WO2025202871A1 - Methods for identifying biomarkers of treatment response and use thereof with a cdk4 inhibitor - Google Patents

Abstract

Methods for identifying a subject eligible to receiving a therapeutic amount of a CDK4 inhibitor, predicting whether a subject is sensitive to treatment with a CDK4 inhibitor and methods of treating cancer are disclosed. The methods are based on measurement of mRNA expression, protein expression, and/or activity of cyclin-dependent kinase 6 (CDK6), cyclin E1 (CCNE1), cyclin D1 (CCND1), and/or Androgen Receptor (AR).

Description

METHODS FOR IDENTIFYING BIOMARKERS OF TREATMENT RESPONSE AND USE THEREOF WITH A CDK4 INHIBITOR

FIELD

The invention features methods, kits, and devices for identifying biomarkers of patient sensitivity to medical treatments and predicting treatment efficacy using the biomarkers.

BACKGROUND

Most cancer drugs are effective in some patients, but not in others. This can be due to genetic variation among tumors and can be observed even among tumors within the same patient. Variable patient response is particularly pronounced with respect to targeted therapeutics. Therefore, the full potential of targeted therapies cannot be realized without suitable tests for determining which patients will benefit from which drugs. According to the National Institutes of Health (NIH), the term "biomarker" is defined as "a characteristic that is objectively measured and evaluated as an indicator of normal biologic or pathogenic processes or pharmacological response to a therapeutic intervention."

Furthermore, the effective treatment of cancer depends, to a large extent, on the accuracy with which malignant tissue can be subtyped according to clinicopathological features that reflect disease aggressiveness.

The development of improved diagnostics based on the discovery of biomarkers has the potential to accelerate new drug development by identifying, in advance, those patients most likely to show a clinical response to a given drug. Such diagnostics have the potential to significantly reduce the size, length and cost of clinical trials. Technologies such as genomics, proteomics and molecular imaging currently enable rapid, sensitive and reliable detection of specific gene mutations, expression levels of particular genes, and other molecular biomarkers. Despite the availability of various technologies for molecular characterization of tumors, the clinical utilization of cancer biomarkers remains largely unrealized because relatively few cancer biomarkers have been discovered. For example, a recent review article states:

“The challenge is discovering cancer biomarkers. Although there have been clinical successes in targeting molecularly defined subsets of several tumor types - such as chronic myeloid leukemia, gastrointestinal stromal tumor, lung cancer and glioblastoma multiforme - using molecularly targeted agents, the ability to apply such successes in a broader context is severely limited by the lack of an efficient strategy to evaluate targeted agents in patients. The problem mainly lies in the inability to select patients with molecularly defined cancers for clinical trials to evaluate these exciting new drugs. The solution requires biomarkers that reliably identify those patients who are most likely to benefit from a particular agent.” (Sawyers, 2008, Nature 452:548-552, at 548.)

Comments such as the foregoing illustrate the recognition of a need for the discovery of clinically useful biomarkers and diagnostic methods based on such biomarkers.

There are three distinct types of cancer biomarkers: (1) prognostic biomarkers, (2) predictive biomarkers, and (3) pharmacodynamic (PD) biomarkers. A prognostic biomarker is used to classify a cancer, e.g., a solid tumor, according to aggressiveness, i.e., rate of growth and/or metastasis, and refractiveness to treatment. This is sometimes called distinguishing "good outcome" tumors from "poor outcome" tumors. A predictive biomarker is used to assess the probability that a particular patient will benefit from treatment with a particular drug. For example, patients with breast cancer in which the ERBB2 (HER2 or NEU) gene is amplified are likely to benefit from treatment with trastuzumab (HERCEPTIN®), whereas patients without ERBB2 gene amplification are unlikely to benefit from treatment with trastuzumab. A PD biomarker is an indication of the effect(s) of a drug on a patient while the patient is taking the drug. Accordingly, PD biomarkers often are used to guide dosage level and dosing frequency, during the early stages of clinical development of a new drug. For a discussion of cancer biomarkers, see, e.g., Sawyers, 2008, Nature 452:548-552.

Despite a large amount of pre-clinical and clinical research focused on CDK inhibitors, the mechanisms responsible for the anti-tumor activity of such inhibitors are not completely understood. As with some types of targeted therapy, some, but not all, patients benefit from PF- 07220060 therapy. Therefore, there is a need for diagnostic methods based on predictive biomarkers that can be used to identify patients with tumors that are likely (or unlikely) to respond to treatment with CDK4 selective inhibitor, PF-07220060.

SUMMARY

As will be discussed in more detail herein, the present application is based, at least in part, on the identification of biomarkers that are predictive of a subject's responsiveness to a method of treating cancer comprising administering a CDK4 selective inhibitor to a patient in need thereof. Thus, certain biomarkers described herein are useful, for example, in identifying and/or selecting a patient or a subset of patients having cancer that are more likely to benefit from treatment with a CDK4 selective inhibitor. In addition, the methods described herein are useful, for example, in selecting appropriate treatment modalities (e.g., therapy comprising a CDK4 selective inhibitor) for a patient having cancer.

According to a first aspect of the invention, there is provided a method of treating cancer in a subject that exhibits modified levels compared to the baseline mRNA expression, protein expression, and/or activity of cyclin-dependent kinase 6 (CDK6), cyclin E1 (CCNE1), cyclin D1 (CCND1), and/or Androgen Receptor (AR), comprising administering to the subject an effective amount of PF-07220060, wherein the subject is eligible for treatment.

According to a second aspect of the invention, there is provided a method of treating cancer in a subject, wherein the subject exhibits: a) an absence or decreased levels compared to the baseline mRNA expression level, and/or protein expression level, and/or activity level of any one of CDK6 and CCNE1 ; and/or b) an increased levels compared to the baseline mRNA expression level, and/or protein expression level, and/or activity level of any one of CCND1 and AR; comprising administering to the subject an effective amount of PF-07220060, wherein the subject is eligible for treatment.

According to a third aspect of the invention, there is provided a method of identifying a subject in need of treatment with PF-07220060, wherein the subject exhibits modified levels compared to the baseline mRNA expression, protein expression, and/or activity of CDK6, CCNE1 , CCND1 , and/or androgen receptor (AR), wherein the subject is eligible for treatment when: a) the level of mRNA expression, protein expression, and/or activity of CDK6 and/or CCNE1 is lower than the baseline, and/or b) the level of mRNA expression, protein expression, and/or activity of CCND1 and/or AR is higher than the baseline.

According to a fourth aspect of the invention, there is provided a method of identifying a subject eligible to receiving a therapeutic amount of PF-07220060, the method comprising: a) identifying a subject eligible for treatment with a therapeutic amount of PF- 07220060; b) evaluating and screening the subject to identify if the subject has inactivating mutations, or other genomic alteration, including deletion, of Retinoblastoma Protein 1 gene (RB1); and c) excluding from treatment with PF-07220060, any subject identified as having inactivating mutations, or other genomic alteration, including deletion, of RB1.

According to a fifth aspect of the invention, there is provided a method of predicting and/or determining the responsiveness of a subject having cancer to PF-07220060 comprising: a) providing a test sample from the subject; b) assaying a level of phosphorylation of RB1 in the test sample during treatment or after administration of PF-07220060; and c). comparing the level of phosphorylation of RB1 in step b) with the level of phosphorylation of RB1 in an untreated patient, wherein the absence of, or a significant decrease in level of phosphorylation of RB1 , in the test sample as compared to test sample of the untreated patient, indicates that the subject is responsive to the treatment with PF-07220060.

According to a sixth aspect of the invention, there is provided a method of predicting and/or determining the responsiveness of a subject having cancer to PF-07220060 comprising: a) providing a test sample from the subject; b) assaying a level of mRNA expression, protein expression and/or activity of CDK6 and CCNE1 in the test sample; and c) comparing the level of mRNA expression, protein expression and/or activity of CDK6 and CCNE1 in an untreated patient, wherein the subject is the subject is responsive to the treatment with PF-07220060 when level of mRNA expression, protein expression and/or activity of CDK6 and CCNE1 is elevated in the test sample compared to a normal control.

According to a seventh aspect of the invention, there is provided a method of predicting and/or determining the responsiveness of a subject having cancer to PF-07220060 comprising: a) providing a test sample from the subject; b) assaying a level of mRNA expression, protein expression and/or activity of CCND1 , and/or androgen receptor (AR), in the test sample; and c) comparing the level of mRNA expression, protein expression and/or activity of CCND1 , and/or AR, in an untreated patient, wherein the subject is the subject is responsive to the treatment with PF-07220060 when level of mRNA expression, protein expression and/or activity of CCND1 , and/or AR, is lower in the test sample compared to a normal control.

According to an eighth aspect of the invention, there is provided a method of predicting dependency of cancer on CDK4 and sensitivity to PF-07220060, wherein: a) a CDK4 to CDK6 (CDK4/CDK6) mRNA and/or protein expression ratio is quantified and wherein the CDK4/CDK6 ratio is a predictor (positive correlation); b) a CDK6 mRNA and/or protein is quantified and wherein CDK6 mRNA and/or protein is a predictor (inverse correlation); c) a CCND1 mRNA and/or protein is quantified and wherein CCNDImRNA and/or protein is a predictor (positive correlation); d) B1 mutations and/or RB1 deletions are identified and wherein RB1 mutations and/or RB1 deletions are predictors (inverse correlation); or e) a CCNE1 mRNA and/or protein is quantified and wherein CCNE1 mRNA and/or protein is a predictor (inverse correlation).

According to a ninth aspect of the invention, there is provided a method for adjusting the dose of PF-07220060 that is administered to a subject during a treatment regimen for cancer that comprises determining the absence of, or a significant decrease in level of phosphorylation of RB1 , and a) Increasing the dose of the PF-07220060 if there is a detectable phosphorylation of RB1 ; or b) maintaining the dose of the PF-07220060 if the level of a phosphorylation of RB1 is sufficiently decreased over time, wherein determining the level of comprises: i. providing a test sample from said subject; and ii. assaying the level of phosphorylation of RB1 in the test sample.

According to a tenth aspect of the invention, there is provided a method of diagnosing a subject having cancer that is responsiveness to PF-07220060, comprising contacting the subject with an agent that detects the presence of CDK6 and/or CCNE1 in the cancer or a sample thereof, wherein modified levels compared to the baseline mRNA expression, protein expression and/or activity of CDK6, CCNE1 , CCND1 , Androgen Receptor (AR) and mutations of Retinoblastoma Protein 1 (RB1), is diagnostic of cancer that is responsiveness to PF-07220060.

According to an eleventh aspect of the invention, there is provided a kit comprising a) an agent for detecting the level of CDK6 and/or CCNE1 ; and b) instructions for using the agent for diagnosing or detecting level of CDK6 and/or CCNE1 , for identifying whether a subject is responsiveness to PF-07220060 for determining the progression of cancer, for assessing the efficacy of a treatment for the cancer, and/or for adjusting the dose of PF-07220060, during the treatment of cancer.

According to a twelfth aspect of the invention, there is provided a diagnostic system comprising: a) an assortment, collection, or compilation of test results data representing the level of CDK6 and/or CCNEIin a plurality of test samples; b) a means for computing an index value using said level, wherein the index value comprises a diagnostic, prognostic, progression, or treatment score; and c) a means for reporting the index value. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a-1b show the predictive biomarkers from human cancer lines in vitro. FIG. 1a, shows significant predictors of CDK4 dependency from internal analysis of Broad’s DepMap/Achilles CRISPR cell line data. FIG. 1b, shows significant predictors of PF-07220060 sensitivity from internal analysis of in-house PF-07220060 breadth of efficacy (BOE) data.

FIGS. 2a-2d show increased expression of CDK6 and CCNE1 negates growth inhibition by PF-07220060 in HR+ HER2- ZR751 multicellular tumor spheroids. FIG. 2a shows growth curves of ZR751 dCas9 VPR control: CDK6 overexpressing breast cancer spheroids during treatment with either DMSO (vehicle) or 300 nM of PF-07220060. FIG. 2b shows growth curves of ZR751 dCas9 VPR control: CCNE1 overexpressing breast cancer spheroids during treatment with either DMSO (vehicle) or 300 nM of PF-07220060. FIG. 2c shows CDK6 protein expression by WB analysis of spheroids. FIG. 2d shows CCNE1 protein expression by WB analysis of spheroids.

FIGS. 3a-3f show increased expression of CDK6 and CCNE1 negates growth inhibition by PF-07220060 in HR+ HER2- T47D multicellular tumor spheroids. FIG. 3a shows growth curves of T47D dCas9 VPR control: CDK6 overexpressing breast cancer spheroids during treatment with either DMSO (vehicle), 300 or 1000 nM of PF-07220060. FIG. 3b shows growth curves of T47D dCas9 VPR control: CCNE1 overexpressing breast cancer spheroids during treatment with either DMSO (vehicle), 300 or 1000 nM of PF-07220060. FIG. 3c shows CDK6 protein expression by WB analysis of spheroids. FIG. 3d shows CCNE1 protein expression by WB analysis of spheroids. FIG. 3e shows quantification of the CDK6 WB protein signals from FIG. 3c. FIG. 3f shows quantification of the CCNE1 WB protein signals from FIG. 3d.

FIGS. 4a-4b show CDK6 expression determines PF-07220060 efficacy in selected HR+ HER2- breast cancer spheroid models. FIG. 4a shows ectopic expression of CDK6 in the HR+ HER2- ZR751 breast cancer spheroid model results in resistance to PF-07220060 induced spheroid growth inhibition. FIG. 4a shows shRNA induced CDK6 depletion in the HR+ HER2- T47D breast cancer spheroid model.

FIGS. 5a-5c show an inverse association between PF-07220060 induced tumor growth inhibition and CDK6 protein level across a panel of xenograft models. FIG. 5a shows linear regression analysis of percentage of tumor growth inhibition as a function of CDK6 protein molecule per cell. PF-07220060 dosing was 60 mg/kg BID. FIG. 5b shows bar graph comparing percentage of tumor growth inhibition obtained from palbociclib and PF-07220060. FIG. 5c shows results of quantification of CDK6 protein molecules per cell in a panel of cancer cell line models by semi-quantitative WB analysis. DETAILED DESCRIPTION

This present invention relates to biomarkers which may be used to evaluate the likelihood that a CDK4 inhibitor would produce an anti-cancer effect in a subject. As such, these biomarkers may be used in methods of treating cancer patients. In particular, methods for monitoring whether a subject will be responsive (e.g., sensitive or resistant) to treatment with a CDK4 inhibitor, PF- 07220060, therapy are disclosed.

The present invention may be understood more readily by reference to the following detailed description of the embodiments and preferred embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. It is further to be understood that unless specifically defined herein, the terminology used herein is to be given its traditional meaning as known in the relevant art.

E1 A method of treating cancer in a subject, as defined above.

E2 The method of embodiment E1 , further comprising administering to the subject an additional therapeutic agent, wherein the additional therapeutic agent comprises an endocrine therapy agent, a second CDK inhibitor, a PI3 kinase inhibitor, or a KAT6 inhibitor.

E3 The method of any one of embodiments E1 or E2, further comprising administering to the subject an additional therapeutic agent, wherein the additional therapeutic agent comprises an endocrine therapy agent.

E4 The method of any one of embodiments E1 to E3, wherein the endocrine therapy agent is an aromatase inhibitor, an androgen receptor inhibitor, a selective estrogen receptor degrader (SERD), or a selective estrogen receptor modulator (SERM).

E5 The method of any one of embodiments E1 to E4, wherein the endocrine therapy agent is selected from the group consisting of letrozole, anastrozole, exemestane, fulvestrant, elacestrant, amcenestrant, giredestrant, RG6171, camizestrant, AZD9496, rintodestrant, ZN-c5, LSZ102, D-0502, LY3484356, SHR9549, tamoxifen, raloxifene, toremifene, lasofoxifene, bazedoxifene and afimoxifene.

E6 The method of any one of embodiments E1 to E5, wherein the endocrine therapy agent is letrozole or fulvestrant. E7 The method of any one of embodiments E1 to E6, further comprising administering to the subject one or more of chemotherapy, immunotherapy, or radiation therapy, or combinations thereof.

E8 The method of any one of embodiments E1 to E7, wherein the cancer is head, neck, lung, prostate, colon, pancreas, esophagus, liver, skin, kidney, adrenal gland, brain, or comprises a lymphoma, breast, endometrium, uterus, ovary, testes, lung, prostate, colon, pancreas, esophagus, liver, skin, kidney, adrenal gland, brain, Pan-cancer, KRAS-NSCLC, ewing sarcoma, or mantle cell lymphoma.

E9 The method of any one of embodiments E1 to E8, wherein the cancer comprises a metastasis or recurring tumor, or neoplasia.

E10 The method of any one of embodiments E1 to E9, wherein the cancer comprises breast cancer or prostate cancer.

E11 The method of any one of embodiments E1 to E10, wherein the cancer is breast cancer.

E12 The method of any one of embodiments E1 to E11 , wherein the breast cancer is HR+ HER2- breast cancer.

E13 A method of predicting and/or determining the responsiveness to PF-07220060 of a subject having cancer, as defined above.

E14 The method of embodiment E13, wherein the test sample is selected from the group consisting of ex vivo and in vivo samples.

E15 The method of any one of embodiments E13 to E14, wherein the test sample comprises a bodily fluid from said subject.

E16 The method of any one of embodiments E13 to E15, wherein the bodily fluid comprises whole blood, a component of whole blood, plasma, or serum.

E17 The method of any one of any one of embodiments E13 to E16, wherein the test sample comprises a tissue biopsy. Each of the embodiments of the present invention described herein may be combined with one or more other embodiments of the present invention described herein which is not inconsistent with the embodiment(s) with which it is combined. In addition, each of the embodiments below describing the invention envisions within its scope the pharmaceutically acceptable salts of the compound of the invention.

List of abbreviations mRNAseq mRNA Sequencing WB Western Blot WES Whole Exome Sequencing RPPA Reverse Phase Protein Array CCND1 Cyclin D1 CCNE1 Cyclin E1 CDK4 Cyclin Dependent Kinase 4 CDK6 Cyclin Dependent Kinase 6 RB1 Retinoblastoma Protein 1 AR Androgen Receptor DRC Dose Response Curve AUC Area Under the Curve EC50 Effective Concentration 50% BOE Breadth of Efficacy NSCLC Non-Small Cell Lung Cancer KS Kolmogorov-Smirnov

Definitions

As used herein, the term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

A "mutant," or "mutation" is any change in DNA or protein sequence that deviates from wild type sequence of the target, e.g., a target disclosed herein. This includes without limitation; single base nucleic acid changes or single amino acid changes, insertions, deletions and truncations of the wild type target gene (including all of its splice forms (i.e. , transcript variants)) and its corresponding protein.

The term "antibody" or "antibody to a target" and the like as used herein refers to whole antibodies that interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) a target epitope and inhibit signal transduction.

All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied (+) or (-) by increments of 0.1 . It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about." It also is to be understood, although not always explicitly stated, that the reagents described herein are merely examples and that equivalents of such are known in the art.

The term "combination" refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present invention and a combination partner (e.g., another drug as explained below, also referred to as "therapeutic agent" or "co-agent") may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic effect. The single components may be packaged in a kit or separately.

The term "pharmaceutical combination" as used herein refers to either a fixed combination in one dosage unit form, or non-fixed combination or a kit of parts for the combined administration where two or more therapeutic agents may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g., synergistic effect.

The term "combination therapy" refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration encompasses co-administration in multiple, or in separate containers (e.g., tablets, capsules, powders, and liquids) for each active ingredient. Powders and/or liquids may be reconstituted or diluted to a desired dose prior to administration. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.

In certain instances, compounds of the present invention are combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, anti-nausea agents (or anti-emetics), pain relievers, cytoprotective agents, and combinations thereof.

General chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5- deoxy- 5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNll®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (actinomycin D, Cosmegan®), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone (Maxidex®), docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin(R), Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5- fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), temozolomide (Temodar®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®). Anti-cancer agents of particular interest for combinations with the compounds of the present invention include fluorouracil (5-Fll), irinotecan and temozolomide.

A compound of the present invention may also be used to advantage in combination with known therapeutic processes, for example, the administration of hormones or especially radiation. A compound of the present invention may in particular be used as a radiosensitizer, especially for the treatment of tumors which exhibit poor sensitivity to radiotherapy.

One or both of the components (e.g., tablets, capsules, powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination" as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g., a compound of the present invention and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, e.g., a compound of the present invention and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

In combination therapy, the compound of the present invention and other anti-cancer agent(s) may be administered either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient.

In one embodiment, the compound of the present invention and the other anticancer agent(s) is generally administered sequentially in any order by infusion or orally. The dosing regimen may vary depending upon the stage of the disease, physical fitness of the patient, safety profiles of the individual drugs, and tolerance of the individual drugs, as well as other criteria well- known to the attending physician and medical practitioner(s) administering the combination. The compound of the present invention and other anti-cancer agent(s) may be administered within minutes of each other, hours, days, or even weeks apart depending upon the particular cycle being used for treatment. In addition, the cycle could include administration of one drug more often than the other during the treatment cycle and at different doses per administration of the drug.

A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

"Gene expression" or alternatively a "gene product" refers to the nucleic acids or amino acids (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

As used herein, "expression" refers to the process by which DNA is transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

"Differentially expressed" as applied to a gene, refers to the differential production of the mRNA transcribed and/or translated from the gene or the protein product encoded by the gene. A differentially expressed gene may be overexpressed or under-expressed as compared to the expression level of a normal or control cell. However, as used herein, overexpression is an increase in gene expression and generally is at least 1.25 fold or, alternatively, at least 1.5 fold or, alternatively, at least 2 fold, or alternatively, at least 3 fold or alternatively, at least 4 fold expression over that detected in a normal or control counterpart cell or tissue. As used herein, under-expression, is a reduction of gene expression and generally is at least 1.25 fold, or alternatively, at least 1.5 fold, or alternatively, at least 2 fold or alternatively, at least 3 fold or alternatively, at least 4 fold expression under that detected in a normal or control counterpart cell or tissue. The term "differentially expressed" also refers to where expression in a cancer cell or cancerous tissue is detected but expression in a control cell or normal tissue (e.g., noncancerous cell or tissue) is undetectable.

A high expression level of the gene can occur because of over expression of the gene or an increase in gene copy number. The gene can also be translated into increased protein levels because of deregulation or absence of a negative regulator. Lastly, high expression of the gene can occur due to increased stabilization or reduced degradation of the protein, resulting in accumulation of the protein.

A "gene expression profile" or "gene signature" refers to a pattern of expression of at least one biomarker that recurs in multiple samples and reflects a property shared by those samples, such as mutation, response to a particular treatment, or activation of a particular biological process or pathway in the cells. A gene expression profile differentiates between samples that share that common property and those that do not with better accuracy than would likely be achieved by assigning the samples to the two groups at random. A gene expression profile may be used to predict whether samples of unknown status share that common property or not. Some variation between the biomarker(s) and the typical profile is to be expected, but the overall similarity of biomarker(s) to the typical profile is such that it is statistically unlikely that the similarity would be observed by chance in samples not sharing the common property that the biomarker(s) reflects.

By “CRISPR” or "CRISPR to a target" or " CRISPR to inhibit a target" and the like is meant a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. By "Cas" is meant a CRISPR -associated protein. By " CRISPR /Cas" system is meant a system derived from CRISPR and Cas which can be used to silence, enhance or mutate the target gene, e.g., a target gene of any of the targets disclosed herein.

Naturally-occurring CRISPR /Cas systems are found in approximately 40 percent of sequenced eubacteria genomes and 90 percent of sequenced archaea. Grissa et al. 2007. BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. 2007. Science 315: 1709-1712; Marragini et al. 2008 Science 322: 1843-1845.

The CRISPR /Cas system has been modified for use in gene editing (silencing, enhancing or changing specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. 2012. Nature 482: 331-8. This is accomplished by introducing into the eukaryotic cell a plasmid containing a specifically designed CRISPR and one or more appropriate Cas.

The CRISPR sequence, sometimes called a CRISPR locus, comprises alternating repeats and spacers. In a naturally-occurring CRISPR, the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence; in the target CRISPR /Cas system, the spacers are derived from the target gene sequence. The repeats generally show some dyad symmetry, implying the formation of a secondary structure such as a hairpin, but they are not truly palindromic.

RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. These comprise a spacer flanked by a repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. 2010. Science 327: 167-170; Makarova et al. 2006 Biology Direct 1 : 7. The spacers thus serve as templates for RNA molecules, analogously to siRNAs. Pennisi 2013. Science 341 : 833-836.

As these naturally occur in many different types of bacteria, the exact arrangements of the CRISPR and structure, function and number of Cas genes and their product differ somewhat from species to species. Haft et al. 2005 PLoS Comput. Biol. 1 : e60; Kunin et al. 2007. Genome Biol. 8: R61 ; Mojica et al. 2005. J. Mol. Evol. 60: 174-182; Bolotin et al. 2005. Microbiol. 151 : 2551-2561 ; Pourcel et al. 2005. Microbiol. 151 : 653-663; and Stern et al. 2010. Trends. Genet. 28: 335-340. For example, the Cse (Cas subtype, E. coli) proteins (e.g., CasA) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer- repeat units that Cascade retains. Brouns et al. 2008. Science 321 : 960-964. In other prokaryotes, Cas6 processes the CRISPR transcript. The CRISPR -based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module) proteins in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. A simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cutting sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing. Pennisi 2013. Science 341 : 833-836.

The CRISPR /Cas system can thus be used to edit a target gene, e.g., a target gene disclosed herein (adding or deleting a basepair), e.g., repairing a damaged target gene, or introducing a premature stop which thus decreases expression of an over-expressed target. The CRISPR /Cas system can alternatively be used like RNA interference, turning off the target gene in a reversible fashion. In a mammalian cell, for example, the RNA can guide the Cas protein to the target promoter, sterically blocking RNA polymerases.

Artificial CRISPR /Cas systems can be generated which inhibit a target, e.g., a target disclosed herein, using technology known in the art, e.g., that described in U.S. Patent App. No. 13/842,859. The present disclosure thus provides a CRISPR /Cas system suitable for editing a target, e.g., any of the targets disclosed herein, for use in the treatment of cancer, such as a cancer disclosed herein. Also provided is a use of a CRISPR /Cas system suitable for editing a target gene, e.g., any of the target genes disclosed herein for the manufacture of a medicament for treating cancer, such as a cancer disclosed herein.

In another embodiment, the present invention provides a method of treating cancer associated with microsatellite instability, gene amplifications, duplications, deletions or mutations, such as a cancer disclosed herein, by administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a CRISPR /Cas system suitable for editing a target, e.g., any of the targets disclosed herein.

In another embodiment, a CRISPR /Cas system suitable for editing a target, e.g., any of the targets disclosed herein, for use in the treatment of cancer is provided. The cancer may be a cancer disclosed herein.

An inhibitory CRISPR system can include a guide RNA (gRNA) comprising a targeting domain, i.e. , a nucleotide sequence that is complementary to a target DNA strand, and a second domain that interacts with an RNA-directed nuclease, e.g., cpfl or Cas molecule, e.g., Cas9 molecule.

As used herein, "macrophage marker protein" means a macrophage cell surface protein, the detection of which is useful for identifying macrophages among the other types of cells present in a tissue sample from a tumor. Exemplary human macrophage marker proteins are CCR2, CD14, CD68, CD163, CSFIR and MSRI. Other macrophage marker proteins can be employed in practicing the present disclosure.

As used herein, "receiver operating characteristic" (ROC) curve means a plot of false positive rate (sensitivity) versus true positive rate (specificity) for a binary classifier system. In construction of an ROC curve, the following definitions apply:

False negative rate "FNR" = 1 - TPR

True positive rate "TPR" = true positive I (true positive + false negative)

False positive rate "FPR" = false positive I (false positive + true negative)

As used herein, "response" or "responding" to treatment means, with regard to a treated tumor, that the tumor displays: (a) slowing of growth, (b) cessation of growth, or (c) regression.

As used herein, "threshold determination analysis" means analysis of a dataset representing a given tumor type, e.g., human renal cell carcinoma, to determine a threshold score for that particular tumor type. The dataset representing a given tumor type can include, for each tumor from a group of such tumors: (a) actual tumor response data (response and non-response to a treatment such as PF-07220060), and (b) macrophage content and/or CD68 expression levels.

As used herein, "threshold score" means a score above which a tumor is classified as being likely sensitive to treatment with a CDK4 inhibitor, such as with PF-07220060.

As used herein, “cyclin-dependent kinase 6 protein” means the human protein encoded by the gene identified by NCBI Symbol ‘CDK6’ and NCBI Gene ID ‘1021’, and allelic variants thereof.

As used herein, "cyclin E1 protein” means the human protein encoded by the gene identified by NCBI Symbol ‘CCNET and NCBI Gene ID ‘898’, and allelic variants thereof.

As used herein, "cyclin D1 protein” means the human protein encoded by the gene identified by NCBI Symbol ‘CCNDT and NCBI Gene ID ‘595’, and allelic variants thereof.

As used herein, "Androgen Receptor protein" means the human protein encoded by the gene identified by NCBI Symbol ‘AR’ and NCBI Gene ID ‘367’, and allelic variants thereof.

As used herein, the term "inhibit", "inhibiting", or "inhibit the growth" or "inhibiting the proliferation" of a cancer cell refers to slowing, interrupting, arresting or stopping the growth of the cancer cell, and does not necessarily indicate a total elimination of the cancer cell growth. The terms "inhibit" and "inhibiting", or the like, denote quantitative differences between two states, refer to at least statistically significant differences between the two states. For example, "an amount effective to inhibit growth of cancer cells" means that the rate of growth of the cells will be at least statistically significantly different from the untreated cells. Such terms are applied herein to, for example, rates of cell proliferation.

As used herein, the term "inhibitor" refers to any compound capable of inhibiting the expression or activity of a target, e.g., a target disclosed herein, that is to say, in particular, any compound inhibiting the transcription of the gene, the maturation of RNA, the translation of mRNA, the posttranslational modification of the protein, the enzymatic activity of the protein, the interaction of same with a substrate, etc. The term also refers to any agent that inhibits or abrogates the normal cellular function of the target protein, either by ATP-competitive inhibition of the active site, allosteric modulation of the protein structure, disruption of protein- protein interactions, or by inhibiting the transcription, translation, or stability of the protein.

As used herein, the terms "neoplastic cells," "neoplastic disease," "neoplasia," "tumor," "tumor cells," "cancer," and "cancer cells," (used interchangeably) refer to cells which exhibit relatively autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation (i.e., de-regulated cell division). Neoplastic cells can be malignant or benign. A "metastatic cell or tissue" means that the cell can invade and destroy neighboring body structures.

A "polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers" or a "set of primers" consisting of an "upstream" and a "downstream" primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermaly stable polymerase enzyme. Methods for PCR are well known in the art, and taught, for example in PCR: A Practical Approach, M. MacPherson et al., IRL Press at Oxford.

A cell is "sensitive," displays "sensitivity" for inhibition, or is "amenable to treatment" with an inhibitor, e.g., an inhibitor to any of the targets disclosed herein, when the cell viability is reduced and/or the rate of cell proliferation is reduced upon treatment with an inhibitor, e.g., an inhibitor to any of the targets disclosed herein, when compared to an untreated control.

The phrases "therapeutically effective amount," "effective amount" or “sufficient amount” mean an amount of a compound or composition described herein that, when administered to a patient in need of such treatment, is sufficient to (i) treat the particular disease, condition, or disorder, (ii) attenuate, ameliorate, or eliminate one or more symptoms of the particular disease, condition, or disorder, or (iii) delay the onset of one or more symptoms of the particular disease, condition, or disorder described herein. The amount of a compound that will correspond to such an amount will vary depending upon factors such as the particular compound or composition, disease condition and its severity, and the identity (e.g., weight) of the mammal in need of treatment, but can nevertheless be routinely determined by one skilled in the art.

As used herein, the term “reducing tumor growth” refers to an inhibition or a reduction in tumor growth or metastasis of a cancer as compared to its growth prior to treatment. The reduction of tumor growth may be a reduction of about 5% or greater (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater), and can be measured by any suitable means known in the art.

By a “reference” is meant any useful reference used to compare protein or mRNA levels or activity. The reference can be any sample, standard, standard curve, or level that is used for comparison purposes. The reference can be a normal reference sample or a reference standard or level. A “reference sample” can be, for example, a control, e.g., a predetermined negative control value, such as a “normal control” or a prior sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., a cell or tissue) from a subject not having a disease; a sample from a subject that is diagnosed with a disease, but not yet treated with a therapeutic agent described herein; a sample from a subject that has been treated by a therapeutic agent described herein; or a sample of a purified protein (e.g., any described herein) at a known normal concentration. By “reference standard or level” is meant a value or number derived from a reference sample. A “normal control value” is a predetermined value indicative of non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range (“between X and Y”), a high threshold (“no higher than X”), or a low threshold (“no lower than X”). A subject having a measured value within the normal control value for a particular biomarker is typically referred to as “within normal limits” for that biomarker. A normal reference standard or level can be a value or number derived from a normal subject not having a disease or disorder (e.g., cancer); or a subject that has been treated with a therapeutic agent described herein. In preferred embodiments, the reference sample, standard, or level is matched to the sample subject sample by at least one of the following criteria: age, weight, sex, disease stage, and overall health. A standard curve of levels of a purified protein, e.g., as described herein, within the normal reference range can also be used as a reference.

As used interchangeably herein, the terms “subject,” “patient,” and “individual” refer to any organism to which a therapeutic agent in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans). A subject may seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition.

The terms "treat," “treated, “treating” or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

As used herein, the term “administration” refers to the administration of a composition (e.g., a compound or a preparation that includes a therapeutic agent as described herein, e.g., a CDK4 inhibitor) to a subject or system. Administration to an animal subject (e.g., to a human) may be by any appropriate route. For example, in some embodiments, administration may be systemic (including intravenous), intratumoral, bronchial, buccal, enteral, interdermal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal, transdermal, vaginal, or vitreal.

The term “cancer” refers to a condition caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas.

As described further herein, a cancer cell, a cancer type, or a subject afflicted with a cancer, is "inhibitor sensitive," "sensitive to treatment with an inhibitor," "sensitive to therapeutic inhibition," or described in similar terms if it is amenable to treatment with an inhibitor for the target, e.g., due to a genetic alteration, or expression or activity level of the target or a target- associated molecule.

A "control cell," "normal cell" or "wild-type" refers to non-cancerous tissue or cells.

A "control tissue," "normal tissue" or "wild-type" refers to non-cancerous tissue or cells. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

By “determining the level of a protein” is meant the detection of a protein, or an mRNA encoding the protein, by methods known in the art either directly or indirectly. “Directly determining” means performing a process (e.g., performing an assay or test on a sample or “analyzing a sample” as that term is defined herein) to obtain the physical entity or value. “Indirectly determining” refers to receiving the physical entity or value from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Methods to measure protein level generally include, but are not limited to, western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property of a protein including, but not limited to, enzymatic activity or interaction with other protein partners. Methods to measure mRNA levels are known in the art.

By “level” is meant a level or activity of a protein, or mRNA encoding the protein, as compared to a reference. The reference can be any useful reference, as defined herein. By a “modified level” or a “decreased level” or an “increased level” of a protein is meant a decrease or increase in protein level, as compared to a reference (e.g., a decrease or an increase by about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or more; a decrease or an increase of more than about 1%, about 5%, about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%, as compared to a reference; a decrease or an increase by less than about 0.01-fold, about 0.02- fold, about O.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or less; or an increase by more than about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold, or more). A level of a protein may be expressed in mass/vol (e.g., g/dL, mg/mL, pg/mL, or ng/mL) or percentage relative to total protein or mRNA in a sample.

In practicing any of the subject methods, the overexpression may be assessed by: (a) detecting a level of mRNA; (b) detecting a level of cDNA produced from reverse transcription of mRNA; (c) detecting a level of polypeptide; (d) detecting a level of cell-free DNA; or (e) a nucleic acid amplification assay, a hybridization assay, sequencing, or a combination thereof. The overexpression may be characterized by an expression level of CCND1 in the cancer that is higher than a reference expression level of CCND1.

As used herein, “endocrine therapy” or “hormone therapy” means an aromatase inhibitor, a selective estrogen receptor degrader (SERD), or a selective estrogen receptor modulator (SERM). In certain embodiments, endocrine therapy includes fulvestrant, tamoxifen, toremifene, anastrozole, exemestane, or letrozole.

The term “antiestrogen” as used herein refers to a class of drugs that prevent estrogens like estradiol from mediating the biological effects in the body. Antiestrogens act by blocking the estrogen receptor (ER) and/or inhibiting or suppressing estrogen production. In an embodiment, an antiestrogen is an aromatase inhibitor, a selective estrogen receptor degrader (SERD) or a selective estrogen receptor modulator (SERM). Examples of an aromatase inhibitor include, but are not limited to, anastrozole. Examples of a SERD include, but are not limited to, fulvestrant. Additional SERDs include elacestrant (RAD-1901 , Radius Health), SAR439859 (Sanofi), RG6171 (Roche), AZD9833 (AstraZeneca), AZD9496 (AstraZeneca), rintodestrant (G1 Therapeutics), ZN-c5 (Zentalis), LSZ102 (Novartis), D-0502 (Inventisbio), LY3484356 (Lilly), and SHR9549 (Jiansu Hengrui Medicine). Examples of a SERM include, but are not limited to, tamoxifen, clomifene and raloxifene. Additional SERMS include toremifene, lasofoxifene, bazedoxifene and afimoxifene.

In an embodiment, the aromatase inhibitor includes letrozole, exemestane, and anastrozole. In an embodiment, the SERM includes tamoxifen, clomifene and raloxifene.

In an embodiment, an antiestrogen of the present invention includes fulvestrant and letrozole. In an embodiment, an antiestrogen of the present invention includes fulvestrant. In an embodiment, an antiestrogen of the present invention includes letrozole.

CDK inhibitors

Cyclin-dependent kinases (CDKs) and related serine/threonine protein kinases are important cellular enzymes that perform essential functions in regulating cell division and proliferation. CDKs 1-4, 6, 10, and 11 have been reported to play a direct role in cell cycle progression, while CDKs 3, 5 and 7-9 may play an indirect role (e.g., through activation of other CDKs, regulation of transcription or neuronal functions). The CDK catalytic units are activated by binding to regulatory subunits, known as cyclins, followed by phosphorylation. Cyclins can be divided into four general classes (Gi, Gi/S, S and M cyclins) whose expression levels vary at different points in the cell cycle. Cyclin B/CDK1 , cyclin A/CDK2, cyclin E/CDK2, cyclin D/CDK4, cyclin D/CDK6, and likely other heterodynes are important regulators of cell cycle progression.

CDK inhibitors have been demonstrated to be useful in treating cancer. Increased activity or temporally abnormal activation of cyclin-dependent kinases has been shown to result in the development of human tumors, and human tumor development is commonly associated with alterations in either the CDK proteins themselves or their regulators (Cordon-Cardo C. Mutations of cell cycle regulators: biological and clinical implications for human neoplasia. Am. J. Pathol. (1995) 147:545-560; Karp J E, Broder S. Molecular foundations of cancer: new targets for intervention. Nat. Med. (1995) 1 :309-320; Hall M, Peters G. Genetic alterations of cyclins, cyclin-dependent kinases, and Cdk inhibitors in human cancer. Adv. Cancer Res. (1996) 68:67- 108).

In an embodiment, CDK4 selective inhibitors of the present invention include “PF- 07220060” which refers to 1 ,5-anhydro-3-({5-chloro-4-[4-fluoro-2-(2-hydroxypropan-2-yl)-1- (propan-2-yl)-1/7-benzimidazol-6-yl]pyrimidin-2-yl}amino)-2,3-dideoxy-D-f/7reo-pentitol, which has the following chemical structure, including hydrates, salts and polymorphs thereof:

PF-07220060 and pharmaceutically acceptable salts thereof, are disclosed in International Publication No. WO 2019/207463, U.S. Patent Nos. 10,766,884 and 11 ,220,494, and US Patent Publication US 2022/0089580; and International Publication No. WO 2022/058871 , the contents of which are incorporated herein by reference in their entirety. Unless indicated otherwise, all references herein to PF-07220060 include references to salts, solvates, hydrates, and complexes thereof, and to solvates, hydrates and complexes of salts thereof, including polymorphs, stereoisomers, and isotopically labelled versions thereof. Diseases and Disorders associated with CDK4

In some embodiments, the disease or disorder associated with CDK4 is a cancerous tumor comprising an aberration that activates the CDK4 kinase activity. This includes, but is not limited to, cancers that are characterized by amplification or overexpression of CCNE1 such as ovarian cancer, uterine carcinosarcoma and breast cancer and p27 inactivation such as breast cancer and melanomas.

In some embodiments, the disease or disorder associated with CDK4 is a cancer with an absence or decreased CCNE1 gene expression, wildtype RB1 gene, increased CCND1 expression or increased CCND1 nuclear localization, increased activity of nuclear hormone receptors and increased CDK4 activity, including CDK4 overexpression. (Choi and Anders, Oncogene, 2014).

In some embodiments, the disease or disorder associated with CDK4 is lung squamous cell carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, breast invasive carcinoma, uterine carcinosarcoma, ovarian serous cystadenocarcinoma, stomach adenocarcinoma, esophageal carcinoma, bladder urothelial carcinoma, mesothelioma, or sarcoma.

In some embodiments, the disease or disorder associated with CDK4 is lung adenocarcinoma, breast invasive carcinoma, uterine carcinosarcoma, ovarian serous cystadenocarcinoma, or stomach adenocarcinoma.

In some embodiments, the disease or disorder associated with CDK4 is an adenocarcinoma, carcinoma, or cystadenocarcinoma.

In some embodiments, the disease or disorder associated with CDK4 is uterine cancer, ovarian cancer, stomach cancer, esophageal cancer, lung cancer, bladder cancer, pancreatic cancer, or breast cancer.

In some embodiments, the disease or disorder associated with CDK4 is a cancer.

In some embodiments, the cancer is characterized by absence or decreased mRNA or protein expression of CCNE1. In some embodiments, the cancer is ovarian cancer or breast cancer, characterized by absence or decreased mRNA or protein expression of CCNE1.

In some embodiments, the breast cancer is chemotherapy or radiotherapy resistant breast cancer, endocrine resistant breast cancer, trastuzumab resistant breast cancer, or breast cancer demonstrating primary or acquired resistance to CDK4/6 inhibition. In some embodiments, the breast cancer is advanced or metastatic breast cancer.

Examples of cancers that are treatable with a CDK4 inhibitor using the methods of the present disclosure include, but are not limited to, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, endometrial cancer, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or urethra, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, Merkel cell carcinoma, and combinations of said cancers. The methods of the present disclosure are also useful for the treatment of metastatic cancers, especially metastatic cancers that express PD-L1 .

In some embodiments, cancers that are treatable with a CDK4 inhibitor using the methods of the present disclosure include, but are not limited to, melanoma (e.g., metastatic malignant melanoma, BRAF and HSP90 inhibition-resistant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), squamous cell head and neck cancer, urothelial cancer (e.g., bladder), and cancers with high microsatellite instability (MSI^hi9h). Additionally, the disclosure includes refractory or recurrent malignancies whose growth may be inhibited using the compounds of the disclosure.

In some embodiments, cancers that are treatable with a CDK4 inhibitor using the methods of the present disclosure include, but are not limited to, solid tumors (e.g., prostate cancer, colon cancer, esophageal cancer, endometrial cancer, ovarian cancer, uterine cancer, renal cancer, hepatic cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, cancers of the head and neck, thyroid cancer, glioblastoma, sarcoma, bladder cancer, etc.), hematological cancers (e.g., lymphoma, leukemia such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), DLBCL, mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsed or refractory NHL and recurrent follicular), Hodgkin lymphoma, multiple myeloma) and combinations of said cancers.

In some embodiments, cancers that are treatable with a CDK4 inhibitor using the methods of the present disclosure include, but are not limited to, cholangiocarcinoma, bile duct cancer, triple negative breast cancer, rhabdomyosarcoma, small cell lung cancer, leiomyosarcoma, hepatocellular carcinoma, brain cancer, brain tumor, astrocytoma, neuroblastoma, neurofibroma, basal cell carcinoma, chondrosarcoma, epithelioid sarcoma, eye cancer, Fallopian tube cancer, gastrointestinal cancer, gastrointestinal stromal tumors, hairy cell leukemia, intestinal cancer, islet cell cancer, oral cancer, mouth cancer, throat cancer, laryngeal cancer, lip cancer, mesothelioma, neck cancer, nasal cavity cancer, ocular cancer, ocular melanoma, pelvic cancer, rectal cancer, renal cell carcinoma, salivary gland cancer, sinus cancer, spinal cancer, tongue cancer, tubular carcinoma, urethral cancer, and ureteral cancer.

In some embodiments, diseases and indications that are treatable with a CDK4 inhibitor using the methods of the present disclosure include, but are not limited to hematological cancers, sarcomas, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, and skin cancers. Exemplary hematological cancers include lymphomas and leukemias such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma, Non-Hodgkin lymphoma (including relapsed or refractory NHL and recurrent follicular), Hodgkin lymphoma, myeloproliferative diseases (e.g., primary myelofibrosis (PMF), polycythemia vera (PV), and essential thrombocytosis (ET)), myelodysplasia syndrome (MDS), T-cell acute lymphoblastic lymphoma (T-ALL) and multiple myeloma (MM).

Exemplary sarcomas include chondrosarcoma, osteosarcoma, rhabdomyosarcoma, angiosarcoma, fibrosarcoma, liposarcoma, myxoma, rhabdomyoma, rhabdosarcoma, fibroma, lipoma, harmatoma, and teratoma.

Exemplary lung cancers include non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bronchogenic carcinoma, squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma, alveolar (bronchiolar) carcinoma, bronchial adenoma, chondromatous hamartoma, and mesothelioma.

Exemplary gastrointestinal cancers include cancers of the esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma), and colorectal cancer.

Exemplary genitourinary tract cancers include cancers of the kidney (adenocarcinoma, Wilm's tumor [nephroblastoma]), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), and testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma).

Exemplary liver cancers include hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma.

Exemplary bone cancers include, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumors.

Exemplary nervous system cancers include cancers of the skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma, glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), and spinal cord (neurofibroma, meningioma, glioma, sarcoma), as well as neuroblastoma and Lhermitte-Duclos disease.

Exemplary gynecological cancers include cancers of the uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma: serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, embryonal rhabdomyosarcoma), and fallopian tubes (carcinoma).

Exemplary skin cancers include melanoma, basal cell carcinoma, Merkel cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, and keloids. In some embodiments, diseases and indications that are treatable using the compounds of the present disclosure include, but are not limited to, triple-negative breast cancer (TNBC), myelodysplastic syndromes, testicular cancer, bile duct cancer, esophageal cancer, and urothelial carcinoma.

In some embodiments, the disease or disorder associated with CDK4 is an infection, e.g., a viral infection, a bacterial infection, a fungus infection or a parasite infection.

Biomarkers and Methods of Predicting Responsiveness to a CDK4 Inhibitor

Provided herein are biomarkers that are useful in predicting responsiveness to a CDK4 inhibitor (improvement in disease status as evidenced by, e.g., disease remission/resolution) of a subject having, suspected of having, or at risk of developing a disease or disorder associated with CDK4. Thus, provided herein are methods of predicting the response to a CDK4 inhibitor of a human subject having, suspected of having, or at risk of developing a disease or disorder associated with CDK4. In certain embodiments, the predictive methods described herein predict that the subject will respond to treatment with the CDK4 inhibitor with at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, at least 95 percent, at least 98 percent, at least 99 percent, or 100 percent accuracy. For example, in some embodiments, if the predictive methods described herein are applied to 10 subjects having, suspected of having, or at risk of developing a disease or disorder associated with CDK4, and 8 of those 10 subjects are predicted to respond to treatment with a CDK4 inhibitor based on a predictive method described herein, and 7 of those 8 subjects do indeed respond to treatment with a CDK4 inhibitor, then the predictive method has an accuracy of 87.5 percent (7 divided by 8). A subject is considered to respond to the CDK4 inhibitor if the subject shows any improvement in disease status as evidenced by, e.g., reduction or alleviation in symptoms, disease remission/resolution, etc.

Evaluating a Biomarker Workflow

In some embodiments, the extent of a phenotypic effect of perturbing a target in a cell line may be established from assay readouts. Assay readouts provide a measure, preferably quantitative, of the effect of a perturbation or any other intervention (such as, for example, a compound treatment or genetic knockout using techniques such as clustered regularly interspaced short palindromic repeats or ‘CRISPR’) on a phenotype of interest. The phenotype could be any phenotype of interest for the endotype being studied, for example glucose uptake, cell viability, and so on. In examples, the assay readouts may be from large-scale experiments, comprising thousands of assay readouts across hundreds of cell lines. Suitably, the assay readouts are sufficient to provide reasonably comprehensive representation of all or most of the possible endotypes of a given disease. In examples, assay readouts may comprise or be derived from CRISPR or CERES screening data or scores, or any other gene effect scores. Assay readouts may additionally or alternatively comprise scores derived from any large-scale genomic and/or chemical perturbation experiments or scores derived from short hairpin ribonucleic acid (shRNA), small molecules screen, genome editing tools, chemical screens, or cellular potency data, for example from pharmacogenomics screening. It will be appreciated that in non-limiting examples, assay readouts may convey information relating to essential genes in a cancer cell line.

Assay readouts may be normalized using a positive and negative control. For example, a negative control could relate to a perturbation that is known to have no effect on the cells while a positive control could relate to a perturbation that is known to kill the cells. It is customary to normalize across the positive and negative controls to achieve comparable assay readout values between experimental batches. In suitable examples, gene effect scores such as CRISPR or CERES scores may be normalized between 0 (which could indicate that the target is not essential for optimal cell proliferation) and 1 (which could indicate that the target is essential for optimal cell proliferation).

In order to determine the degree of specificity of a target to a given endotype, an endotype specificity score may be calculated. As described above, the approach is to determine the extent to which the target produces a greater phenotypic effect in assays that map to the given endotype compared with assays that do not map to the given endotype. Affinity scores provide a measure of which assays map to the endotype of interest, and which do not, and assay readouts provide a measure of the phenotypic effect of perturbation of the target on the cell lines of the respective matching and unmatching assay.

A non-limiting example of a calculation of an endotype specificity score will now be described. In this example, the disease is a cancer and the assay readouts are target efficacy scores such as CERES scores or CRISPR scores computed from CRISPR gene essentiality screens, as described herein.

In-vitro sensitivity to CDK4 depletion

A number of patient stratification strategies could be employed to find patients likely to be sensitive to CDK4 target, e.g., CDK4 selective inhibitor, PF-07220060, depletion, including but not limited to: testing for microsatellite instability, screening for mutations, amplifications, duplications or deletions of target genes, and testing for target or target-associated molecule expression (e.g., mRNA or protein) or activity (e.g., enzyme activity).

Once a subject has been assayed for target status and predicted to be sensitive to treatment with a CDK4 selective inhibitor, PF-07220060, administration of PF-07220060 to a subject can be affected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents may be empirically adjusted.

Different CDK4 inhibitors can be administered. In one embodiment, more than one inhibitor of a CDK4, is chosen and administered to the subject. Cyclin-dependent kinase 6 (CDK6), cyclin E1 (CCNE1), cyclin D1 (CCND1) expression, e.g., RNA or protein, or activity can then be assayed for after administration of PF-07220060. This assay can also be done at multiple timepoints after administration of the different inhibitor. For example, a first inhibitor could be administered to the patient and cyclin-dependent kinase 6 (CDK6), cyclin E1 (CCNE1), cyclin D1 (CCND1) expression, e.g., RNA or protein, or activity assayed for at 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 16 hours, 24 hours, 48 hours, 3 days, 1 week, 2 weeks, 3 weeks, or 1 month or several months after administration. A second inhibitor could then be administered and target expression, e.g., RNA or protein, or activity can be assayed for again at 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 16 hours, 24 hours, 48 hours, 3 days, 1 week, 2 weeks, 3 weeks, or 1 month or several months after administration of the second inhibitor.

It is well known in the art that cancers can become resistant to chemotherapeutic treatment, especially when that treatment is prolonged. Determining the presence of CDK6, CCNE1 , CCND1 expression, e.g., RNA or protein, or activity can be done after prolonged treatment with any chemotherapeutic to determine if the cancer would be sensitive to PF- 07220060. If the subject has been previously treated with another chemotherapeutic or another inhibitor, it is useful to assay for CDK6, CCNE1 , CCND1 expression, e.g., RNA or protein, or activity to determine if the tumor is sensitive to the inhibitor of the target. This assay can be especially beneficial to the patient if the cancer goes into remission and then re-grows or has metastasized to a different site.

In one embodiment, an in-vitro sensitivity to CDK4 depletion was assessed using publicly available whole-genome CRISPR drop out screening across 766 cell lines heavily annotated cell lines across 27 diverse cancer lineages Unbiased machine learning procedures published by Ryu McMillan were then used to assign features predictive of CDK4 dependency using data derived from mRNAseq, reverse phase protein arrays (RPPA), and whole exome sequencing (WES).

In some embodiments, protein and/or mRNA expression of CDK6, CCND1 , AR, CCNE1 as well as mutations in RB1 unbiasedly predicted sensitivity to CDK4 depletion in Achilles, either by negative correlation (CDK6 mRNA, RB1 mutations, CCNE1 mRNA) or positive correlation (CCND1 mRNA, AR protein). In certain embodiments, AR predicted sensitivity selectively in the context of breast and prostate cancers. The CDK4 selective inhibitor, PF-07220060, was profiled across 650 cell lines in 9-point dose-response assays. In a particular embodiment, a high correlation between dependency calculated in Achilles and response metrics (AUC, EC50) to CDK4 inhibition in the BOE screening was demonstrated. In some such embodiments, markers that predict sensitivity in Achilles maintained predictive capacity to CDK inhibition.

In another such embodiment, increased expression of CDK6 or CCNE1 negated growth inhibition by PF-07220060 in multicellular tumor spheroid models of HR+ HER2- breast cancer. Conversely, depletion of CDK6 by shRNA sensitized these spheroids to PF-07220060. In specific embodiments, there was an inverse correlation between PF-07220060 induced tumor growth inhibition and the level of the CDK6 protein in xenograft derived cell lines. Taken together, CDK6 expression emerged as a key determinant of tumor cell response to PF-07220060: high CDK6 expression is causally linked to tumor cell resistance to PF-07220060; conversely, absence, or low CDK6 expressing cancer cell lines or xenografts tend to be among the most sensitive to PF- 07220060.

Tissue Sample

A tissue sample from a tumor in a human patient can be used as a source of RNA, a source of protein, or a source of thin sections for immunohistochemistry (IHC), so level of CDK6, CCNE1 , CCND1 , and/or Androgen Receptor (AR), expression in the sample can be determined as described in the present disclosure. The tissue sample can be obtained by using conventional tumor biopsy instruments and procedures. Endoscopic biopsy, excisional biopsy, incisional biopsy, fine needle biopsy, punch biopsy, shave biopsy and skin biopsy are examples of recognized medical procedures that can be used by one of skill in the art to obtain tumor samples. The tumor tissue sample should be large enough to provide sufficient RNA, protein, or thin sections for measuring marker gene, e.g., expression level or visualizing macrophages by IHC, e.g., CDK6, CCNE1 , CCND1 , and/or AR positive cell expression.

Body fluid samples can be obtained from a subject using any of the methods known in the art. Methods for extracting cellular DNA from body fluid samples are well known in the art. Typically, cells are lysed with detergents. After cell lysis, proteins are removed from DNA using various proteases. DNA is then extracted with phenol, precipitated in alcohol, and dissolved in an aqueous solution. Methods for extracting acellular DNA from body fluid samples are also known in the art. Commonly, a cellular DNA in a body fluid sample is separated from cells, precipitated in alcohol, and dissolved in an aqueous solution.

Generally, a solid tumor sample can be a test sample of cells or tissue that are obtained from a subject with cancer by biopsy or surgical resection. A sample of cells or tissue can be removed by needle aspiration biopsy. For this, a fine needle attached to a syringe is inserted through the skin and into the tissue of interest. The needle is typically guided to the region of interest using ultrasound or computed tomography (CT) imaging. Once the needle is inserted into the tissue, a vacuum is created with the syringe such that cells or fluid may be sucked through the needle and collected in the syringe. A sample of cells or tissue can also be removed by incisional or core biopsy. For this, a cone, a cylinder, or a tiny bit of tissue is removed from the region of interest. CT imaging, ultrasound, or an endoscope is generally used to guide this type of biopsy. More particularly, the entire cancerous lesion may be removed by excisional biopsy or surgical resection. In the present invention, the test samples is typically a sample of cells removed as part of surgical resection or circulating tumor derived DNA.

The test sample of, for example tissue, may also be stored in, e.g., RNAIater® (Ambion; Austin T ex.) or flash frozen and stored at -80 degrees centigrade for later use. The biopsied tissue sample may also be fixed with a fixative, such as formaldehyde, paraformaldehyde, or acetic acid/ethanol. The fixed tissue sample may be embedded in wax (paraffin) or a plastic resin. The embedded tissue sample (or frozen tissue sample) may be cut into thin sections. RNA or protein may also be extracted from a fixed or wax-embedded tissue sample.

Cancers amenable for treatment according to the present invention include any of the cancers disclosed herein, as well as other possible cancer types with microsatellite instability, gene amplifications, duplications, deletions or mutations.

Measurement of Gene

Detection of gene expression can be by any appropriate method, including for example, detecting the quantity of mRNA transcribed from the gene or the quantity of cDNA produced from the reverse transcription of the mRNA transcribed from the gene or the quantity of the polypeptide or protein encoded by the gene. These methods can be performed on a sample by sample basis or modified for high throughput analysis. For example, using Affymetrix™ 11133 microarray chips.

In one aspect, gene expression is detected and quantitated by hybridization to a probe that specifically hybridizes to the appropriate probe for that biomarker. The probes also can be attached to a solid support for use in high throughput screening assays using methods known in the art. WO 97/10365 and U.S. Pat. Nos. 5,405,783; 5,412,087 and 5,445,934, for example, disclose the construction of high density oligonucleotide chips which can contain one or more of the sequences disclosed herein. Using the methods disclosed in U.S. Pat. Nos. 5,405,783; 5,412,087 and 5,445,934, the probes of this invention are synthesized on a derivatized glass surface. Photoprotected nucleoside phosphoramidites are coupled to the glass surface, selectively deprotected by photolysis through a photolithographic mask, and reacted with a second protected nucleoside phosphoramidite. The coupling/deprotection process is repeated until the desired probe is complete.

In one aspect, the expression level of a gene is determined through exposure of a nucleic acid sample to the probe-modified chip. Extracted nucleic acid is labeled, for example, with a fluorescent tag, preferably during an amplification step. Hybridization of the labeled sample is performed at an appropriate stringency level. The degree of probe-nucleic acid hybridization is quantitatively measured using a detection device. See U.S. Pat. Nos. 5,578,832 and 5,631 ,734.

Alternatively any one of gene copy number, transcription, or translation can be determined using known techniques. For example, an amplification method such as PCR may be useful. General procedures for PCR are taught in MacPherson et al., PCR: A Practical Approach, (IRL Press at Oxford University Press (1991)). However, PCR conditions used for each application reaction are empirically determined. A number of parameters influence the success of a reaction. Among them are annealing temperature and time, extension time, Mg²⁺ and /or ATP concentration, pH, and the relative concentration of primers, templates, and deoxyribonucleotides. After amplification, the resulting DNA fragments can be detected by agarose gel electrophoresis followed by visualization with ethidium bromide staining and ultraviolet illumination.

In one embodiment, the hybridized nucleic acids are detected by detecting one or more labels attached to the sample nucleic acids. The labels can be incorporated by any of a number of means well known to those of skill in the art. However, in one aspect, the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acid. Thus, for example, polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide a labeled amplification product. In a separate embodiment, transcription amplification, as described above, using a labeled nucleotide (e.g., fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.

Alternatively, a label may be added directly to the original nucleic acid sample (e.g., mRNA, polyA, mRNA, cDNA, etc.) or to the amplification product after the amplification is completed. Means of attaching labels to nucleic acids are well known to those of skill in the art and include, for example nick translation or end-labeling (e.g., with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore).

Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 1251 , 35S, 14C, or 32P) enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Detection of labels is well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.

The detectable label may be added to the target (sample) nucleic acid(s) prior to, or after the hybridization, such as described in WO 97/10365. These detectable labels are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization. In contrast, "indirect labels" are joined to the hybrid duplex after hybridization. Generally, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. For example, the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected. For a detailed review of methods of labeling nucleic acids and detecting labeled hybridized nucleic acids see Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization with Nucleic Acid Probes, P.

RNA Analysis

Conventional microarray analysis and quantitative polymerase chain reaction (QPCR) are examples of methods for determining the level of macrophage marker gene expression at the mRNA level. In some embodiments of the disclosure, RNA is extracted from the cells, tumor or tissue of interest using standard protocols. In other embodiments, RNA analysis is performed using techniques that do not require RNA isolation. RNA Isolation

Methods for rapid and efficient extraction of eukaryotic mRNA, i.e., poly(a) RNA, from tissue samples are well established and known to those of skill in the art. See, e.g., Ausubel et al., 1997, Current Protocols of Molecular Biology, John Wiley and Sons. The tissue sample can be fresh, frozen or fixed paraffin-embedded (FFPE) samples such as clinical study tumor specimens. In general, RNA isolated from fresh or frozen tissue samples tends to be less fragmented than RNA from FFPE samples. FFPE samples of tumor material, however, are more readily available, and FFPE samples are suitable sources of RNA for use in methods of the present disclosure. For a discussion of FFPE samples as sources of RNA for gene expression profiling by RT-PCR, see, e.g., Clark-Langone et al., 2007, BMC Genomics 8:279. Also see, De Andres et al., 1995, Biotechniques 18:42044; and Baker et al., U.S. Patent Application Publication No. 2005/0095634. The use of commercially available kits with vendor's instructions for RNA extraction and preparation is widespread and common. Commercial vendors of various RNA isolation products and complete kits include Qiagen (Valencia, CA), Invitrogen (Carlsbad, CA), Ambion (Austin, TX) and Exiqon (Woburn, MA).

In general, RNA isolation begins with tissue/cell disruption. During tissue/cell disruption it is desirable to minimize RNA degradation by RNases. One approach to limiting RNase activity during the RNA isolation process is to ensure that a denaturant is in contact with cellular contents as soon as the cells are disrupted. Another common practice is to include one or more proteases in the RNA isolation process. Optionally, fresh tissue samples are immersed in an RNA stabilization solution, at room temperature, as soon as they are collected. The stabilization solution rapidly permeates the cells, stabilizing the RNA for storage at 4 degrees centigrade, for subsequent isolation.

In some protocols, total RNA is isolated from disrupted tumor material by cesium chloride density gradient centrifugation. In general, mRNA makes up approximately 1 percent to 5 percent of total cellular RNA. Immobilized Oligo(dT), e.g., oligo(dT) cellulose, is commonly used to separate mRNA from ribosomal RNA and transfer RNA. If stored after isolation, RNA must be stored in under RNase-free conditions. Methods for stable storage of isolated RNA are known in the art. Various commercial products for stable storage of RNA are available.

RNA-Seq Analysis

Provided herein, RNAs are not limited as long as they are RNAs that can be analyzed by RNA-Seq analysis. The RNAs may include mRNAs, untranslated RNAs, microRNA, and so on.

As described herein, the RNAs are not limited as long as they are present in organisms. The organisms are not limited as long as they are multicellular organisms having organs. The organisms may be plants or animals. In some such embodiments, the animals are mammals such as humans, mice, rats, dogs, cats, rabbits, cows, horses, goats, sheep and pigs, or birds such as chickens. In specific embodiments, mammals are humans, mice, dogs, cats, cows, horses and pigs. In a particular embodiment of each of the foregoing, the mammal is a human.

In some embodiments of the each of the foregoing, the organisms include both diseased and non-diseased organisms.

The cells to be analyzed are not limited as long as they are present in organs of the organisms. In specific embodiments, the organs are organs with known cellular composition therein.

An organ means an assembly of several tissues present in an organism and having a certain independent form and a specific function. For example, when the organisms are mammals, the term “organ” may include circulatory system organs (heart, artery, vein, lymph duct, etc.), respiratory system organs (nasal cavity, paranasal sinus, larynx, trachea, bronchi, lung, etc.), gastrointestinal system organs (lip, cheek, palate, tooth, gum, tongue, salivary gland, pharynx, esophagus, stomach, duodenum, jejunum, ileum, cecum, appendix, ascending colon, transverse colon, sigmoid colon, rectum, anus, liver, gallbladder, bile duct, biliary tract, pancreas, pancreatic duct, etc.), urinary system organs (urethra, bladder, ureter, kidney), nervous system organs (cerebrum, cerebellum, mesencephalon, brain stem, spinal cord, peripheral nerve, autonomic nerve, etc.), female reproductive system organs (ovary, oviduct, uterus, vagina, etc.), breast, male reproductive system organs (penis, prostate, testicle, epididymis, vas deferens), endocrine system organs (hypothalamus, pituitary gland, pineal body, thyroid gland, parathyroid gland, adrenal gland, etc.), integumentary system organs (skin, hair, nail, etc.), hematopoietic system organs (blood, bone marrow, spleen, etc.), immune system organs (lymph node, tonsil, thymus, etc.), bone and soft tissue organs (bone, cartilage, skeletal muscle, connective tissue, ligament, tendon, diaphragm, peritoneum, pleura, adipose tissue (brown adipose, white adipose), etc.), sensory system organs (eyeball, palpebra, lacrimal gland, external ear, middle ear, inner ear, cochlea, etc.), and so on. In the present invention, the tissue of interest is preferably that of heart, cerebrum, lung, kidney, adipose tissue, liver, skeletal muscle, testicle, spleen, thymus, bone marrow, pancreas, or skin (including epidermis above the subcutaneous tissue, papillary layer and plexiform layer). Preferred organs are aorta, brain, fat, heart, kidney, large intestine, liver, lung, bone marrow, pancreas, skin, skeletal muscle, spleen and thymus.

In one embodiment, the RNA-Seq analysis is a transcriptome analysis, which is a method for analyzing the expressed genes or the number of counts (also called the number of read counts) thereof by comprehensively acquiring reads including sequence information from RNAs present in a sample of interest and mapping the reads on a reference sequence. The number of counts corresponds to the gene expression level. The count data for RNA-Seq analysis may include the gene names of expressed genes and/or registration numbers thereof in a gene database, and the numbers of counts of reads of respective genes.

In a particular embodiment of each of the foregoing, RNA-Seq analysis can be performed using a DNA sequencer called next generation sequencer or third generation sequencer. Examples of next generation sequencers include MiSeq9 (trademark), HiSeq (trademark), NextSeq (trademark) and MiSeq (trademark) available from Illumina, Inc. (San Diego, Calif.); Ion Proton (trademark) and Ion PGM (trademark) available from Thermo Fisher Scientific (Waltham, Mass.); GS FLX+ (trademark) and GS Junior (trademark) available from Roche (Basel, Switzerland), and so on. Examples of third generation sequencers include PacBio Sequel (tradename) and so on.

A count data set for scRNA-Seq analysis is a set of count data generated based on gene expressions predicted by expression analysis of genes expressed in individual cells of an organism and/or a computer analysis method. For example, a count data set for scRNA-Seq analysis may be count data acquired from real individual cells by RNA-Seq analysis. Also, a count data set for scRNA-Seq analysis may be a count data set predicted by performing, for example, deconvolution on count data acquired from a whole organ by RNA-Seq analysis based on reference cell composition ratios by a computer analysis method according to the method described in Non-Patent Documents 6 to 19. As a method for predicting a count data set for scRNA-Seq analysis, a method called Complete Deconvolution for Sequencing data (CDSeq) (Non-Patent Document 19), for example, may be preferred.

In some embodiments, the method of the present invention comprises RNA-Seq analysis. For example, gene expression level of CDK6, CCNE1 , CCND1 , and/or AR is measured RNA- Seq analysis.

Microarray

The mRNA expression level of one or more genes encoding macrophage marker proteins such as CDK6, CCNE1 , CCND1 , and/or AR, can be measured using conventional DNA microarray expression profiling technology. A DNA microarray is a collection of specific DNA segments or probes affixed to a solid surface or substrate such as glass, plastic or silicon, with each specific DNA segment occupying a known location in the array. Hybridization with a sample of labeled RNA, usually under stringent hybridization conditions, allows detection and quantitation of RNA molecules corresponding to each probe in the array. After stringent washing to remove non-specifically bound sample material, the microarray is scanned by confocal laser microscopy or other suitable detection method. Modern commercial DNA microarrays, often known as DNA chips, typically contain tens of thousands of probes, and thus can measure expression of tens of thousands of genes simultaneously. Such microarrays can be used in practicing the present disclosure. Alternatively, custom chips containing as few probes as those needed to measure expression of one or more genes encoding macrophage marker proteins, such as CDK6, CCNE1 , CCND1 , and/or AR, plus necessary controls or standards, e.g., for data normalization, can be used in practicing the disclosure.

To facilitate data normalization, a two-color microarray reader can be used. In a two-color (two-channel) system, samples are labeled with a first fluorophore that emits at a first wavelength, while an RNA or cDNA standard is labeled with a second fluorophore that emits at a different wavelength. For example, Cy3 (570 nm) and Cy5 (670 nm) often are employed together in two- color microarray systems.

DNA microarray technology is well-developed, commercially available, and widely employed. Therefore, in performing methods disclosed herein, a person of ordinary skill in the art can use microarray technology to measure expression levels of genes encoding macrophage marker proteins such as CDK6, CCNE1 , CCND1 , and/or AR without undue experimentation. DNA microarray chips, reagents (such as those for RNA or cDNA preparation, RNA or cDNA labeling, hybridization and washing solutions), instruments (such as microarray readers) and protocols are well known in the art and available from various commercial sources. Commercial vendors of microarray systems include Agilent Technologies (Santa Clara, CA) and Affymetrix (Santa Clara, CA), but other PCR systems can be used as well.

Quantitative RT-PCR

The level of mRNA representing individual genes encoding proteins such as CDK6, CCNE1 , CCND1 , and/or AR can be measured using conventional quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) technology. Advantages of qRT-PCR include sensitivity, flexibility, quantitative accuracy, and ability to discriminate between closely related mRNAs. Guidance concerning the processing of tissue samples for quantitative PCR is available from various sources, including manufacturers and vendors of commercial products for qRT-PCR (e.g., Qiagen (Valencia, CA) and Ambion (Austin, TX)). Instrument systems for automated performance of qRT-PCR are commercially available and used routinely in many laboratories. An example of a well-known commercial system is the Applied Biosystems 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).

Once mRNA is isolated, the first step in gene expression profiling by RT- PCR is the reverse transcription of the mRNA template into cDNA, which is then exponentially amplified in a PCR reaction. Two commonly used reverse transcriptases are avilo myeloblastosis virus reverse transcriptase (AM -RT) and Moloney murine leukemia virus reverse transcriptase (MMLV-RT). The reverse transcription reaction typically is primed with specific primers, random hexamers, or oligo(dT) primers. The resulting cDNA product can be used as a template in the subsequent polymerase chain reaction.

The PCR step is carried out using a thermostable DNA-dependent DNA polymerase. The polymerase most commonly used in PCR systems is a Thermus aquaticus (Taq) polymerase. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification, i.e., regions of the cDNAs reverse transcribed from genes encoding macrophage marker proteins, such as CD68. Therefore, when qRT-PCR is employed in the present disclosure primers specific to each marker gene are based on the cDNA sequence of the gene. Commercial technologies such as SYBR® green or TaqMan®(Applied Biosystems, Foster City, CA) can be used in accordance with the vendor's instructions. Messenger RNA levels can be normalized for differences in loading among samples by comparing the levels of housekeeping genes such as beta-actin or GAPDH. The level of mRNA expression can be expressed relative to any single control sample such as mRNA from normal, non-tumor tissue or cells. Alternatively, it can be expressed relative to mRNA from a pool of tumor samples, or tumor cell lines, or from a commercially available set of control mRNA.

Detection of proteins

Expression level of cyclin-dependent kinase 6 (CDK6), cyclin E1 (CCNE1), cyclin D1 (CCND1), and/or Androgen Receptor (AR), can be determined by examining protein expression or the protein product. Determining the protein level involves measuring the amount of any immunospecific binding that occurs between an antibody that selectively recognizes and binds to the polypeptide of the biomarker in a sample obtained from a patient and comparing this to the amount of immunospecific binding of at least one biomarker in a control sample. The amount of protein expression of the target can be increased or reduced when compared with control expression.

A variety of techniques are available in the art for protein analysis. They include but are not limited to radioimmunoassays, ELISA (enzyme linked immunosorbent assays), "sandwich" immunoassays, immunoradiometric assays, in situ immunoassays (using e.g., colloidal gold, enzyme or radioisotope labels), Western blot analysis, immunoprecipitation assays, immunofluorescent assays, flow cytometry, immunohistochemistry, HPLC, mass spectrometry, confocal microscopy, enzymatic assays, surface plasmon resonance and PAGE- SDS.

In one embodiment, a method of determining if a subject afflicted with a cancer will respond to therapeutic treatment with a CDK4 inhibitor, e.g., PF-07220060, is provided.

In one embodiment, a method of determining the sensitivity of a cancer cell associated with the loss of target function through a CDK4 inhibitor, e.g., PF-07220060, is provided.

ELISA

Performing an ELISA, e.g., CDK6, CCNE1 , CCND1 , and/or AR ELISA, requires at least one antibody against the protein, i.e. , the detection antibody. In an exemplary embodiment, the protein is a CDK6, CCNE1 , CCND1 , and/or AR protein from a sample to be analyzed is immobilized on a solid support such as a polystyrene microtiter plate. This immobilization can be by non-specific binding of the CDK6, CCNE1 , CCND1 , and/or AR, e.g., through adsorption to the surface. Alternatively, immobilization can be by specific binding, e.g., through binding of CDK6, CCNE1 , CCND1 , and/or AR protein from the sample by a capture antibody (anti-cyclin-dependent kinase 6 (CDK6), anti- cyclin E1 (CCNE1) antibody, anti-cyclin D1 (CCND1) antibody, and/or antiAndrogen Receptor (AR) antibody) different from the detection antibody), in a "sandwich" ELISA. After the CDK6, CCNE1 , CCND1 , and/or AR is immobilized, the detection antibody is added, and the detection antibody forms a complex with the boundCDK6, CCNE1 , CCND1 , and/or AR . The detection antibody is linked to an enzyme, either directly or indirectly, e.g., through a secondary antibody that specifically recognizes the detection antibody. Typically, between each step, the plate, with bound CDK6, CCNE1 , CCND1 , and/or AR, is washed with a mild detergent solution. Typical ELISA protocols also include one or more blocking steps, which involve use of a non- specifically binding protein such as bovine serum albumin to block unwanted non-specific binding of protein reagents to the plate. After a final wash step, the plate is developed by addition of an appropriate enzyme substrate, to produce a visible signal, which indicates the quantity of CDK6, CCNE1 , CCND1 , and/or AR in the sample. The substrate can be, e.g., a chromogenic substrate or a fluorogenic substrate. ELISA methods, reagents and equipment are well-known in the art and commercially available.

It is understood that the expression levels of other macrophage marker proteins, e.g., CCR2, CD14, CD163, CSF1 R, and MSRI , as well as other macrophage specific marker proteins can be measured by ELISA using detecting antibodies specific for each macrophage marker protein.

The number of macrophages in a given cell population can be determined (e.g., visualized) by immunohistochemistry. In addition, the percentage and density of cells in a sample that are positive for a given biomarker protein, such as CDK6, CCNE1 , CCND1 , and/or AR, can be determined by immunochemistry. Assaying a macrophage marker protein by IHC, e.g., CDK6, CCNE1 , CCND1 , and/or AR IHC, requires at least one antibody against a macrophage marker protein, e.g., at least one anti- CDK6, anti- CCNE1 antibody, anti-CCND1 antibody, and/or anti- AR antibody. Numerous anti-CDK6, anti-CCNE1 antibody, anti-CCND1 antibody, and/or anti-AR antibodies suitable for IHC are commercially available. For example, suitable antibodies can be purchased from Dako North America, Inc. (Carpinteria, CA), abeam (Cambridge, MA), Abnova (Walnut, CA), R and D Systems (Minneapolis, MN) or Invitrogen (Carlsbad, CA). Using standard techniques, the anti-CDK6, anti-CCNE1 antibody, anti-CCND1 antibody, and/or anti-ARantibody can be used to detect the presence of CDK6, CCNE1 , CCND1 , and/or AR protein in sections, e.g., 5 micron sections, obtained from tumors, including paraffin-embedded and frozen tumor sections. Typically, the tumor sections are initially treated in such a way as to retrieve the antigenic structure of proteins that were fixed in the initial process of collecting and preserving the tumor material. Slides are then blocked to prevent non-specific binding by the anti-CDK6, anti-CCNE1 antibody, anti-CCND1 antibody, and/or anti-ARdetection antibody. The presence of CDK6, CCNE1 , CCND1 , and/or AR protein is then detected by binding of the anti- cyclin- dependent kinase 6 (CDK6), anti- cyclin E1 (CCNE1) antibody, anti-cyclin D1 (CCND1) antibody, and/or anti-Androgen Receptor (AR) antibody to the CDK6, CCNE1 , CCND1 , and/or AR protein, respectively. The detection (primary) antibody is linked to an enzyme, either directly or indirectly, e.g., through a secondary antibody or polymer that specifically recognizes the detection (primary) antibody. Typically, the tumor sections are washed and blocked with nonspecific protein such as bovine serum albumin between steps. The slide is developed using an appropriate enzyme substrate to produce a visible signal. The samples can be counterstained with hematoxylin.

It is understood that the expression of other macrophage marker proteins, e.g., CCR2, CD14, CD163, CSF1 R, and MSR1 , as well as other macrophage specific marker proteins can be detected by IHC in a similar manner using antibodies specific for each macrophage marker protein.

Data Interpretation

A macrophage score for a tumor can be interpreted with respect to a threshold score. A macrophage score, or the expression level of a particular biomarker such as CDK6, CCNE1 , CCND1 , and/or AR, that is equal to or higher than the threshold score can be interpreted as predictive of the tumor being likely to be sensitive (responsive) to treatment with a CDK4 inhibitor, such as with PF-07220060. Alternatively, macrophage scores, or the expression level of a particular biomarker such as CDK6, CCNE1 , CCND1 , and/or AR, equal to or lower than the threshold score can be interpreted as predictive of a tumor being likely to be resistant (non- responsive) to treatment with a CDK4 inhibitor, such as PF-07220060.

An optimum threshold macrophage score, or CDK6, CCNE1 , CCND1 , and/or AR expression level, can be determined (or at least approximated) empirically by performing a threshold determination analysis. Preferably, threshold determination analysis includes receiver operator characteristic (ROC) curve analysis. ROC curve analysis is an established statistical technique, the application of which is within ordinary skill in the art. For a discussion of ROC curve analysis, see generally Zweig et al., 1993, "Receiver operating characteristic (ROC) plots: a fundamental evaluation tool in clinical medicine," Clin. Chem. 39:561-577; and Pepe, 2003, The statistical evaluation of medical tests or classification and prediction, Oxford Press, New York. Macrophage scores, CDK6, CCNE1 , CCND1 , and/or AR expression levels, and the optimum threshold scores may vary from tumor type to tumor type. Therefore, a threshold determination analysis preferably is performed on one or more datasets representing any given tumor type to be tested using the present disclosure. The dataset used for threshold determination analysis includes: (a) actual response data (response or non-response), and (b) a macrophage score or CDK6, CCNE1 , CCND1 , and/or AR expression level for each tumor sample from a group of tumors. Once a macrophage score or CDK6, CCNE1 , CCND1 , and/or AR expression level threshold is determined with respect to a given tumor type, that threshold can be applied to interpret macrophage scores or CDK6, CCNE1 , CCND1 , and/or AR expression levels from tumors of that tumor type.

The ROC curve analysis can be performed as follows. Any sample with a macrophage score or CDK6, CCNE1 , CCND1 , and/or AR expression level greater than or equal to the threshold is identified as a responder (sensitive). Alternatively, any sample with a macrophage score or CDK6, CCNE1 , CCND1 , and/or AR expression level less than or equal to the threshold is identified as a non-responder (resistant). For every macrophage score or CDK6, CCNE1 , CCND1 , and/or AR expression level from a tested set of samples, "responders" and "nonresponders" (hypothetical calls) are classified using that score as the threshold. This process enables calculation of TPR (y vector) and FPR (x vector) for each potential threshold, through comparison of hypothetical calls against the actual response data for the data set. Then an ROC curve is constructed by making a dot plot, using the TPR vector, and FPR vector. If the ROC curve is above the diagonal from (0, 0) point to (1 .0, 0.5) point, it shows that the macrophage test result is a better test than random.

The ROC curve can be used to identify the best operating point. The best operating point is the one that yields the best balance between the cost of false positives weighed against the cost of false negatives. These costs need not be equal. The average expected cost of classification at point x,y in the ROC space is determined by the following formula.

C = (1-p) alpha*x + p*beta(l-y) wherein: alpha = cost of a false positive, beta = cost of missing a positive (false negative), and p = proportion of positive cases.

False positives and false negatives can be weighted differently by assigning different values for alpha and beta. For example, if it is decided to include more patients in the responder group at the cost of treating more patients who are non-responders, one can put more weight on alpha. In this case, it is assumed that the cost of false positive and false negative is the same (alpha equals to beta). Therefore, the average expected cost of classification at point x, y in the ROC space is:

C’ = (l-p)*x + p*(l-y). The smallest C’ can be calculated after using all pairs of false positive and false negative (x, y). The optimum score threshold is calculated as the score of the (x, y) at C’.

In addition to predicting whether a tumor will be sensitive or resistant to a CDK4 inhibitor, such as PF-07220060, a macrophage score or CDK6, CCNE1 , CCND1 , and/or AR expression level provides an approximate, but useful, indication of how likely a tumor is to be sensitive or resistant.

In one embodiment, a method of determining if a subject afflicted with a cancer will respond to therapeutic treatment with a CDK4 inhibitor, e.g., PF-07220060, is provided, comprising: contacting a sample obtained from said subject with a reagent capable of detecting human cancer cells exhibiting expression.

In some embodiments of the methods of the disclosure, macrophage marker gene expression such as CDK6, CCNE1 , CCND1 , and/or AR can be detected at the protein level. Examples of methods for measuring the level of macrophage marker gene expression at the protein level include enzyme linked immunosorbent assay (ELISA) and IHC analysis.

Administration and Dosing

The dosage of the CDK4 inhibitor, e.g., PF-07220060, described herein, and/or compositions including the CDK4 inhibitor, e.g., PF-07220060, described herein, can vary depending on many factors, such as the pharmacodynamic properties of the agent or compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the agent or compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The CDK4 inhibitor, e.g., PF- 07220060, described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In a preferred embodiment, the daily dose of a CDK4 inhibitor or a pharmaceutically acceptable salt thereof, is administered orally.

A CDK4 inhibitor, or a pharmaceutically acceptable salt, may be present in a pharmaceutical composition which includes a pharmaceutically acceptable excipient. "Pharmaceutically acceptable excipient" refers to a component that may be included in the compositions described herein, is physiologically suitable for pharmaceutical use, and causes no significant adverse effects nor therapeutic effects to a subject. The term ’excipient’ is used herein to describe any ingredient other than the compound(s) of the invention. The choice of excipient will to a large extent depend on factors such as the mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

The amount of a CDK4 inhibitor, or a pharmaceutically acceptable salt, in the pharmaceutical compositions may be any amounts disclosed herein. The compounds of the method, use or combination of the present invention may be formulated prior to administration. The formulation will preferably be adapted to the particular mode of administration. These compounds may be formulated with pharmaceutically acceptable excipients as known in the art and administered in a wide variety of dosage forms as known in the art. Dosage unit forms or pharmaceutical compositions suitable for oral administration include, but are not limited to tablets, capsules, such as gelatin capsules, pills, powders, granules, aqueous and nonaqueous oral solutions and suspensions, packaged in containers adapted for subdivision into individual doses.

In another embodiment, the dosage of a compound or pharmaceutical composition described herein may vary within the range depending upon the dosage form employed and the route of administration utilized. In another embodiment, an amount of a compound or pharmaceutical composition described herein administered to a subject may be dependent upon factors known to a skilled artisan, including bioactivity and bioavailability of the compound (e.g., half-life and stability of the compound in the body), chemical properties of the compound (e.g., molecular weight, hydrophobility and solubility), route and frequency of administration, and the like. Further, it will be understood that the specific dose of a pharmaceutical composition comprising a compound as disclosed herein may depend on a variety of factors including physical condition of the subject (e.g., age, gender, weight), and medical history of the subject (e.g., medications being taken, health condition other diseases or disorders). The precise dose of a pharmaceutical composition administered to a subject may be determined by methods known to a skilled artisan such as a pharmacologist, or an anesthesiologist.

In an embodiment, the CDK4 inhibitor, for example, PF-07220060, described herein, is administered at a daily dosage of from about 1 mg to about 1000 mg per day. In another embodiment, the CDK4 inhibitor is administered at a daily dosage from about 10 mg to about 1000 mg per day. In another embodiment, the CDK4 inhibitor is administered at a dosage of from about 25 mg to about 900 mg per day. In another embodiment, the CDK4 inhibitor is administered at a dosage of from about 50 mg to about 800 mg per day. In another embodiment the CDK4 inhibitor is administered at dosages of about: 1 , 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 260, 270, 275, 280, 290, 300, 325, 350, 375, 400, 425, 450, 475 or 500 mg on a QD, BID schedule. In certain embodiments, the CDK4 inhibitor is administered at a dosage of about 50 mg QD, about 50 mg BID, about 75 mg QD, about 75 mg BID, about 200 mg QD, about 200 mg BID, about 300 mg QD, about 300 mg BID, about 400 mg QD, about 400 mg BID, or about 500 mg QD.

In an embodiment, PF-07220060, described herein, is administered once or twice daily to comprise a complete cycle of 28 days.

Repetition of the 28-day cycles is continued during treatment with the combination of the present invention. Repetition of the administration or dosing regimens may be conducted as necessary to achieve the desired reduction or diminution of cancer cells. A “continuous dosing schedule”, as used herein, is an administration or dosing regimen without dose interruptions, e.g., without days off treatment. Repetition of 28-day treatment cycles without dose interruptions between the treatment cycles is an example of a continuous dosing schedule. In an embodiment, the compounds of the combination of the present invention may be administered in a continuous dosing schedule. In an embodiment, the compounds of the combination of the present invention may be administered concurrently in a continuous dosing schedule.

Method of Treatment

In one embodiment, the disclosure provides a method of treating a cancer in a subject in need thereof, which includes administering to the subject an amount of a cyclin-dependent kinase 4 (CDK4) inhibitor as described herein, wherein the subject exhibits modified levels compared to the baseline mRNA expression, protein expression, and/or activity of CDK6, CCNE1 , CCND1 , and/or AR, comprising administering to the subject an effective amount of PF-07220060

The term “locally advanced”, as used herein, as it relates to cancer, may or may not be treated with curative intent. The term “metastatic” as used herein, as it relates to cancer, cannot be treated with curative intent. Those skilled in the art will be able to recognize and diagnose locally advanced and metastatic cancer in a patient.

For convenience, certain well-known abbreviations, may be used herein, including: castration resistant prostate cancer (CRPC), estrogen receptor positive (ER+), human epidermal growth factor receptor 2 negative (HER2-), hormone receptor (HR), human epidermal growth factor receptor 2 positive (HER2+), non-small cell lung cancer (NSCLC) and progesterone receptor (PR).

In one embodiment, the cancer is selected from the group consisting of lung cancer, mesothelioma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, hepatic carcinoma, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin’s disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, hematology malignancy, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, spinal axis tumors, glioblastoma, brain stem glioma, pituitary adenoma, head and neck cancer, or a combination of two or more of the foregoing cancers. Another embodiment relates to methods of treating cancer in a patient. Another embodiment relates to the treatment of cancer in a patient comprising administering to the patient an amount of the compounds described herein that are effective in treating the cancer.

In one embodiment, the cancer is breast, lung, colon, brain, head and neck, prostate, stomach, pancreatic, ovarian, melanoma, endocrine, uterine, testicular, or bladder cancer.

In one embodiment, the cancer is breast, lung, prostate, pancreatic, or ovarian cancer.

In one embodiment, the cancer is breast, lung, or prostate cancer.

In one embodiment, the cancer is breast cancer.

In one embodiment, the breast cancer is HR+ breast cancer.

In one embodiment, the HR+ breast cancer is PR+ and/or ER+ breast cancer.

In one embodiment, the breast cancer is PR+ breast cancer.

In one embodiment, the breast cancer is ER+ breast cancer.

In one embodiment, the breast cancer is ER+ HER2- breast cancer.

In one embodiment, the breast cancer is ER+ HER2+ breast cancer.

In one embodiment, the breast cancer is locally advanced or metastatic ER+ breast cancer.

In one embodiment, the breast cancer is locally advanced or metastatic ER+ HER2- breast cancer.

In one embodiment, the breast cancer is locally advanced or metastatic ER+ HER2+ breast cancer.

In one embodiment, the lung cancer is non-small cell lung cancer.

In one embodiment, the lung cancer is locally advanced or metastatic non-small cell lung cancer.

In one embodiment, the prostate cancer is castration resistant prostate cancer.

In one embodiment, the prostate cancer is locally advanced or metastatic castration resistant prostate cancer.

Another embodiment relates to methods of treating solid tumors in a patient. Another embodiment relates to the treatment of solid tumors in a patient comprising administering to the patient an amount of the compounds described herein that are effective in treating the solid tumor.

In one embodiment, the solid tumor is breast, lung, colon, brain, head and neck, prostate, stomach, pancreatic, ovarian, melanoma, endocrine, uterine, testicular, or bladder.

In one embodiment, the solid tumor is breast, lung, prostate, pancreatic, or ovarian.

In one embodiment, the solid tumor is breast, lung, or prostate.

In one embodiment, the solid tumor is breast cancer, and in a further embodiment, the breast cancer is HR+ breast cancer, and in a still further embodiment the HR+ breast cancer is PR+ and/or ER+ breast cancer ER+ breast cancer.

In one embodiment, the solid tumor is breast cancer, and in a further embodiment, the breast cancer is ER+ HER2- breast cancer. In one embodiment, the solid tumor is breast cancer, and in a further embodiment, the breast cancer is ER+ HER2+ breast cancer.

In one embodiment, the solid tumor is breast cancer, and in a further embodiment, the breast cancer is locally advanced or metastatic ER+ HER2- breast cancer.

In one embodiment, the solid tumor is breast cancer, and in a further embodiment, the breast cancer is locally advanced or metastatic ER+ HER2+ breast cancer.

In one embodiment, the solid tumor is lung cancer, and in a further embodiment the lung cancer is non-small cell lung cancer.

In one embodiment, the solid tumor is lung cancer, and in a further embodiment the lung cancer is locally advanced or metastatic non-small cell lung cancer.

In one embodiment, the solid tumor is prostate cancer, and in a further embodiment the prostate cancer is castration resistant prostate cancer.

In one embodiment, the solid tumor is prostate cancer, and in a further embodiment the prostate cancer is locally advanced or metastatic castration resistant prostate cancer.

Another embodiment relates to methods of treating hematologic tumors in a patient. Another embodiment relates to the treatment of hematologic tumors in a patient comprising administering to the patient an amount of the compounds described herein that is effective in treating the hematologic tumor.

In one embodiment, the hematologic tumor is leukemia, lymphoma or multiple myeloma.

In one embodiment, the hematologic tumor is leukemia or lymphoma.

Another embodiment relates to methods of treating cancer in a patient with locally advanced or metastatic ER+HER2- breast cancer, CRPC, or NSCLC whose disease progressed on or is intolerant to standard therapy.

Another embodiment relates to methods of treating cancer in a patient with locally advanced or metastatic ER+HER2- breast cancer, CRPC, or NSCLC whose disease progressed on or is intolerant to standard therapy.

Another embodiment relates to methods of treating cancer in a patient with locally advanced or metastatic 2L+ ER+HER2 breast cancer who has progressed after at least one prior line of treatment with an endocrine therapy and CDK4/6 inhibitor. In an embodiment thereof, the patient is administered a combination of a CDK4 inhibitor and fulvestrant.

Another embodiment relates to methods of treating cancer in a patient with locally advanced or metastatic 2L+ ER+HER2 breast cancer who has progressed after at least one prior line of treatment with an endocrine therapy and CDK4/6 inhibitor.

Another embodiment relates to methods of treating cancer in a patient with advanced or metastatic 2L+ ER+HER2- breast cancer who has progressed after at least one prior line of CDK4/6 inhibitor and one line of endocrine therapy. In an embodiment thereof, the patient is administered a CDK4 inhibitor. Another embodiment relates to methods of treating cancer in a patient with advanced or metastatic 2-4L fulvestrant-naive ER+HER2- breast cancer whose disease has progressed after one line of a CDK4/6 inhibitor and one line of endocrine therapy and who must not have received more than three lines of systemic therapies in advanced or metastatic setting. In an embodiment thereof, the patient is administered a CDK4 inhibitor and fulvestrant.

Another embodiment relates to methods of treating cancer in a patient who has received prior CDK4/6i plus non-steroidal aromatase inhibitor (NSAI).

Another embodiment relates to methods of treating cancer in a patient with advanced or metastatic breast cancer who has received prior CDK4/6i plus non-steroidal aromatase inhibitor (NSAI).

Another embodiment relates to methods of treating cancer in a patient with advanced or metastatic breast cancer who has received two prior lines of systemic therapies. In a further embodiment, one of the two prior lines of systemic therapy is CDK4/6i plus NSAI. In a further further embodiment, one of the two prior lines of systemic therapy is an approved treatment targeting estrogen receptor 1 (ESR1), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), AKT1 , phosphatase and tensin homolog (PTEN), or breast cancer gene (BRCA).

Another embodiment relates to methods of treating cancer in a patient with advanced or metastatic breast cancer who has not received any prior systemic therapies but has received CDK4/6i plus NSAI as the most recent adjuvant therapy.

T est Kits

In one aspect of the present invention, kits that include one or more compound of the present invention and a combination partner as disclosed herein are provided.

Representative kits include (a) a compound of the present invention or a pharmaceutically acceptable salt thereof, (b) at least one combination partner, e.g., as indicated above, whereby such kit may comprise a package insert or other labeling including directions for administration.

The disclosure further includes a diagnostic test kit comprising certain components for performing methods of the present disclosure. A diagnostic test kit enhances convenience, speed and reproducibility in the performance of diagnostic assays. For example, in an exemplary qRT- PCR-based embodiment of the disclosure, a basic diagnostic test kit includes PCR primers for analyzing expression of macrophage markers, e.g., cyclin-dependent kinase 6 (CDK6), cyclin E1 (CCNE1), cyclin D1 (CCND1), and/or Androgen Receptor (AR). In other embodiments, a more elaborate test kit contains not only PCR primers, but also buffers, reagents and detailed instructions for measuring CDK6, CCNE1 , CCND1 , and/or AR expression levels, using PCR technology. In some embodiments, the kit includes a test protocol and all the consumable components needed for the test, except the RNA sample(s). In an exemplary DNA microarray-based embodiment of the disclosure, a test kit includes a microfluidic card (array) designed for use with a particular instrument. Optionally, the microfluidic card is a custom-made device designed specifically for measurement of macrophage marker gene expression. Such custom microfluidic cards are commercially available. For example, the TaqMan Array is a 384-well microfluidic card (array) designed for use with the Applied Biosystems 7900HT Fast Real Time PCR System (Applied Biosystems, Foster City, CA). An exemplary fluidic card may include any combination of probes for measuring CCR2, CD14, CD68, CD163, CSF1 R and/or MSR1 expression plus necessary controls or standards, e.g., for data normalization. Other macrophage marker proteins can also be included on a fluidic card for practicing the disclosure.

In some embodiments of the disclosure, the test kit contains materials for determining tumor macrophage content by IHC. An IHC kit, for example, may contain a primary antibody against a human macrophage marker, e.g., a mouse anti-human CDK6, CCNE1 , CCND1 , and/or AR antibody, and a secondary antibody conjugated to a reporter enzyme, e.g., horseradish peroxidase. In some embodiments, the secondary antibody is replaced with a conjugated polymer that specifically recognizes the primary antibody.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

EXAMPLES

The present disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or content of the disclosure in any way.

General methods

Profiling and Selectivity

In-vitro profiling of CDK4 sensitivity either through genetic knockout or chemical inhibition has an advantage in that profiling can be accomplished across a large number of cancer cell line models. This enables the statistical power required to assign predictive features of response. Hypotheses generated in 2D can then be tested in more complex models that better recapitulate patient response (i.e., 3D and in-vivo models). Recent screening efforts have attempted, on a genome scale, to understand differences in cell line dependencies after genetic deletion of 18, 119 genes using whole genome CRISPR screens across 766 cell lines profiled in 27 diverse cancer lineages. Additionally, cell line sensitivity to CDK4 inhibition was assessed through BOE profiling of PF-07220060 across a panel of 650 cell lines. These datasets were used to understand differential lineage sensitivities to CDK4 inhibition and to identify features predictive of response pan-cancer and in a lineage specific manner.

Equipment

The envision plate reader was used to read SRB assay plates.

Software

All analyses are completed in R v. 3.6.0.

Cell culture

All cell lines were maintained in a humidified environment in the presence of 5% CO2 at 37°C and cultured according to manufacturer specifications.

Example 1

Chemical Profiling

Cells were collected when confluence reached 50-80%, counted using the Vi-Cell, diluted to proper seeding density and seeded into 96-well clear bottom plates in 90 uL complete media per well. Seeding densities were determined based on growth rate. Compound was added 24 hours post-seeding at 3-fold serial dilutions ranging from 0.45 nM to 3 uM (9 doses total). At 7 days post-compound treatment, cell viability was determined using a sulforhodamine B (SRB) assay (abeam). Briefly, 25uL/well of 50% TCA was added into the 96 well plates at 4 degrees for 1hour. Plates were washed with 200 uL/well of ddFW 4x. H2O as discarded and 50uL/well of 0.2% SRB in 1% acetic acid was added followed by incubation in the dark at room temperature for 30 minutes. 0.2% SRB was discarded, and the plate was washed using 200 uL/well of 1% acetic acid 4x. 50 uL/well of 10nM Tris-base buffer (pH 8.5) was added followed by vigorous shaking for 10 minutes. Absorbance was read at 570 nm by the Envision.

Example 2

Computational Methods

Elastic Net

The elastic net analysis was performed as described in McMillan et al, 2018, supra, PMID 29681454. In order to assign predictive biomarkers to each chemical, a penalized linear regression model, the elastic net, was used. Each dataset was individually and separately input into the elastic net. Candidate predictive features were selected from normalized measures of gene expression (RNAseq), protein expression (RPPA), and binary measures of gene mutational statuses (Whole Exome Sequencing). mRNA measures are likely to be the most variable across laboratories are subject to fluctuations from an in-vitro to an in-vivo system and are the most difficult to assess in patient samples. Thus, mRNA measures were only considered inputs into the elastic net if there was at least a two-fold difference between lowest and highest expressing cells. The elastic net assigns biomarkers to a response vector of activity scores by solving a basic linear regression problem as follows:

Let |X be the matrix of predictive features where n is the number of cell lines included in the training dataset and p is the number of features and let y £ be the vector of binary sensitivity values for the same cell line panel. Columns of the predictive features matrix and y were normalized to have a mean of zero and a standard deviation of 1. The elastic net attempts to find which weighted linear combination of the columns of the predictive features matrix can best approximate y, or it solves the following equation for w:

\ar.qmin_w [||v — Xuz||5]

The elastic net solves the above by enforcing a penalty to the solution that makes the solution both unique and sparse so that only the features that best approximate y are left with non-zero weight values. It does this by combining L1-norm and L2-norm regularization parameters so that the elastic net formulation to the above problem is given by: rqmin_w [||y - Xw||, + AfalM? + (1 - a) IMIill where |cr, are two adjustable parameters such that p controls the degree of the overall penalty and |a controls the degree to which the L1 norm and L2 norm constraints are applied so that when |a=0, only the L1 penalty is applied, and when only the L2 penalty is applied. In order to determine the optimal values of alpha and lambda to use in the model, we did 1000 iterations of 10-fold cross-validation where, in each iteration, the cells were randomly re-sampled into different groups. The values of alpha and lambda were chosen to be those that resulted in the minimum mean squared error for each fold. The data was subjected to a series of 1000 bootstrap permutations in which the cell lines were sampled with replacement, and features were assigned to each bootstrapped dataset. Features with a higher frequency of representation across bootstrapped permutations correspond to those with the highest confidence of being associated with drug response as opposed as being assigned due to overfitting of the model. Features were assigned to a chemical that were present in > 85% of permutations. Higher weighted features in the elastic net correspond to those with better mathematical predictive capacity and lowly weighted features may be assigned due to model overfitting. Cutoff values were assigned for weight values to select the most predictive features while still maintaining sparcity in the model. As weight magnitudes and distributions vary depending on input feature set and chemical sensitivity distributions, cutoffs were selected to be tailored to each chemical/feature set combination by selecting weight values +/- 2 standard deviations from the mean. Weight distributions follow a Gaussian distribution, so this cutoff corresponds to the top 5% of the most predictive features being assigned to each chemical. As input into the elastic net, both AUC and EC50 values as measures of sensitivities were used. Additionally, as log transformation of the sensitivity vector could better identify exceptional responders to a compound in some instances, both the linear and log transformed sensitivity measure as input to the algorithm were used. Non-transformed CERES scores (CDK4 gene dependency scores) were used as input from the Achilles dataset. In all cases, lower AUC/EC50 or CERES scores derived from Achilles correspond to higher sensitivities. The elastic net was run using the glmnet package in R.

ROC curves

Receiver operator curves (ROC) were used to assess significance of the elastic net model assigned to each response vector (BOE and Achilles CERES scores) and was performed as described in McMillan et al, 2018, supra, PMID 29681454. For each feature set, features were associated to a sensitivity vector and predicted sensitivities according to the procedures outlined above. To generate ROC curves for the sum of all markers across all feature sets, weights were first normalized within dataset so the weight with the highest absolute value was given a value of +/-1. A cutoff of Sj = 0 was used to binarize cell lines into predicted sensitive and resistant classes. Each response vector in our dataset was converted to a z-score. Cell lines were considered to be sensitive if z<=-0.5 and were considered to be resistant if z>0.5. The ROCR package in R was then used to calculate specificity (100 - false positive rate) and sensitivity (true positive rate) and plot the values. As input to the ROCR package, ‘true positives’ were considered to be those whose predictions were correct (sensitive cells predicted to be sensitive and resistant cells predicted to be resistant). Area under the ROC curve was calculated with and p-value was calculated to test the hypothesis that the area under the ROC curve is greater than 0.5 (random) using the ROCR R package.

Dose response curve (DRC) fitting

Chemical response for each cell line was converted to percent viability by normalizing to DMSO using the following equation:

^Xnorm=100*^{Xtreatment x}dmso

Where x indicates the CyQuant value at a particular dose. The DRC package were used in R to fit a standard 4 parameter log-logistic fit to the data and discover EC50 values. As imputed EC50 values have shown to be problematic in re-tests of large drug screening datasets, the values were not imputed. Rather, if the imputed EC50 value is greater than the top tested dose, an EC50 of the top dose was assigned. Additionally, to correct for low EC50 values being assigned to chemicals in which the response is shallow, an EC50 value of the top tested dose was assigned if the chemical does not result in at least 30% reduction in SRB values. To account for artificial inflation/deflation of AUC/EC50 associated with DMSO normalization, curves were artificially shifted so that the maximum values were always at the value x=100. AUC values were calculated by determining area under the curve of the log fitted hill equation through standard integral analysis using the midpoint rule. AUC values were normalized to the maximum dose range, so values range between 0-~1 , with 1 a value of 1 indicating the full curve (i.e. no response). The assigned EC50 or AUC value is then the mean between the replicates.

Example 3

In-vitro Selective Dependencies to CDK4 Depletion

To gain an understanding of in-vitro selective dependencies to CDK4 depletion, Broad’s DepMap CRISPR cell line data was utilized. A regularized machine learning protocols were applied to isolate mRNA, mutation or protein features that best predict sensitivities from this data. The expression of cell cycle regulators, including mRNA and/or protein expression of CDK6, CCNE1 , CCND1 , AR, and mutations and mRNA/protein expression of RB1 were among the top features significantly predicting sensitivity to CDK4 depletion pan-cancer (FIG. 1a). Using the weighted regression model to predict sensitivity in each lineage, showed differential predictive capacity of the markers across lineage. Interestingly, AR was the top predictive marker selectively in breast cancer, while CCND1 and CDK6 mRNA selectively predicted sensitivity in NSCLC and some other tumor types. Similar results were obtained when the weighted linear regression model was applied to the BOE cell line profiling study, where a large collection of human cancer cell lines, derived from a broad range of tumor types, was treated with PF-07220060 (FIG. 1b). CDK6 and CCNE1 findings were evaluated in multicellular tumor spheroid models of HR+ HER2- breast cancer. Increased endogenous expression of either gene by dCas-VPR-mediated transcriptional activation negates PF-07220060 induced spheroid growth inhibition (FIGS. 2 and 3). Likewise, ectopic expression of CDK6 leads to resistance to PF-07220060 induced spheroid growth inhibition (FIG. 4). Conversely, depletion of CDK6 by shRNA sensitizes HR+ HER2- breast cancer spheroids to PF-07220060 (FIG. 4). Consistent with this, the level of the CDK6 protein was inversely correlated with the tumor growth inhibition efficacy of PF-07220060 across a panel of selected xenograft models, comprising a variety of tumor types, including HR+ HER2- breast and prostate cancer, and mantle cell lymphoma (FIG. 5).

Claims

CLAIMS What is claimed is:

1. A method of treating cancer in a subject that exhibits modified levels compared to the baseline mRNA expression, protein expression, and/or activity of cyclin-dependent kinase 6 (CDK6), cyclin E1 (CCNE1), cyclin D1 (CCND1), and/or Androgen Receptor (AR), comprising administering to the subject an effective amount of PF-07220060, wherein the subject is eligible for treatment.

2. A method of treating cancer in a subject, wherein the subject exhibits: a. an absence or decreased levels compared to the baseline mRNA expression level, and/or protein expression level, and/or activity level of any one of CDK6 and CCNE1 ; and/or b. an increased levels compared to the baseline mRNA expression level, and/or protein expression level, and/or activity level of any one of CCND1 and AR; comprising administering to the subject an effective amount of PF-07220060, wherein the subject is eligible for treatment.

3. The method of any one of claim 1 or 2, further comprising administering to the subject an additional therapeutic agent, wherein the additional therapeutic agent comprises an endocrine therapy agent, a second CDK inhibitor, a PI3 kinase inhibitor, or a KAT6 inhibitor.

4. The method of any one of claims 1 to 3, further comprising administering to the subject an additional therapeutic agent, wherein the additional therapeutic agent comprises an endocrine therapy agent.

5. The method of any one of claims 1 to 4, wherein the endocrine therapy agent is an aromatase inhibitor, an androgen receptor inhibitor, a selective estrogen receptor degrader (SERD), or a selective estrogen receptor modulator (SERM).

6. The method of any one of claims 1 to 5, wherein the endocrine therapy agent is selected from the group consisting of letrozole, anastrozole, exemestane, fulvestrant, elacestrant, amcenestrant, giredestrant, RG6171 , camizestrant, AZD9496, rintodestrant, ZN-c5, LSZ102, D- 0502, LY3484356, SHR9549, tamoxifen, raloxifene, toremifene, lasofoxifene, bazedoxifene and afimoxifene.

7. The method of any one of claims 1 to 6, wherein the endocrine therapy agent is letrozole or fulvestrant.

8. The method of any one of claims 1 to 7, further comprising administering to the subject one or more of chemotherapy, immunotherapy, or radiation therapy, or combinations thereof.

9. The method of any one of claims 1 to 8, wherein the cancer is head, neck, lung, prostate, colon, pancreas, esophagus, liver, skin, kidney, adrenal gland, brain, or comprises a lymphoma, breast, endometrium, uterus, ovary, testes, lung, prostate, colon, pancreas, esophagus, liver, skin, kidney, adrenal gland, brain, Pan-cancer, KRAS-NSCLC, ewing sarcoma, or mantle cell lymphoma.

10. The method of any one of claims 1 to 9, wherein the cancer comprises a metastasis or recurring tumor, or neoplasia.

11 . The method of any one of claims 1 to 10, wherein the cancer comprises breast cancer or prostate cancer.

12. The method of any one of claims 1 to 11 , wherein the cancer is breast cancer.

13. The method of any one of claims 1 to 12, wherein the breast cancer is HR+ HER2- breast cancer.

14. A method of identifying a subject in need of treatment with PF-07220060, wherein the subject exhibits modified levels compared to the baseline mRNA expression, protein expression, and/or activity of CDK6, CCNE1 , CCND1 , and/or androgen receptor (AR), wherein the subject is eligible for treatment when: a. the level of mRNA expression, protein expression, and/or activity of CDK6 and/or CCNE1 is lower than the baseline, and/or b. the level of mRNA expression, protein expression, and/or activity of CCND1 and/or AR is higher than the baseline.

15. A method of identifying a subject eligible to receiving a therapeutic amount of PF- 07220060, the method comprising: a. identifying a subject eligible for treatment with a therapeutic amount of PF- 07220060; b. evaluating and screening the subject to identify if the subject has inactivating mutations, or other genomic alteration, including deletion, of Retinoblastoma Protein 1 gene (RB1); and c. excluding from treatment with PF-07220060, any subject identified as having inactivating mutations, or other genomic alteration, including deletion, of RB1.

16. A method of predicting and/or determining the responsiveness to PF-07220060 of a subject having cancer comprising: a. providing a test sample from the subject; b. assaying a level of phosphorylation of RB1 in the test sample during treatment or after administration of PF-07220060; and c. comparing the level of phosphorylation of RB1 in step b with the level of phosphorylation of RB1 in an untreated patient, wherein the absence of, or a significant decrease in level of phosphorylation of RB1 , in the test sample as compared to test sample of the untreated patient, indicates that the subject is responsive to the treatment with PF-07220060.

17. A method of predicting and/or determining the responsiveness to PF-07220060 of a subject having cancer comprising: a. providing a test sample from the subject; b. assaying a level of mRNA expression, protein expression and/or activity of CDK6 and CCNE1 in the test sample; and c. comparing the level of mRNA expression, protein expression and/or activity of CDK6 and CCNE1 in an untreated patient, wherein the subject is responsive to the treatment with PF-07220060 when level of mRNA expression, protein expression and/or activity of CDK6 and CCNE1 is elevated in the test sample compared to a normal control.

18. A method of predicting and/or determining the responsiveness to PF-07220060 of a subject having cancer comprising: a. providing a test sample from the subject; b. assaying a level of mRNA expression, protein expression and/or activity of CCND1 , and/or androgen receptor (AR), in the test sample; and c. comparing the level of mRNA expression, protein expression and/or activity of CCND1 , and/or AR, in an untreated patient, wherein the subject is the subject is responsive to the treatment with PF-07220060 when level of mRNA expression, protein expression and/or activity of CCND1 , and/or AR, is lower in the test sample compared to a normal control.

19. The method of any one of claims 16 to 18, wherein the test sample is selected from the group consisting of ex vivo and in vivo samples.

20. The method of claim 19, wherein the test sample comprises a bodily fluid from said subject.

21. The method of claim 20, wherein the bodily fluid comprises whole blood, a component of whole blood, plasma, or serum.

22. The method of any one of claims 16 to 18, wherein the test sample comprises a tissue biopsy.

23. The method of any one of claims 16 to 22, wherein the assay comprises contacting the test sample with an antibody that recognizes and/or binds CDK6, CCNE1 , CCND1 , AR and/or RB1 , wherein an increased mRNA and/or protein expression of CDK6, CCNE1 , CCND1 , AR and/or RB1 indicates responsiveness to PF-07220060.

24. A method of predicting dependency of cancer on CDK4 and sensitivity to PF-07220060, wherein: a) a CDK4 to CDK6 (CDK4/CDK6) mRNA and/or protein expression ratio is quantified and wherein the CDK4/CDK6 ratio is a predictor (positive correlation); b) a CDK6 mRNA and/or protein is quantified and wherein CDK6 mRNA and/or protein is a predictor (inverse correlation); c) a CCND1 mRNA and/or protein is quantified and wherein CCNDImRNA and/or protein is a predictor (positive correlation); d) RB1 mutations and/or RB1 deletions are identified and wherein RB1 mutations and/or RB1 deletions are predictors (inverse correlation); or e) a CCNE1 mRNA and/or protein is quantified and wherein CCNE1 mRNA and/or protein is a predictor (inverse correlation).

25. A method for adjusting the dose of PF-07220060 that is administered to a subject during a treatment regimen for cancer that comprises determining the absence of, or a significant decrease in level of phosphorylation of RB1, and a. increasing the dose of the PF-07220060 if there is a detectable phosphorylation of RB1 ; or b. maintaining the dose of the PF-07220060 if the level of a phosphorylation of RB1 is sufficiently decreased over time, wherein determining the level of comprises: i. providing a test sample from said subject; and ii. assaying the level of phosphorylation of RB1 in the test sample.

26. A method of diagnosing a subject having cancer that is responsiveness to PF-07220060, comprising contacting the subject with an agent that detects the presence of CDK6 and/or CCNE1 in the cancer or a sample thereof, wherein modified levels compared to the baseline mRNA expression, protein expression and/or activity of CDK6, CCNE1 , CCND1 , Androgen Receptor (AR) and mutations of Retinoblastoma Protein 1 (RB1), is diagnostic of cancer that is responsiveness to PF-07220060.

27. A kit comprising a. an agent for detecting the level of CDK6 and/or CCNE1 ; and b. instructions for using the agent for diagnosing or detecting level of CDK6 and/or CCNE1 , for identifying whether a subject is responsiveness to PF-07220060 for determining the progression of cancer, for assessing the efficacy of a treatment for the cancer, and/or for adjusting the dose of PF-07220060, during the treatment of cancer.

28. A diagnostic system comprising: a. an assortment, collection, or compilation of test results data representing the level of CDK6 and/or CCNEI in a plurality of test samples; b. a means for computing an index value using said level, wherein the index value comprises a diagnostic, prognostic, progression, or treatment score; and c. a means for reporting the index value.