to killing by heat.
  • the efficiency of killing is amplified by pre-treatment of cells with either rapamycin or metformin.
  • a compound blocking the mTOR pathway is, for example, a compound blocking or down- regulating mTOR, ml_ST8/B-Gbl, SIN1 , raptor, rictor, or other components of the mTOR complexes or other components of the mTOR pathway, such as Rheb, LST8, S6K1 , Akt1 , Akt2, RSK1 , ERK1/2.
  • Compounds activating the mTOR pathway are e.g. compounds blocking or down-regulating negative regulators of the mTOR pathway such as Tsd , Tsc2 or AMPK, REDD1/2, GSK-3.
  • Such compounds render cells sensitive to the mTOR pathway blockers listed above, in particular to rapamycin or rapamycin-like compounds.
  • examples for such compounds are anti-sense RNA, siRNA or shRNA down-regulating any of the mentioned proteins implicated in the mTOR pathway or components of the mTOR complexes, in particular down-regulating Tsc2 or PTEN.
  • Other compounds blocking or down-regulating the above targets are likewise preferred, for example organic compounds of a molecular weight below 1500, preferably below 500. Such compounds may be found by the screening method described herein below.
  • mTOR pathway means mammalian Target of fiapamycin signaling pathway and includes upstream pathways signaling down to mTOR and pathways signaling downstream from mTOR to their various effector molecules regulating gene expression and protein synthesis as defined by A. Soulard and M.N. Hall, Cell 129:434 (2007).
  • TOR is a highly conserved protein kinase and a central controller of cell growth and metabolism.
  • the mammalian TOR (mTOR) signaling pathway controls cell growth and metabolism in response to nutrients (e.g., amino acids), growth factors (e.g., insulin, IGF- 1 , PDGF), and cellular energy status (ATP).
  • nutrients e.g., amino acids
  • growth factors e.g., insulin, IGF- 1 , PDGF
  • ATP cellular energy status
  • Nutrients are the dominant mTOR input as high levels of amino acids can compensate for an absence of the other mTOR inputs but not vice versa, mTOR activates cell growth by positively and negatively regulating several anabolic and catabolic process, respectively, that collectively determine mass accumulation.
  • the anabolic processes include transcription, protein synthesis, ribosome biogenesis, nutrient transport, and mitochondrial metabolism.
  • mTOR negatively regulates catabolic processes such as mRNA degradation, ubiquitin-dependent proteolysis, autophagy and apoptosis.
  • mTOR is found in two functionally and structurally distinct multiprotein complexes (mTORCI and mTORC2 in mammals), each of which signals via a different set of effector pathways.
  • mTORCI is rapamycin sensitive, whereas mTORC2 is rapamycin insensitive.
  • the best-characterized phosphorylation substrates of mTOR are S6K and 4E-BP1 , via which mTORCI controls translation, and Akt/PKB, via which mTORC2 controls cell survival and likely other processes.
  • S6K and 4E-BP1 via which mTORCI controls translation
  • Akt/PKB via which mTORC2 controls cell survival and likely other processes.
  • the two mTOR complexes and the overall architecture of the mTOR signaling network appear to be conserved from yeast to human.
  • mTOR plays a key role in development and aging, and is implicated in many major diseases including cancer, cardiovascular disease, inflammatory disease, and metabolic disorders.
  • mTOR complex means two structurally distinct multiprotein complexes, mTORCI and mTORC2, each of which signals via a different set of effector pathways.
  • mTORCI is rapamycin sensitive whereas mTORC2 is rapamycin insensitive.
  • Japanese is a macrolide also named sirolimus, and is 23,27-epoxy-3H-pyrido[2,1 -c] [1 ,4]oxaazacyclohentricontine. As described above, rapamycin blocks the mTOR pathway by interaction with mTORC1.
  • a "rapamycin-like compound” is, for example, RAD001 (Novartis), CCI-779 (Wyeth)
  • rapamycin-like compounds As described for rapamycin, rapamycin-like compounds also block the mTOR pathway.
  • Metformin is 1 ,1 -dimethylbiguanide, usually in the form of the hydrochloride salt, but the free base or other acid addition salts may be used as well. Metformin inhibits mTOR- dependent translation initiation via the mTOR pathway component AMPK, a kinase with mTOR inhibitory activity, which acts by phosphorylating and thus activating the negative mTOR regulator Tsc2. The net effect of metformin is to inhibit mTOR via activating AMPK, which activates Tsc2.
  • a “metformin-like compound” is, for example, AICA-R (5-aminoimidazole-4-carboxamide riboside) and drugs modulating glucose metabolism, for example 2-deoxy-glucose, glitazones, sulfonylureas, meglitinides, thiazolidinediones, alpha-glucosidase inhibitors, incretins, glucagon-like peptide agonists, DPP4 inhibitors, or amylin analogs.
  • Preferred metformin-like compounds are AICA-R and glitazones. As described for metformin, metformin-like compounds also block the mTOR pathway.
  • a compound blocking the mTOR pathway in combination with metformin or a metformin-like compound does not include the combination of metformin with itself, however, includes combinations of metformin with a metformin-like compound, or of one metformin-like compound with another metformin-like compound.
  • a particular shRNA down-regulating TSC2 is an oligonucleotide comprising the target sequence CCCUUAUAUCACUAAGGGUUU (SEQ ID NO:1 ) in mouse or CCCUUUCAUCACUAAGGGCCU (SEQ ID NO: 2) in human.
  • a particular shRNA down-regulating PTEN is an oligonucleotide comprising the target sequence CGACUUAGACUUGACCUAUAU (SEQ ID N0:3) in mouse and human.
  • a particular shRNA down-regulating rictor is an oligonucleotide comprising the target sequence CGGUUCAUACAAGAGUUAUUU (SEQ ID N0:4) in mouse and AGGUUCAUACAAGAAUUAUUU (SEQ ID NO:5) in human.
  • a particular shRNA down-regulating raptor is an oligonucleotide comprising the target sequence GCCCGAGUCUGUGAAUGUAAU (SEQ ID NO:6) in mouse and GCCUGAGUCGGUAAAUGUGCU (SEQ ID NO:7) in human.
  • Neoplastic diseases considered are, for example, epithelial neoplasms, squamous cell neoplasms, basal cell neoplasms, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous neoplasms, ducal-, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, specialized gonadal neoplasms, paragangliomas and gangliomal tumors, naevi and melanomas, soft tissue tumors and sarcomas, fibromatous neoplasms, myxomatous neoplasms, lipomatous neoplasms, myomatous neo
  • organ-specific cancers selected from renal cell cancer, endometrial carcinoma, glioblastoma, head and neck cancer, colon cancer, pancreatic cancer, gastric cancer, hepatocarcinoma, ovarian cancer, thyroid carcinoma, breast cancer, prostate cancer, and lung cancer.
  • TSC Tuberous sclerosis complex
  • LAM lymphangioleiomyoma
  • hamartoma syndromes Cowden's disease
  • Lhermitte-Duclos disease Bannayan syndrome
  • Proteus syndrome Proteus syndrome.
  • Hyperproliferative conditions considered are, for example, lymphoproliferative disorders, dysplasias and metaplasias of different tissues, psoriasis, atherosclerosis, restenosis after angioplastic surgery, angiogenesis and particularly tumor angiogenesis, and cancers such as gastro-intestinal stromal tumors and Menetriers syndrome.
  • mTOR is implicated are, for example, aging, type-ll diabetes and other metabolic conditions, cardiovascular disease, and autosomal-dominant polycystic kidney disease.
  • Aging processes which can be positively influenced by blocking the mTOR pathway are, for example, type Il diabetes, Parkinson, and Alzheimer disease.
  • Cardiovascular diseases are, for example, conditions involving endothelial proliferation as in atherosclerosis leading to thrombotic diseases and stroke.
  • the invention further relates to the use of a compound blocking or activating the mTOR pathway for the manufacture of a medicament for the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, in combination with rapamycin or a rapamycin-like compound, and/or in combination with metformin or a metformin-like compound, and optionally in combination with an inhibitor of the MAPK, Wnt, Jak-Stat, PI-3K, JUNK or hedgehog pathway and/or a DNA damaging agent, 2'-deoxy-glucose, or an inducer of hypoxia.
  • Inhibitors of the MAPK pathway considered are, for example, BAY 43-9006, PD98059, U0126, SB202190, SB203580, FR167653, ZM336372, and GW5074.
  • Inhibitors of the Wnt pathway considered are, for example, fumagillin.
  • Inhibitors of the Jak-Stat pathway considered are, for example, WP1034, WP1066, AG490, and CP690550.
  • Inhibitors of the PI-3K pathway considered are, for example, wortmannin, LY294002, PM 03, XL147, SF1 126, and TGX221.
  • Inhibitors of the JUNK pathway considered are, for example, CC401 , SP600125, 4-hydroxynonenal, JNK inhibitor IX, ALX 159-600, and AS601245.
  • Inhibitors of the hedgehog pathway considered are, for example, CUR61414, jervine, and cyclopamine Hh inhibitor.
  • DNA damaging agents considered are, for example, etoposide, cisplatin, doxorubicin, tamoxifen, bleomycin, irinotecan, topotecan and a) antimetabolites such as 5-fluorouracil (ICN), gemcitabine HCI (Gemzar, EIi Lilly), b) alkylating agents such as oxaliplatin (EloxantinT"', Sanofi-Synthelabo), dacarbazin (Detimedac, Medac), cyclo- phosphamide (Endoxan TM, Asta) and carboplatin (Paraplatin, Bristol-Meyers Squibb), c) cell-cycle inhibitor such as vinorelbine (Navelbine TM, Robapharm), vinblastine (VelbeT"", EIi Lilly), docetaxel (TaxotereT"", Aventis), d) DNA breaker (topo-isome
  • Inducers of hypoxia considered are, for example, desferrioxamine, cobalt chloride, Revlimid (Celgene), and anti-angiogenic ABT-510 (Abbott).
  • Pyrogenic substances considered are, for example bacterial polysaccharides, cytokines including but not limited to interferons, IL-6 and IL-1 , and prostaglandins.
  • the invention is based on the following experimental observations:
  • pathway addiction in cancer is very relevant to therapy, as it implies that blocking a pathway to which the tumor cell is addicted would lead to the elimination of that cell.
  • This concept has implications for strategies of chemotherapy, because if one forces the tumor cell into a given pathway ("forced addiction"), as is shown in the present invention by use of RNA interference, one can sensitize the cell extraordinarly to inhibitors (blockers) of that pathway.
  • the mTOR specific inhibitor rapamycin is perhaps the most specific kinase inhibitor known and well tolerated when applied as immunosuppressant to transplantation patients, with recently also promising results when its derivatives (rapamycin-like compounds) are applied as anticancer drugs, for example in kidney cancers.
  • mTOR phosphorylates p70 ribosomal S6 protein kinase and 4E-BP1 , a repressor of the translation initiation factor elF-4E, and thus exerts a powerful controlling effect on translation.
  • a known negative inhibitor of mTOR is Tsc2, a GTPase-activating protein (i.e. an activator protein of an enzyme with guanosyl triphosphatase activity) that inhibits Rheb, a G-protein (which is a protein with guanosyl triphosphatase activity) stimulating the kinase activity of TOR.
  • the GTPase Tsc2 which forms a functional complex with protein Tsc1 , is inhibited by growth signals via protein kinase B (PKB, also called akt) and ERK (extracellular regulated kinase) with net stimulation of mTOR, and stimulated via the phosphatase PTEN, the kinase LKB1 and the kinase AMPK (AMP-dependent kinase) with net inhibition of mTOR, the latter ones being tumor suppressor genes.
  • PTEN protein kinase B
  • AMPK AMP-dependent kinase
  • Chemical modulation of these mTOR regulators contributes to addiction to mTOR and induces sensitivity to rapamycin or rapamycin-like substances, i.e. chemically forced addiction to mTOR with resultant sensitivity to mTOR pathway blockers (inhibitors).
  • mutants from IL-3 dependent mast cells obtained by frame-shift mutagenesis [K.F. Kiser et al., Oncogene 25:6595-6603 (2006)] were exposed to rapamycin, a mTOR inhibitor originally used as an immunosuppressant which inhibits proliferation of certain cells at nM concentrations, and derivatives of which are in clinical trials as antineoplastic agents.
  • Mutant line D2c is highly sensitive to rapamycin with an IC 50 of less than 1 nM, while precursor 15V4 cells, which are v-H-ras expressing PB-3c, are not inhibited (Fig 1A).
  • D2c cells may have acquired rapamycin sensitivity concomitantly with loss of IL-3 dependence following ICR191 mutagenesis via activation of the mTOR pathway. It is known that IL-3 independence in D2c cells results from recessive mutation (Kiser et al., be. cit), and it is demonstrated in the present invention that the rapamycin sensitivity of these cells is also recessive. Somatic hybrid cells generated previously by cell fusion between D2c and clone 20 were used, the latter being an IL-3 dependent subclone of PB-3c with tumor suppressor activity in cell fusion experiments [A.P.
  • mTOR pathway blockade is sufficient for therapy, while in most cancers rescue pathways (in the model described in this invention the IL-3 pathway) must be blocked in addition to the mTOR pathway.
  • rapamycin is an extremelyly specific inhibitor of mTOR
  • mTOR overexpression could transform 15V4 cells to IL-3 independent growth in order to confirm further that IL-3 independence can be brought about by forced mTOR pathway activation.
  • an AU-tagged murine mTOR construct was introduced by retroviral infection in 15V4 cells, and other cells were infected with an empty control retrovirus. After puromycin selection and removal of IL-3, IL-3 independently growing mTOR overexpressing 15V4 cells are obtained, while the control cells do not survive more than 2 days without IL-3. These mTOR overexpressing and IL-3 independent cells are also rapamycin sensitive (Figure 2). The finding that mTOR overexpression activates mTOR signaling in 15V4 cells and permits rapamycin sensitive IL-3 independence provides additional evidence for the claim that hyperactivation of mTOR signaling transforms 15V4 cells to IL-3 independence.
  • Rapamycin inhibition occurs via apoptosis
  • Table 1 Determination of live and dead (apoptotic or necrotic) cells following 48 h growth in presence of rapamycin and in presence or absence of IL-3.
  • IL-3 presence (+) or absence (-) of interleukin-3 r: 50 nM rapamycin treatment (+) or DMSO control treatment (-)
  • A percentage (%) of living cells
  • B percentage of early apoptotic cells
  • C percentage of dead late apoptotic cells
  • D percentage of dead necrotic cells. Numbers representative from at least four experiments.
  • the number of dead cells increases dramatically upon addition of rapamycin in 6.5 and D2c cells, but not in precursor line 15V4.
  • Cell death is antagonized in 6.5 but not in D2c cells by addition of IL-3.
  • IL-3 dependent 15V4 precursor cells only 4% of late apoptotic cells were found in the presence of IL-3 when treated for 48 h with rapamycin, while in 6.5 and D2c cells this value was 3.7 and 54%, respectively.
  • both mutants had around 80% of late apoptotic cells after rapamycin treatment.
  • Both 6.5 and D2c cells undergo apoptosis as defined by Pl-staining and annexin V positivity in response to rapamycin, while only cells from mutant 6.5 is rescued from apoptosis by IL-3, in keeping with the findings from Figure 1.
  • IL-3 activates an alternative pathway, which protects against apoptosis.
  • Apoptosis is the mechanism by which the mutants respond to blocking mTOR. In all mutants tested except D2c this mechanism can be overcome by providing exogenous IL-3.
  • mTOR pathway addiction can be overcome in most mutants by providing a growth- factor activating a parallel growth pathway. For the concept of mTOR pathway addiction, this means that parallel pathways must be silenced in order to reveal pathway addiction.
  • Outer left column indicates cell lines + base-line signal - loss of signal +++ increased signal
  • Data are from at least 3 independent determinations
  • MAPK was found to be hyperactive in clone D2c, shown by increased phosphorylation of ERK1/2-Thr202/Tyr204.
  • the mRNA of Tsc2 in D2c and of Pten in clones 6.5, 6.8 and 7.2 was sequenced and compared to the 15V4 sequence.
  • D2c has a frame shift insertion in codon 50 of Tsc2, which creates a premature translational stop in codon 51 .
  • 6.5 has a frame shift insertion in codon 44 of Pten, which creates a premature stop codon in 51.
  • 6.8 has a frame shift insertion in codon 56 of Pten, which creates a premature stop codon in 62.
  • 7.2 has a frame shift insertion in codon 132 of one Pten allele, which creates a premature stop codon in 149 and a frame shift insertion in codon 320 of the other Pten allele, which creates a premature stop codon in 326.
  • Clone D2c carries a frame shift in Tsc2 and clones 6.5, 6.8 and 7.2 carry distinct frame shifts in the Pten gene. All frame shift insertions identified in Tsc2 and Pten created premature translation termination codons, and generated truncated non-functional proteins. These data support the idea that by inhibiting PTEN or Tsc2, for example by shRNA or by other organic molecules, one can trigger forced addiction to the mTOR pathway in cells.
  • TSC2 or PTEN were downregulated by shRNA following retroviral infection in 15V4 cells, and these cells tested for IL-3 independent growth.
  • shRNA sequences against each target were tested in 15V4 cells by lentiviral infection followed by puromycin selection.
  • a correlation was observed between the increase in survival rate in medium lacking IL-3 and the knockdown efficiency of either TSC2 or PTEN, which indicates that loss of function of TSC2 or PTEN promotes IL-3 independence in 15V4 cells.
  • IL-3 independent knockdown cells were tested for rapamycin sensitivity (Fig. 3B).
  • the IL-3 independent TSC2 or PTEN knockdown cells had acquired sensitivity to rapamycin.
  • TSC2 knockdown cells were only sensitive to rapamycin in the absence of IL-3 but not in its presence. In other words, forced addiction to mTOR was overcome, like in all clones except D2c, by IL-3.
  • This difference in rapamycin sensitivity between the TSC2 mutant D2c and the TSC2 knockdown cells may either result from a difference in TSC2 inhibition between these cells or from additional features present only in D2c, as for example the observed activation of MAPK signaling.
  • Rapamycin sensitive cells require raptor or rictor for growth
  • TSC2 deficient 15V4 cells transform to IL-3 independence primarily via signaling from mTORCI
  • PTEN deficient cells primarily transform via mT0RC2.
  • raptor and rictor efficient knockdown sequences were tested by Western blotting, and used to determine proliferation rates after downregulation of mTORCI or mT0RC2.
  • IL-3 dependent precursor 15V4 cells grew almost equally well when raptor or rictor was downregulated (Figure 4, upper panel). From D2c cells, no outgrowth occurred when raptor was downregulated, in presence or absence of IL-3, indicating that expression of raptor is crucial for mTOR dependent growth of these cells (Fig. 4, lower panel). 6.5 cells, in contrast, were very sensitive to downregulation of rictor, which was growth inhibitory, but inhibition was rescued by IL-3 (Fig. 4, middle panel).
  • Rapamycin sensitive cells are also sensitive to metformin, alone or in combination with rapamycin
  • IL-3 dependent precursor cells 15V4 are not sensitive to metformin (Fig. 5A), nor, as shown in Figure 1 , to rapamycin. Rapamycin sensitive clone 6.5 is, however, sensitive to metformin (Fig. 5D, 5E).
  • Fig. 5D, 5E a synergistic effect is observed in cells growing without IL-3.
  • D2c cells lacking Tsc2 are sensitive to rapamycin, but not to metformin, in presence or absence of IL-3 (Fig. 5B, 5C).
  • Table 3 Determination of live and dead (apoptotic or necrotic) cells following growth in presence of metformin and in presence or absence of IL-3.
  • Outer left column indicates cell lines m: presence (+) or absence (-) of 5 mM metformin t: time (hours) after removal of interleukin-3
  • A percentage (%) of living cells
  • B percentage of early apoptotic cells
  • C percentage of dead late apoptotic cells
  • D percentage of dead necrotic cells
  • haemopoietic IL-3 dependent cells can be transformed to IL-3 independent growth by downregulation of Tsc2 or PTEN achieved by stable transfection of shRNA.
  • Two or more shRNA sequences are active per gene, indicating that this is not an off-target effect.
  • the effect requires the presence of an activated ras gene, as PB-3c clone 15 could not be transformed, in contrast to isogenic cells infected with v-H-ras (clone 15V4).
  • Transformed cells become addicted for growth to the mTOR pathway, as treatment with rapamycin leads to rapid and powerful onset of apoptosis.
  • Rapamycin-sensitive cells express the marker gene fructose- 1 ,6-bisphosphatase, a key regulator of gluconeogenesis
  • Loss-of-function mutations in precursor 15V4 cells enable IL-3 independent but rapamycin-sensitive growth via activation of the mTOR pathway, and inhibition by rapamycin can in all but mutant D2c be relieved by addition of IL-3. Importantly, fusion of mutants with parental cells restore not only the IL-3 requirement but also rapamycin sensitivity.
  • Microarray experiments using Affymetrix chips allows identification of signature transcripts related to IL-3 independence and rapamycin sensitivity.
  • Transcripts were identified, which are a) overexpressed in a mutant compared to precursor 15V4, b) downregulated following cell fusion, and c) sensitive to rapamycin.
  • Three mutants and their fusion hybrids with IL-3 dependent PB-3c clone 20 were analyzed (Fig. 6).
  • the mutants 6.5, 6.8 and H1.2 displayed between 180 and 227 transcripts that were elevated two-fold or more when compared to precursor 15V4 cells. 12 overexpressed genes were common to all mutants (Fig. 6A).
  • FBP2 fructose- 1 ,6-bisphosphatase-2
  • FBP2 is a biomarker to identify tumor cells which, when growing autonomously in the absence of growth factor, are sensitive to rapamycin or other inhibitors of the mTOR pathway. Also, FBP2 expression is under negative control by the tumor suppressor PTEN.
  • PTEN deficient, rapamycin sensitive cells expressing fructose 1 ,6-bisphosphatase are susceptible to killing by hyperthermia
  • FBPase fructose 1 ,6-bisphosphatase 2
  • PFK1 constitutively expressed glycolytic enzyme phosphofructokinase
  • mutants with elevated FBP2 expression are sensitive to thermotherapy, and FBP2 or other key regulatory enzymes involved in glucose metabolism such as hexokinase, phosphofructokinase-1 (PFK-1 ), fructose-1 ,6- bisphosphate aldolase, pyruvate kinase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1 ,6-bisphosphatase 1 (FBPase-1 ), glucose-6-phosphatase, phosphofructokinase-2 (PFK-2), or fructose-2,6-bisphosphatase 2 (FBPase-2) are a diagnostic marker predicting thermotherapy response.
  • Rapamycin synergises with hyperthermia in killing thermo-sensitive mutant cells The FBP2 expressing mutants 6.5 and 7.2 were preincubated overnight with rapamycin in the presence of IL-3.
  • IL-3 signaling protects rapamycin sensitive cells from entering apoptosis and the pre-incubation serves to fully abolish mTOR signaling and suppress any relevant downstream effectors.
  • D2c and D5a an IL-3 independent 15V4 derivative
  • Metformin snyergizes with hyperthermia in killing thermosensitive mutant cells FBP2 expressing cells (6.5, 7.2, VG59) and, for control, 15V4 and D2c cells were incubated overnight with 5 mM metformin and incubated in parallel at 37 0 C and 44.5 0 C. Viability of cells was determined after 4 h as described above. FBP2 expressing cells showed a substantial drop in viability when incubated at 44.5 0 C.
  • Table 6 Synergism between metformin and hyperthermia in killing thermo-sensitive FBP2 expressing mutant cell lines.
  • pre-malignant or malignant cells in a patient are forced to grow in a mTOR addicted fashion, possibly through activation of the mTOR pathway by inhibition of negative regulators, these cells will now be extraordinarly sensitive to rapamycin and rapamycin-like compounds or other inhibitors of the mTOR pathway, provided that rescue pathways are also shut-off.
  • the choice of which other inhibitors should be co-applied with the mTOR inhibitor will depend on the tumor type, in particular on the question of which pathway is active in the given tumor cell.
  • Known oncogenic pathways include the MAPK, Wnt, Jak- Stat, PI-3K, JUNK and hedgehog. For these pathways, a number of more or less specific inhibitors are known.
  • the invention further relates to a method for screening for a compound blocking the mTOR pathway.
  • compounds for example compounds from a compound library, or selected single compounds for which there is already an indication of possible usefulness in tumor treatment, can be easily tested, and an indication obtained whether these compounds should be applied in the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated.
  • the method is also suitable for high-throughput screening.
  • Compounds are tested on particular IL-3 dependent, rapamycin-insensitive cells, and in parallel, in mutants of such cells, which are rapamycin-sensitive.
  • Such cells are known and may be obtained, e.g. as described by K.F. Kiser et al., Oncogene 25:6595-6603 (2006), from immortalized bone-marrow-derived mast cell lines.
  • the invention relates to a method for screening for a compound blocking the mTOR pathway, wherein
  • a compound to be tested is added to an IL-3 dependent, rapamycin-insensitive cell and in parallel to a first rapamycin-sensitive mutant cell of said IL-3 dependent, rapamycin-insensitive cell, and compounds selected, which do not inhibit said IL-3 dependent, rapamycin-insensitive cell, but inhibiting said first rapamycin-sensitive mutant cell,
  • step (b) compounds selected from step (a) are retested for inhibitory activity on a second IL-3 independent mutant of said IL-3 dependent, rapamycin-insensitive cell, and compounds selected, which are inhibiting said second IL-3 independent mutant cell.
  • the method is based on the fact that IL-3 dependent 15V4 cells are insensitive to mTOR pathway blockade exerted by rapamycin, while several mutant derivatives (6.5, 6.8, 7.2, D2c, H1.2) are sensitive to rapamycin due to their addiction to the mTOR pathway. This offers the possibility to perform a dual screen of 15V4 and e.g. D2c alongside each other and screen for compounds inhibiting the latter but not the former. Toxic compounds are readily eliminated as they are equally toxic to both cells.
  • Selected hits are tested further on the additional mTOR addicted and rapamycin sensitive clones (e.g. 6.5, 6.8, 7.2, H1.2) and selected if active, and simultaneously tested on other IL-3 independent clones without mTOR pathway addiction (D5a, 15bcr-abl) and rejected if active. This allows rapid elimination of false positives and selection of specific candidates.
  • additional mTOR addicted and rapamycin sensitive clones e.g. 6.5, 6.8, 7.2, H1.2
  • D5a, 15bcr-abl mTOR pathway addiction
  • 10'0OO cells are plated per well in Iscove's Modified Dulbecco's Medium with 10% FCS, and compounds diluted in a series from 10 ⁇ M to 0.01 ⁇ M.
  • Controls contain solely solvent (typically 0.1% DMSO).
  • Cell proliferation is measured between days 1 -3 by XTT or Alamar blue assay and absorption read by a photometer or a fluorescence reader, respectively. The assay is suitable for high throughput screening. Hits are re-tested in triplicates.
  • the invention further relates to a method to predict sensitivity of a tumor to a compound blocking the mTOR pathway combined with application of thermal energy.
  • rapamycin-sensitive cells express the marker gene fructose-1 ,6,-bisphosphatase, and that such cells are susceptible to killing by hyperthermia.
  • marker genes are also considered, in particular hexokinase, phosphofructokinase-1 (PFK-1 ), fructose-1 , 6-bisphosphate aldolase, pyruvate kinase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1 , 6-bisphosphatase 1 (FBPase-1 ), glucose-6-phosphatase, phosphofructokinase- 2 (PFK-2), fructose-2, 6-bisphosphatase 2 (FBPase-2).
  • PFK-1 phosphofructokinase-1
  • FBPase-1 6-bisphosphatase 1
  • PFK-2 phosphofructokinase-2
  • FBPase-2 6-bisphosphatase 2
  • the marker gene fructose-1 ,6,-bisphosphatase can easily be measured, for example by PCR procedures allowing quantitative determination of FBP2 mRNA expression, or by determination of FBP2 protein levels with standard procedures for protein detection and quantification, in particular Western blotting or ELISA or by enzymological assays using the cognate substrate fructose-1 ,6-bisphosphate.
  • the other mentioned marker genes and the corresponding enzyme proteins may be determined in the same way using their analogous substrates in enzymological procedures.
  • mTOR pathway is activated in tumor cells under analysis. This may be performed, using biopsy material, by determining the phosphorylation status of the mTOR targets S6 kinase phosphorylated at Thr-389 or 4E-BP1 phosphorylated at multiple sites including Thr-69/70 (both proteins are phosphorylated by mTORCI ) or PKB/akt phosphorylated at Ser-473 (phosphorylated by mTORC2). Phosphorylation of said mTOR targets is recognized by Western blotting using suitable commercially available phospho- and protein-specific antibodies. Normal adjacent tissue serves as negative control. Another indication of mTOR pathway activation is autophosphorylation of mTOR, also determined by Western blotting.
  • the invention relates to a method to predict sensitivity of a tumor to a compound blocking the mTOR pathway combined with application of thermal energy, wherein (a) FBP2 expression is measured in tumor cells and in adjacent non-tumor cells by (i) determination of FBP2 mRNA expression by quantitative PCR procedure, (ii) semiquantitative determination of FBP2 protein levels by Western blotting of cell lysates,
  • mTOR activation is measured in tumor cells by determination of the phosphorylation status of S6 kinase and/or 4E-BP1 and/or PKB/akt;
  • step (c) the tumor is judged sensitive to a compound blocking the mTOR pathway combined with application of thermal energy, if the level of FBP2 expression determined in step (a) is increased more than 3-fold in tumor cells in comparison to adjacent non-tumor cells and if evidence for mTOR activation is obtained in step (b).
  • the test is based on the fact that the expression of the gluconeogenic enzyme FBP2 triggers a futile cycle with consumption of energy.
  • Known examples constitute tumors addicted to the mTOR pathway after mutation of PTEN.
  • the test thus measures the expression of FBP2 by real-time PCR together with determination of mTOR activation by detection of the phosphorylation status of S6 kinase (Thr-389), or 4E-BP1 (Thr-69/70 plus additional sites), or of PKB/akt (Ser-473), optionally together with the expression of PTEN by Western blot.
  • S6 kinase Thr-389
  • 4E-BP1 Thr-69/70 plus additional sites
  • PKB/akt Ser-473
  • compositions that comprise a compound blocking or activating the mTOR pathway, optionally together with the other components mentioned herein before, as active ingredient(s) and that can be used especially in the treatment of the diseases mentioned.
  • Compositions for enteral administration such as nasal, buccal, rectal or, especially, oral administration, and for (the particularly preferred) parenteral administration, such as intravenous, intramuscular or subcutaneous administration, to warm-blooded animals, especially humans, are preferred.
  • compositions comprise the active ingredient(s) alone or, preferably, together with a pharmaceutically acceptable carrier.
  • the dosage of the active ingredient(s) depends upon the disease to be treated and upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, and the mode of administration.
  • compositions for parenteral administration, preference is given to solutions of the active ingredients, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example in the case of lyophilized compositions comprising the active ingredient(s) alone or together with a carrier, for example mannitol, can be made up before use.
  • the pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes.
  • the said solutions or suspensions may comprise viscosity-increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers, e.g. Tween 80 (polyoxyethylene (20) sorbitan mono-oleate).
  • Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or microbicides, such as sorbic acid or benzoic acid.
  • Suspensions in oil comprise as the oil component the vegetable, synthetic, or semisynthetic oils customary for injection purposes.
  • liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms.
  • the alcohol component of these fatty acid esters has a maximum of 6 carbon atoms and is a monovalent or polyvalent, for example a mono-, di-or trivalent, alcohol, especially glycol and glycerol.
  • vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and groundnut oil are especially useful.
  • injectable preparations are usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers. Solutions such as are used, for example, for parenteral administration can also be employed as infusion solutions.
  • compositions for enteral, in particular oral use are preferred.
  • Such preparations for oral use are, e.g. tablets, capsules containing granules, mini-tablets or other solid, semi-solid or liquid forms, solutions to be taken as drops or as syrup, and the like.
  • Suitable carriers for solid oral preparations are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate.
  • Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
  • Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl- methylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient.
  • compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the hard capsules may contain the active ingredient(s) in the form of granules, for example in admixture with fillers, such as corn starch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers.
  • the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxy- ethylene sorbitan fatty acid ester type, may also be added.
  • suitable liquid excipients such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxy- ethylene sorbitan fatty acid ester type, may also be added.
  • compositions suitable for rectal administration are, for example, suppositories that consist of a combination of the active ingredient(s) and a suppository base.
  • Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.
  • the pharmaceutical compositions comprise from approximately 1 % to approximately 95% of the active ingredient(s), single-dose administration forms comprising in the preferred embodiment from approximately 20% to approximately 90% active ingredient(s) and forms that are not of single-dose type comprising in the preferred embodiment from approximately 5% to approximately 20% active ingredient(s).
  • Unit dose forms are, for example, coated and uncoated tablets, ampoules, vials, suppositories, or capsules. Examples are capsules containing from about 0.05 g to about 1.0 g active ingredient(s).
  • Further dosage forms are, for example, ointments, creams, pastes, foams, tinctures, lipsticks, drops, sprays, and the like.
  • compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilizing processes.
  • Method of treatment for example by means of conventional mixing, granulating, coating, dissolving or lyophilizing processes.
  • the invention further relates to a method of treating neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, by administering a therapeutically effective amount of a compound blocking or activating the mTOR pathway in combination with rapamycin or a rapamycin-like compound, and/or in combination with metformin or a metformin-like compound, and optionally in combination with an inhibitor of the MAPK, Wnt, Jak-Stat, PI-3K, JUNK or hedgehog pathway and/or a DNA damaging agent, 2'-deoxy-glucose, or an inducer of hypoxia.
  • the invention relates also to a method of treating neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, by administering a therapeutically effective amount of a compound blocking or activating the mTOR pathway, in particular of rapamycin, a rapamycin-like compound, metformin, or a metformin-like compound, in combination with thermotherapy.
  • a compound blocking or activating the mTOR pathway in particular of rapamycin, a rapamycin-like compound, metformin, or a metformin-like compound, in combination with thermotherapy.
  • the mTOR pathway is activated, just as performed in the experimental work described herein by shRNA, by another inhibitor of Tsc2, for example a pharmaceutically acceptable chemical compound other than shRNA.
  • Tsc2 depends for mTOR pathway activation on complex formation with Tsc1 (which is a tumor suppressor similar to Tsc2)
  • an inhibitor of Tsc1 will have the same effect.
  • Other negative regulators of mTOR besides PTEN and Tsc2 (or Tsc1 ) will provide similar drug targets, and inhibitors of NF1 , GAP, or stimulators of LKB1 and AMPK will have the same effect, either by themselves or combined with a Tsc2 or Tsd inhibitor.
  • phase III Before the actual therapeutic phase III with triggering of apoptosis by mTOR pathway inhibitors is induced, it may be necessary to block alternative rescue pathways, e.g. by inhibitors of the MAPK, Wnt, Jak- Stat, and PI-3K-PKB pathway. This leads to cell death upon mTOR activation and application of mTOR pathway inhibitors.
  • the combination of phases I and III can be termed "cell death via forced mTOR pathway addiction", by the proposed combination of inhibitors.
  • phase I a physiological inhibitor (PTEN or Tsc2), which is a tumor suppressor s inhibited, which seems paradoxical in cancer therapy, as conventional wisdom argues that tumor suppressors, if targeted in cancer, should be reinstalled (by genetic transfer or a compound which restores lost function).
  • PTEN a physiological inhibitor
  • Tsc2 or PTEN renders cells rapamycin or metformin sensitive
  • the inhibitor (Phase I) for pathway activation is inhibited, and in phase Il compounds which target activators (oncogenes) stimulating mTOR bypassing pathways are combined. This is a strategy that has not been proposed before.
  • rapamycin or its recently described analogues such as RAD001 (Novartis) or CCI-779 (Wyeth Pharmaceuticals) or AP23573 (Ariad Pharmaceuticals), or other mTOR pathway inhibitors.
  • Phase IV aims at amplifying the apoptotic effect. It may, in some tumors, not be required, but provides an attractive synergistic option to amplify the effect.
  • the rationale is based on the observation that cells addicted to the mTOR pathway, or cells carrying mutations in the PTEN-PKB-mTOR pathway are exquisitively sensitive to applied heat energy, or to rapamycin or metformin, or to a combination of heat and said drugs.
  • rapamycin is known to exert a synergistic effect when combined with DNA damaging agents such as etoposide.
  • An interesting alternative way to amplify the apoptotic response is to install in the tumor cell either hypoxic conditions or low ATP levels, as mTOR induction under those conditions will be particularly damaging for cells.
  • Therapeutic RNA interference can be achieved by a) lentiviral infection expressing the suitable shRNA sequences; b) in vivo transfection of DNA expressing the suitable shRNA sequences; c) in vivo transfection of shRNA (small hairpin RNA).
  • a compound blocking the mTOR pathway administered in combination with one or more of the mentioned combination partners may be administered as a fixed combination, staggered or independently of one another.
  • the combination partners not contained in a fixed pharmaceutical preparation may be in combined packages comprising the required amounts of each combination partner.
  • the compounds may be administered in addition to radiotherapy or surgical intervention or heat application.
  • Cell culture reagents, rapamycin and puromycin are obtained from Sigma; HRP-coupled anti-mouse and anti-rabbit secondary antibodies from Pierce; anti-PKB-pS473, anti-S6K- pT389, anti-S6K, anti-ERK1/2-pT202/Y204, anti-ERKI/2, anti-PTEN from Cell Signaling Technology; anti-PKB from Santa Cruz; anti-actin (MAB1501 ) from Chemicon; polyclonal anti-TSC1 and anti-TSC2 are generated as described [M. van S strengenhorst et al., Hum MoI Genet. 7:1053-57 (1998)].
  • IL-3 dependent PB-3c mast cells and its subclones are cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum, 2mM L-Glutamine, 100 U ml "1 penicillin, 100 ⁇ g ml "1 streptomycin and 50 ⁇ M ⁇ -mercaptoethanol.
  • IL-3 dependent cells require the addition of conditioned medium (CM) from X63-mll_3 cells.
  • Clone 20 and clone 15 are subclones of PB-3c; clone 20, but not clone 15 is able to suppress autocrine tumor formation upon cell fusion [A.P.
  • 15V4 clone is a v-H-ras expressing clone 15; 15V4wt18 is 15V4 expressing GFP; D2c, 6.5, 6.8, 7.2, 8.8 and H1 .2 are IL-3 independent clones that were isolated from 15V4 after frameshift mutagen ICR191 treatment and IL-3 removal; 20xD2c is a fusion hybrid between 20 and D2c.
  • 15V4wt18 cells are treated with 2 ⁇ g/ml ICR191 (Sigma) for 2 h at 37 ⁇ O in IL-3 containing medium, and allowed to recover for 24 h.
  • IL-3-independent clones are selected by IL-3 removal and viable cells are enriched by Ficoll-Paque (Amersham Pharmacia). For IL-3 removal, the cells are washed twice with the IL-3 free medium. For cell fusion, PB-3c-20puro cells and IL-3-independent 15V4wt18 cells are mixed and fused by electroporation in serum-free IMDM at 220V and 900 ⁇ F. Hybrids are selected in IL-3 containing medium supplemented with puromycin (2 ⁇ g/ml) and hygromycin (1000 U/ml). Hybrid cells are subsequently cultured in the absence and presence of IL-3 to assess dominance or recessivity of mutations.
  • Plasmids AU1 -mTORwt [A. Sekulic et al., Cancer Res. 60:3504-13 (2000)] is subcloned from pcDNA3.1 with the Notl and a blunted Hindi 11 restriction sites into the Notl and a blunted BamHI restriction sites of a modified pMSCVpuro version, harbouring BamHI and Notl sites.
  • PlatE cells For production of retroviral particles, 4 x 10 6 PlatE cells [S. Morita et al., Gene Ther. 7:1063-66 (2000)] are transfected with 15 ⁇ g of retroviral pMSCVpuro plasmids (BD Biosciences Clontech, Palo Alto, CA, USA) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers protocol. Virus secreting platE cells are cultured in IMDM containing 10 % calf serum. At 72 h after transfection, virus- containing supernatant is harvested and filtered using a 0.45 ⁇ m pore filter.
  • 1 x 10 6 15V4 cells are infected with 1 ml viral supernatant containing 10 ⁇ g/ml hexadimethrin (Polybrene, Fluka, Buchs, Switzerland). After 24 h, infected cells are selected with puromycin (2 ⁇ g/ml) for at least 36 h.
  • Lentiviral particles are obtained from Sigma at a titre of 1 -3 x 10 7 transfection units (TU) per ml. 5 x 10 5 15v4, 6.5 or D2c cells are infected with 5 x 10 5 TU of lentiviral supernatant in presence of 10 ⁇ g/ml hexadimethrin. After 24 h, infected cells are selected with puromycin (2 ⁇ g/ml) for at least 36 h.
  • the target sequences are CAACAAGAUGAAGAGCACCAA (SEQ ID NO:8) for a non-targeting control; CCCUUAUAUCACUAAGGGUUU (SEQ ID NO:1 ) targeting murine TSC2; CGACUUAGACUUGACCUAUAU (SEQ ID NO:3) targeting murine PTEN; CGGUUCAUACAAGAGUUAUUU (SEQ ID NO:4) targeting murine Rictor and GCCCGAGUCUGUGAAUGUAAU (SEQ ID NO:6) targeting murine Raptor.
  • cells are washed with PBS and then lysed in ice-cold TNE buffer (50 mM Tris-HCI pH 8.0, 150 mM NaCI, 0.5 mM EDTA, 1% Triton X-100) supplemented with protease and phosphatase inhibitors (1x Roche complete protease inhibitor cocktail, 1 ⁇ g/ml Aprotinin, 1 ⁇ g/ml Pefabloc, 1 mM PMSF, 10 mM NaF, 10 mM NaN 3 , 10 mM
  • RNA sequences of cDNA and genomic DNA RNA is extracted with Trizol (Invitrogen) after the manufacturer's guidelines.
  • cDNA is prepared from the extracted RNA, using M-MLV Reverse Transcriptase (Promega) and oligo dT(15) primers following the manufacturer's guidelines.
  • Genomic DNA is extracted with the DNeasy Blood & Tissue kit (Qiagen).
  • the sequences of interest from cDNA and genomic DNA are amplified with gene specific primers, using HotStar Taq Plus polymerase (Qiagen) following the manufacturer's protocol, gel purified and then sequenced, using gene specific primers (Microsynth).
  • 10 4 cells per well are plated in 150 ⁇ l medium containing indicated concentrations of inhibitors in 96-well microtiter plates.
  • the cells are labelled either for 4 h at 37 ⁇ € with cell proliferation kit Il (XTT) (Roche Diagnostics GmbH) or for 6 h at 37 ⁇ € with Alamar Blue (Biosource) according to the manufacturers protocols.
  • XTT cell proliferation kit Il
  • Biosource Alamar Blue
  • the absorption or fluorescence is measured with a safire 2 (Tecan) instrument, and normalized to inhibitor-free controls. Standard deviations are calculated using the mean value of triplicates from usually three independent experiments.
  • FACS analysis is performed after staining with 5 ⁇ g/ml propidium iodide in PBS (Sigma) or Annexin V-FITC (Calbiochem) following the manufacturer's guidelines. Plotting of the different fluorescence signals versus the sidescatter signal allows gating and counting of cells.
  • RNA labeling of RNA was performed as described in the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, USA). Double-stranded cDNA was synthesized according to the One-Cycle cDNA Synthesis Kit (Affymetrix, Cat# 900431 ), starting from 5 ⁇ g of total RNA. The material was purified with the Sample Cleanup Module (Affymetrix, Cat# 900371 ). The purified cDNA was used for an in vitro transcription reaction by using the IVT labeling kit (Affymetrix, Cat# 900449) to synthesize cRNA in the presence of a biotin-conjugated ribonucleotide analog.
  • RNA Nano 6000 Chips (2100 Bioanalyzer, Agilent).
  • the cRNA targets were incubated at 94 0 C for 35 min in the provided Fragmentation Buffer and the resulting fragments of 50-150 nucleotides were again monitored using the Bioanalyzer. All synthesis reactions were carried out using a PCR thermo-block (T 1 Thermocycler, Biometra, G ⁇ ttingen, Germany) to ensure the highest possible degree of temperature control and reproducibility.
  • the hybridization cocktail (130 ⁇ l) containing fragmented biotin-labeled target cRNA at a final concentration of 0.05 ⁇ g/ ⁇ l was transferred into Affymetrix Mouse Genome 430A 2.0 and incubated at 45 0 C on a rotator in a hybridization oven 640 (Affymetrix) for 16 h at 60 rpm.
  • the arrays were washed and stained on a Fluidics Station 450 (Affymetrix) by using the Hybridization Wash and Stain Kit (Affymetrix, Cat# 900720). To increase the signal strength the antibody amplification protocol was used (FS450_0002).
  • the GeneChips were processed with an Affymetrix GeneChip ® Scanner 3000 7G (Affymetrix) by using the current default settings. DAT image files of the microarrays were generated using GeneChip Operating Software (GCOS 1 .4, Affymetrix).
  • GCOS 1 .4, Affymetrix GeneChip Operating Software
  • Cytoplasmic RNA was prepared as described [N.N. Gough, Annal. Biochem. 173:93-95,
  • Sense primer for FBP2 was ⁇ '-ATGAATGAGCAATGGAGATGG- 3' (SEQ ID NO:9); the antisense primer ⁇ '-CCGTATCTTTCTTTACCCCTGG-S' (SEQ ID NO:10).
  • 18S rRNA was amplified with sense primer 5'-CGGCTACCACATCC AAGGAA-3' (SEQ ID NO:1 1 ) and antisense primer ⁇ '-GCTGGAATTACCGCGGCT-S' (SEQ ID NO:12).
  • FBP2 mRNA FBP2 mRNA
  • Reverse transcription 1 ⁇ g cytoplasmic RNA using iScript (Bio-Rad); reaction conditions are according to the manufacturer.
  • Real time PCR is performed using cDNA dilutions for FBP2 (1 :40), for PTEN (1 :40) and for 18S (1 :4000). Reaction volume is 20 ⁇ l in 96 well plates.
  • RQ Kit IQ-Sybergreen Supermix (Bio-Rad), ThermoCycler mylQ (Bio-Rad).
  • Amplification 3 min 95 0 C initial denaturing (10 sec 95 0 C / 20 sec 55 0 C / 10 sec 72 0 C), 40 cycles. Melting curves: 1 min 95 0 C denaturing, 1 min 55 0 C annealing, 55 0 C to 9O 0 C / 0.5 0 C, intervals 10 sec ramping. Data analysis is performed with Bio-Rad IQ5 analysis software.

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Abstract

The invention relates to compounds blocking or activating the mTOR pathway for use as a medicament in combination with rapamycin or a rapamycin-like compound and/or with metformin or a metformin-like compound and/or with thermal energy, for the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated. Experiments are described which demonstrate that mutational loss of Tsc2 or PTEN or down-regulating Tsc2 or PTEN by shRNA sensitizes cells for rapamycin and metformin and for thermal treatment, with synergies of these three forms of treatments. The invention further relates to a method to predict sensitivity of a tumor to the combination of mTOR pathway inhibition combined with application of thermal energy.

Description

Chemotherapy of neoplastic diseases using combinations of rapamycin and compounds modulating mTOR pathway alone or in combination with heat
Background of the invention
Strategies in cancer therapy are often based on inhibition of dominantly acting oncogene functions or on restoring lost tumor suppressor functions. Several tumor suppressor functions have been recognized and are the basis for extensive research in applying this knowledge to develop medicaments or gene therapy protocols for treatment of neoplastic diseases.
Rapamycin is known to inhibit T-cell proliferation at nanomolar concentrations, and its derivatives are more recently also in experimental clinical use as potential anti-neoplastic agents. However, it is observed that several cell types are not sensitive to treatment. Biomarkers which predict responsiveness of cancer cells to rapamycin or its derivatives are needed.
K.F. Kiser et al., Oncogene 25:6595-6603 (2006) describe the isolation and characterization of dominant and recessive IL-3-independent hematopoietic transformants of an immortalized bone-marrow-derived mast cell line. It was recognized that these transformants may be useful to develop new cancer treatment strategies, however their full potential was not yet exploited.
Metformin, a drug used for the treatment of type Il diabetes, applied over prolonged times to diabetic patients appears to reduce the risk of cancer [J. M. M. Evans et al., BMJ
330:1304-1305 (2005)]. However, the cellular mode of action and the cellular targets of metformin are not known.
M. Zakikhani et al., Cancer Res 66:10269-10273 (2006) report that metformin acts as a growth inhibitor of epithelial cells and inhibits phosphorylation of the mTOR target S6 kinase. The effect is not specific for cancer cells, as non-transformed cells (MCF-10A) are also inhibited. Only partial inhibition of growth is reported, and no evidence for cell death (apoptosis) is presented.
R.J. Dowling et al., Cancer Res 67:10804-10812 (2007) show that metformin inhibits mTOR-dependent translation initiation via the mTOR pathway components AMPK (adenosyl monophosphate activated kinase) and its target Tsc2 (tubreous sclerosis complex 2), and suggest that this may be a mechanism how metformin inhibits cancer cells. Growth inhibition reported is only partial. More than 40% of cells survive after treatment, indicating that under those conditions metformin alone is not adequate for cancer treatment.
I. B. Sahra et al., Oncogene 1 -1 1 (2008) report that metformin exerts an antitumoral effect via blocking the cell cycle but not inducing apoptosis. The mechanism is independent of AMPK (adenosyl-monophosphate activated protein kinase).
US patent application 2007/10280918 describes a pharmaceutical composition for treating cancer comprising a combination of a PP2A methylating agent and an active principle selected from a large number of enzyme inhibitor compounds also naming rapamycin and metformin.
Applying heat to cancer cells (thermotherapy) is an experimental mode of therapy of promising, yet limited success, where the underlying cellular determinants for sensitivity are not known. There is a dual need for a) sensitizing cancer cells to this form of treatment and b) for a diagnostic test predicting thermosensitivity of a given cancer.
Summary of the invention
The invention relates to compounds either blocking or activating the mTOR pathway for use in the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, in combination with rapamycin or a rapamycin- like compound and/or in combination with metformin or a metformin-like compound and/or in combination with thermal energy, and optionally in combination with further suitable inhibitor compounds. mTOR pathway activating compounds sensitize cells to rapamycin and exert therapeutic synergism with rapamycin. Compounds blocking the pathway are therapeutic on their own and additionally synergize with rapamycin and with mTOR pathway activators.
Compounds blocking or activating the mTOR pathway are, for example, compounds blocking or downregulating Tsc2, pten, Tsc1 , raptor, rictor, or blocking or downregulating other components of the mTOR complexes 1 and 2, or blocking or downregulating other components of the mTOR pathway including downstream elements of the mTOR pathway. Particular examples of such compounds are anti-sense RNA, siRNA or shRNA downregulating Tsd , Tsc2 or pten. Rapamycin or a rapamycin-like compound is a further example of a compound blocking the mTOR pathway.
Metformin exerts a powerful synergistic effect with rapamycin on some but not other cells. The invention therefore, in particular, relates to compounds blocking or activating the mTOR pathway such as rapamycin or a rapamycin-like compound in combination with metformin or a metformin-like compound, and to other compounds blocking or activating the mTOR pathway in combination with rapamycin or a rapamycin-like compounds and/or in combination with metformin or a metformin-like compound for use in the treatment of the mentioned diseases.
The invention further relates to the use of a compound blocking or activating the mTOR pathway for the manufacture of a medicament for the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, in combination with rapamycin or a rapamycin-like compound and/or in combination with metformin or a metformin-like compound and/or in combination with thermal energy, and optionally in combination with an inhibitor of the MAPK, Wnt, Jak-Stat, Pl-3k, JUNK or hedgehog pathway and/or a DNA damaging agent, 2'-deoxy-glucose, an inducer of hypoxia, or a pyrogenic compound.
The invention further relates to pharmaceutical preparations comprising a compound blocking or activating the mTOR pathway and one or several of the mentioned further inhibitors.
The invention further relates to a method of treating neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, comprising administering a therapeutically effective amount of a compound blocking or activating the mTOR pathway in combination with rapamycin or a rapamycin-like compound and/or in combination with thermotherapy and/or in combination with metformin or a metformin-like compound, and optionally in combination with an inhibitor of the MAPK, Wnt, Jak-Stat, Pl- 3K, JUNK or hedgehog pathway and/or a DNA damaging agent, 2'-deoxy-glucose, an inducer of hypoxia, or a pyrogenic compound. In particular, the invention relates to a method of treating the mentioned diseases with compounds blocking or activating the mTOR pathway, such as rapamycin or a rapamycin-like compound or metformin or a metformin-like compound in combination with thermotherapy. The invention further relates to a method for screening for a compound blocking the mTOR pathway using known IL-3 dependent cells that are insensitive to mTOR pathway blockade exerted by rapamycin, and in parallel known IL-3 independent mutant cells that are sensitive to rapamycin due to their addiction to the mTOR pathway. Such mutants can be obtained by frameshift mutagen (ICR191 ) treatment of IL-3 dependent cells followed by selection of independent cells via IL-3 removal [K. F. Kiser et al., Oncogene 25:6595-6603 (2006)]. Suitable cells are identified by establishing rapamycin sensitivity of growth.
The invention further relates to a method to predict sensitivity of a tumor to the combination of mTOR pathway inhibition combined with application of thermal energy, based on the fact that cells with mutations in Pten display elevated levels of the gluconeogenic enzyme fructose-1 ,6-bisphosphatase 2 (FBP2) and are heat sensitive due to an energy burning "futile cycle".
Brief description of the Figures
Figure 1: Growth inhibition by rapamycin and rescue by IL-3
X-axis: nanomolar concentration of rapamycin; Y-axis: % of cell growth. Indicated cell lines were grown with indicated concentrations of rapamycin (X-axis in nM) for 48 h in presence or absence of IL-3 (as indicated), and growth (Y-axis in %) was determined.
Fig. 1A: Rapamycin inhibits the mutant line D2c, but not its non-mutagenized precursor
15V4 nor IL-3 dependent clone cl20. Importantly, cell hybrid 20xD2c is rapamycin insensitive, indicating that drug sensitivity is recessive and was acquired by a loss-of- function mutation restorable by cell fusion. All cells are tested in presence of IL-3.
Fig. 1 B: Five additional IL-3 independent mutant lines, all derived from 15V4, are likewise sensitive to rapamycin. Drug inhibition is rescued in all cases by the presence of IL-3 during growth (dashed lines).
Fig. 1 C: Clone D2c is different from all clones, in that it is not rescued by the addition of IL-3.
Figure 2: Growth of cells transformed by mTOR are sensitive to rapamycin. X-axis: nanomolar concentration of rapamycin; Y-axis: % of cell growth. Following transformation of IL-3 dependent 15V4 cells by an mTOR construct and isolation of IL-3 independent cells, the sensitivity to rapamycin was determined following 48 h incubation with indicated concentrations of rapamycin. The fact that cells die by this mTOR antagonist proves that 15V4 can become addicted to mTOR-dependent growth by activation of the mTOR pathway.
Figure 3: Transformation of 15V4 to IL-3 independent growth by siRNA targeting PTEN or TSC2.
Fig. 3A: 15V4 cells were infected with lentiviruses encoding small hairpin RNA (shRNA) targeting PTEN (shPTEN), TSC2 (shTSC2), or a non-targeting control sequence (shCTRL) and selected for puromycin resistance for 3 days. Then, IL-3 was removed and the number of viable cells determined at the indicated times (days without IL-3: 0, 1 , 2, 3, 4, 7, 13, 18, 23). Results indicate that downregulation of both PTEN and TSC2 is sufficient to transform cells to IL-3 independent growth.
Fig. 3B: X-axis: nanomolar concentration of rapamycin; Y-axis: % of cell growth. The upper panel shows sensitivity of shPTEN cells described in 3A to rapamycin treatment. The lower panel shows sensitivity of shTSC2 cells described in 3A to rapamycin treatment. Drug inhibition is rescued in shPTEN and shTSC2 cells by the presence of IL-3 during growth.
The sensitivity to rapamycin was determined following 48 h incubation with indicated concentrations of rapamycin. The fact that cells die by this mTOR antagonist proves that 15V4 can become addicted to mTOR-dependent growth by activation of the mTOR pathway.
Figure 4: IL-3 independent growth of 6.5 and D2c specifically requires mTOR complex 1
(mTORCI) or mTORC2.
X-axis: time in hours; Y-axis: cells per milliliter. Precursor cells 15V4 (upper panel), mutants 6.5 (middle panel) and D2c (lower panel) were infected with lentiviruses carrying shRNA targeting raptor (shRap), a component of mTORCI , or rictor (shRic), a component of mTORC2, or non-targeting (shCon). After selection with puromycin for 2 days, IL-3 was removed (-IL-3) or not (+IL-3) and the number of viable cells determined over time.
Note that in precursor cells, neither downregulation of rictor nor of raptor had a significant effect on growth. In contrast, downregulation of rictor in 6.5 cells reduced growth
(reversed by addition of IL-3). In D2c cells, no growth was observed when raptor was downregulated.
These results indicate that mTORCI is vital for growth of D2c, and mTORC2 for 6.5. Figure 5: Metformin inhibits growth of 6.5, but not of normal precursor cells 15V4 X-axis: millimolar concentration of metformin; Y-axis: % of cell growth (note the logarithmic scale). Cells were incubated at the indicated nM concentrations of rapamycin (R) and metformin, alone or in combination, and growth was determined after 48 h. Panel A: precursor 15V4 cells are insensitive even to the combination of drugs. Panels B, C: D2c is sensitive to rapamycin, but hardly to metformin. No change in presence of IL-3.
Panels D, E: rapamycin sensitive 6.5 cells are sensitive to metformin alone, with a very strong synergistic effect when combined with rapamycin. Drug inhibition is rescued in 6.5 cells by the presence of IL-3 during growth.
Figure 6: Identification of marker gene FBP2 by Affymetrix DNA microchip analysis. RNA expression from triplicate cultures are analyzed. All cells including mutants were grown in the presence of IL-3 before RNA isolation. Indicated are the number of transcripts which show a change factor of at least two-fold.
A: Comparison of mutants 6.5, 6.8 and H1 .2 with precursor 15V4. Numbers outside the circles indicate the number of transcripts up- or downregulated in the respective mutant compared to precursor line 15V4. Arrows pointing up: upregulated transcripts; arrows pointing down: downregulated transcripts. For example, in clone 6.5, 180 transcripts are upregulated, and 193 transcripts are downregulated. Note that 12 transcripts are upregulated and 24 transcripts are downregulated in all three mutants. B: Comparison of mutants 6.5, 6.8 and H1 .2 with their backfusion hybrids 6.5 x 20, 6.8 x 20 and H1.2 x 20. Arrows pointing up: upregulated transcripts in mutant versus hybrid; arrows pointing down: downregulated transcripts in mutant versus hybrid. Note that 69 transcripts are elevated in all mutants versus their hybrids, while 32 transcripts are downregulated in all hybrids.
C: Comparison of mutants 6.5 and H1.2 grown for 48 h in presence or absence of 10 nM rapamycin and in presence of IL-3 to prevent apoptosis. Arrows pointing up: transcripts upregulated by rapamycin; arrows pointing down: transcripts downregulated by rapamycin. Note that 57 transcripts are sensitive to rapamycin.
D: Relative expression level of FBP2 expression in precursor cells, mutants (with and without rapamycin) and hybrids. Y-axis: arbitrary units of expression. X-axis: "1 " = clone 20; "2" = 15V4; "3" = 6.5; "4" = hybrid 6.5x20; "5" = 6.5 treated with 10 nM rapamycin; "6" = 6.8; "7" = hybrid 6.8x20; "8" = H1 .2; "9" = H1.2x20 treated with 10 nM rapamycin; "10" = hybrid H1 .2x20. Figure 7: Analysis of FBP2 expression by real-time quantitative PCR X-axis: indicated cell lines. Y-axis: relative number of transcripts with 15V4 set as 1 (exponential units).
A: Expression of FBP2. Cells used are as indicated. 6.5GFP = 6.5 transfected with GFP for control; 6.5PTEN = 6.5 transfected with PTEN cDNA to restore PTEN function. B: Expression of FBP2 and of PTEN (note downregulation by shRNA). 15V4shbGlo = 15V4 transfected with beta-globin-targeting shRNA sequence; 15V4shPTEN = 15V4 transfected with PTEN-targeting shRNA sequence.
Detailed description of the invention
The invention relates to compounds modulating the mTOR pathway either as activators thus sensitizing cells to rapamycin (rapamycin sensitizers) or as inhibitors thus acting in synergy with rapamycin, for use as a medicament in combination with rapamycin or a rapamycin-like compound and/or in combination with metformin or a metformin-like compound and/or in combination with thermal energy, for the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated.
The invention further relates to the application of thermal energy together with the aforesaid inhibitors of the mTOR pathway, based on the observation that mTOR pathway activation, particularly due to Pten mutation, renders tumor cells sensitive to killing by heat. The efficiency of killing is amplified by pre-treatment of cells with either rapamycin or metformin.
A compound blocking the mTOR pathway is, for example, a compound blocking or down- regulating mTOR, ml_ST8/B-Gbl, SIN1 , raptor, rictor, or other components of the mTOR complexes or other components of the mTOR pathway, such as Rheb, LST8, S6K1 , Akt1 , Akt2, RSK1 , ERK1/2. Compounds activating the mTOR pathway are e.g. compounds blocking or down-regulating negative regulators of the mTOR pathway such as Tsd , Tsc2 or AMPK, REDD1/2, GSK-3. Such compounds render cells sensitive to the mTOR pathway blockers listed above, in particular to rapamycin or rapamycin-like compounds. Examples for such compounds are anti-sense RNA, siRNA or shRNA down-regulating any of the mentioned proteins implicated in the mTOR pathway or components of the mTOR complexes, in particular down-regulating Tsc2 or PTEN. Other compounds blocking or down-regulating the above targets are likewise preferred, for example organic compounds of a molecular weight below 1500, preferably below 500. Such compounds may be found by the screening method described herein below.
"mTOR pathway" means mammalian Target of fiapamycin signaling pathway and includes upstream pathways signaling down to mTOR and pathways signaling downstream from mTOR to their various effector molecules regulating gene expression and protein synthesis as defined by A. Soulard and M.N. Hall, Cell 129:434 (2007). "TOR" is a highly conserved protein kinase and a central controller of cell growth and metabolism. The mammalian TOR (mTOR) signaling pathway controls cell growth and metabolism in response to nutrients (e.g., amino acids), growth factors (e.g., insulin, IGF- 1 , PDGF), and cellular energy status (ATP). Nutrients are the dominant mTOR input as high levels of amino acids can compensate for an absence of the other mTOR inputs but not vice versa, mTOR activates cell growth by positively and negatively regulating several anabolic and catabolic process, respectively, that collectively determine mass accumulation. The anabolic processes include transcription, protein synthesis, ribosome biogenesis, nutrient transport, and mitochondrial metabolism. Conversely, mTOR negatively regulates catabolic processes such as mRNA degradation, ubiquitin-dependent proteolysis, autophagy and apoptosis. mTOR is found in two functionally and structurally distinct multiprotein complexes (mTORCI and mTORC2 in mammals), each of which signals via a different set of effector pathways. mTORCI is rapamycin sensitive, whereas mTORC2 is rapamycin insensitive. The best-characterized phosphorylation substrates of mTOR are S6K and 4E-BP1 , via which mTORCI controls translation, and Akt/PKB, via which mTORC2 controls cell survival and likely other processes. Like mTOR itself, the two mTOR complexes and the overall architecture of the mTOR signaling network appear to be conserved from yeast to human. As a central controller of cell growth and metabolism, mTOR plays a key role in development and aging, and is implicated in many major diseases including cancer, cardiovascular disease, inflammatory disease, and metabolic disorders. Indeed, it has been estimated that mTOR is upregulated and the cause of malignancy in 70% of all tumors. Pharmacological modulation of pathways directed to mTORCI and mTORC2 as well as the signals emanating from these two complexes provide potential targets for a broad spectrum of diseases.
"mTOR complex" means two structurally distinct multiprotein complexes, mTORCI and mTORC2, each of which signals via a different set of effector pathways. mTORCI is rapamycin sensitive whereas mTORC2 is rapamycin insensitive. "Rapamycin" is a macrolide also named sirolimus, and is 23,27-epoxy-3H-pyrido[2,1 -c] [1 ,4]oxaazacyclohentricontine. As described above, rapamycin blocks the mTOR pathway by interaction with mTORC1.
A "rapamycin-like compound" is, for example, RAD001 (Novartis), CCI-779 (Wyeth
Ayerst), AP23573/Deforolimus, AP21967, or AP1510 (Ariad Pharmaceuticals/Merck). As described for rapamycin, rapamycin-like compounds also block the mTOR pathway.
It is understood that in definitions of the type "A compound blocking the mTOR pathway in combination with rapamycin or a rapamycin-like compound", such a definition does not include the combination of rapamycin with itself, however, includes combinations of rapamycin with a rapamycin-like compound, or of one rapamycin-like compound with another rapamycin-like compound.
"Metformin" is 1 ,1 -dimethylbiguanide, usually in the form of the hydrochloride salt, but the free base or other acid addition salts may be used as well. Metformin inhibits mTOR- dependent translation initiation via the mTOR pathway component AMPK, a kinase with mTOR inhibitory activity, which acts by phosphorylating and thus activating the negative mTOR regulator Tsc2. The net effect of metformin is to inhibit mTOR via activating AMPK, which activates Tsc2.
A "metformin-like compound" is, for example, AICA-R (5-aminoimidazole-4-carboxamide riboside) and drugs modulating glucose metabolism, for example 2-deoxy-glucose, glitazones, sulfonylureas, meglitinides, thiazolidinediones, alpha-glucosidase inhibitors, incretins, glucagon-like peptide agonists, DPP4 inhibitors, or amylin analogs. Preferred metformin-like compounds are AICA-R and glitazones. As described for metformin, metformin-like compounds also block the mTOR pathway.
It is understood that in definitions of the type "A compound blocking the mTOR pathway in combination with metformin or a metformin-like compound", such a definition does not include the combination of metformin with itself, however, includes combinations of metformin with a metformin-like compound, or of one metformin-like compound with another metformin-like compound. A particular shRNA down-regulating TSC2 is an oligonucleotide comprising the target sequence CCCUUAUAUCACUAAGGGUUU (SEQ ID NO:1 ) in mouse or CCCUUUCAUCACUAAGGGCCU (SEQ ID NO: 2) in human.
A particular shRNA down-regulating PTEN is an oligonucleotide comprising the target sequence CGACUUAGACUUGACCUAUAU (SEQ ID N0:3) in mouse and human.
A particular shRNA down-regulating rictor is an oligonucleotide comprising the target sequence CGGUUCAUACAAGAGUUAUUU (SEQ ID N0:4) in mouse and AGGUUCAUACAAGAAUUAUUU (SEQ ID NO:5) in human.
A particular shRNA down-regulating raptor is an oligonucleotide comprising the target sequence GCCCGAGUCUGUGAAUGUAAU (SEQ ID NO:6) in mouse and GCCUGAGUCGGUAAAUGUGCU (SEQ ID NO:7) in human.
Neoplastic diseases considered are, for example, epithelial neoplasms, squamous cell neoplasms, basal cell neoplasms, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous neoplasms, ducal-, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, specialized gonadal neoplasms, paragangliomas and gangliomal tumors, naevi and melanomas, soft tissue tumors and sarcomas, fibromatous neoplasms, myxomatous neoplasms, lipomatous neoplasms, myomatous neoplasms, complex mixed and stromal neoplasms, fibroepithelial neoplasms, synovial like neoplasms, mesothelial neoplasms, germ cell neoplasms, trophoblastic neoplasms, mesonephromas, blood vessel tumors, lymphatic vessel tumors, osseous and chondromatous neoplasms, giant cell tumors, miscellaneous bone tumors, odontogenic tumors, gliomas, neuro- epitheliomatous neoplasms, meningiomas, nerve sheath tumors, granular cell tumors and alveolar soft part sarcomas, Hodgkin's and non Hodgkin's lymphomas, other lymphoreticular neoplasms, plasma cell tumors, mast cell tumors, immunoproliferative diseases, leukemias, miscellaneous myeloproliferative disorders, lymphoproliferative disorders and myelodysplastic syndromes. Considered are, in particular, the following organ-specific cancers selected from renal cell cancer, endometrial carcinoma, glioblastoma, head and neck cancer, colon cancer, pancreatic cancer, gastric cancer, hepatocarcinoma, ovarian cancer, thyroid carcinoma, breast cancer, prostate cancer, and lung cancer. More particularly the following neoplastic diseases are considered: Tuberous sclerosis complex (TSC), lymphangioleiomyoma (LAM), Peutz-Jegher's syndrome, hamartoma syndromes, Cowden's disease, Lhermitte-Duclos disease, Bannayan syndrome, and Proteus syndrome.
Hyperproliferative conditions considered are, for example, lymphoproliferative disorders, dysplasias and metaplasias of different tissues, psoriasis, atherosclerosis, restenosis after angioplastic surgery, angiogenesis and particularly tumor angiogenesis, and cancers such as gastro-intestinal stromal tumors and Menetriers syndrome.
Other processes wherein mTOR is implicated are, for example, aging, type-ll diabetes and other metabolic conditions, cardiovascular disease, and autosomal-dominant polycystic kidney disease.
Aging processes which can be positively influenced by blocking the mTOR pathway are, for example, type Il diabetes, Parkinson, and Alzheimer disease.
Cardiovascular diseases are, for example, conditions involving endothelial proliferation as in atherosclerosis leading to thrombotic diseases and stroke.
The invention further relates to the use of a compound blocking or activating the mTOR pathway for the manufacture of a medicament for the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, in combination with rapamycin or a rapamycin-like compound, and/or in combination with metformin or a metformin-like compound, and optionally in combination with an inhibitor of the MAPK, Wnt, Jak-Stat, PI-3K, JUNK or hedgehog pathway and/or a DNA damaging agent, 2'-deoxy-glucose, or an inducer of hypoxia.
Inhibitors of the MAPK pathway considered are, for example, BAY 43-9006, PD98059, U0126, SB202190, SB203580, FR167653, ZM336372, and GW5074.
Inhibitors of the Wnt pathway considered are, for example, fumagillin.
Inhibitors of the Jak-Stat pathway considered are, for example, WP1034, WP1066, AG490, and CP690550. Inhibitors of the PI-3K pathway considered are, for example, wortmannin, LY294002, PM 03, XL147, SF1 126, and TGX221.
Inhibitors of the JUNK pathway considered are, for example, CC401 , SP600125, 4-hydroxynonenal, JNK inhibitor IX, ALX 159-600, and AS601245.
Inhibitors of the hedgehog pathway considered are, for example, CUR61414, jervine, and cyclopamine Hh inhibitor.
DNA damaging agents considered are, for example, etoposide, cisplatin, doxorubicin, tamoxifen, bleomycin, irinotecan, topotecan and a) antimetabolites such as 5-fluorouracil (ICN), gemcitabine HCI (Gemzar, EIi Lilly), b) alkylating agents such as oxaliplatin (EloxantinT"', Sanofi-Synthelabo), dacarbazin (Detimedac, Medac), cyclo- phosphamide (Endoxan TM, Asta) and carboplatin (Paraplatin, Bristol-Meyers Squibb), c) cell-cycle inhibitor such as vinorelbine (Navelbine TM, Robapharm), vinblastine (VelbeT"", EIi Lilly), docetaxel (TaxotereT"", Aventis), d) DNA breaker (topo-isomerase inhibitor, intercalator, strand breaker) such as doxorubicin HCI (Adriblastin TM, Pharmacia-Upjohn), bleomycin (Asta-Medica), irinotecan (Campto, Aventis), etoposide phosphate (Etopophos, Bristol- Meyers Squibb), topotecan HCI, (Hycamtin, GlaxoSmithKline), e) mixtures thereof, f) compounds interfering with the signal transduction pathway, such as caspase activity modifiers, agonists and antagonists of cell death receptors, modifiers of nucleases, phosphatases and kinases such as imatinib mesylate (Gleevec™, Novartis), dexamethasone, phorbol myristate acetate, cyclosporin A, quercetin, tamoxifen (Alexis Corporation, Switzerland).
Inducers of hypoxia considered are, for example, desferrioxamine, cobalt chloride, Revlimid (Celgene), and anti-angiogenic ABT-510 (Abbott).
Pyrogenic substances considered are, for example bacterial polysaccharides, cytokines including but not limited to interferons, IL-6 and IL-1 , and prostaglandins.
The invention is based on the following experimental observations:
TOR and pathway addiction So called pathway addiction in cancer is very relevant to therapy, as it implies that blocking a pathway to which the tumor cell is addicted would lead to the elimination of that cell. This concept has implications for strategies of chemotherapy, because if one forces the tumor cell into a given pathway ("forced addiction"), as is shown in the present invention by use of RNA interference, one can sensitize the cell exquisitely to inhibitors (blockers) of that pathway. In the case of the mTOR pathway, the mTOR specific inhibitor rapamycin is perhaps the most specific kinase inhibitor known and well tolerated when applied as immunosuppressant to transplantation patients, with recently also promising results when its derivatives (rapamycin-like compounds) are applied as anticancer drugs, for example in kidney cancers. mTOR phosphorylates p70 ribosomal S6 protein kinase and 4E-BP1 , a repressor of the translation initiation factor elF-4E, and thus exerts a powerful controlling effect on translation. A known negative inhibitor of mTOR is Tsc2, a GTPase-activating protein (i.e. an activator protein of an enzyme with guanosyl triphosphatase activity) that inhibits Rheb, a G-protein (which is a protein with guanosyl triphosphatase activity) stimulating the kinase activity of TOR. The GTPase Tsc2, which forms a functional complex with protein Tsc1 , is inhibited by growth signals via protein kinase B (PKB, also called akt) and ERK (extracellular regulated kinase) with net stimulation of mTOR, and stimulated via the phosphatase PTEN, the kinase LKB1 and the kinase AMPK (AMP-dependent kinase) with net inhibition of mTOR, the latter ones being tumor suppressor genes. Chemical modulation of these mTOR regulators contributes to addiction to mTOR and induces sensitivity to rapamycin or rapamycin-like substances, i.e. chemically forced addiction to mTOR with resultant sensitivity to mTOR pathway blockers (inhibitors).
IL-3-independent mutants with addiction to the mTOR pathway
The known non-autocrine growth autonomous mutants from IL-3 dependent mast cells obtained by frame-shift mutagenesis [K.F. Kiser et al., Oncogene 25:6595-6603 (2006)] were exposed to rapamycin, a mTOR inhibitor originally used as an immunosuppressant which inhibits proliferation of certain cells at nM concentrations, and derivatives of which are in clinical trials as antineoplastic agents. Mutant line D2c is highly sensitive to rapamycin with an IC50 of less than 1 nM, while precursor 15V4 cells, which are v-H-ras expressing PB-3c, are not inhibited (Fig 1A). These results demonstrate that D2c cells may have acquired rapamycin sensitivity concomitantly with loss of IL-3 dependence following ICR191 mutagenesis via activation of the mTOR pathway. It is known that IL-3 independence in D2c cells results from recessive mutation (Kiser et al., be. cit), and it is demonstrated in the present invention that the rapamycin sensitivity of these cells is also recessive. Somatic hybrid cells generated previously by cell fusion between D2c and clone 20 were used, the latter being an IL-3 dependent subclone of PB-3c with tumor suppressor activity in cell fusion experiments [A.P. Nair et al., Oncogene, 7:1963-1972 (1992)] to test for loss of drug sensitivity. Indeed, the IL-3 dependent hybrid cells (20xD2c) are again insensitive to rapamycin (Fig. 1 A), indicating that rapamycin sensitivity and loss of IL-3 dependence resulted from a possibly common loss-of-function mutation in D2c. These observations led to the present invention claiming that addiction to the mTOR pathway can be induced by a chemical mutagen and that inhibitors of the missing function can likewise induce pathway addiction in a non-mutagenized cell. To prove these claims a number of independent isolates were generated by repeating ICR191 treatment of 15V4 cells and selection for IL-3 independence. Five additional isolates were obtained (6.5, 6.8, 7.2, 8.8 and H1.2) that were again highly sensitive to rapamycin (Fig. 1 B, solid lines). Three of them (6.5, 6.8, H1.2) were subjected to cell fusion with PB-3c clone 20. All somatic hybrids were again IL-3 dependent and rapamycin insensitive, which confirms the claim and further suggests that a loss-of-function mutation activates the mTOR pathway in these growth autonomous mutants. Importantly, rapamycin sensitivity indicates that the mutants are addicted to the mTOR pathway.
Rescue of mTOR addiction by growth-factor IL-3
As all clones analyzed are selected for IL-3 independence via ICR191 mutagenesis and tested for rapamycin sensitivity in the absence of IL-3, they were further tested whether addition of the cytokine to the cell cultures would have an effect on drug sensitivity by relieving pathway addiction via activating an alternative growth pathway. In fact, four mutants are rescued from rapamycin inhibition by addition of IL-3, while D2c is inhibited by rapamycin even in the presence of IL-3 (Fig. 1 B, C, dashed lines). The implication for cancer chemotherapy is that in tumors for which D2c is a model, mTOR pathway blockade is sufficient for therapy, while in most cancers rescue pathways (in the model described in this invention the IL-3 pathway) must be blocked in addition to the mTOR pathway.
Transformation to rapamycin sensitive growth by overexpression of mTOR
As rapamycin is an exquisitely specific inhibitor of mTOR, it was tested whether mTOR overexpression could transform 15V4 cells to IL-3 independent growth in order to confirm further that IL-3 independence can be brought about by forced mTOR pathway activation. For overexpression, an AU-tagged murine mTOR construct was introduced by retroviral infection in 15V4 cells, and other cells were infected with an empty control retrovirus. After puromycin selection and removal of IL-3, IL-3 independently growing mTOR overexpressing 15V4 cells are obtained, while the control cells do not survive more than 2 days without IL-3. These mTOR overexpressing and IL-3 independent cells are also rapamycin sensitive (Figure 2). The finding that mTOR overexpression activates mTOR signaling in 15V4 cells and permits rapamycin sensitive IL-3 independence provides additional evidence for the claim that hyperactivation of mTOR signaling transforms 15V4 cells to IL-3 independence.
Rapamycin inhibition occurs via apoptosis
By microscopic analysis it was observed that rapamycin induces actual cell death in the panel of rapamycin sensitive mutants. To further explore whether the mechanism of rapamycin induced cell death occurs through apoptosis in these cells, propidium iodide staining in combination with annexin V staining was applied to determine late apoptotic cells. The results are summarized in Table 1 , documenting the percentage of live, of early apoptotic and of late apoptotic cells.
Table 1 : Determination of live and dead (apoptotic or necrotic) cells following 48 h growth in presence of rapamycin and in presence or absence of IL-3.
Figure imgf000016_0001
Outer left column indicates cell lines
IL-3: presence (+) or absence (-) of interleukin-3 r: 50 nM rapamycin treatment (+) or DMSO control treatment (-)
A: percentage (%) of living cells; B: percentage of early apoptotic cells; C: percentage of dead late apoptotic cells; D: percentage of dead necrotic cells. Numbers representative from at least four experiments.
The number of dead cells (determined by the presence of annexin V and staining with propidium iodide) increases dramatically upon addition of rapamycin in 6.5 and D2c cells, but not in precursor line 15V4. Cell death is antagonized in 6.5 but not in D2c cells by addition of IL-3. Together, these data indicate that these cell lines are representative models to study induction of cell death by inhibitors of the mTOR pathway. In IL-3 dependent 15V4 precursor cells, only 4% of late apoptotic cells were found in the presence of IL-3 when treated for 48 h with rapamycin, while in 6.5 and D2c cells this value was 3.7 and 54%, respectively. In the absence of IL-3, both mutants had around 80% of late apoptotic cells after rapamycin treatment. Both 6.5 and D2c cells undergo apoptosis as defined by Pl-staining and annexin V positivity in response to rapamycin, while only cells from mutant 6.5 is rescued from apoptosis by IL-3, in keeping with the findings from Figure 1. These findings demonstrate that in 6.5 cells, like in precursor 15V4 cells, IL-3 activates an alternative pathway, which protects against apoptosis. Apoptosis is the mechanism by which the mutants respond to blocking mTOR. In all mutants tested except D2c this mechanism can be overcome by providing exogenous IL-3. In other words, mTOR pathway addiction can be overcome in most mutants by providing a growth- factor activating a parallel growth pathway. For the concept of mTOR pathway addiction, this means that parallel pathways must be silenced in order to reveal pathway addiction.
Loss of PTEN or TSC2 leads to rapamycin sensitive growth
The finding that loss-of-function mutations transform 15V4 cells to IL-3 independent but mTOR dependent proliferation suggests that negative regulators of mTOR have become deficient in these autonomously growing cells. Therefore, further components of the mTOR signaling network were tested in seven rapamycin-sensitive clones (6.4, 6.5, 6.8, 7.2, 8.8, H1.2 and D2c) in comparison to 15V4 cells. The data are summarized in Table 2.
Table 2: Characterization of IL-3 independent mutants by Western blotting
Figure imgf000017_0001
Outer left column indicates cell lines + base-line signal - loss of signal +++ increased signal Data are from at least 3 independent determinations
By Western analysis, two different mTOR activation patterns are found in the panel of mutants. In clone D2c, hyperactivation of mTORCI is observed, shown by increased phosphorylation of the mTORCI specific readout S6K-Thr389 (Table 2) in these cells. In contrast, hyperactivation of mTORC2 is observed in clones 6.5, 6.8 and 7.2, shown by increased phosphorylation of the mTORC2 specific readout PKB-Ser473. So far, there is no indication for an activation of mTOR signaling in clones 6.4, 8.8 and H1.2. In accordance with the different mTOR activation pattern, there are also protein deficiencies in negative mTOR regulators in the mutant cell panel. In clone D2c, a reduced Western blot signal is observed for the tumor suppressor TSC2 (Table 2). In clones 6.5, 6.8 and 7.2, the tumor suppressor PTEN is undetectable. Other components of the mTOR signaling network, including LKB1 , NF1 , PHLPP, CTMP, Rheb, FoxO1/3, PKCalpha, PRAS40 and FKBP38, appear unaffected. None of the examined mutants shows an increase in phosphorylation of STAT5-Tyr694, a signal that reflects activation of the Jak- Stat pathway. In addition to mTOR activation, MAPK was found to be hyperactive in clone D2c, shown by increased phosphorylation of ERK1/2-Thr202/Tyr204. The absence of the TSC2 or PTEN protein signals in clones D2c, 6.5, 6.8 and 7.2, and the correlation with mTOR activation in these cells suggests that ICR191 treatment induced frame shifts in Tsc2 of clone D2c and in Pten of clones 6.5, 6.8 and 7.2. The mRNA of Tsc2 in D2c and of Pten in clones 6.5, 6.8 and 7.2 was sequenced and compared to the 15V4 sequence. D2c has a frame shift insertion in codon 50 of Tsc2, which creates a premature translational stop in codon 51 . 6.5 has a frame shift insertion in codon 44 of Pten, which creates a premature stop codon in 51. 6.8 has a frame shift insertion in codon 56 of Pten, which creates a premature stop codon in 62. 7.2 has a frame shift insertion in codon 132 of one Pten allele, which creates a premature stop codon in 149 and a frame shift insertion in codon 320 of the other Pten allele, which creates a premature stop codon in 326. Clone D2c carries a frame shift in Tsc2 and clones 6.5, 6.8 and 7.2 carry distinct frame shifts in the Pten gene. All frame shift insertions identified in Tsc2 and Pten created premature translation termination codons, and generated truncated non-functional proteins. These data support the idea that by inhibiting PTEN or Tsc2, for example by shRNA or by other organic molecules, one can trigger forced addiction to the mTOR pathway in cells.
Transformation to rapamycin sensitive growth by RNA interference targeting PTEN or TSC2 Loss of TSC2 or PTEN function is sufficient for the transformation of 15V4 cells to IL-3 independence. TSC2 or PTEN were downregulated by shRNA following retroviral infection in 15V4 cells, and these cells tested for IL-3 independent growth. To obtain efficient knockdowns of either TSC2 or PTEN, five different shRNA sequences against each target were tested in 15V4 cells by lentiviral infection followed by puromycin selection. A correlation was observed between the increase in survival rate in medium lacking IL-3 and the knockdown efficiency of either TSC2 or PTEN, which indicates that loss of function of TSC2 or PTEN promotes IL-3 independence in 15V4 cells. The results of Figure 3 show that by introducing shRNA sequences specific for TSC2 or PTEN, 15V4 cells grew out vigorously upon removal of IL-3 after a period of 6 to 8 days whereas control shRNA expressing cells did not survive more than 2 days without IL-3. The finding that the shRNA-mediated knockdown of either negative regulator of mTOR, TSC2 or PTEN, is sufficient for the transformation of 15V4 cells to IL-3 independent growth and survival provided experimental evidence for the physiological relevance of the identified Tsc2 and Pten mutations in IL-3 independent growth of clones D2c, 6.5, 6.8 and 7.2. To verify whether IL-3 independent growth of 15V4 cells upon TSC2 or PTEN knockdown was mediated by mTOR, these IL-3 independent knockdown cells were tested for rapamycin sensitivity (Fig. 3B). As with the previous frame shift mutants, the IL-3 independent TSC2 or PTEN knockdown cells had acquired sensitivity to rapamycin. Unlike the rapamycin sensitivity in the TSC2 mutant D2c, TSC2 knockdown cells were only sensitive to rapamycin in the absence of IL-3 but not in its presence. In other words, forced addiction to mTOR was overcome, like in all clones except D2c, by IL-3. This difference in rapamycin sensitivity between the TSC2 mutant D2c and the TSC2 knockdown cells may either result from a difference in TSC2 inhibition between these cells or from additional features present only in D2c, as for example the observed activation of MAPK signaling.
Rapamycin sensitive cells require raptor or rictor for growth
TSC2 deficient 15V4 cells transform to IL-3 independence primarily via signaling from mTORCI , and PTEN deficient cells primarily transform via mT0RC2. The dependency of the different cell types on growth signals from the two mTOR complexes, mTORCI and mTORC2, was tested by knocking down either the mTORCI specific subunit raptor or the mTORC2 specific subunit rictor in the mutants D2c and 6.5, respectively. For both targets, raptor and rictor, efficient knockdown sequences were tested by Western blotting, and used to determine proliferation rates after downregulation of mTORCI or mT0RC2. IL-3 dependent precursor 15V4 cells grew almost equally well when raptor or rictor was downregulated (Figure 4, upper panel). From D2c cells, no outgrowth occurred when raptor was downregulated, in presence or absence of IL-3, indicating that expression of raptor is crucial for mTOR dependent growth of these cells (Fig. 4, lower panel). 6.5 cells, in contrast, were very sensitive to downregulation of rictor, which was growth inhibitory, but inhibition was rescued by IL-3 (Fig. 4, middle panel). These data provide evidence that transformation of TSC2 deficient D2c cells depends on mTORCI signaling while transformation of PTEN deficient 6.5 cells depends on mTORC2, suggesting that loss of TSC2 transforms cells primarily via mTORCI but loss of PTEN acts via mTORC2. Importantly, these results show a) that raptor and rictor, depending on the mutant type, are required for mTOR addicted growth, and b) are potential drug targets. As a consequence compounds directed against raptor, rictor or similar components of the mTOR signaling pathway are useful to treat cancer, alone or in combination with rapamycin, in cells addicted to the mTOR pathway.
Rapamycin sensitive cells are also sensitive to metformin, alone or in combination with rapamycin
IL-3 dependent precursor cells 15V4 are not sensitive to metformin (Fig. 5A), nor, as shown in Figure 1 , to rapamycin. Rapamycin sensitive clone 6.5 is, however, sensitive to metformin (Fig. 5D, 5E). Notably, when combined with 0.5 or 1 nM rapamycin, a synergistic effect is observed in cells growing without IL-3. However, when the growth factor is added, the inhibitory effect of both drugs is abolished. D2c cells lacking Tsc2 are sensitive to rapamycin, but not to metformin, in presence or absence of IL-3 (Fig. 5B, 5C).
Table 3: Determination of live and dead (apoptotic or necrotic) cells following growth in presence of metformin and in presence or absence of IL-3.
Figure imgf000020_0001
Outer left column indicates cell lines m: presence (+) or absence (-) of 5 mM metformin t: time (hours) after removal of interleukin-3
A: percentage (%) of living cells, B: percentage of early apoptotic cells, C: percentage of dead late apoptotic cells, D: percentage of dead necrotic cells
Shown is a representative experiment from at least four experiments performed
The data of Table 3 show that the number of dead cells (determined by the presence of annexin V and staining with propidium iodide) increases dramatically upon addition of metformin in 6.5 but not in D2c cells. These data indicate that metformin acts in synergy with rapamycin on induction of apoptosis specifically in 6.5 cells, and that these cells are a representative model to identify synergistic drugs for mTOR pathway inhibition in combination with rapamycin analogs. Metformin and similar drugs modulating glucose metabolism can be used to treat cancers growing in dependence of mTOR pathway activation, for which 6.5 cells are a model. Particularly efficient is the combination of rapamycin with metformin.
It is surprising to find that haemopoietic IL-3 dependent cells can be transformed to IL-3 independent growth by downregulation of Tsc2 or PTEN achieved by stable transfection of shRNA. Two or more shRNA sequences are active per gene, indicating that this is not an off-target effect. Interestingly, the effect requires the presence of an activated ras gene, as PB-3c clone 15 could not be transformed, in contrast to isogenic cells infected with v-H-ras (clone 15V4). Transformed cells become addicted for growth to the mTOR pathway, as treatment with rapamycin leads to rapid and powerful onset of apoptosis. In contrast, cells not expressing Tsc2 specific shRNA do not die by rapamycin exposure, but remain viable. Induction of apoptosis in infected cells, however, can be bypassed by adding IL-3. This indicates that following IL-3 receptor activation, stimulation of the Jak- Stat pathway is able to circumvent mTOR pathway addiction by activating a genetic program where proliferation is independent of mTOR activation. It is predicted that by adding to these cells an inhibitor of the Jak-Stat pathway one could abrogate this rescue effect of IL-3. Combined treatment of shRNA transformed cells with rapamycin and Jak- Stat inhibitors will kill cells even in the presence of IL-3.
Rapamycin-sensitive cells express the marker gene fructose- 1 ,6-bisphosphatase, a key regulator of gluconeogenesis
Loss-of-function mutations in precursor 15V4 cells enable IL-3 independent but rapamycin-sensitive growth via activation of the mTOR pathway, and inhibition by rapamycin can in all but mutant D2c be relieved by addition of IL-3. Importantly, fusion of mutants with parental cells restore not only the IL-3 requirement but also rapamycin sensitivity.
Microarray experiments using Affymetrix chips allows identification of signature transcripts related to IL-3 independence and rapamycin sensitivity. Transcripts were identified, which are a) overexpressed in a mutant compared to precursor 15V4, b) downregulated following cell fusion, and c) sensitive to rapamycin. Three mutants and their fusion hybrids with IL-3 dependent PB-3c clone 20 were analyzed (Fig. 6). The mutants 6.5, 6.8 and H1.2 displayed between 180 and 227 transcripts that were elevated two-fold or more when compared to precursor 15V4 cells. 12 overexpressed genes were common to all mutants (Fig. 6A). In the corresponding hybrids, between 165 and 327 genes were downregulated following cell fusion, of which 32 were common to all the mutants (Fig. 6B). To identify rapamycin-sensitive transcripts, the mutants 6.5 and H1.2 were examined. As IL-3 prevents rapamycin-induced cell death in these cells, this enables analysis of the effect of rapamycin in the protective presence of the cytokine, i.e. in the absence of ongoing apoptosis. 138 (mutant 6.5) and 175 (mutant H1.2) transcripts were down- regulated by rapamycin (two-fold change or more), with 57 transcripts downregulated in both clones (Fig. 6C). From 24'600 probe sets on the chip, about 12O00 were found to be expressed in the cells analyzed. From those, a single transcript that was upregulated in all three mutants was identified, downregulated in all three hybrids, and also sensitive to rapamycin: The transcript encoding fructose- 1 ,6-bisphosphatase-2 (FBP2) (Fig. 6D). This metabolic enzyme catalyzes the second to last step in gluconeogenesis and is a regulatory gene regulating the decision between glycolysis and gluconeogenesis. Hence, FBP2 is a marker gene of rapamycin-sensitive malignant cell transformation related to metabolism.
Overexpression observed by microarray analysis was confirmed by real-time PCR on RNA from 6.5, 6.8, the precursor line 15V4, and 7.2, an additional mutant with a frame- shift mutation in PTEN included in the analysis. All three mutants show substantial overexpression of FBP2 compared to precursor 15V4 (Fig. 7A). 6.5 cells were transfected with a plasmid carrying the PTEN structural gene or, for control, GFP. Expression of PTEN led to downregulation of FBP2 confirming that FBP2 is a marker of cell transformation. In yet another approach, using infection with shRNA-carrying lentiviruses, a sequence carrying small hairpin (sh) sequences specific for PTEN were introduced into precursor cells 15V4, or for control, sh sequences for beta-globin. The expected downregulation of PTEN mRNA was confirmed by real-time PCR (Fig. 7B). Notably, FBP2 expression was strongly amplified by the downregulation of PTEN. Hence, FBP2 is a biomarker to identify tumor cells which, when growing autonomously in the absence of growth factor, are sensitive to rapamycin or other inhibitors of the mTOR pathway. Also, FBP2 expression is under negative control by the tumor suppressor PTEN.
PTEN deficient, rapamycin sensitive cells expressing fructose 1 ,6-bisphosphatase are susceptible to killing by hyperthermia
Co-expression of fructose 1 ,6-bisphosphatase 2 (FBPase, EC 3.1 .3.1 1 ) together with the constitutively expressed glycolytic enzyme phosphofructokinase (PFK1 , EC 2.7 A A 1 ) initiates a "futile cycle", whereby the reaction catalyzed by PFK: ATP + D-fructose-6-phosphate = ADP + D-fructose-1 ,6-bisphosphate is simultaneously reversed by FBPase activity:
D-fructose-1 ,6-bisphosphate + H2O = D-fructose-6-phosphate + P1
The net effect of such a futile substrate cycle is prodigious consumption of ATP and heat generation. This effect is physiologically relevant in the liver for maintenance of core body temperature, and to maintain optimum working temperature in skeletal muscle. Transformed cells have slightly elevated temperatures due to increased metabolic rates (Warburg effect). In addition cells expressing FBPase have even higher temperatures due to the thermogenic consequence of the futile cycle. The increased cellular temperature renders transformed cells more sensitive to killing by hyperthermia (45.50C) as compared to non-transformed cells and offers an alternative therapeutic option. Experiments performed on parallel cultures of 6.5, 7.2 and VG59 (all confirmed by Q-PCR to highly express FBP2) showed a marked drop in viability after 4 h in cultures kept at 45.5^ while paired cultures at 37<O showed no reduction in cell viability (Table 4). Non-FBP2 expressing cells such as mutant D2c and the precursor 15V4 were comparatively more resistant to hyperthermic killing. In addition, re-introduction of PTEN into 6.5 reduced heat sensitivity (Table 4). Therefore mutants with elevated FBP2 expression are sensitive to thermotherapy, and FBP2 or other key regulatory enzymes involved in glucose metabolism such as hexokinase, phosphofructokinase-1 (PFK-1 ), fructose-1 ,6- bisphosphate aldolase, pyruvate kinase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1 ,6-bisphosphatase 1 (FBPase-1 ), glucose-6-phosphatase, phosphofructokinase-2 (PFK-2), or fructose-2,6-bisphosphatase 2 (FBPase-2) are a diagnostic marker predicting thermotherapy response.
Table 4: Hyperthermic killing of FB P2 expressing cells
%
37° 45.5°
FB P2 PTEN Oh 2h 4h Oh 2h 4h
6.5 +++ — 92 91 86 92 76 8
6.5GFP +++ — 90 ND 90 90 ND 7
6.5PTEN + + 98 ND 98 98 ND 56
7.2 +++ — 96 95 94 96 94 45
VG59 +++ + 96 95 93 96 75 16
15V4 - + 98 98 95 98 96 80
D2c - + 95 94 95 95 93 91
100,000 cells were cultured in the absence of IL3 at 37°C and 45.50C and viable cell counts were performed at 0, 2 and 4 hours. Cell viability was determined by propidium iodide staining followed by FACS analysis.
Outer left column shows the various cell lines tested. + present - absent +++ strongly present % viable cells
ND not determined
Rapamycin synergises with hyperthermia in killing thermo-sensitive mutant cells The FBP2 expressing mutants 6.5 and 7.2 were preincubated overnight with rapamycin in the presence of IL-3. IL-3 signaling protects rapamycin sensitive cells from entering apoptosis and the pre-incubation serves to fully abolish mTOR signaling and suppress any relevant downstream effectors. As controls, D2c and D5a (an IL-3 independent 15V4 derivative) cells were similarly treated. IL-3 was removed by washing and cultures incubated at 370C and 45.50C in the presence/absence of rapamycin. As seen in Table 5, there is minimal loss of viability at 370C even with addition of rapamycin. However under hyperthermic conditions, there is a marked drop in viability after 4 hours and this effect is potentiated by rapamycin. Non-FBP2 expressing controls, D2c and D5a, do not exhibit thermosensitivity nor any additional effect with rapamycin. Therefore FBP2 expression is a valuable indicator for rapamycin enhanced sensitivity to hyperthermic therapy. Table 5: Synergism between rapamycin and hyperthermia in killing thermo-sensitive FBP2 expressing mutant cell lines.
Figure imgf000025_0001
Cells were precultured in medium with or without 10 nM rapamycin (Rapa) overnight. Cell pellets were collected and washed with fresh medium, counted, and each culture was split into medium with or without 10 nM rapamycin. Cultures (100,000 cells) were incubated in parallel at 370C and 44.50C and aliquots taken for viability measurement after 4 hours. Cell viability was determined by propidium iodide staining followed by FACS analysis. Shown is the mean values from three experiments. Outer left column shows the various cell lines tested. + present - absent
% viable cells
Metformin snyergizes with hyperthermia in killing thermosensitive mutant cells FBP2 expressing cells (6.5, 7.2, VG59) and, for control, 15V4 and D2c cells were incubated overnight with 5 mM metformin and incubated in parallel at 370C and 44.50C. Viability of cells was determined after 4 h as described above. FBP2 expressing cells showed a substantial drop in viability when incubated at 44.50C.
Table 6: Synergism between metformin and hyperthermia in killing thermo-sensitive FBP2 expressing mutant cell lines.
< /o
37° 44.5°
FBP2 — Met — Met
6.5 +++ 95 54 24 2
7.2 +++ 97 83 60 19
D2c - 97 90 92 82
D5a - 99 88 88 48
Cells were precultured with or without 5 mM metformin (Met) overnight. Cell pellets were counted and each culture was split into medium with or without 5 mM metformin. Cultures (100,000 cells) were incubated in parallel at 370C and 44.50C and aliquots taken for viability measurement after 4 hours. Cell viability was determined by propidium iodide staining followed by FACS analysis. Shown is the mean value from three experiments. Outer left column shows the various cell lines tested + present - absent % viable cells These findings have important implications for novel strategies in cancer chemotherapy. If pre-malignant or malignant cells in a patient are forced to grow in a mTOR addicted fashion, possibly through activation of the mTOR pathway by inhibition of negative regulators, these cells will now be exquisitely sensitive to rapamycin and rapamycin-like compounds or other inhibitors of the mTOR pathway, provided that rescue pathways are also shut-off. The choice of which other inhibitors should be co-applied with the mTOR inhibitor will depend on the tumor type, in particular on the question of which pathway is active in the given tumor cell. Known oncogenic pathways include the MAPK, Wnt, Jak- Stat, PI-3K, JUNK and hedgehog. For these pathways, a number of more or less specific inhibitors are known.
Method for screening for a compound blocking the mTOR pathway
The invention further relates to a method for screening for a compound blocking the mTOR pathway. In such a method, compounds, for example compounds from a compound library, or selected single compounds for which there is already an indication of possible usefulness in tumor treatment, can be easily tested, and an indication obtained whether these compounds should be applied in the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated. The method is also suitable for high-throughput screening. Compounds are tested on particular IL-3 dependent, rapamycin-insensitive cells, and in parallel, in mutants of such cells, which are rapamycin-sensitive. Such cells are known and may be obtained, e.g. as described by K.F. Kiser et al., Oncogene 25:6595-6603 (2006), from immortalized bone-marrow-derived mast cell lines.
In particular the invention relates to a method for screening for a compound blocking the mTOR pathway, wherein
(a) a compound to be tested is added to an IL-3 dependent, rapamycin-insensitive cell and in parallel to a first rapamycin-sensitive mutant cell of said IL-3 dependent, rapamycin-insensitive cell, and compounds selected, which do not inhibit said IL-3 dependent, rapamycin-insensitive cell, but inhibiting said first rapamycin-sensitive mutant cell,
(b) compounds selected from step (a) are retested for inhibitory activity on a second IL-3 independent mutant of said IL-3 dependent, rapamycin-insensitive cell, and compounds selected, which are inhibiting said second IL-3 independent mutant cell. The method is based on the fact that IL-3 dependent 15V4 cells are insensitive to mTOR pathway blockade exerted by rapamycin, while several mutant derivatives (6.5, 6.8, 7.2, D2c, H1.2) are sensitive to rapamycin due to their addiction to the mTOR pathway. This offers the possibility to perform a dual screen of 15V4 and e.g. D2c alongside each other and screen for compounds inhibiting the latter but not the former. Toxic compounds are readily eliminated as they are equally toxic to both cells. Selected hits are tested further on the additional mTOR addicted and rapamycin sensitive clones (e.g. 6.5, 6.8, 7.2, H1.2) and selected if active, and simultaneously tested on other IL-3 independent clones without mTOR pathway addiction (D5a, 15bcr-abl) and rejected if active. This allows rapid elimination of false positives and selection of specific candidates.
Using microtiter plates, 10'0OO cells are plated per well in Iscove's Modified Dulbecco's Medium with 10% FCS, and compounds diluted in a series from 10 μM to 0.01 μM. Controls contain solely solvent (typically 0.1% DMSO). Cell proliferation is measured between days 1 -3 by XTT or Alamar blue assay and absorption read by a photometer or a fluorescence reader, respectively. The assay is suitable for high throughput screening. Hits are re-tested in triplicates.
Method to predict sensitivity of a tumor to the combination of mTOR pathway inhibition combined with application of thermal energy.
The invention further relates to a method to predict sensitivity of a tumor to a compound blocking the mTOR pathway combined with application of thermal energy.
The particular steps of such a method are based on the observations explained above in the corresponding paragraphs, wherein it is described that rapamycin-sensitive cells express the marker gene fructose-1 ,6,-bisphosphatase, and that such cells are susceptible to killing by hyperthermia. Other marker genes are also considered, in particular hexokinase, phosphofructokinase-1 (PFK-1 ), fructose-1 , 6-bisphosphate aldolase, pyruvate kinase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1 , 6-bisphosphatase 1 (FBPase-1 ), glucose-6-phosphatase, phosphofructokinase- 2 (PFK-2), fructose-2, 6-bisphosphatase 2 (FBPase-2).
The marker gene fructose-1 ,6,-bisphosphatase can easily be measured, for example by PCR procedures allowing quantitative determination of FBP2 mRNA expression, or by determination of FBP2 protein levels with standard procedures for protein detection and quantification, in particular Western blotting or ELISA or by enzymological assays using the cognate substrate fructose-1 ,6-bisphosphate. The other mentioned marker genes and the corresponding enzyme proteins may be determined in the same way using their analogous substrates in enzymological procedures.
To exclude false positives, it is advisable to determine in parallel, whether the mTOR pathway is activated in tumor cells under analysis. This may be performed, using biopsy material, by determining the phosphorylation status of the mTOR targets S6 kinase phosphorylated at Thr-389 or 4E-BP1 phosphorylated at multiple sites including Thr-69/70 (both proteins are phosphorylated by mTORCI ) or PKB/akt phosphorylated at Ser-473 (phosphorylated by mTORC2). Phosphorylation of said mTOR targets is recognized by Western blotting using suitable commercially available phospho- and protein-specific antibodies. Normal adjacent tissue serves as negative control. Another indication of mTOR pathway activation is autophosphorylation of mTOR, also determined by Western blotting.
In particular, the invention relates to a method to predict sensitivity of a tumor to a compound blocking the mTOR pathway combined with application of thermal energy, wherein (a) FBP2 expression is measured in tumor cells and in adjacent non-tumor cells by (i) determination of FBP2 mRNA expression by quantitative PCR procedure, (ii) semiquantitative determination of FBP2 protein levels by Western blotting of cell lysates,
(iii) determination of FBP2 protein levels by ELISA, and/or (iv) enzymatic assay using fructose-1 ,6-bisphosphate as substrate; and
(b) mTOR activation is measured in tumor cells by determination of the phosphorylation status of S6 kinase and/or 4E-BP1 and/or PKB/akt; and
(c) the tumor is judged sensitive to a compound blocking the mTOR pathway combined with application of thermal energy, if the level of FBP2 expression determined in step (a) is increased more than 3-fold in tumor cells in comparison to adjacent non-tumor cells and if evidence for mTOR activation is obtained in step (b).
The test is based on the fact that the expression of the gluconeogenic enzyme FBP2 triggers a futile cycle with consumption of energy. Known examples constitute tumors addicted to the mTOR pathway after mutation of PTEN. The test thus measures the expression of FBP2 by real-time PCR together with determination of mTOR activation by detection of the phosphorylation status of S6 kinase (Thr-389), or 4E-BP1 (Thr-69/70 plus additional sites), or of PKB/akt (Ser-473), optionally together with the expression of PTEN by Western blot. For control, tumor-adjacent healthy tissue is examined. Tumor cells lacking PTEN expression and/or showing elevated levels of mTOR activity with elevated FBP2 levels (e.g. 3-fold, preferably 10-fold) can be identified as being responsive to mTOR inhibition and thermotherapy alone or in combination.
Pharmaceutical compositions
The present invention relates also to pharmaceutical compositions that comprise a compound blocking or activating the mTOR pathway, optionally together with the other components mentioned herein before, as active ingredient(s) and that can be used especially in the treatment of the diseases mentioned. Compositions for enteral administration, such as nasal, buccal, rectal or, especially, oral administration, and for (the particularly preferred) parenteral administration, such as intravenous, intramuscular or subcutaneous administration, to warm-blooded animals, especially humans, are preferred.
The compositions comprise the active ingredient(s) alone or, preferably, together with a pharmaceutically acceptable carrier. The dosage of the active ingredient(s) depends upon the disease to be treated and upon the species, its age, weight, and individual condition, the individual pharmacokinetic data, and the mode of administration.
For parenteral administration, preference is given to solutions of the active ingredients, and also suspensions or dispersions, especially isotonic aqueous solutions, dispersions or suspensions which, for example in the case of lyophilized compositions comprising the active ingredient(s) alone or together with a carrier, for example mannitol, can be made up before use. The pharmaceutical compositions may be sterilized and/or may comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes. The said solutions or suspensions may comprise viscosity-increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers, e.g. Tween 80 (polyoxyethylene (20) sorbitan mono-oleate). Preferred preservatives are, for example, antioxidants, such as ascorbic acid, or microbicides, such as sorbic acid or benzoic acid. Suspensions in oil comprise as the oil component the vegetable, synthetic, or semisynthetic oils customary for injection purposes. In respect of such, special mention may be made of liquid fatty acid esters that contain as the acid component a long-chained fatty acid having from 8 to 22, especially from 12 to 22, carbon atoms. The alcohol component of these fatty acid esters has a maximum of 6 carbon atoms and is a monovalent or polyvalent, for example a mono-, di-or trivalent, alcohol, especially glycol and glycerol. As mixtures of fatty acid esters, vegetable oils such as cottonseed oil, almond oil, olive oil, castor oil, sesame oil, soybean oil and groundnut oil are especially useful.
The manufacture of injectable preparations is usually carried out under sterile conditions, as is the filling, for example, into ampoules or vials, and the sealing of the containers. Solutions such as are used, for example, for parenteral administration can also be employed as infusion solutions.
If the compound blocking the mTOR pathway and the optional other active ingredients are organic compounds of a molecular weight below 1000, pharmaceutical preparations for enteral, in particular oral use, are preferred. Such preparations for oral use are, e.g. tablets, capsules containing granules, mini-tablets or other solid, semi-solid or liquid forms, solutions to be taken as drops or as syrup, and the like. Suitable carriers for solid oral preparations are especially fillers, such as sugars, for example lactose, saccharose, mannitol or sorbitol, cellulose preparations, and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, and also binders, such as starches, for example corn, wheat, rice or potato starch, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, for example silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.
Tablet cores can be provided with suitable, optionally enteric, coatings through the use of, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropyl- methylcellulose phthalate. Dyes or pigments may be added to the tablets or tablet coatings, for example for identification purposes or to indicate different doses of active ingredient.
Pharmaceutical compositions for oral administration also include hard capsules consisting of gelatin, and also soft, sealed capsules consisting of gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules may contain the active ingredient(s) in the form of granules, for example in admixture with fillers, such as corn starch, binders, and/or glidants, such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid excipients, such as fatty oils, paraffin oil or liquid polyethylene glycols or fatty acid esters of ethylene or propylene glycol, to which stabilizers and detergents, for example of the polyoxy- ethylene sorbitan fatty acid ester type, may also be added.
Pharmaceutical compositions suitable for rectal administration are, for example, suppositories that consist of a combination of the active ingredient(s) and a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.
The pharmaceutical compositions comprise from approximately 1 % to approximately 95% of the active ingredient(s), single-dose administration forms comprising in the preferred embodiment from approximately 20% to approximately 90% active ingredient(s) and forms that are not of single-dose type comprising in the preferred embodiment from approximately 5% to approximately 20% active ingredient(s). Unit dose forms are, for example, coated and uncoated tablets, ampoules, vials, suppositories, or capsules. Examples are capsules containing from about 0.05 g to about 1.0 g active ingredient(s).
Further dosage forms are, for example, ointments, creams, pastes, foams, tinctures, lipsticks, drops, sprays, and the like.
The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by means of conventional mixing, granulating, coating, dissolving or lyophilizing processes. Method of treatment
The invention further relates to a method of treating neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, by administering a therapeutically effective amount of a compound blocking or activating the mTOR pathway in combination with rapamycin or a rapamycin-like compound, and/or in combination with metformin or a metformin-like compound, and optionally in combination with an inhibitor of the MAPK, Wnt, Jak-Stat, PI-3K, JUNK or hedgehog pathway and/or a DNA damaging agent, 2'-deoxy-glucose, or an inducer of hypoxia.
Based on the fact that cells with mutations in Pten overexpress the enzyme FBP2 and render cells sensitive to hyperthermia, the invention relates also to a method of treating neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, by administering a therapeutically effective amount of a compound blocking or activating the mTOR pathway, in particular of rapamycin, a rapamycin-like compound, metformin, or a metformin-like compound, in combination with thermotherapy.
Based on the results demonstrating that one can force a cell to grow in a mTOR addicted fashion by applying a Tsc2 or PTEN inhibitor, the following particular method of treatment, a four-phase combination strategy, for the treatment of neoplastic diseases is envisaged:
Figure imgf000032_0001
In therapy phase I, the mTOR pathway is activated, just as performed in the experimental work described herein by shRNA, by another inhibitor of Tsc2, for example a pharmaceutically acceptable chemical compound other than shRNA. As Tsc2 depends for mTOR pathway activation on complex formation with Tsc1 (which is a tumor suppressor similar to Tsc2), an inhibitor of Tsc1 will have the same effect. Other negative regulators of mTOR besides PTEN and Tsc2 (or Tsc1 ) will provide similar drug targets, and inhibitors of NF1 , GAP, or stimulators of LKB1 and AMPK will have the same effect, either by themselves or combined with a Tsc2 or Tsd inhibitor. Before the actual therapeutic phase III with triggering of apoptosis by mTOR pathway inhibitors is induced, it may be necessary to block alternative rescue pathways, e.g. by inhibitors of the MAPK, Wnt, Jak- Stat, and PI-3K-PKB pathway. This leads to cell death upon mTOR activation and application of mTOR pathway inhibitors. The combination of phases I and III can be termed "cell death via forced mTOR pathway addiction", by the proposed combination of inhibitors. It is notable that in phase I, a physiological inhibitor (PTEN or Tsc2), which is a tumor suppressor s inhibited, which seems paradoxical in cancer therapy, as conventional wisdom argues that tumor suppressors, if targeted in cancer, should be reinstalled (by genetic transfer or a compound which restores lost function). Here, based on the observation that shRNA targeting Tsc2 or PTEN renders cells rapamycin or metformin sensitive, the inhibitor (Phase I) for pathway activation is inhibited, and in phase Il compounds which target activators (oncogenes) stimulating mTOR bypassing pathways are combined. This is a strategy that has not been proposed before. The actual mTOR pathway inhibition can then be achieved by rapamycin, or its recently described analogues such as RAD001 (Novartis) or CCI-779 (Wyeth Pharmaceuticals) or AP23573 (Ariad Pharmaceuticals), or other mTOR pathway inhibitors. Phase IV aims at amplifying the apoptotic effect. It may, in some tumors, not be required, but provides an attractive synergistic option to amplify the effect. The rationale is based on the observation that cells addicted to the mTOR pathway, or cells carrying mutations in the PTEN-PKB-mTOR pathway are exquisitively sensitive to applied heat energy, or to rapamycin or metformin, or to a combination of heat and said drugs. A second rationale is that rapamycin is known to exert a synergistic effect when combined with DNA damaging agents such as etoposide. An interesting alternative way to amplify the apoptotic response is to install in the tumor cell either hypoxic conditions or low ATP levels, as mTOR induction under those conditions will be particularly damaging for cells.
Therapeutic RNA interference can be achieved by a) lentiviral infection expressing the suitable shRNA sequences; b) in vivo transfection of DNA expressing the suitable shRNA sequences; c) in vivo transfection of shRNA (small hairpin RNA). A compound blocking the mTOR pathway administered in combination with one or more of the mentioned combination partners may be administered as a fixed combination, staggered or independently of one another. The combination partners not contained in a fixed pharmaceutical preparation may be in combined packages comprising the required amounts of each combination partner. The compounds may be administered in addition to radiotherapy or surgical intervention or heat application.
Experimental part
Chemicals and Antibodies
Cell culture reagents, rapamycin and puromycin are obtained from Sigma; HRP-coupled anti-mouse and anti-rabbit secondary antibodies from Pierce; anti-PKB-pS473, anti-S6K- pT389, anti-S6K, anti-ERK1/2-pT202/Y204, anti-ERKI/2, anti-PTEN from Cell Signaling Technology; anti-PKB from Santa Cruz; anti-actin (MAB1501 ) from Chemicon; polyclonal anti-TSC1 and anti-TSC2 are generated as described [M. van Slegtenhorst et al., Hum MoI Genet. 7:1053-57 (1998)].
Cell culture and cell lines
IL-3 dependent PB-3c mast cells and its subclones are cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum, 2mM L-Glutamine, 100 U ml"1 penicillin, 100 μg ml"1 streptomycin and 50 μM β-mercaptoethanol. IL-3 dependent cells require the addition of conditioned medium (CM) from X63-mll_3 cells. Clone 20 and clone 15 are subclones of PB-3c; clone 20, but not clone 15 is able to suppress autocrine tumor formation upon cell fusion [A.P. Nair et al., Oncogene 7:1963- 1972 (1992)]. 15V4 clone is a v-H-ras expressing clone 15; 15V4wt18 is 15V4 expressing GFP; D2c, 6.5, 6.8, 7.2, 8.8 and H1 .2 are IL-3 independent clones that were isolated from 15V4 after frameshift mutagen ICR191 treatment and IL-3 removal; 20xD2c is a fusion hybrid between 20 and D2c. For frame shift mutagenesis, 15V4wt18 cells are treated with 2 μg/ml ICR191 (Sigma) for 2 h at 37<O in IL-3 containing medium, and allowed to recover for 24 h. This procedure is repeated over several rounds. IL-3-independent clones are selected by IL-3 removal and viable cells are enriched by Ficoll-Paque (Amersham Pharmacia). For IL-3 removal, the cells are washed twice with the IL-3 free medium. For cell fusion, PB-3c-20puro cells and IL-3-independent 15V4wt18 cells are mixed and fused by electroporation in serum-free IMDM at 220V and 900 μF. Hybrids are selected in IL-3 containing medium supplemented with puromycin (2 μg/ml) and hygromycin (1000 U/ml). Hybrid cells are subsequently cultured in the absence and presence of IL-3 to assess dominance or recessivity of mutations.
Plasmids AU1 -mTORwt [A. Sekulic et al., Cancer Res. 60:3504-13 (2000)] is subcloned from pcDNA3.1 with the Notl and a blunted Hindi 11 restriction sites into the Notl and a blunted BamHI restriction sites of a modified pMSCVpuro version, harbouring BamHI and Notl sites.
Viral infections
For production of retroviral particles, 4 x 106 PlatE cells [S. Morita et al., Gene Ther. 7:1063-66 (2000)] are transfected with 15 μg of retroviral pMSCVpuro plasmids (BD Biosciences Clontech, Palo Alto, CA, USA) using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturers protocol. Virus secreting platE cells are cultured in IMDM containing 10 % calf serum. At 72 h after transfection, virus- containing supernatant is harvested and filtered using a 0.45 μm pore filter. 1 x 106 15V4 cells are infected with 1 ml viral supernatant containing 10 μg/ml hexadimethrin (Polybrene, Fluka, Buchs, Switzerland). After 24 h, infected cells are selected with puromycin (2 μg/ml) for at least 36 h.
Lentiviral particles are obtained from Sigma at a titre of 1 -3 x 107 transfection units (TU) per ml. 5 x 105 15v4, 6.5 or D2c cells are infected with 5 x 105TU of lentiviral supernatant in presence of 10 μg/ml hexadimethrin. After 24 h, infected cells are selected with puromycin (2 μg/ml) for at least 36 h. For shRNA mediated knockdown, the target sequences are CAACAAGAUGAAGAGCACCAA (SEQ ID NO:8) for a non-targeting control; CCCUUAUAUCACUAAGGGUUU (SEQ ID NO:1 ) targeting murine TSC2; CGACUUAGACUUGACCUAUAU (SEQ ID NO:3) targeting murine PTEN; CGGUUCAUACAAGAGUUAUUU (SEQ ID NO:4) targeting murine Rictor and GCCCGAGUCUGUGAAUGUAAU (SEQ ID NO:6) targeting murine Raptor.
Cell lysis and Western blotting
For cell lysis, cells are washed with PBS and then lysed in ice-cold TNE buffer (50 mM Tris-HCI pH 8.0, 150 mM NaCI, 0.5 mM EDTA, 1% Triton X-100) supplemented with protease and phosphatase inhibitors (1x Roche complete protease inhibitor cocktail, 1 μg/ml Aprotinin, 1 μg/ml Pefabloc, 1 mM PMSF, 10 mM NaF, 10 mM NaN3, 10 mM
NaPPi, 10 mM beta-glycerophosphate, 10 mM p-nitrophenylphosphate). After 10 minutes of incubation, cell debris is removed by centrifugation for 15 minutes at 12000 g. The protein concentration is determined with a Bradford assay and samples are equalized. For Western blotting, SDS-PAGE sample buffer is added and the probes are heated for 5 minutes at 950C. For Western blotting, 20-40 μg of protein per lane are electrophoresed in SDS-PAGE mini gels and transferred to nitrocellulose. The manufacturer's guidelines are followed for antibody incubations. ECL Western and ECL Advance Western kits (Amersham) are used for chemiluminescence detection.
Sequencing of cDNA and genomic DNA RNA is extracted with Trizol (Invitrogen) after the manufacturer's guidelines. cDNA is prepared from the extracted RNA, using M-MLV Reverse Transcriptase (Promega) and oligo dT(15) primers following the manufacturer's guidelines. Genomic DNA is extracted with the DNeasy Blood & Tissue kit (Qiagen). The sequences of interest from cDNA and genomic DNA are amplified with gene specific primers, using HotStar Taq Plus polymerase (Qiagen) following the manufacturer's protocol, gel purified and then sequenced, using gene specific primers (Microsynth).
XTT and Alamar Blue assays
104 cells per well are plated in 150μl medium containing indicated concentrations of inhibitors in 96-well microtiter plates. For XTT absorption assays or Alamar Blue fluorescence assays, the cells are labelled either for 4 h at 37<€ with cell proliferation kit Il (XTT) (Roche Diagnostics GmbH) or for 6 h at 37<€ with Alamar Blue (Biosource) according to the manufacturers protocols. The absorption or fluorescence is measured with a safire2 (Tecan) instrument, and normalized to inhibitor-free controls. Standard deviations are calculated using the mean value of triplicates from usually three independent experiments.
FACS analysis
For the viability assessment of cells, FACS analysis is performed after staining with 5 μg/ml propidium iodide in PBS (Sigma) or Annexin V-FITC (Calbiochem) following the manufacturer's guidelines. Plotting of the different fluorescence signals versus the sidescatter signal allows gating and counting of cells.
Proliferation rates To measure proliferation rates of infected puromycin resistant 15V4, 6.5 or D2c cells, 50,000 viable cells were seeded and then counted every 24 hours. cRNA Target Synthesis and Gene Chip Hybridization
Biotin labeling of RNA was performed as described in the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, USA). Double-stranded cDNA was synthesized according to the One-Cycle cDNA Synthesis Kit (Affymetrix, Cat# 900431 ), starting from 5 μg of total RNA. The material was purified with the Sample Cleanup Module (Affymetrix, Cat# 900371 ). The purified cDNA was used for an in vitro transcription reaction by using the IVT labeling kit (Affymetrix, Cat# 900449) to synthesize cRNA in the presence of a biotin-conjugated ribonucleotide analog. Approximately 80 μg of labeled cRNA from each reaction was purified by using the Sample Cleanup Module, and average size of the cRNA molecules was assessed on RNA Nano 6000 Chips (2100 Bioanalyzer, Agilent). The cRNA targets were incubated at 940C for 35 min in the provided Fragmentation Buffer and the resulting fragments of 50-150 nucleotides were again monitored using the Bioanalyzer. All synthesis reactions were carried out using a PCR thermo-block (T 1 Thermocycler, Biometra, Gόttingen, Germany) to ensure the highest possible degree of temperature control and reproducibility. The hybridization cocktail (130 μl) containing fragmented biotin-labeled target cRNA at a final concentration of 0.05 μg/μl was transferred into Affymetrix Mouse Genome 430A 2.0 and incubated at 450C on a rotator in a hybridization oven 640 (Affymetrix) for 16 h at 60 rpm. The arrays were washed and stained on a Fluidics Station 450 (Affymetrix) by using the Hybridization Wash and Stain Kit (Affymetrix, Cat# 900720). To increase the signal strength the antibody amplification protocol was used (FS450_0002). The GeneChips were processed with an Affymetrix GeneChip® Scanner 3000 7G (Affymetrix) by using the current default settings. DAT image files of the microarrays were generated using GeneChip Operating Software (GCOS 1 .4, Affymetrix).
Real time PCR
Cytoplasmic RNA was prepared as described [N.N. Gough, Annal. Biochem. 173:93-95,
(1988)] and reverse transcribed with M-MLV reverse transcriptase (New England Biolabs). PCR was performed using the QuantiTect SYBR Green PCR kit (Qiagen) following the protocol of the supplier. Sense primer for FBP2 was δ'-ATGAATGAGCAATGGAGATGG- 3' (SEQ ID NO:9); the antisense primer δ'-CCGTATCTTTCTTTACCCCTGG-S' (SEQ ID NO:10). For control, 18S rRNA was amplified with sense primer 5'-CGGCTACCACATCC AAGGAA-3' (SEQ ID NO:1 1 ) and antisense primer δ'-GCTGGAATTACCGCGGCT-S' (SEQ ID NO:12). For reaction and analysis, ABI PRISM 7700 equipment was used. Expression levels of FBP2 mRNA were corrected for 18S rRNA. Reverse transcription: 1 μg cytoplasmic RNA using iScript (Bio-Rad); reaction conditions are according to the manufacturer. Real time PCR is performed using cDNA dilutions for FBP2 (1 :40), for PTEN (1 :40) and for 18S (1 :4000). Reaction volume is 20 μl in 96 well plates. RQ Kit : IQ-Sybergreen Supermix (Bio-Rad), ThermoCycler mylQ (Bio-Rad). Amplification: 3 min 950C initial denaturing (10 sec 950C / 20 sec 550C / 10 sec 720C), 40 cycles. Melting curves: 1 min 950C denaturing, 1 min 550C annealing, 550C to 9O0C / 0.50C, intervals 10 sec ramping. Data analysis is performed with Bio-Rad IQ5 analysis software.

Claims

Claims
1. A compound blocking or activating the mTOR pathway in combination with rapamycin or a rapamycin-like compound and/or in combination with metformin or a metformin-like compound, for use in the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated.
2. The compound according to claim 1 down-regulating Tsc2, PTEN, raptor or rictor.
3. The compound according to claim 1 down-regulating Tsc2.
4. The compound according to claim 1 down-regulating Tsc1 , NF1 , GAP, PTEN, LKB1 or AMPK.
5. The compound according to claim 1 or 2, which is an anti-sense RNA, siRNA or shRNA down-regulating Tsc2, PTEN, raptor or rictor.
6. The compound according to claim 5, which is a shRNA down-regulating Tsc2 or PTEN.
7. The compound according to claim 5, which is a shRNA down-regulating Tsc2.
8. The compound according to claim 5, which comprises an oligonucleotide sequence selected from CCCUUAUAUCACUAAGGGUUU (SEQ ID NO:1 ), CCCUUUCAUCACUAAGGGCCU (SEQ ID NO:2), CGACUUAGACUUGACCUAUAU (SEQ ID NO:3), CGGUUCAUACAAGAGUUAUUU (SEQ ID NO:4)
AGGUUCAUACAAGAAUUAUUU (SEQ ID NO:5), GCCCGAGUCUGUGAAUGUAAU (SEQ ID NO:6), and GCCUGAGUCGGUAAAUGUGCU (SEQ ID NO:7).
9. The compound according to claim 1 , which is rapamycin, in combination with metformin or a metformin-like compound.
10. Use of a compound blocking or activating the mTOR pathway for the manufacture of a medicament further comprising rapamycin or a rapamycin-like compound and/or metformin or a metformin-like compound, for the treatment of neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated.
1 1. The use according to claim 10, wherein the medicament for the treatment of other processes wherein mTOR is implicated is a medicament for the treatment of aging, diabetes and other metabolic conditions, or cardiovascular disease.
12. The use according to claim 10 to 1 1 , wherein the medicament further comprises an inhibitor of the MAPK, Wnt, Jak-Stat, PI-3K, JUNK or hedgehog pathway.
13. The use according to any of claims 10 to 12, wherein the medicament further comprises a DNA damaging agent, 2'-deoxy-glucose, or an inducer of hypoxia.
14. A pharmaceutical composition comprising a compound blocking or activating the mTOR pathway and rapamycin or a rapamycin-like compound and/or metformin or a metformin-like compound.
15. A method of treating neoplastic diseases, hyperproliferative conditions or other processes wherein mTOR is implicated, comprising administering a therapeutically effective amount of a compound blocking or activating the mTOR pathway in combination with rapamycin or a rapamycin-like compound and/or in combination with metformin or a metformin-like compound and/or in combination with thermal energy.
16. The method according to claim 15 comprising administering a therapeutically effective amount of a compound blocking or activating the mTOR pathway in combination with thermal energy.
17. The method of claim 16 consisting of the steps
(I) activation of mTOR pathway by administration of inhibitors of Tsc2 or Tsc1 or PTEN, if desired combined with or replaced by inhibitors of other negative mTOR regulators;
(II) inhibition of rescue pathway by administration of inhibitors of MAPK, Wnt, Jak-Stat, or PI-3K-PKB pathway; (III) induction of apoptosis by administration of rapamycin or a rapamycin-like compound alone or in combination with metformin; and
(IV) amplification of cell killing by administration of heat, a pyrogenic compound, a DNA damaging agent, 2'-deoxy-glucose, or an inducer of hypoxia.
18. A method for screening for a compound blocking the mTOR pathway, wherein (a) a compound to be tested is added to an IL-3 dependent, rapamycin-insensitive cell and in parallel to a first rapamycin-sensitive mutant cell of said IL-3 dependent, rapamycin-insensitive cell, and compounds selected, which are not inhibiting said IL-3 dependent, rapamycin-insensitive cell, but inhibiting said first rapamycin-sensitive mutant cell,
(b) compounds selected from step (a) are retested for inhibitory activity on a second IL-3 independent mutant of said IL-3 dependent, rapamycin-insensitive cell, and compounds selected, which are inhibiting said second IL-3 independent mutant cell.
19. A method to predict sensitivity of a tumor to a compound blocking the mTOR pathway combined with application of thermal energy, wherein
(a) expression of a marker gene selected from fructose-1 ,6-bisphosphatase 1 , hexokinase, phosphofructokinase-1 , fructose- 1 ,6-bisphosphate aldolase, pyruvate kinase, pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1 ,6-bisphosphatase 2, glucose-6-phosphatase, phosphofructokinase-2, and fructose-2,6-bisphosphatase is measured in the tumor cells and in adjacent non-tumor cells;
(b) mTOR activation is confirmed in the tumor cells; and
(c) the tumor is judged sensitive to a compound blocking the mTOR pathway combined with application of thermal energy, if the level of the marker gene expression determined in step (a) is increased more than 3-fold in tumor cells in comparison to adjacent non- tumor cells and if evidence for mTOR activation is obtained in step (b).
20. The method according to claim 19, wherein
(a) FBP2 expression is measured in tumor cells and in adjacent non-tumor cells by (i) determination of FBP2 mRNA expression by quantitative PCR procedure,
(ii) semiquantitative determination of FBP2 protein levels by Western blotting of cell lysates,
(iii) determination of FBP2 protein levels by ELISA, and/or (iv) enzymatic assay using fructose-1 ,6-bisphosphate as substrate; and (b) mTOR activation is measured in tumor cells by determination of the phosphorylation status of S6 kinase and/or 4E-BP1 and/or PKB/akt; and
(c) the tumor is judged sensitive to a compound blocking the mTOR pathway combined with application of thermal energy, if the level of FBP2 expression determined in step (a) is increased more than 3-fold in tumor cells in comparison to adjacent non-tumor cells and if evidence for mTOR activation is obtained in step (b).
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