WO2021150945A1 - Méthodes de diagnostic et de traitement du cancer - Google Patents

Méthodes de diagnostic et de traitement du cancer Download PDF

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WO2021150945A1
WO2021150945A1 PCT/US2021/014697 US2021014697W WO2021150945A1 WO 2021150945 A1 WO2021150945 A1 WO 2021150945A1 US 2021014697 W US2021014697 W US 2021014697W WO 2021150945 A1 WO2021150945 A1 WO 2021150945A1
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cancer
cdk4
cdk6
cell
tumor
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Poulikos Poulikakos
Xuewei Wu
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Icahn School Of Medicine At Mount Sinai
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism

Definitions

  • This disclosure relates generally to methods of diagnosing and treating cancer, based on CDK6 and CDK4 levels in tumors.
  • Cyclin-dependent kinase 4 (CDK4, also known as CMM3 and PSK-J3) (Matsushime et al, (1992) Cell Oct 16;71(2):323-34) and cyclin-dependent kinase 6 (CDK6, also known as MCPH12 and PLSTIRE) (Meyerson M. and Harlow E., (1994) Mol Cell Biol. Mar;14(3):2077-86) are related serine/threonine kinases (referred to together as “CDK4/6”) that play a critical role in the cyclin D/CDK4/6/Rb/E2F signaling pathway (“CDK4/6/Rb signaling”) (Sherr C.J.
  • CDK4/6 regulate cell cycle progression by phosphorylating and inactivating the tumor suppressor Retinoblastoma protein (Rb) and thus have been targeted by small molecule inhibitors for cancer therapy (Sherr C.J. et al, (2016) Cancer Discov 6, 353-367; O'Leary B. Nature Reviews. Clinical Oncology (2016) 13, 417-430).
  • CDK4/6 inhibitors (“CDK4/6i”) are established therapeutics for metastatic breast cancer. CDK4/6 inhibitors (CDK4/6i) in combination with hormonal therapy showed significant clinical activity in Rb-proficient metastatic ER positive breast cancers (B.
  • CDK4/6 Since the activity of CDK4/6 requires a functional RB protein, tumors that do not express functional Rb are resistant to these drugs (E. S. Knudsen, E.S. and Witkiewicz, A.K. (2017) Trends Cancer 3, 39-55). However, in many tumor types predominantly expressing wild-type RBI (lung adenocarcinomas, melanomas, colon cancers etc) preclinical and clinical studies have shown only modest effectiveness of CDK4/6i (Hamilton, E. et al, (2016) Cancer Treat Rev 45, 129-138; Gong X. et al, (2017) Cancer Cell 32, 761-776; Kim S.
  • the present disclosure relates in general to the use of CDK6 and/or the CDK4:CDK6 ratio as a predictive biomarker for drug response of tumors to CDK4/6- directed therapies. Described herein are methods of treating subjects with particular CDK4/6-related therapies based on the subject’s CDK6 level and/or CDK4 to CDK6 ratio. Also described herein are kits for measuring CDK6 and/or CDK4 levels.
  • the disclosure features a method for treating a cancer in a human subject, the method comprising: (a) obtaining a tumor sample from the subject; (b) determining a level of cyclin dependent kinase 6 (CDK6) in the tumor sample; and (c) comparing the level of CDK6 as determined in (b) with a reference level in a control; wherein when the level of CDK6 in the tumor is lower than the reference level of CDK6 in the control, the tumor is classified as a CDK4-dependent tumor and treated with a therapeutically effective amount of one or more of a CDK4/6 inhibitor and a CDK4/6- directed PROTAC.
  • CDK6 cyclin dependent kinase 6
  • the disclosure features for treating a cancer in a human subject, the method comprising: (a) obtaining a tumor sample from the subject; (b) determining levels of CDK6 and CDK4 in the tumor sample; and (c) determining the ratio of CDK4 to CDK6 levels; wherein (i) when the CDK6 level is below a threshold level and/or the CDK4 to CDK6 ratio is above a threshold ratio, the tumor is classified as a CDK4-dependent tumor and the subject is treated with a composition comprising a therapeutically effective amount of a CDK4/6-directed PROTAC; or (ii) when the CDK6 level is above a threshold level and/or the CDK4 to CDK6 ratio is below a threshold ratio, the tumor is classified as a CDK6-dependent tumor and the subject is treated with a composition comprising a therapeutically effective amount of a CDK4/6 inhibitor.
  • the CDK6 threshold level in the biological sample is below about 5.64 CPM, as determined by
  • the CDK4/6 inhibitor is one or more agents selected from a group consisting of abemaciclib (Verzenio), dinaciclib. palbociclib (Ibrance), ribociclib (Kisqali), trilaciclib (G1T28), G1T38, and the group of CDK4/6 inhibitor compounds referred to in International Patent Application Publications W02016040858, WO2011130232, WO2011101409, W02016025650, and W02013006532 and the CDK4/6-directed PROTAC is one or more agents selected from MS 140, the PROTACs referred to in FIGs. 14-18, and bivalent compounds referred to in International Patent Application Publication WO2018106870.
  • the CDK4/6 inhibitor and/or CDK4/6-directed PROTAC is administered in combination with one or more additional therapeutic regimens selected from the group consisting of surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and immunotherapy.
  • the targeted therapy is with one or more agents selected from a group consisting of MEK inhibitors, ERK inhibitors, hormonal therapy, and RAS(G12C) inhibitors.
  • the cancer is a solid tumor cancer.
  • the cancer is a cancer expressing functional retinoblastoma protein (Rb).
  • the disclosure provides a method of treating a cancer in a human subject in need thereof, comprising administering to the human subject a therapeutically effective amount of a cancer therapy, wherein the human subject has been previously determined to have, in a biological sample obtained from the human subject, at least one of (a) a CDK6 level prior to initiation of the cancer therapy that is lower than a reference level in a control, and (b) a ratio of CDK4 to CDK6 levels prior to initiation of the cancer therapy that is higher than a reference level in a control.
  • the disclosure provides a method for treating a human subject with cancer, comprising (a) measuring a level of CDK6 and optionally measuring a level of CDK4, and further, optionally determining a ratio of the CDK4 to CDK6 levels in a biological sample taken from the subject; and (b) treating the subject with a therapeutically effective amount of a cancer therapy if the measured levels of CDK6 and optionally the ratio of CDK4 to CDK6 indicate that the subject is a candidate for receiving the cancer therapy.
  • the cancer therapy is one or more agents selected from a group consisting of abemaciclib (Verzenio), palbociclib (Ibrance), ribociclib (Kisqali), trilaciclib (G1T28), G1T38, and the group of CDK4/6 inhibitor compounds referred to in International Patent Application Publications W02016040858, WO2011130232, WO2011101409, W02016025650, and W02013006532, MS140, the PROTACs referred to in FIGs. 14-18, and the group of bivalent compounds referred to in International Patent Application Publication W02018106870.
  • the cancer therapy is administered in combination with one or more additional therapeutic regimens selected from the group consisting of surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and immunotherapy.
  • the targeted therapy is with one or more agents selected from a group consisting of MEK inhibitors, ERK inhibitors, hormonal therapy, and RAS(G12C) inhibitors.
  • the cancer is a solid tumor cancer or a leukemia. In some embodiments, the cancer is a cancer expressing functional retinoblastoma protein (Rb).
  • Rb functional retinoblastoma protein
  • the solid tumor cancer is selected from a group consisting of non-small-cell lung carcinoma (NSCLC), lung cancer, breast cancer, ewing sarcoma, central nervous system neoplasm, skin cancer, head and neck cancer, ovarian cancer, colon cancer, anal cancer, stomach cancer, gastrointestinal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, testicular cancer, brain stem glioma, pituitary cancer, adrenocortical cancer, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, lymphoma, liver cancer, kidney cancer, bone cancer, bladder cancer, colorectal cancer, endometrial cancer, renal cell cancer, pancreatic cancer, prostate cancer, thyroid
  • NSCLC non-small-
  • the leukemia is acute myeloid leukemia (AML) and T-cell acute lymphocytic leukemia (T-ALL), Mantle Cell Lymphoma (MCL), and Multiple Myeloma (MM).
  • AML acute myeloid leukemia
  • T-ALL T-cell acute lymphocytic leukemia
  • MCL Mantle Cell Lymphoma
  • MM Multiple Myeloma
  • the disclosure provides a method for predicting prognosis of a human subject with cancer, comprising (a) obtaining a biological sample of the cancer from the subject; (b) determining a level of CDK6 in the biological sample; and (c) comparing the level of CDK6 as determined in (b) with a reference level of CDK6 in a control; wherein a lower level of CDK6 in the biological sample compared to the CDK6 reference level in the control is an indication that the cancer in the subject will be successfully treated with a CDK4/6 inhibitor.
  • the disclosure provides a method of predicting response to a cancer therapy or predicting disease progression in a human subject with cancer comprising: (a) obtaining a biological sample from the subject; (b) determining levels of CDK4 and CDK6 in the sample and obtaining the ratio of CDK4/CDK6; (c) based on the determinations of step (b), determining a probability of response to the cancer therapy or a future risk of cancer progression in the subject.
  • the levels of CDK6 and/or CDK4 are measured by determining one or more of the mRNA levels, cDNA levels and protein levels of CDK6 and/or CDK4.
  • the disclosure provides a method of identifying a compound capable of treating cancer, or identifying a compound capable of reducing the risk of developing cancer, or identifying a compound capable of reducing the risk of cancer recurrence or development of metastatic cancer, comprising: (a) providing a cell expressing CDK6; (b) contacting the cell with a candidate compound; and (c) determining whether the candidate compound reduces the expression or activity of CDK6; wherein the reduction observed in the presence of the compound indicates that the compound is capable of treating cancer, or reducing the risk of developing cancer, or reducing the risk of cancer recurrence or development of metastatic cancer.
  • the disclosure provides a kit comprising means for quantifying the levels of CDK6 and/or CDK4.
  • the kit comprises reagents for specifically measuring the levels of CDK6 and/or CDK4 in a biological sample.
  • the reagents are nucleic acid molecules or antibodies.
  • FIG. 1A shows a bar graph of substantial growth inhibition (GI90) values based on cell growth crystal violet assays for the indicated cell lines treated with increasing concentrations of Palbociclib (PB) for 10-15 days.
  • the CDK4/6i- sensitive or resistant cell lines are indicated.
  • FIG. IB shows immunoblots of the indicated antibodies in lysates of the indicated cell lines treated with 1 mM PB for 24, 48 and 72 hr.
  • FIG. 1C shows immunoblots of the indicated antibodies in lysates of the indicated cell lines treated with 3 mM Ribociclib (RB) for 24, 48 and 72 hr.
  • FIG. ID shows immunoblots of the indicated antibodies in lysates of the indicated cell lines treated with 0.3 mM Abemaciclib (AB) for 24, 48 and 72 hr.
  • FIG. IE shows immunoblots of the indicated antibodies for cell cycle regulators (pRb, CDK6, CDK4, pT172-CDK4, CRBN, CDK2, cyclins D1-D3, cyclin E2, p21, p27, pi 6) and the control (actin) in lysates of the indicated cell lines.
  • FIG. 2A shows A549 and A375 cell lines expressing Doxycycline (Dox)- inducible short hairpin CDK4 (shCDK4) or shCDK6 treated with 0.1 pg/ml doxycycline for 36 hr, followed by the indicated concentrations of PB for 24 hr.
  • Cell lysates were immunoblotted with the indicated antibodies for cell cycle regulators and the control (actin).
  • FIG. 2B shows cell growth crystal violet assay for A549 and A375 cell lines expressing Dox-inducible shCDK4 or shCDK6 in the presence or absence of 0.1 pg/ml doxycycline and increasing concentrations of PB for 10 days.
  • FIG. 2C top panel shows cell growth crystal violet assay for the indicated cell lines (A549, SK-MEL-2, Calu-6, and HCT116) expressing Dox-inducible shCDK4 or shCDK6 in the presence or absence of 0.1 pg/ml doxycycline for 10 days followed by crystal violet staining.
  • the bottom panel shows immunoblots of the indicated antibodies for cell cycle regulators and control (actin) in lysates of the indicated cell lines treated with or without 0.1 pg/ml doxycycline for 72 hr.
  • FIG. 2D top panel shows cell growth crystal violet assay for the indicated cell lines (A375, A549, Calu-6, and HCT116) expressing Dox-inducible shCDK6 or shRNA- resistant form of CDK6 (shr-CDK6) in the presence or absence of 0.05 pg/ml doxycycline for 10 days followed by crystal violet staining.
  • the bottom panel shows immunoblots of the indicated antibodies for cell cycle regulators and control (actin) in lysates of the indicated cell lines treated with or without 0.05 pg/ml doxycycline for 72 hr.
  • 2E top panel shows cell scatterplots of DEMETER score (DepMap RNAi; DEMETER2 Data v5) and expression for CDK4 and CDK6.
  • the bottom panel shows scatterplots of CERES (DepMap CRISPR;Public 20Q1) score and the mass spectrometry- based proteomics levels of CDK4 and CDK6 (Nusinow DP et al, 2020). All expression values are in log2(TPM +1). Proteomic levels are shown as normalized log2 -transformed ratios to the bridge sample in each Tandem Mass Tags (TMT) 10-plex as previously described (Nusinow DP and Gygi SP, 2020; BIORXIV: doi:10.1101/2020.02.03.932384). Cell lines harboring COSMIC hotspot mutations to RBI are annotated in light dots. P- values were calculated based on linear regression analysis.
  • FIG. 3A shows immunoblots of the indicated antibodies for cell cycle regulators and control (actin) in lysates of the indicated NSCLC cell lines transfected with control short interfering RNA (siRNA), siCDK4 and siCDK6 for 72 hr.
  • HI 792, H2087, H2291, HCC827, and HI 915, are CDK4/6i-sensitive cell lines.
  • Calu6, and HC1666 are CDK4/6i-resistant cell lines.
  • FIG. 3B shows immunoblots of the indicated antibodies for cell cycle regulators and control (actin) in lysates of the indicated NSCLC cell lines.
  • the ratio of CDK4:CDK6 in the various cell lines is also indicated.
  • MCF7, H1792, H358, H2087, H2291, and HCC827 are CDK4/6i-sensitive cell lines.
  • Calu6, A549, PC9 and HC1666 are CDK4/6i-resistant cell lines.
  • FIG. 3C shows immunoblots of the indicated antibodies for cell cycle regulators in lysates of the indicated NSCLC cell lines treated with increasing concentrations of PB for 24 hr.
  • H358, H2291, H2087, H1792, and HCC827 are CDK4/6i-sensitive cell lines.
  • PC9 is a CDK4/6i-resistant cell line.
  • FIG. 3D shows cell growth crystal violet assays for the indicated NSCLC cell lines treated with increasing concentrations of PB for 10-15 days and stained with crystal violet.
  • H358, H2291, H2087, H1792, and HCC827 are CDK4/6i-sensitive cell lines.
  • PC9 is a CDK4/6i-resistant cell line.
  • FIG. 3E shows progression-free survival analysis of NSCLC/RAS-mutant patients that received abemaciclib in the JUNIPER trial based on CDK6-low versus CDK6-medium/high tumors.
  • ITT Translational Research population; Abema: Abemaciclib
  • FIG. 3F shows overall survival analysis of NSCLC/RAS-mutant patients that received abemaciclib in the JUNIPER trial based on CDK6-low versus CDK6- medium/high tumors.
  • HR Hazard Ratio
  • ITT Translational Research population
  • Abema Abemaciclib
  • FIG. 4 A shows the chemical structure of the bifunctional CDK4/6 inhibitor- degrader MS140.
  • FIG. 4B shows the IC50 of in vitro kinase activity assays for PB and MS 140 against CDK4/cyclin D1 and CDK6/cyclin Dl.
  • FIG. 4C shows immunoblots of the indicated antibodies for CDK4, CDK6 and control (actin) in lysates of Colo205 cells pretreated with either the proteasome inhibitor 100 nM bortezomib (BOR), 10 mM PB, 10 pM pomalidomide (POM), the Nedd8- activating enzyme inhibitor 1 pM MLN4924 (MLN), or control (DMSO) for 4 hr, followed by treatment with MS 140 (100 nM/3 hr).
  • BOR proteasome inhibitor 100 nM bortezomib
  • POM 10 mM PB
  • POM 10 pM pomalidomide
  • MN Nedd8- activating enzyme inhibitor 1 pM MLN4924
  • DMSO control
  • FIG. 4D shows immunoblots of the indicated antibodies for CDK4, CRBN and control (actin) in lysates of ZR-75-1 wild-type and CRBN-deficient cells (ZR-75-1 sgCRBN) treated with the indicated concentrations of MS 140 for 5 hr.
  • FIG. 4E shows immunoblots of the indicated antibodies and control (actin) in lysates of Colo205 cells treated with DMSO (control), MS140-ve and MS 140 for 5 hr.
  • FIG. 4F shows a volcano plot of the protein log2 ratios which represents the quantitative dynamics of 4,822 proteins in MS 140 and MS 140 negative control (140-ve) treated Colo205 samples (0.3 pM, 5 hr). The experiment was conducted in duplicate.
  • FIG. 4G shows a cell growth crystal violet assay in the presence of varying concentrations of PB or MS 140 for 10-15 days.
  • FIG. 4H shows immunoblots of the indicated antibodies and control (actin) in lysates of the indicated cell lines treated with increasing concentrations of PB or MS 140 for 24 hr.
  • FIG. 41 shows immunoblots of the indicated antibodies and control (actin) in lysates of KMS-12-PE and Pfeiffer cells treated with increasing concentrations of PB or MS 140 for 24 hr. Lysates were immunoblotted with the indicated antibodies.
  • FIG. 4J shows cell viability of KMS-12-PE and Pfeiffer cells treated with PB or MS140 for 72-96 hr. Cell viability was assayed using 0.1 mg/ml resazurin solution. IC50 values were determined by nonlinear regression curve fit in Graphpad Prism in six replicates.
  • FIG. 4K shows immunoblots of the indicated antibodies and control (actin) in lysates of tumor samples from mice carrying JeKo-1 xenografts.
  • the mice were treated with vehicle or MS140 (25 mg/kg, b.i.d) or PB (50 mg/kg, q.d.) for 3 days.
  • FIG. 5A shows cell growth crystal violet assays for the indicated cell lines treated with increasing concentrations of PB or MS 140 for 10-12 days.
  • FIG. 5B shows immunoblots of the indicated antibodies in lysates of A375, SKMEL2 and Calu6 cells were treated with increasing concentrations of PB or MS 140 for 24 hr.
  • FIG. 5C shows immunoblots of the indicated antibodies in lysates of A549 or A375 expressing Dox-inducible shCDK6 were treated with either the indicated concentrations of doxycycline (A375: 0.1 pg/ml) or MS 140 for 72 hr.
  • FIG. 5D shows cell growth crystal violet assays for the indicated cell lines treated with either doxycycline or MS 140 for 10 days. Colonies were stained with crystal violet.
  • FIG. 5E shows immunoblots of the indicated antibodies in lysates of Rb- proficient cell lines treated with MS 140 (3 nM) for the indicated time points.
  • FIG. 5F shows graphs of relative band intensities (%) derived by immunoblot analysis of CDK6 and actin expression using Image J.
  • the indicated cell lines were treated with PB (1 pM/2 hr) followed by a Cellular Thermal Shift Assay (CETSA).
  • FIG. 5G shows graphs of relative band intensities (%) derived by immunoblot analysis of CDK6 and actin expression using Image J.
  • the indicated cell lines were treated with MS140-ve (15 pM/2 hr) followed by CETSA assay.
  • FIG. 5H shows immunoblots of the indicated antibodies in lysates of MV4-11 and SKMEL2 cells pretreated with increasing concentrations of PB for 2 hr and the lysates subjected to a desthiobiotin-ADP enrichment assay for CDK6.
  • FIG. 6A shows immunoblots of the indicated antibodies for cell cycle regulators in lysates from CDK6-dependent cell lines.
  • FIG. 6B top panel shows a volcano plot of the CDK6-interacting proteins in KMS-12-PE and Calu6. Proteins in large circles were annotated as HSP90/CDC37- related. The bottom panel shows a comparison of total peptide-spectrum match (PSM) for the HSP90 protein family and CDC37 in KMS-12-PE and Calu6.
  • PSM total peptide-spectrum match
  • FIG. 6C shows immunoblots from the lysates of indicated cell lines which were either subjected to co-immunoprecipitation with a CDK6 antibody followed by immunoblotting with HSP90, CDC37 and CDK6, or immunoblotted with the indicated antibodies.
  • FIG. 6D shows immunoblots of the indicated antibodies in lysates of cell lines treated with the HSP90 inhibitor Ganetespib (GAN, 30 nM) at the indicated time points.
  • FIG. 6E shows immunoblots representing CDK6 thermal stability assay (CETSA) in lysates of CDK4/6i- sensitive (KMS-12-PE, MV4-11, Pfeiffer, and Colo205) and CDK4/6i-resistant (Calu6, A375, A549, SKMEL2 and the Rb-null BT549) cell lines heat-treated at increasing temperature end points.
  • CETSA CDK6 thermal stability assay
  • FIG. 6F shows immunoblots of the indicated antibodies in lysates of indicated cell lines treated with 100 pg/ml CHX at the indicated time points.
  • FIG. 6G shows immunoblots of the indicated antibodies in lysates of A375 cells transfected with either WT or CDK6 (S178P) followed by treatment with increasing concentrations of MS 140 for 24 hr.
  • FIG. 6H shows relative mRNA expression by qPCR analysis of the indicated Rb/E2F target genes in tumors, kidney and liver from mice bearing JeKo-1 tumors treated with vehicle or MS140 (25 mg/kg, b.i.d) or PB (50 mg/kg, q.d.) for 3 days.
  • Data represents mean ⁇ S.D. of triplicates.
  • FIG. 7 shows a model of CDK6 association with the HSP90 complex affecting tumor cell sensitivity to CDK4/6 inhibitors and degraders.
  • FIG. 7A depicts that in CDK4/6 inhibitor and degrader-sensitive cells, CDK6 is associated with the HSP90 complex. CDK4/6 inhibitors or CDK6 degraders bind strongly to CDK6, and promote CDK6 inhibition or both CDK6 inhibition and degradation respectively.
  • FIG. 7B depicts that in CDK4/6 inhibitor and degrader-resistant cells, CDK6 is weakly associated with the HSP90 complex. In these cells, CDK4/6 inhibitors and degraders bind CDK6 weakly, and thus fail to promote CDK6 inhibition or both inhibition and degradation, respectively.
  • FIG. 8A shows cell growth crystal violet assays for the indicated cell lines treated with increasing concentrations of PB for 10-16 days and stained with crystal violet.
  • CDK4/6i-sensitive cell lines are in the left panel
  • CDK4/6i-resistant cell lines are in the right panel.
  • FIG. 8B shows immunoblots of the indicated antibodies in lysates from MCF7 and HCT116 cell lines treated with 1 mM PB for 24, 48 and 72 hr.
  • FIG. 8C shows immunoblots of the indicated antibodies in lysates from Colo205 cells were treated with 1 pM PB at the indicated time points.
  • FIG. 9A shows immunoblots of the indicated antibodies in lysates from A673 and TC-71 cells transfected with non-targeting control or siCDK4 or siCDK6 for 72 hr.
  • FIG. 9B shows the relationship between CDK4 and CDK6 expression (CCLE RNA-seq) and DepMap CRISPR-Cas9 single-gene knockout scores (CERES; 20Q1 public dataset). All expression values are in log2(TPM +1). Cell lines harboring COSMIC hotspot mutations to RBI are annotated in light dots. P- values were calculated based on linear regression analysis.
  • FIG. 10A shows immunoblots of the indicated antibodies in lysates from cell lines treated with increasing concentrations of PB for 24 hr.
  • FIG. 10B shows GI90 values of PB and CDK4/6 dependency in NSCLC cell lines.
  • Cell lines NCIH358, NCIH2087, NCIH1792, NCIH2291, AND HCC827 are CDK4/6- sensitive cell lines; Calu6, A549, HI 666, and PC9 are CDK4/6-resistant cell lines.
  • FIG. 11A shows immunoblots of the indicated antibodies in lysates from Colo205 cells were treated with increasing concentrations of MS 140 for 5 hr.
  • FIG. 11B shows immunoblots of the indicated antibodies in lysates from Colo205 treated with MS 140 (0.5 mM) for the indicated time points.
  • FIG. llC shows immunoblots of the indicated antibodies in lysates from T47D cells pretreated with either the proteasome inhibitor 100 nM bortezomib (BOR), 10 pM PB, 10 pM pomalidomide (POM) or 1 pM MLN4924 (MLN) for 4 hr, followed by treatment with MS 140 (100 nM/3 hr).
  • BOR bortezomib
  • POM 10 pM pomalidomide
  • MN 1 pM MLN4924
  • FIG. 11D shows the chemical structure of the MS 140 negative control (MS 140- ve) that does not bind CRBN.
  • FIG. HE shows immunoblots of the indicated antibodies in lysates from U87MG and MCF7 treated with increasing concentrations of PB or MS 140 for 24 hr.
  • FIG. HF shows immunoblots of the indicated antibodies in lysates from H358 cells treated with increasing concentrations of MS 140 for 24 hr.
  • FIG. HG shows cell viability (%) of Mantle Cell Lymphoma (MCL) cells treated with PB or MS 140 for 72-96 hr.
  • the cell viability was assayed using 0.1 mg/ml resazurin solution.
  • IC50 values were determined by nonlinear regression curve fit in Graphpad Prism in six replicates.
  • FIG. HH shows immunoblots of the indicated antibodies in lysates from MCL cell lines treated with increasing concentrations of PB or MS 140 for 24 hr.
  • FIG. HI shows immunoblots of the indicated antibodies in lysates from MCL cell lines treated with 0.1 pM PB or MS 140 at different time points.
  • FIG. 11 J shows GLo values of PB and MS 140 in hematologic cancer cell lines.
  • FIG. HK shows immunoblots of the indicated antibodies in lysates from tumor samples in mice carrying Colo205 xenografts.
  • the mice were treated with vehicle or MS 140 (30 mg/kg, b.i.d) for 3 days.
  • FIG. 11L shows scatter plot of fold change for an efficacy assay in Colo205 tumor xenografts in nude mice treated with vehicle or MS140 (30 mg/kg, b.i.d) for 21 days.
  • Data represent mean ⁇ S.D., unpaired two-tailed t-test.
  • FIG. 12A shows immunoblots of the indicated antibodies in lysates from MV4-11 and A375 treated with MS140 (3 nM) or YKL-06-102 (3 nM) or BSJ-02-162 (3nM) at different time points.
  • FIG. 12B shows immunoblots of the indicated antibodies in lysates from KMS- 12-PE and Calu6 treated MS140 (3 nM) or YKL-06-102 (3 nM) or BSJ-02-162 (3 nM) at different time points. Lysates were subjected to immunoblotting with the indicated antibodies.
  • FIG. 13A shows immunoblots of the indicated antibodies in lysates from the indicated cell lines were either subjected to co-immunoprecipitation with a CDK6 antibody followed by immunoblotting with or immunoblotted with the indicated antibodies.
  • FIG. 13B shows immunoblots of the indicated antibodies in lysates from the indicated cell lines treated with increasing concentrations of Ganetespib (GAN) for 24 hr.
  • GAN Ganetespib
  • FIG. 13C shows immunoblots of the indicated antibodies in lysates from the indicated cell lines treated with 40 nM Luminespib (LUM) at the indicated time points.
  • FIG. 13D shows immunoblots of the indicated antibodies in lysates from CDK4- dependent cell lines treated with 30 nM GAN for the indicated time points.
  • FIG. 13E shows immunoblots of the indicated antibodies in lysates from KMS- 12-PE and Calu6 treated with increasing concentrations of GAN for 24 hr.
  • FIG. 13F shows immunoblots of the indicated antibodies in lysates from A375 cells ectopically expressing V5-CDK6 or V5-CDK6 S178p immunoprecipitated with a V5 antibody. The immunoprecipitates were subjected to kinase assay with recombinant Rb protein as substrate.
  • FIG. 13G shows immunoblots of the indicated antibodies in lysates from KMS- 12-PE and BT549 cells treated with increasing concentrations of MS140 for 24 hr. Lysates were subjected to immunoblotting with the indicated antibodies.
  • Data represent mean ⁇ S.D., paired two- tailed t-test.
  • FIG. 14A shows the chemical structures of various palbociclib-based PROTACs. The linkers are depicted for each named structure.
  • FIG. 14B shows the chemical structures of various ribociclib-based PROTACs. The linkers are depicted for each named structure.
  • FIG. 14C shows the chemical structures of abemaciclib-based PROTACs. The alternative linkers are depicted for the named PROTAC.
  • FIG. 15 shows the chemical structures of various palbociclib-based PROTACs. The linkers are depicted for each named structure.
  • FIG. 16 shows the chemical structures of various palbociclib-based PROTACs. The linkers are depicted for each named structure.
  • FIG. 17 shows the chemical structures of various palbociclib-based PROTACs. The linkers are depicted for each named structure.
  • FIG. 18 shows the chemical structures of three different PROTACs.
  • the articles a, an, and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
  • description referring to “about X” includes description of “X.”
  • Numeric ranges are inclusive of the numbers defining the range. As used herein, the term “about” permits a variation of ⁇ 10% within the range of the significant digit.
  • the terms “treat”, “treating” and “treatment” and variations thereof refer to partially or completely alleviating, inhibiting, ameliorating, or relieving the disease or condition from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder (e.g., cancer) are ameliorated or otherwise beneficially altered.
  • a disease or disorder e.g., cancer
  • amelioration of the symptoms of a particular disorder refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with treatment by the compositions and methods of the present disclosure.
  • the treatment can promote or result in, for example, a decrease in the number of tumor cells (e.g., in a subject) relative to the number of tumor cells prior to treatment; a decrease in the viability (e.g., the average/mean viability) of tumor cells (e.g., in a subject) relative to the viability of tumor cells prior to treatment; a decrease in the rate of growth of tumor cells; a decrease in the rate of local or distant tumor metastasis; or reductions in one or more symptoms associated with one or more tumors in a subject relative to the subject's symptoms prior to treatment.
  • the term “treating cancer” or “treating a tumor” means causing a partial or complete decrease in the rate of growth of a tumor, and/or in the size of the tumor and/or in the rate of local or distant tumor metastasis, and/or the overall tumor burden in a subject, and/or any decrease in tumor survival, in the presence of a compound (e.g., an CDK4/6 inhibitor/degrader) described herein.
  • a compound e.g., an CDK4/6 inhibitor/degrader
  • “treat” and its variations refers to slowing the progression or reversing the progression of cancer relative to an untreated control.
  • Exemplary CDK4/6-mediated cancers that can be treated with the methods of this disclosure, include but are not limited to, solid tumors (e.g., breast cancer (e.g., ER+ breast cancer) and prostate cancer), leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, and myeloid leukemia), lymphoma (e.g., Burkitt's lymphoma, cutaneous T- cell lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin's lymphoma, mantel cell lymphoma, and non-Hodgkin's lymphoma (NHL)), adrenocortical cancer, AIDS
  • Ewing sarcoma soft tissue sarcoma, Sezary syndrome, squamous cell carcinoma, squamous neck cancer, synovial sarcoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer, trophoblastic tumors, urethral cancer, uterine cancer, fallopian tube cancer, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.
  • biological sample refers to a sample obtained from a subject.
  • biological samples include any clinical samples useful in the detection of cancer in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as: blood; derivatives and fractions of blood, such as serum; biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin; swabs; skin scrapes; urine; sputum; cerebrospinal fluid; prostate fluid; pus; or bone marrow aspirates.
  • a sample includes a solid tumor biopsy obtained from a human subject.
  • a sample includes cells, for example a group of cells collected as part of a tissue section.
  • the term “expressed” or “expression” refers to the transcription from a gene to a ribonucleic acid (RNA) molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene.
  • RNA ribonucleic acid
  • the term “expressed” or “expression” may refer to the translation from the RNA molecule to give a protein, a polypeptide or a portion thereof.
  • CDK6 and/or CDK4 gene expression can be detected as, e.g., protein or RNA expression of a target gene. That is, the presence or expression level (amount) of a gene can be determined by detecting and/or measuring the level of mRNA or protein expression of the gene.
  • gene expression can be detected as the activity of a protein encoded by the CDK6 gene.
  • gene expression can be detected as the activity of a protein encoded by the CDK4 gene.
  • the expression of a gene can be determined by detecting and/or measuring expression or concentration of a protein encoded by the gene.
  • Methods of determining protein expression/concentration are well known in the art.
  • a generally used method involves the use of antibodies specific for the target protein of interest.
  • methods of determining protein expression include, but are not limited to, western blot or dot blot analysis, immunohistochemistry (e.g., quantitative immunohistochemistry), immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT; Coligan, J. E., et al., eds. (1995) Current Protocols in Immunology.
  • radioimmunoassay radioimmunoassay
  • chemiluminescent immunoassay electrochemiluminescence immunoassay
  • latex turbidimetric immunoassay latex photometric immunoassay
  • immuno-chromatographic assay immuno-chromatographic assay
  • antibody array analysis see, e.g., U.S. Publication Nos. 2003/0013208 and 2004/171068, the disclosures of each of which are incorporated herein by reference in their entirety. Further description of many of the methods above and additional methods for detecting protein expression can be found in, e.g., Sambrook et al. (supra).
  • the presence or amount of CDK6 and/or CDK4 protein expression of the CDK6 and/or CDK4 gene can be determined using a western blotting technique.
  • a lysate can be prepared from a surface skin sample, or the surface skin sample itself, can be contacted with Laemmli buffer and subjected to sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE-resolved proteins, separated by size, can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to immunoblotting techniques using a detectably-labeled antibody specific to the protein of interest.
  • a filter membrane e.g., nitrocellulose
  • the presence or amount of bound detectably-labeled antibody indicates the presence or amount of protein in the surface skin sample.
  • the SimplePlex platform is used to measure the levels of CDK6 and/or CDK4.
  • SimplePlex is commercially available from Protein Simple (San Jose, CA, USA) (See Dysinger M, et al. J. Immunol. Methods. 451:1-10, 2017).
  • an immunoassay can be used for detecting and/or measuring the protein expression of a gene (e.g., CDK6 and/or CDK4 genes).
  • a gene e.g., CDK6 and/or CDK4 genes.
  • an immunoassay can be performed with an antibody that bears a detection moiety (e.g., a fluorescent agent or enzyme).
  • Proteins from a surface skin sample can be conjugated directly to a solid-phase matrix (e.g., a multi-well assay plate, nitrocellulose, agarose, sepharose, encoded particles, or magnetic beads) or it can be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin) that attaches to a solid-phase matrix upon binding to a second member of the specific binding pair (e.g., streptavidin or biotin).
  • a specific binding pair e.g., biotin or streptavidin
  • Such attachment to a solid-phase matrix allows the proteins to be purified away from other interfering or irrelevant components of the surface skin sample prior to contact with the detection antibody and also allows for subsequent washing of unbound antibody.
  • the presence or amount of bound detectably-labeled antibody indicates the presence or amount of protein in the surface skin sample.
  • the present disclosure includes polyclonal antibodies, as well as monoclonal antibodies.
  • the antiserum obtained by immunizing animals such as rabbits with a protein or fragment thereof of this disclosure (i.e., a protein or an immunological fragment thereof of CDK6 and/or CDK4 protein), as well polyclonal and monoclonal antibodies of all classes, human antibodies, and humanized antibodies produced by genetic recombination, are also included.
  • an intact protein or its partial peptide may be used as the antigen for immunization.
  • partial peptides of the proteins for example, the amino (N)-terminal fragment of the protein and the carboxy (C)-terminal fragment can be given.
  • a gene encoding a protein of interest e.g., CDK6 and/or CDK4 or a fragment thereof (e.g., an immunological fragment) is inserted into a known expression vector, and, by transforming the host cells with the vector described herein, the desired protein or a fragment thereof is recovered from outside or inside the host cells using standard methods.
  • This protein can be used as the sensitizing antigen.
  • cells expressing the protein, cell lysates, or a chemically synthesized protein of the disclosures may be also used as a sensitizing antigen.
  • the mammal that is immunized by the sensitizing antigen is not restricted; however, it is preferable to select animals by considering the compatibility with the parent cells used in cell fusion.
  • animals belonging to the orders rodentia, lagomorpha, or primates are used.
  • animals belonging to the order of rodentia that may be used include, for example, mice, rats, and hamsters.
  • animals belonging to the order of lagomorpha that may be used include, for example, rabbits.
  • animals belonging to the order of primates that may be used include, for example, monkeys.
  • monkeys to be used include the infraorder catarrhini (old world monkeys), for example, Macaca fascicularis, rhesus monkeys, sacred baboons, and chimpanzees.
  • the sensitizing antigen is injected intraperitoneally or subcutaneously into mammals.
  • the sensitizing antigen is suitably diluted and suspended in physiological saline, phosphate-buffered saline (PBS), and so on, and mixed with a suitable amount of general adjuvant if desired, for example, with Freund’s complete adjuvant.
  • PBS phosphate-buffered saline
  • the solution is emulsified and injected into the mammal.
  • the sensitizing antigen suitably mixed with Freund’s incomplete adjuvant is preferably given several times every 4 to 21 days.
  • a suitable carrier can also be used when immunizing and animal with the sensitizing antigen.
  • the elevation in the level of serum antibody is detected by usual methods.
  • Polyclonal antibodies against the proteins of the present disclosure can be prepared as follows. After verifying that the desired serum antibody level has been reached, blood is withdrawn from the mammal sensitized with antigen. Serum is isolated from this blood using conventional methods. The serum containing the polyclonal antibody may be used as the polyclonal antibody, or according to needs, the polyclonal antibody-containing fraction may be further isolated from the serum. For example, a fraction of antibodies that specifically recognize the protein of the invention may be prepared by using an affinity column to which the protein is coupled. Then, the fraction may be further purified by using a Protein A or Protein G column in order to prepare immunoglobulin G or M.
  • immunocytes are taken from the mammal and used for cell fusion.
  • splenocytes can be mentioned as preferable immunocytes.
  • parent cells fused with the above immunocytes mammalian myeloma cells are preferably used. More preferably, myeloma cells that have acquired the feature, which can be used to distinguish fusion cells by agents, are used as the parent cell.
  • the cell fusion between the above immunocytes and myeloma cells can be conducted according to known methods, for example, the method by Milstein et al. (Galfre et al, Methods Enzymol. 73:3-46, 1981).
  • the hybridoma obtained from cell fusion is selected by culturing the cells in a standard selection medium, for example, HAT culture medium (medium containing hypoxanthine, aminopterin, and thymidine).
  • HAT culture medium medium containing hypoxanthine, aminopterin, and thymidine.
  • the culture in this HAT medium is continued for a period sufficient enough for cells (non-fusion cells) other than the objective hybridoma to perish, usually from a few days to a few weeks.
  • the usual limiting dilution method is carried out, and the hybridoma producing the objective antibody is screened and cloned.
  • a hybridoma producing the objective human antibodies having the activity to bind to proteins can be obtained by the method of sensitizing human lymphocytes, for example, human lymphocytes infected with the EB virus, with proteins, protein-expressing cells, or lysates thereof in vitro and fusing the sensitized lymphocytes with myeloma cells derived from human, for example, U266, having a permanent cell division ability.
  • the monoclonal antibodies obtained by transplanting the obtained hybridomas into the abdominal cavity of a mouse and extracting ascites can be purified by, for example, ammonium sulfate precipitation, protein A or protein G column, DEAE ion exchange chromatography, an affinity column to which the protein of the present disclosure is coupled, and so on.
  • Monoclonal antibodies can be also obtained as recombinant antibodies produced by using the genetic engineering technique (see, for example, Borrebaeck C. A.K. and Larrick, J.W., THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD (1990)).
  • Recombinant antibodies are produced by cloning the encoding DNA from immunocytes, such as hybridoma or antibody-producing sensitized lymphocytes, incorporating into a suitable vector, and introducing this vector into a host to produce the antibody.
  • the present disclosure encompasses such recombinant antibodies as well.
  • Antibodies or antibody fragments specific for a protein encoded by one or more biomarkers can also be generated by in vitro methods such as phage display.
  • the antibody of the present disclosure may be an antibody fragment or modified-antibody, so long as it binds to a protein encoded by a biomarker of the disclosure (CDK6 and/or CDK4).
  • Fab, F (ab’) 2, Fv, or single chain Fv (scFv) in which the H chain Fv and the L chain Fv are suitably linked by a linker Huston et al, (1998) Proc. Natl. Acad. Sci.
  • antibody fragments are generated by treating antibodies with enzymes, for example, papain or pepsin.
  • they may be generated by constructing a gene encoding an antibody fragment, introducing this into an expression vector, and expressing this vector in suitable host cells (see, for example, Co et al, J.
  • the antibodies may be conjugated to various molecules, such as fluorescent substances, radioactive substances, and luminescent substances. Methods to attach such moieties to an antibody are already established and conventional in the field (see, e.g., US 5,057,313 and 5,156,840). Examples of methods that assay the antigen-binding activity of the antibodies include, for example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/or immunofluorescence.
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • a protein encoded by a biomarker of the invention is added to a plate coated with the antibodies of the present disclosure, and then, the antibody sample, for example, culture supernatants of antibody- producing cells, or purified antibodies are added. Then, secondary antibody recognizing the primary antibody, which is labeled by alkaline phosphatase and such enzymes, is added, the plate is incubated and washed, and the absorbance is measured to evaluate the antigen-binding activity after adding an enzyme substrate such as p-nitrophenyl phosphate.
  • a protein fragment for example, a fragment comprising a C- terminus, or a fragment comprising an N-terminus may be used.
  • BIAcore Pharmacia
  • the antibody and a sample presumed to contain a protein of the disclosure are contacted, and the protein encoded by a biomarker of the disclosure is detected or assayed by detecting or assaying the immune complex formed between the above-mentioned antibody and the protein.
  • Mass spectrometry based quantitation assay methods for example, but not limited to, multiple reaction monitoring (MRM)-based approaches in combination with stable- isotope labeled internal standards, are an alternative to immunoassays for quantitative measurement of proteins. These approaches do not require the use of antibodies and so the analysis can be performed in a cost- and time- efficient manner (see, for example, Addona et al., Nat. Biotechnol, 27:633-641, 2009; Kuzyk et al, Mol. Cell Proteomics, 8:1860-1877, 2009; Paulovich et al, Proteomics Clin. Appl, 2:1386-1402, 2008).
  • MRM offers superior multiplexing capabilities, allowing for the simultaneous quantification of numerous proteins in parallel. The basic theory of these methods has been well-established and widely utilized for drug metabolism and pharmacokinetics analysis of small molecules.
  • the expression level of CDK6 and/or CDK4 is determined by measuring RNA levels.
  • a variety of suitable methods can be employed to detect and/or measure the level of mRNA expression of a gene.
  • mRNA expression can be determined using Northern blot or dot blot analysis, reverse transcriptase-PCR (RT-PCR; e.g., quantitative RT-PCR), in situ hybridization (e.g., quantitative in situ hybridization) or nucleic acid array (e.g., oligonucleotide arrays or gene chips) analysis. Details of such methods are described below and in, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual Second Edition vol. 1, 2 and 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, Nov. 1989;
  • the presence or amount of one or more discrete mRNA populations in a biological sample can be determined by isolating total mRNA from the biological sample (see, e.g., Sambrook et al. (supra) and U.S.
  • Patent No. 6,812,341 subjecting the isolated mRNA to agarose gel electrophoresis to separate the mRNA by size.
  • the size-separated mRNAs are then transferred (e.g., by diffusion) to a solid support such as a nitrocellulose membrane.
  • the presence or amount of one or more mRNA populations in the surface skin sample can then be determined using one or more detectably-labeled-polynucleotide probes, complementary to the mRNA sequence of interest, which bind to and thus render detectable their corresponding mRNA populations.
  • Detectable-labels include, e.g., fluorescent (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, allophycocyanin (APC), or phycoerythrin), luminescent (e.g., europium, terbium, QdotTM nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,
  • radiological e.g., 1251, 1311, 35S, 32P, 33P, or 3H
  • enzymatic horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase
  • the presence or amount of discrete populations of mRNA (e.g., mRNA encoded by the CDK6 and/or CDK4 genes) in a biological sample can be determined using nucleic acid (or oligonucleotide) arrays.
  • isolated mRNA from a biological sample e.g., a tumor sample
  • RT-PCR with, e.g., random hexamer or oligo(dT)-primer mediated first strand synthesis.
  • the amplicons can be fragmented into shorter segments.
  • the RT-PCR step can be used to detectably- label the amplicons, or, optionally, the amplicons can be detectably-labeled subsequent to the RT-PCR step.
  • the detectable-label can be enzymatically (e.g., by nick- translation or kinase such as T4 polynucleotide kinase) or chemically conjugated to the amplicons using any of a variety of suitable techniques (see, e.g., Sambrook et al, supra).
  • the detectably-labeled-amplicons are then contacted with a plurality of polynucleotide probe sets, each set containing one or more of a polynucleotide (e.g., an oligonucleotide) probe specific for (and capable of binding to) a corresponding amplicon, and where the plurality contains many probe sets each corresponding to a different amplicon.
  • a polynucleotide e.g., an oligonucleotide
  • the probe sets are bound to a solid support and the position of each probe set is predetermined on the solid support.
  • the binding of a detectably-labeled amplicon to a corresponding probe of a probe set indicates the presence or amount of a target mRNA in the surface skin sample. Additional methods for detecting mRNA expression using nucleic acid arrays are described in, e.g., U.S. Patent Nos. 5,445,934; 6,027,880; 6,057,100; 6,156,501; 6,261,776; and 6,576,424; the disclosures of each of which are incorporated herein by reference in their entirety.
  • Methods of detecting and/or for quantifying a detectable label depend on the nature of the label.
  • the products of reactions catalyzed by appropriate enzymes can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light.
  • detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.
  • Methods for detecting or measuring gene expression can optionally be performed in formats that allow for rapid preparation, processing, and analysis of multiple samples. This can be, for example, in multi-welled assay plates (e.g., 96 wells or 386 wells) or arrays (e.g., nucleic acid chips or protein chips).
  • Stock solutions for various reagents can be provided manually or robotically, and subsequent sample preparation (e.g., RT-PCR, labeling, or cell fixation), pipetting, diluting, mixing, distribution, washing, incubating (e.g., hybridization), sample readout, data collection (optical data) and/or analysis (computer aided image analysis) can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting the signal generated from the assay. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorimeters, and devices that measure radioisotope decay.
  • Exemplary high-throughput cell-based assays can utilize ArrayScan® VTI HCS Reader or KineticScan® HCS Reader technology (Cellomics Inc., Pittsburgh, PA).
  • the expression level of the CDK6 and/or CDK4 gene biomarkers of this disclosure can be assessed and/or measured.
  • any part of the nucleic acid sequence of the genes can be used, e.g., as hybridization polynucleotide probes or primers (e.g., for amplification or reverse transcription).
  • the probes and primers can be oligonucleotides of sufficient length to provide specific hybridization to an RNA, DNA, cDNA, or fragments thereof isolated from a surface skin sample.
  • varying hybridization conditions can be employed to achieve varying degrees of selectivity of a probe or primer towards target sequence.
  • the primers and probes can be detectably-labeled with reagents that facilitate detection (e.g., fluorescent labels, chemical labels (see, e.g., U.S. Patent Nos. 4,582,789 and 4,563,417), or modified bases).
  • reagents e.g., fluorescent labels, chemical labels (see, e.g., U.S. Patent Nos. 4,582,789 and 4,563,417), or modified bases).
  • nucleic acid molecule In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double- stranded structure under the particular hybridization conditions (e.g., solvent and salt concentrations) employed.
  • Hybridization can be used to assess homology between two nucleic acid sequences.
  • a nucleic acid sequence described herein, or a fragment thereof can be used as a hybridization probe according to standard hybridization techniques.
  • the hybridization of a probe of interest e.g., a probe containing a portion of a nucleotide sequence described herein or its complement
  • DNA, RNA, cDNA, or fragments thereof from a test source is an indication of the presence of DNA or RNA corresponding to the probe in the test source.
  • Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons,
  • Moderate hybridization conditions are defined as hybridization in 2X sodium chloride/sodium citrate (SSC) at 30°C, followed by a wash in 1 X SSC,
  • Primers can be used in in a variety of PCR-type methods. For example, polymerase chain reaction (PCR) techniques can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA.
  • the PCR primers are designed to flank the region that one is interested in amplifying. Primers can be located near the 5' end, the 3' end or anywhere within the nucleotide sequence that is to be amplified.
  • the amplicon length is dictated by the experimental goals. For qPCR, the target length is closer to 100 base pairs and for standard PCR, it is near 500 base pairs.
  • PCR primers can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3’ to 5’ direction using phosphoramidite technology) or as a series of oligonucleotides.
  • one or more pairs of long oligonucleotides can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed.
  • a short segment of complementarity e.g., about 15 nucleotides
  • DNA polymerase is used to extend the oligonucleotides, resulting in a single, double- stranded nucleic acid molecule per oligonucleotide pair.
  • nucleic acid sequences or fragments thereof can be used in nucleic acid arrays for detection and/or quantitation of gene expression.
  • nucleic acid and amino acid sequences of human CDK6 and CDK4 biomarkers of this disclosure that can be measured using methods of this disclosure include the following sequences and variants thereof:
  • SEQ ID NO: 1 human CDK6 nucleic acid sequence (NM_001259.8)
  • SEQ ID NO: 2 human CDK6 amino acid sequence (NP_001138778.1)
  • SEQ ID NO: 3 human CDK4 nucleic acid sequence (NM_000075.4)
  • SEQ ID NO: 4 human CDK4 amino acid sequence (NP_000066. 1) MATSRYEPVAEIGVGAYGTVYKARDPHSGHFVALKSVRVPNGGGGGGGLPISTV REVALLRRLEAFEHPNVVRLMDVCATSRTDREIKVTLVFEHVDQDLRTYLDKAPP PGLPAETIKDLMRQFLRGLDFLHANCIVHRDLKPENILVTSGGTVKLADFGLARIY S Y QM ALTP VVVTLWYRAPEVLLQ ST YATP VDMW S VGCIFAEMFRRKPLF CGN SE ADQLGKIFDLIGLPPEDDWPRDVSLPRGAFPPRGPRPVQSVVPEMEESGAQLLLE
  • the ratio (quotient) of CDK4 to CDK6 is determined by conventional means known in the art in which the level of one protein or RNA amount (CDK4) is divided by the level of the other protein or RNA amount (CDK6) to obtain the mathematical ratio of CDK4 to CDK6.
  • the methods of the present disclosure can involve, measuring the expression level (e.g., mRNA, cDNA, or protein concentration) of CDK6 and/or CDK4 in a biological sample from a subject (e.g., a human subject with a tumor), wherein the expression level of CDK6 and/or CDK4 gene or protein, and/or the CDK4:CDK6 ratio compared to a control, predicts whether a CDK4/6-based therapy (e.g., a CDK4/6 inhibitor and/or a CDK4/6-directed PROTAC) will be successful in treating a subject with that tumor.
  • a CDK4/6-based therapy e.g., a CDK4/6 inhibitor and/or a CDK4/6-directed PROTAC
  • the level of CDK6 and/or CDK4:CDK6 ratio in a tumor compared to a control or threshold level can predict the sensitivity of that tumor to a CDK4/6 inhibitor; the subject response to CDK4/6 inhibitors; and whether or not a subject will be a responder to treatment comprising a CDK4/6-based therapy.
  • control refers to a single biological sample or multiple biological samples from which an average amount or level of biomarker (e.g., CDK6 and/or CDK4) can be determined.
  • a “reference level in a control” refers to the average amount or level of that biomarker in single sample or a set of biological samples.
  • the amount of the biomarker that is measured in the sample may be relative or absolute. In some embodiments the relative expression of mRNA or cDNA is measured in the test sample versus the control sample. In other embodiments, the absolute amount of protein biomarker is measured in the test sample versus the control sample.
  • control refers to a threshold level of CDK6 mRNA as determined by quantitative PCR or RNA-seq, or CDK6 protein levels, as determined by Immunohistochemistry or mass spectrometry analysis.
  • the level/concentration of CDK6 in a tumor sample from a subject is measured by any RNA sequencing technology known in the art (see e.g., Kukurba KR and Montgomery SB, (2015) Cold Spring Harb Protoc. Nov; (11): 951-969; and Hrdlickova R et al, (2017) Wiley Interdiscip Rev RNA. Jan; 8(1):
  • RNA sequencing when the level/concentration of CDK6 in a tumor sample as measured by RNA sequencing is lower than 5.37 counts per million reads (CPM), the tumor is classified as “CDK6-low”. Conversely, when the level/concentration of CDK6 in a tumor sample as measured by RNA sequencing is higher than 5.37 CPM, the tumor is classified as “CDK6-hgh”.
  • the “threshold” expression level/concentration for a CDK4/6 mRNA, cDNAor protein and/or the threshold ratio of CDK4 to CDK6 may also be pre-established by an analysis of mRNA, cDNA, or protein expression in biological samples (e.g., tumor samples) from one or more (e.g., two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, or 40 or more) human subjects who have the same tumors.
  • This pre- established reference value (which may be an average or median expression level/concentration taken from multiple subjects that have tumors) may then be used for the “control” concentration/expression level of the protein or nucleic acid in the comparison with the test sample.
  • the subject is predicted to be responsive to CDK4/6 inhibitor therapy and/or CDK4/6-directed PROTAC therapy if the expression level of the CDK6 being analyzed is lower than the pre-established reference and/or the ratio of CDK4 to CDK6 is higher than the pre-established reference.
  • the “control” is a pre-determined cut-off value.
  • the methods described herein include determining if the concentration of CDK6 and/or CDK4 to CDK6 ratio falls above or below a predetermined cut-off value (the threshold level).
  • a cut-off value is typically a concentration of a protein above or below which is considered predictive of something - e.g., likely to be responsive to a therapy of interest.
  • a reference concentration of CDK6 mRNA, cDNAor protein is identified as a cut-off value, and/or CDK4 to CDK6 ratio above or below of which is predictive of a subject who shows responsiveness to a cancer therapy.
  • cut-off values are not absolute in that clinical correlations can still remain significant over a range of values on either side of the cutoff; however, it is possible to select an optimal cut-off value (e.g. varying H-scores) of concentration of CDK6 and/or CDK4 mRNA, cDNAor protein for a particular sample type. Cut-off values determined for use in the methods described herein can be compared with, e.g., published ranges of CDK6 and/or CDK4 concentrations, but can be individualized to the methodology used and patient population. It is understood that improvements in optimal cut-off values could be determined depending on the sophistication of statistical methods used and on the number and source of samples used to determine reference level values for the different proteins, genes, and sample types. Therefore, established cut-off values can be adjusted up or down, on the basis of periodic reevaluations or changes in methodology or population distribution.
  • control or threshold levels of CDK6 and/or CDK4 mRNA and/or protein levels are established using cell lines or xenografts. In some embodiments, the control or threshold levels of CDK6 and/or CDK4 protein levels are established using immunohistochemistry in preclinical samples.
  • the reference concentration of biomarkers of the present disclosure can be determined by a variety of methods.
  • the reference level can be determined by comparison of the concentration of protein of interest in, e.g., populations of subjects (e.g., patients) that are responsive to a CDK4/6-directed therapy or not responsive to a CDK4/6-directed therapy. This can be accomplished, for example, by histogram analysis, in which an entire cohort of patients is graphically presented, wherein a first axis represents the concentration of a protein of interest and a second axis represents the number of subjects in the cohort whose sample contain one or more concentrations.
  • the reference concentration of a protein can then be made based on an amount or concentration which best distinguishes these separate groups.
  • the reference level can be a single number, equally applicable to every subject, or the reference level can vary, according to specific subpopulations of subjects. For example, older subjects can have a different reference level than younger subjects. In addition, a subject with more severe disease can have a different reference value than one with a milder form of the disease (e.g., early stage vs late stage cancer).
  • the pre-established cut-off value can be a CDK6 and/or CDK4 protein concentration that is determined based on receiver operating characteristic (ROC) analysis.
  • ROC curves are used to determine a cut-off value for a clinical test.
  • a biological sample e.g., a tumor sample
  • the test will find some, but not all, responders to respond to a CDK4/6-directed therapy.
  • the ratio of the responders found by the test to the total number of responders is the true positive rate (also known as sensitivity).
  • the test will find some, but not all, non-responders to not respond to a CDK4/6-directed therapy.
  • the ratio of the non responders found by the test to the total number of non-responders is the true negative rate (also known as specificity).
  • the ROC curve analysis of the CDK4/6-directed therapy responsiveness test will find a cut off value that will minimize the number of false positives and false negatives.
  • a ROC is a graphical plot which illustrates the performance of a binary class stratifier system as its discrimination threshold is varied. It is created by plotting the fraction of true positives out of the positives versus the fraction of false positives out of the negatives, at various threshold settings.
  • the CDK6 and/or CDK4 protein concentration is determined based on ROC analysis predicting response to a CDK4/6-driven therapy with a positive predictive value, wherein a concentration of a protein of interest (e.g., CDK6 and/or CDK4) equal to or below the pre-established cut off value is a low concentration of the protein of interest and a value higher than the pre-established cut-off value is a high concentration of the protein of interest.
  • a concentration of a protein of interest e.g., CDK6 and/or CDK4
  • the CDK4 to CDK6 protein ratio is determined based on ROC analysis predicting response to a CDK4/6-driven therapy with a positive predictive value, wherein a CDK4 to CDK6 protein ratio equal to or below the pre-established cut off value is a low CDK4 to CDK6 ratio and a value higher than the pre-established cut off value is a high CDK4 to CDK6 ratio.
  • the positive predictive value is the proportion of positive test results that are true positives; it reflects the probability that a positive test reflects the underlying condition being tested for. Methods of constructing ROC curves and determining positive predictive values are well known in the art.
  • the pre-established cut-off value can be a CDK6 and/or CDK4 protein concentration that is determined based on simulation models predicting responsiveness to CDK4/6-driven therapy, and wherein a concentration of CDK6 and/or CDK4 equal to or below the pre-established cut-off value is a low concentration of the CDK6 and/or CDK4 and a value higher than the pre-established cut-off value is a high concentration of CDK6 and/or CDK4.
  • the methods described herein can be used to treat proliferative disorders such as cancer, and in particular, a cancer expressing functional retinoblastoma protein (Rb)- proficient cancer.
  • the methods described herein can be used to treat a subject suffering from an Rb-positive cancer or other Rb-positive abnormal cellular proliferative disorder.
  • the cancer or cellular proliferation disorder is a CDK4/6-dependent cancer that requires the activity of CDK4/6 for replication or proliferation, or which may be treated by a CDK4/6 inhibitor and/or a CDK4/6-directed PROTAC. Cancers and disorders of such type can be characterized by the presence of a functional Retinoblastoma protein.
  • Rb-positive abnormal cellular proliferation disorders refer to disorders or diseases caused by uncontrolled or abnormal cellular division which are characterized by the presence of a functional Retinoblastoma protein, which can include cancers.
  • the methods described herein can be used to treat a non-cancerous Rb- positive abnormal cellular proliferation disorder.
  • disorders may include non-malignant lymphoproliferation, non-malignant breast neoplasms, psoriasis, arthritis, dermatitis, pre-cancerous colon lesions or pulps, angiogenesis disorders, immune mediated and non-immune mediated inflammatory diseases, arthritis, age-related macular degeneration, diabetes, and other non-cancerous or benign cellular proliferation disorders.
  • Targeted cancers suitable for treatment with the methods described herein include but are not limited to Rb-positive: estrogen-receptor positive cancer, HER2- negative advanced breast cancer, late-line metastatic breast cancer, liposarcoma, non small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma positive breast cancer as well as retinoblastoma positive endometrial, vaginal and ovarian cancers and lung and bronchial cancers, adenocarcinoma of the colon, adenocarcinoma of the rectum, central nervous system germ cell tumors, teratomas, estrogen receptor-negative breast cancer, estrogen receptor-positive breast cancer, familial testicular germ cell tumors, HER2 -negative breast cancer, HER2- positive breast cancer, male breast cancer, ovarian immature teratomas, ovarian mature teratoma, ovarian monodermal and highly specialized teratomas,
  • the targeted cancers included estrogen-receptor positive, HER2-negative advanced breast cancer, late-line metastatic breast cancer, liposarcoma, non-small cell lung cancer, liver cancer, ovarian cancer, glioblastoma, refractory solid tumors, retinoblastoma positive breast cancer as well as retinoblastoma positive endometrial, vaginal and ovarian cancers and lung and bronchial cancers, metastatic colorectal cancer, metastatic melanoma with CDK4 mutation or amplification, or cisplatin-refractory, and unresectable germ cell tumors.
  • the Rb-positive cancer suitable to treat with the methods described herein include but are not limited to non-small-cell lung carcinoma (NSCLC), colorectal carcinoma (CRC), melanoma, Acute lymphoblastic leukemia (ALL), T-cell acute lymphocytic leukemia (T-ALL), Mantle Cell Lymphoma (MCL), and Multiple Myeloma (MM).
  • NSCLC non-small-cell lung carcinoma
  • CRC colorectal carcinoma
  • melanoma melanoma
  • ALL Acute lymphoblastic leukemia
  • T-ALL T-cell acute lymphocytic leukemia
  • MCL Mantle Cell Lymphoma
  • MM Multiple Myeloma
  • the Rb-positive cancer is selected from an Rb- positive carcinoma, sarcoma, including, but not limited to, lung cancer, 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, 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, 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, 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 C
  • the Rb-positive cancer is selected from the group consisting of Rb-positive: fibrosarcoma, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, hemangio sarcoma, angiosarcoma, lymphangiosarcoma.
  • Mesothelioma Mesothelioma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma; epidermoid carcinoma, malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, glioma anaplastic; glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cyst
  • the Rb-positive cancer or disorder includes a blood disorder or a hematologic malignancy, including, but not limited to, myeloid disorder, lymphoid disorder, leukemia, lymphoma, myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), mast cell disorder, and myeloma (e.g., multiple myeloma), among others.
  • Abnormal proliferation of T-cells, B-cells, and/or NK-cells can result in a wide range of diseases such as cancer, proliferative disorders and inflammatory/immune diseases.
  • a host for example a human, afflicted with any of these disorders can be treated with the methods described herein to achieve a decrease in symptoms or a decrease in the underlying disease.
  • T-cell or NK-cell lymphoma examples include T-cell or NK-cell lymphoma, for example, but not limited to: peripheral T-cell lymphoma; anaplastic large cell lymphoma, for example anaplastic lymphoma kinase (ALK) positive, ALK negative anaplastic large cell lymphoma, or primary cutaneous anaplastic large cell lymphoma; angioimmunoblastic lymphoma; cutaneous T-cell lymphoma, for example mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, primary cutaneous CD30+ T-cell lymphoproliferative disorder; primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma; primary cutaneous gamma-delta T-cell lymphoma; primary cutaneous small/medium CD4+ T-cell lymphoma, and lymphomatoid papulosis; Adult T- cell Leukemia/Lymphoma (AT
  • the methods disclosed herein can be used to treat a subject with a lymphoma or lymphocytic or myelocytic proliferation disorder or abnormality.
  • a Hodgkin Lymphoma including, not limited to: Nodular Sclerosis Classical Hodgkin's Lymphoma (CHL); Mixed Cellularity CHL; Lymphocyte-depletion CHL; Lymphocyte-rich CHL; Lymphocyte Predominant Hodgkin Lymphoma; or Nodular Lymphocyte Predominant HL
  • a Non-Hodgkin Lymphoma including, but not limited to: an AIDS-Related Lymphoma; Anaplastic Large-Cell Lymphoma;
  • Angioimmunoblastic Lymphoma Blastic NK-Cell Lymphoma; Burkitf s Lymphoma; Burkitt-like Lymphoma (Small Non-Cleaved Cell Lymphoma); Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma; Cutaneous T-Cell Lymphoma; Diffuse Large B-Cell Lymphoma; Enteropathy-Type T-Cell Lymphoma; Follicular Lymphoma; Hepatosplenic Gamma-Delta T-Cell Lymphoma; Lymphoblastic Lymphoma; Mantle Cell Lymphoma; Marginal Zone Lymphoma; Nasal T-Cell Lymphoma; Pediatric Lymphoma; Peripheral T-Cell Lymphomas; Primary Central Nervous System Lymphoma; T-Cell Leukemias; Transformed Lymphomas; Treatment-Related T-Cell Lymphomas; or Walden
  • the methods disclosed herein can be used to treat a subject with a specific B-cell lymphoma or proliferative disorder including, but not limited to: multiple myeloma; Diffuse large B cell lymphoma; Follicular lymphoma; Mucosa- Associated Lymphatic Tissue lymphoma (MALT); Small cell lymphocytic lymphoma; Mediastinal large B cell lymphoma; Nodal marginal zone B cell lymphoma (NMZL); Splenic marginal zone lymphoma (SMZL); Intravascular large B-cell lymphoma; Primary effusion lymphoma; or Lymphomatoid granulomatosis;; B-cell prolymphocytic leukemia; Hairy cell leukemia; Splenic lymphoma/leukemia, unclassifiable; Splenic diffuse red pulp small B-cell lymphoma; Hairy cell leukemia-variant; Lymphoplasmacytic lymphoma; Heavy chain diseases,
  • the methods disclosed herein can be used to treat a subject with an acute or chronic leukemia of a lymphocytic or myelogenous origin, including, but not limited to: Acute lymphoblastic leukemia (ALL); Acute myelogenous leukemia (AML); Chronic lymphocytic leukemia (CLL); Chronic myelogenous leukemia (CIVIL); juvenile myelomonocytic leukemia (JMML); hairy cell leukemia (HCL); acute promyelocytic leukemia (a subtype of AML); large granular lymphocytic leukemia; or Adult T-cell chronic leukemia.
  • ALL Acute lymphoblastic leukemia
  • AML Acute myelogenous leukemia
  • CLL Chronic lymphocytic leukemia
  • CIVIL Chronic myelogenous leukemia
  • JMML juvenile myelomonocytic leukemia
  • HCL hairy cell leukemia
  • the patient suffers from an acute myelogenous leukemia, for example an undifferentiated AML (MO); myeloblastic leukemia (Ml; with/without minimal cell maturation); myeloblastic leukemia (M2; with cell maturation); promyelocytic leukemia (M3 or M3 variant [M3V]); myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]); monocytic leukemia (M5); erythroleukemia (M6); or megakaryoblastic leukemia (M7).
  • MO undifferentiated AML
  • Ml myeloblastic leukemia
  • M2 myeloblastic leukemia
  • M2 myeloblastic leukemia
  • M3V promyelocytic leukemia
  • M4 or M4 variant with eosinophilia [M4E] myelomonocytic leukemia
  • M5 mono
  • the presence or normal functioning of the retinoblastoma (Rb) tumor suppressor protein (Rb-positive) can be determined through any of the standard assays known to one of ordinary skill in the art, including but not limited to Western Blot, ELISA (enzyme linked immunoadsorbent assay), IHC (immunohistochemistry), and FACS (fluorescent activated cell sorting).
  • the selection of the assay will depend upon the tissue, cell line or surrogate tissue sample that is utilized e.g., for example Western Blot and ELISA may be used with any or all types of tissues, cell lines or surrogate tissues, whereas the IHC method would be more appropriate wherein the tissue utilized in the methods of the present invention was a tumor biopsy.
  • FACs analysis would be most applicable to samples that were single cell suspensions such as cell lines and isolated peripheral blood mononuclear cells. See for example, US 20070212736 “Functional Immunohistochemical Cell Cycle Analysis as a Prognostic Indicator for Cancer”.
  • molecular genetic testing may be used for determination of retinoblastoma gene status.
  • Molecular genetic testing for retinoblastoma includes the following as described in Lohmann and Gallie “Retinoblastoma. Gene Reviews” (2010): “A comprehensive, sensitive and economical approach for the detection of mutations in the RBI gene in retinoblastoma” Journal of Genetics, 88(4), 517-527 (2009).
  • the cancer to be treated is a solid tumor cancer or a leukemia.
  • Solid tumor cancers include, but are not limited to, non-small-cell lung carcinoma (NSCLC), lung cancer, breast cancer, ewing sarcoma, central nervous system neoplasm, skin cancer, head and neck cancer, ovarian cancer, colon cancer, anal cancer, stomach cancer, gastrointestinal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, testicular cancer, brain stem glioma, pituitary cancer, adrenocortical cancer, gallbladder cancer, multiple myeloma, cholangiocarcinoma, fibrosarcoma, lymphoma, liver cancer, kidney cancer, bone cancer, bladder cancer, colorectal cancer, endometrial cancer, renal
  • CDK4/6 inhibitors and CDK4/6-directed PROTACs may be collectively referred to as CDK4-6-directed therapies in this disclosure.
  • the CDK4/6 inhibitors suitable for use in the methods described herein are compounds which are capable of interfering with the enzymatic activity of CDK4, CDK6, or both CDK4 and CDK6.
  • an "inhibitor” refers to an agent that restrains, retards, or otherwise causes inhibition of a physiological, chemical or enzymatic action or function.
  • An inhibitor can cause an at least 5% decrease in enzyme activity.
  • An inhibitor can also or alternately refer to a drug, compound, or agent that prevents or reduces the expression, transcription, or translation of a gene or protein.
  • An inhibitor can reduce or prevent the function of a protein, e.g., by binding to or activating/inactivating another protein or receptor.
  • Examplary CDK4/6 inhibitors are disclosed in International Patent Application Publications W02016040858, WO2011130232, WO2011101409, W02016025650, and W02013006532, incorporated by reference in their entirety.
  • CDK4/6 inhibitors suitable for use include, but are not limited to the following compounds and pharmaceutically acceptable derivatives and prodrugs thereof:
  • the CDK4/6-directed PROTACs suitable for use in the methods described herein are heterobifunctional small molecules which selectively degrade CDK4, CDK6, or both CDK4 and CDK6 ("PROteolysis TArgeting Chimeras" or "PROTACs") are useful in the treatment of CDK4/6- mediated cancers.
  • the CDK4/6-directed PROTACs also known as CDK4/6 degraders/disruptors
  • useful in the methods of this disclosure include, but are not limited to MS 140 (FIG. 4A) and other molecules disclosed in International Patent Application Publication W02018106870PCT, disclosed herein in its entirety. These PROTACs comprise a CDK4/6 ligand (or targeting moiety) conjugated to a degradation tag.
  • Linkage of the CDK4/6 ligand to the degradation tag can be direct, or indirect via a linker.
  • the CDK4/6 ligand or targeting moiety can be a CDK4/6 inhibitor (e.g., abemaciclib, palbociclib, ribociclib, trilaciclib (G1T28), G1T38, SHR6390, and analogs thereof.
  • the degradation/disruption tags of the present disclosure include, e.g., thalidomide, pomalidomide, lenalidomide, VHL-1, adamantane, 1 -((4,4, 5,5,5- pentafluoropentyl)sulfmyl)nonane, nutlin-3a, RG7112, RG7338, AMG 232, AA-115, bestatin, MV-1, LCL161, and/or analogs thereof.
  • the CDK4/6-directed PROTACs that are suitable for use in the methods described herein include the PROTACs shown in FIGs. 14-18. These PROTACs are described in Brand M. et al., (2019) Cell Chem. Biol Feb 21;26(2):300-306; Jiang B, et al. Angew Chem Int Ed Engl. 2019 May 6;58(19):6321-6326. doi:
  • This disclosure features methods of predicting the response of a subject with cancer (e.g., a Rb-proficient cancer) to any particular CDK4/6 therapy and predicting the prognosis of a subject with cancer.
  • the method involves measuring the CDK6 and/or CDK4 levels obtained from a biological sample from the subject (e.g., a tumor). Based on the levels of CDK6 and/or the CDK4/CDK6 ratio, the probability of response to the cancer therapy, a future risk of cancer progression in the subject is determined in the subject and/or an indication of whether the cancer will be successfully treated with a particular cancer therapy (e.g., a CDK4/6 inhibitor).
  • a particular cancer therapy e.g., a CDK4/6 inhibitor
  • a specific tumor phenotype in a subject with cancer can be categorized as “CDK4-high/CDK6-low” as determined by assessing RNA and/or protein levels of CDK6 and CDK4 in the tumor as described elsewhere.
  • Such a tumor is classified as a CDK4 dependent tumor and is likely to be more sensitive to CDK4/6- directed PROTACs, than to CDK4/6 inhibitors.
  • a tumor phenotype in a subject with cancer can be categorized as “CDK4-low/CDK6-high” as determined by assessing RNA and/or protein levels of CDK6 and CDK4 in the tumor as described elsewhere.
  • a tumor is classified as a CDK6 dependent tumor and is likely to be more sensitive to CDK4/6-directed PROTACs, than to CDK4/6 inhibitors.
  • a CDK6 dependent tumor can be sensitive to CDK4/6 driven therapy (including CDK4/6 inhibitors and CDK4/6-directed PROTACs) if CDK6 binds strongly to heat shock protein (Hsp90) and Cell Division Cycle 37 (HSP90/CDC37) as shown in FIG. 7.
  • a CDK6 dependent tumor can be resistant to CDK4/6 driven therapy if CDK6 binds weakly to HSP90/CDC37 as shown in FIGS. 6-7
  • the methods disclosed herein enable the characterization of a tumor based on biomarker levels (e.g., CDK6 and/or CDK4) in a subject’s biological sample, followed by treatment of said subject with an appropriate cancer therapy.
  • biomarker levels e.g., CDK6 and/or CDK4
  • the methods disclosed herein enable the assessment whether or not a subject having or suspected of having cancer is likely to respond to a cancer therapy.
  • a subject having or suspected of having cancer who is likely to respond to the cancer therapy can be administered the cancer therapy.
  • a subject having or suspected of having cancer who is not likely to respond to a cancer therapy can be administered a different cancer therapy that is suitable for treatment of cancer.
  • the methods of this disclosure also enable the stratification of cancerous tumors into tumors that are more likely to benefit, and tumors that are less likely to benefit, from treatment comprising a particular cancer therapy.
  • the ability to select one or more tumors being considered for treatment with a cancer therapy is beneficial for administering an effective treatment to the subject with the one or more tumors.
  • the subjects who are considered for treatment comprising a cancer therapy include, but are not limited to, subjects having, or suspected of having, cancer.
  • the subject to be treated with a cancer therapy has, is suspected of having, or is likely to develop one or more tumors.
  • the subject having cancer is more likely to respond to a cancer therapy (based on levels of CDK6 and/or CDK4 biomarkers, and/or the ratio of CDK4 to CDK6), the subject can then be administered an effective amount of the cancer therapy.
  • An effective amount of the compound can suitably be determined by a health care practitioner taking into account, for example, the characteristics of the patient (age, sex, weight, race, etc.), the progression of the disease, and prior exposure to the drug. If the subject is less likely to respond to one cancer therapy, the subject can then be optionally administered a different cancer therapy.
  • a biological sample used in a method described herein can be obtained from a human subject of any age, including a fetus, an infant, a child, an adolescent, or an adult, such as an adult having, or suspected of having, cancer.
  • a medical practitioner e.g., a doctor
  • Methods of administering cancer therapies are known in the art. It is understood that any therapy described herein can include one or more additional therapeutic agents. That is, any therapy described herein can be co administered (administered in combination) with one or more additional therapeutic agents such as, but not limited to, other cancer therapies described herein.
  • any therapy described herein can include one or more agents for treating one or more side-effects of a therapy comprising the cancer therapy.
  • Combination therapies can be, e.g., simultaneous or successive.
  • a cancer therapy and the additional therapeutic agent(s) can be administered at the same time or at different times.
  • the one or more additional therapeutic agents can be administered first in time and the cancer therapy can be administered second in time.
  • the one or more additional cancer therapies include but are not limited to surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and immunotherapy.
  • the therapy can replace or augment a previously or currently administered therapy.
  • a previously or currently administered therapy For example, upon treating with a CDK4/6 inhibitor, administration of a non-CDK4/6 inhibitor therapy can cease or diminish, e.g., be administered at lower levels.
  • Administration of the previous therapy can be maintained while the therapy comprising CDK4/6 inhibitor is administered.
  • a previous therapy can be maintained until the level of CDK4/6 inhibitor reaches a level sufficient to provide a therapeutic effect.
  • cancer therapy CDK4/6-driven therapies for Rb-proficient cancers
  • additional therapeutic regimens including, but not limited to surgery, chemotherapy, radiation therapy, hormone therapy, targeted therapy, and immunotherapy.
  • therapeutic regiments are well-known in the art. See e.g., Chessum N et al, Prog Med Chem 2015, Lee YT et al; Eur J Pharmacol 2018; Chin HM et al, Journal of Immunotherapy and Precision Oncology (2019) 2 (1): 10-16.
  • Targeted therapy regimens include the use of agents such as MEK inhibitors, ERK inhibitors, hormonal therapy, and RAS(G12C) inhibitors.
  • MEK inhibitors include, but are not limited to, trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, CI-1040, PD035901, and TAK-733.
  • ERK inhibitors include, but are not limited to, ulixertinib, BVD-523, CC-90003, GDC-0994, and MK-8533.
  • RAS(G12C) inhibitors include but are not limited to AMG 510 and MRTX849.
  • kits can include an antibody or antibodies that can be used to detect one or more of the biomarkers disclosed herein or their concentration or expression levels.
  • the kit can include an antibody that specifically binds CDK6 and/or CDK4.
  • the antibodies in the kit may be monoclonal or polyclonal and can be further conjugated with a detectable label.
  • the kit includes probes that can be used to identify or detect CDK6 and/or CDK4.
  • the kit includes any of the nucleic acid arrays.
  • the kit includes probes and antibodies that can be used to identify or detect CDK6 and/or CDK4 or their expression or expression levels.
  • kits can, optionally, contain instructions for detecting and/or measuring the concentration of one or more proteins or the levels of mRNA in a biological sample (e.g., a tumor sample).
  • the kits can optionally include, e.g., a control (e.g., a concentration standard for the protein being assessed) or control labeled-amplicon set containing known amounts of one or more amplicons recognized by nucleic acid probes of the array.
  • the control can be an insert (e.g., a paper insert or electronic medium such as a CD, DVD, or floppy disk) containing an expression level or expression level ranges of one or more proteins or RNAs (e.g., of CDK6/CDK4).
  • kits can include one or more reagents for processing a biological sample (e.g., calibration reagents, buffers, diluents, color reagents, reagents to stop a reaction).
  • a kit can include reagents for isolating a protein from a tumor sample and/or reagents for detecting the presence and/or amount of a protein in a tumor sample (e.g., an antibody that binds to the CDK6 that is the subject of the detection assay and/or an antibody that binds the antibody that binds to the CDK6).
  • the kit includes at least one microplate (e.g., a 96 well plate; i.e., 12 strips of 8 wells).
  • the microplate can be provided with its corresponding plate cover.
  • the microplate can be polystyrene or of any other suitable material.
  • the microplate can have the antibody that is used to identify the presence of a particular biomarker (e.g., CDK6) coated inside each well.
  • the antibody may be conjugated to a detectable label.
  • kits can include a software package for analyzing the results of, e.g., expression profile or a microarray analysis.
  • kits can also include one or more antibodies for detecting the protein expression of the genes described herein (e.g., CDK6/CDK4).
  • a kit can include (or in some cases consist of) one or a plurality of antibodies capable of specifically binding to one or more proteins encoded by any of the genes described herein and optionally, instructions for detecting and/or measuring the concentration of one or more proteins and/or a detection antibody comprising a detectably-labeled antibody that is capable of binding to at least one antibody of the plurality.
  • the kits can include antibodies that recognize CDK6, CDK4, or both.
  • the kits can include antibodies that recognize CDK6.
  • the kits can include antibodies that recognize CDK4.
  • the kit can also optionally include one or more unit doses of a cancer therapy.
  • kits described herein can also, optionally, include instructions for administering a cancer therapy, where the concentration of one or more proteins or expression level of one or more RNAs predicts that a subject having or suspected of having a cancerous tumor will respond to a cancer therapy.
  • the kit comprises one or more of the following:
  • a microplate e.g., a 96 well plate.
  • the microplate can be coated with an anti- CDK6 antibody that is conjugated with a detectable label.
  • the anti-CDK6 antibody may monoclonal or polyclonal.
  • the antibody can be e.g., from mouse, rabbit, rat, or guinea pig.
  • the detectable label can be e.g., horse radish peroxidase, biotin, a fluorescent moiety, a radioactive moiety, a histidine tag, or a peptide tag.
  • the microplate can be provided with a cover.
  • a vial containing anti-CDK6 conjugated with a detectable label can be e.g., horse radish peroxidase, biotin, a fluorescent moiety, a histidine tag, a peptide tag.
  • the vial can also include a preservative.
  • CDK6 can be a recombinant human CDK6.
  • a vial containing wash buffer (vi) a vial containing wash buffer.
  • the buffer may be provided as a concentrate.
  • Cell lines HEK293T, HEK293FT, Huh-7, U87MG, A375 and A673 were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and penicillin/streptomycin, MCF7, T47D, ZR-75-1, CAMA-1, NCI-H358, Colo205, SK- MEL-1, IGROV-1, TC-71, Mino, JeKo-1, Granta 519, Z- 138, REC-1, KMS-12-PE, MV4-11, MOLM-14, Pfeiffer, SK-MEL-2, A549, Calu-6, NCI-H1792, NCI-H2087, NCI- H2291, NCI-H1915, NCI-H1666, NCI-H1395, HCC827, PC9, HCT15, HCT116, RKO, LoVo, HT-29, SW620 and BT549 were maintained in RPMI1640 supplemente
  • Huh-7 cells were provided by Dr. Amaia Lujambio.
  • Z-138 cells were provided by Dr. E. Premkumar Reddy.
  • REC-1, KMS-12-PE and Pfeiffer were gifts from Dr. Samir Parekh.
  • MV-4-11 and MOLM-14 cells were provided by Dr. Iannis Aifantis (New York Uni veri sty).
  • Mino, JeKo-1, Granta 519 were gifts from Dr. Shannon M. Buckley (University of Kansas).
  • A673 and TC-71 were provided by Dr. Christine A. Pratilas (Johns Hopkins).
  • HEK293FT cells were a kind gift from Dr.
  • Tet-pLKO-puro Plasmids Tet-pLKO-puro (item #21915) was purchased from Addgene.
  • shCDK4 SEQ ID NO: 5; TRCN0000018364: 5' - CCG GGATGACTG GCC TCG AGATGT ACT CGA GTA CAT CTC GAG GCC AGT CAT CTT TTT G -3' and SEQ ID NO: 6;
  • TRCN0000010520 5' - CCG GAC AGT TCG TGA GGT GGC TTT ACT CGA GTA AAG CCA CCT CAC GAA CTG TTT TTT G -3'), shCDK6 (SEQ ID NO: 7; TRCN0000010081: 5'- CCG GGT CTC ACC CAT ACT TCC AGG ACT CGA GTC CTG GAA GTA TGG GTG AGA CTT TTT G-3' and SEQ ID NO: 8;
  • A673, TC-71 and NSCLC cell lines were transfected with SMARTpool ON- TARGETplus Non-targeting pool, or SMARTpool ON-TARGETplus CDK4-siRNA (L- 003238-00-0005), or SMARTpool ON-TARGETplus CDK6-siRNA(L-003240-00-0005) (Dharmacon) using Lipofectamine RNAiMAX (Thermo Fisher).
  • NP40 buffer 50mM Tris pH 7.5, 1% NP40, 150mM NaCl, 10% Glycerol, lmM EDTA
  • protease and phosphatase inhibitors (Roche). Lysates were centrifuged at 15,000 rpm for 10 min and the protein concentration was quantified using BCA (Thermo Fisher). Protein G agarose (Thermo Fisher) was used for immunoprecipitations.
  • the following antibodies were used: phospho-Rb (Ser807/811), Rb (4H1), PLK1 (208G4), cyclin D2 (D52F9), cyclin E2, Aktl (2H10), JAK2 (D2E12), DHFR and B-Actin (13E5) (Cell Signaling), CDK4 (H-22), CDK6 (C-21; DCS-83), CDK2 (D-12), CDK7 (C-19), CDK9 (D-7), p 15/16 (C-7), pl6 (C-20), pl8 (118.2), p57 (KP39), cyclin A (H-432; B-8), cyclin D1 (M- 20; A- 12), cyclin D3 (D-7), cyclin El (HE12), CDC37 (H-271; E-4), Hsp70 (W27) and Hsp90a/B (F-8) (Santa Cruz), p27 and c-Raf (BD Transduction Laboratories), p21 and
  • Palbociclib, MS 140 and ribociclib were assayed by Reaction Biology Corporation for in vitro kinase activity. Briefly, skinase substrates were added to base reaction buffer [20 mM Hepes (pH 7.5), 10 mM MgC12, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3V04, 2 mM DTT, 1% DMSO] Differential CDK4/6 complexed with cyclin D1 were then diluted in the substrate solution.
  • base reaction buffer 20 mM Hepes (pH 7.5), 10 mM MgC12, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3V04, 2 mM DTT, 1% DMSO
  • kinase activity data were presented as % kinase activity in samples relative to DMSO control. IC50 values were calculated using GraphPad Prism 5.
  • Lentivirus was produced by transfecting HEK293FT cells with a lentiviral transfer vector, psPAX2 and pMD.G at a 5:4: 1 ratio using Lipofectamine 2000 (Thermo Fisher). The viral supernatant was collected 72 h after transfection and filtered through a 0.45pm filter unit (Millipore). For Dox-inducible stable CDK4/6 knockdown cell lines, cells were transduced with shCDK4 or shCDK6 lentivirus in the presence of 8 pg/ml polybrene (EMD Millipore) and selected with 2 pg/ml puromycin (Thermo Fisher) for 5- 7 days.
  • EMD Millipore polybrene
  • Thermo Fisher puromycin
  • Cell lysates were prepared and labeled according to the manual instructions for the Pierce Kinase Enrichment Kits and ActivX Probes (Thermo Scientific). Briefly, cells were pre-treated with PB for 2 hr, lysed and centrifuged at 16,000 x g for 10 min at 4 oC. The supernatant was desalted through Zeba Spin Desalting Columns. 1 mg of total cell lysates in 500 pi were used for ATP competition reaction with a final concentration of 5 pM desthiobiotin-ADP probe for 10 min at room temperature. Samples were mixed with 500 pi 8 M urea and 50 pi streptavidin agarose for 1 hr at room temperature on a rotator.
  • Human CRBN gRNA (5’-CACCGTAAACAGACATGGCCGGCGA-3’) was designed using the following website: http://crispr.mit.edu/ and cloned into BsmBl- digested lentiCRISPRv2 (Addgene, #52961). ZR-75-1 cells were transduced with sgCRBN lentivirus in the presence of 8 pg/ml polybrene followed by selecting with 2 pg/ml puromycin for 5 days. Cells were plated at 0.3 cells/well in a 96-well plate. After 2-3 weeks, individual clones were expanded.
  • CRBN homozygous knockout clones were validated by genotyping with primers (F : 5’- AAG TCA TGC TAA GGG CTG GAA C - 3’, R: 5’- GGATGG GTT TCC TGT TCT TAA TAG -3’) and Western Blotting.
  • Colo205 cells were treated with 0.3 mM MS 140 or MS140-ve for 5 hr in duplicate.
  • Cell pellets were lysed in lysis buffer containing 8 M urea, 50 mM Tris, pH 8.0, 75 mM NaCl, 1 mM MgC12, and 500 units Benzonase. Proteins were reduced with DTT and alkylated with iodoacetamide. After precipitation, proteins were first digested with LysC for 4 hr at 37°C. The solution was diluted 4-fold with 25 mM Tris, pH 8.0, 1 mM CaC12 and further digested with trypsin (Promega) for 12 hr at 37 °C. Peptides were desalted on Sep-Pak Light Cl 8 cartridges (Waters, Milford, MA) and dissolved in 30% acetonitrile, 0.1% TFA before loading on a 300-m Source 15S (GE Healthcare,
  • Samples were desalted using PepClean Cl 8 spin columns (Pierce) according to the manufacturer’s directions and resuspended in aqueous 0.1% formic acid.
  • Sample analysis was performed via reversed phase LC-MS/MS using a Proxeon 1000 nano-LC system coupled to a Q Exactive mass spectrometer (Thermo Scientific, San Jose, CA).
  • the Proxeon system was configured to trap peptides using a 3 -cm long, 100-m inner diameter Cl 8 column at 5 1/min liquid flow that was diverted from the analytical column via a vent valve, whereas elution was performed by switching the valve to place the trap column in line with a 15-cm long, 75-m inner diameter, 3.5-m, 300- ⁇ particle Cl 8 analytical column.
  • Analytical separation of all the tryptic peptides was achieved with a linear gradient of 2-30% buffer B over 240 min at a 250 nl/min flow rate, where buffer A was aqueous 0.1% formic acid, and buffer B was acetonitrile in 0.1% formic acid.
  • LC- MS experiments were also performed in a data-dependent mode with full MS (externally calibrated to a mass accuracy of 5 ppm and a resolution of 70,000 at m/z 200) followed by high energy collision-activated dissociation-MS/MS of the top 20 most intense ions.
  • High energy collision-activated dissociation-MS/MS was used to dissociate peptides at a normalized collision energy of 27 eV in the presence of nitrogen bath gas atoms. All five bRPLC fractions were derived from three process technical replicates of each tumor sample and were subjected to two independent LC-MS runs resulting in the production of 20 LC-MS runs for global peptide analysis.
  • Peptide inference was made with a false discovery rate (FDR) of 1%, and peptides were assigned to proteins with a protein FDR of 5%.
  • FDR false discovery rate
  • a precursor ion mass tolerance of 20 ppm was used for the first search that allowed for m/z retention time recalibration of precursor ions that were then subjected to a main search using a precursor ion mass tolerance of 6 ppm and a product ion mass tolerance 0.5 Da.
  • Search parameters included up to two missed cleavages at Lys/Arg on the sequence, oxidation of methionine, and protein N- terminal acetylation as a dynamic modification. Carbamidomethylation of cysteine residues was considered as a static modification.
  • Peptide identifications are reported by filtering of reverse and contaminant entries and assigning to their leading razor protein.
  • LFQ was performed based on peak area.
  • the measured area under the curve of m/z and the retention time-aligned extracted ion chromatogram of a peptide were performed via the label-free quantitation module found in MaxQuant version 1.3.0.5 (30). All replicates for each PDX were included in the LFQ experimental design with peptide- level quantitation performed using unique and razor peptide features corresponding to identifications filtered with a posterior error probability of 0.06, peptide FDR of 0.01, and protein FDR of 0.05.
  • the MaxQuant peptide and protein groups files were processed and stored in an Oracle database, and statistical analysis, model building, and visualization of a majority of data were performed based on Statistical Analysis Software code and R script that was developed in-house.
  • the affinity purified proteins were reduced, alkylated, and digested with trypsin directly on the beads. Briefly, the beads were resuspended in lOOuL lOOmM ammonium bicarbonate. Proteins were reduced with 2pl of 0.2M dithiothreitol (Sigma) for one hour at 57°C at pH 7.5, alkylated with 2m1 of 0.5M iodoacetamide (Sigma) for 45 minutes at room temperature in the dark, and digested using 200ng sequencing grade trypsin (Promega) overnight at room temperature with gentle shaking.
  • the solution was transferred to a new tube and the digestion stopped by adding 100 ul of a 5% formic acid and 0.2% trifluoroacetic acid (TFA) R2 50 pm Poros (Applied Biosystems) beads slurry in water.
  • the samples were allowed to shake at 4°C for 3 hour.
  • the beads were loaded onto Cl 8 ziptips (Millipore), equilibrated with 0.1% TFA, using a microcentrifuge for 30 seconds at 6,000 rpm.
  • the beads were washed with 0.5% acetic acid.
  • Peptides were eluted with 40% acetonitrile in 0.5% acetic acid followed by 80% acetonitrile in 0.5% acetic acid.
  • the organic solvent was removed using a SpeedVac concentrator and the sample reconstituted in 0.5% acetic acid.
  • MS/MS spectra were searched against the Uniprot human reference proteome database containing common contaminant proteins using Sequest within Proteome
  • the search parameters were as follows: precursor mass tolerance ⁇ 10 ppm, fragment mass tolerance ⁇ 0.02 Da, digestion parameters trypsin allowing two missed cleavages, fixed modification of carbamidomethyl on cysteine, variable modification of oxidation on methionine, variable modification of deamidation on glutamine and asparagine, and a 1% peptide and protein FDR searched against a decoy database. The results were filtered to only include proteins identified by at least two unique peptides.
  • Cell viability assay Cells were seeded at 3-10 x 103 cells/well in 96-well plates. 24 hr after seeding, cells were treated with palbociclib and MS140 at a range of concentrations for 72 hr. 10 pi of 0.1 mg/ml resazurin (Sigma- Aldrich) was added to cells and incubated for 2-3 hr at 37°C. Cell viability was determined by measuring the fluorescence at 560 nm excitation wavelength and 590 nm emission wavelength using a Molecular Devices Spectramax M5 plate reader. IC50 values were calculated using log-transformed, normalized data in GraphPad Prism 5.0.
  • Cells were seeded at 1-10 x 103 cells/well in six-well plates. The next day, cells were treated with the increasing concentrations of palbociclib and MS 140 for 10-15 days. Cell culture medium was replaced every 2 days in the presence or absence of inhibitors. Cells were fixed with 10% formalin solution (Sigma- Aldrich) for 5 min at RT followed by 0.05% crystal violet for 25 min. cells were de-stained with tap water and air-dried.
  • Complementary DNA was synthesized with a Superscript IV First-Strand kit (Thermo Fisher).
  • Quantitative real-time PCR was performed using a Fast SYBR Green Master Mix kit (Thermo Fisher) with a 7500 Fast realtime PCR system (Applied Biosystems).
  • PCR primers are as follows: h GAPDH forward: 5’-ACA ACT TTG GTATCG TGG AAG G- 3’, reverse: 5’- GCC ATC ACG CCA C AG TTT C-3’; h PLK1 forward: 5’- CAC CAG CAC GTC GTA GGA TTC-3 ’, reverse: 5’- CCG TAG GTA GTATCG GGC CTC-3’; h CCNA2 forward: 5’- CGC TGG CGG TAC TGA AGT C-3’, reverse: 5’- CGC TGG CGG TAC TGA AGT C- 3’; hAURKB forward: 5’- CAG AAG AGC TGC AC A TTT GAC G -3’, reverse: 5’- CCT TGA GCC CTA AGA GCA GAT TT -3’; h CDC45 forward: 5’- CTT GAAGTT CCC GCC TAT GA A G -3’, reverse: 5’- GCA TGG TTT GCT CCACTATCT
  • m GAPDH forward 5’- AGG TCG GTG TGAACG GAT TTG -3’, reverse: 5’- TGT AGA CCA TGT AGT TGAGGT CA-3’; mAURKB forward: 5’- CAG AAG GAG AAC GCC TAC CC 3’, reverse: 5’- GAG AGC AAG CGC AGA TGT C -3’; m CCNA2 forward: 5’- GCC TTC ACC ATT CAT GTG GAT -3’, reverse: 5’- TTG CTC CGG GTA AAG AGA CAG -3’; mE2Fl forward: 5’- CTC GAC TCC TCG CAG ATC G -3’, reverse: 5’- GAT CCA GCC TCC GTT TCACC -3’; mPCNA forward: 5’- TTT GAG GCA CGC CTG ATC C, reverse: 5’- GGAGAC GTG AGACGAGTC CAT -3’; mPLKl forward: 5
  • RNA interference (RNAi) dependency data were obtained from the 20Q1 public Avana dataset containing genome-scale CRISPR knockout screens for 18,333 genes in 739 cell lines. The gene dependencies were estimated for each gene and cell lines by the CERES algorithm (Meyers RM et al, 2017).
  • RNA interference (RNAi) dependency data were derived from combination of the Broad Institute Project Achilles, Novartis Project Drive, and Marcotte et al. database (Robert McDonald 3 rd et al. (2017), Cell Jul 27;170(3):577- 592. elO; Tshemiak A. et al., Cell. 2017 Jul 27;170(3):564-576; Richard Marcotte R. et al.
  • mice obtained from Envigo Laboratories were injected subcutaneously with 1 x 107 JeKo-1 cells in 1:1 PBS/ Matrigel GFR membrane Matrix (Corning) or 5 x 106 Colo205 cells in PBS.
  • Mice were treated with vehicle (5% DMSO and 95% PEG 300) or MS 140 (25 or 30 mg/kg) intraperitoneally, twice daily, or palbociclib (50 or 60 mg/kg) orally once daily for 3 days when tumors reach around 100 mm3. 5 h after the last dose, tumors were collected for further analysis. Liver and kidney were collected for qPCR analysis.
  • HPLC spectra for all compounds were acquired using an Agilent 1200 Series system with DAD detector. Chromatography was performed on a 2.1 c 150 mm Zorbax 300SB-C18 5 pm column with water containing 0.1% formic acid as solvent A and acetonitrile containing 0.1% formic acid as solvent B at a flow rate of 0.4 mL/min. The gradient program was as follows: 1% B (0-1 min), 1-99% B (1-4 min), and 99% B (4-8 min). High resolution mass spectra (HRMS) data were acquired in positive ion mode using an Agilent G1969AAPI-TOF with an electrospray ionization (ESI) source.
  • HRMS high resolution mass spectra
  • Example 1 Intrinsic resistance to CDK4/6i is associated with incomplete inhibition of Rb/E2F and expression of CDK6
  • CDK4/6i-sensitive PB concentration-dependent effects of PB on the growth of cancer cell lines derived from a variety of Rb-proficient tumor types are assessed. Large variations in cell line response to CDK4/6i are observed, consistent with previous reports (Gong X. et al, (2017) Cancer Cell 32, 761-776; Kim S. et al, (2016) Oncotarget 9, 35226-35240; Ruscetti M. et al., (2016). Science 362, 1416-1422).
  • CDK4/6i-sensitive CDK4/6i-sensitive PB concentrations under 500nM and in many cases as low as 80 nM are sufficient for substantial (over 90%) growth inhibition.
  • CDK4/6i-resistant CDK4/6i-resistant
  • PB concentrations even as high as 2mM have only modest effects on cell growth (FIG. 1A and FIG. 8A).
  • the CDK4/6i-S group include ER+ positive breast cancer cell lines, as expected (Finn R.S. et al, (2009) Breast Cancer Res 11, R77), as well as cancer cell lines from several other tumor types.
  • most Rb-proficient cell lines derived from common solid tumor types including non-small cell lung carcinoma (NSCLC), melanoma and colorectal carcinoma (CRC) showed a CDK4/6i-R phenotype (FIG. 1A and FIG. 8A)
  • CDK4/6i-R tumor cells indicating that incomplete inhibition of Rb/E2F signaling in CDK4/6i-R tumor cells is a general property of CDK4/6i.
  • most CDK4/6i-S cell lines are found to express CDK6 at very low levels, whereas the CDK4/6i-R cell lines express both CDK4 and CDK6 (FIG. IE).
  • Example 2 Tumors expressing both CDK4 and CDK6 depend selectively on CDK6
  • CDK4 or CDK6 Overexpression of a target is a mechanism that can drive resistance to small- molecule inhibitors.
  • shRNA-mediated knockdown of CDK4 or CDK6 is conducted to assess the sensitization of PB-unresponsive cells to CDK4/6i.
  • knockdown of CDK4 is found to reduce Rb/E2F signaling (FIG. 9A).
  • inducible CDK6 knockdown is found to suppress phospho-Rb and downstream E2F signaling (FIG. 2A), as well as cell growth (FIG. 2B), which is further suppressed by treatment with PB.
  • CDK6 but not CDK4
  • CDK6 but not CDK4
  • ectopic expression of an shRNA-resistant mutant of CDK6 is found to rescue cell growth and Rb/E2F output inhibition promoted by CDK6 shRNA-mediated knock down (FIG. 2D), thus establishing the specific role of CDK6 in driving Rb/E2F signaling and cell growth in these cells.
  • CDK4-T172 an established marker of CDK4 activation (Kato, J.Y. et al, (1994) Mol Cell Biol 14, 2713-2721), is readily detected in the CDK4/6i-S cells, but is substantially lower in the CDK4/6i-R cells (FIG. IE). All of these findings suggest that despite expression of both CDK4 and CDK6, Rb/E2F signaling is driven selectively by CDK6 in CDK4/6i-R tumor cells.
  • the AVANA Dependency map is an online research and analysis tool developed by the Broad Institute, based on data derived from profiling hundreds of cancer cell line models for genomic information and sensitivity to genetic and small molecule perturbations.
  • CDK4 expression is found to be relatively uniform across cancer cell lines, however CDK6 expression is highly variable.
  • CDK6 expression and protein levels are found to be predictive of dependence on CDK6, with high levels of CDK6 correlating with dependence on CDK6 and low CDK6 levels correlating with dependence on CDK4, however CDK4 protein or mRNA expression levels are not predictive of dependence on CDK4 (FIG. 2E and FIG. 9B).
  • Example 3 Low expression of CDK6 predicts for sensitivity to CDK4/6i in NSCLC
  • NSCLC Non-small-cell lung carcinoma
  • RNA expression data is analyzed from tumors from the phase III JUNIPER trial (NCT02152631), in which abemaciclib is evaluated in KRASmutated, advanced NSCLC patients (21), alongside erlotinib, a tyrosine kinase inhibitor.
  • NSCLC tumors with low CDK6 expression show significantly longer PFS and OS, compared to patients with tumors expressing high or intermediate levels of CDK6 in the abemacicib arm (FIGS. 3E-F).
  • CDK6 expression can be a predictive biomarker for tumor cell response to CDK4/6i in many tumor types, including a substantial portion of NSCLC, for which there are currently no available targeted therapeutics.
  • Example 4 A CDK4/6-degrader (PROTAC) is more effective than CDK4/6i in CDK4/6i-S tumor cells
  • CDK4/6 might result in more potent inhibition of Rb/E2F output and growth suppression of CDK4/6i-R tumor cells.
  • hetero-bifunctional small molecules are developed (Gadd M.S., et al, (2017) Nat Chem Biol 13, 514-521) that both inhibit CDK4/6 kinase activity as well as target CDK4/6 proteins for degradation.
  • the solvent- exposed piperazine is used as the linker attachment point.
  • CDK4/6-directed PROTACs are synthesized by linking PB to pomalidomide, a moiety with high affinity for the E3 ligase cereblon (CRBN), a component of a cullin-RING ubiquitin ligase complex (Bondeson D.P et al, (2015). Nat Chem Biol 11, 611-617; Winter G. E. et al, (2015) Science 348, 1376-1381; E. S. Fischer E.S. et al, (2014) Nature 512, 49-53; Ito T. et al, (2010) Science 327, 1345-1350; Chamberlain P. P.
  • MS 140 is identified (FIG. 4A), as a highly potent CDK4/6 kinase inhibitor in vitro (FIG. 4B) that markedly suppressed Rb/E2F signaling and reduces CDK4 and CDK6 protein levels in a concentration and time-dependent manner (FIGS. 11A-B).
  • Degradation of CDK4/6 proteins by MS 140 is specific, as shown by its abrogation upon pretreatment with excess PB, or pomalidomide (FIG. 4C and FIG. 11C).
  • CDK4/6 protein degradation by MS 140 is also confirmed to be specifically mediated by the proteasome by its abrogation upon pretreatment with the proteasome inhibitor bortezomib, or with the Nedd8-activating enzyme inhibitor MLN4924 (FIG. 4C and FIG. 11C). Further, MS 140 fails to degrade CDK4 in cells in which CRBN had been knocked-out using CRISPR/Cas9 technology (FIG. 4D), confirming that target degradation by MS 140 was specifically mediated by CRBN. As a negative control, MS140-ve (FIG. 11D), a methyl analog of MS 140 is designed and synthesized, which is predicted not to bind CRBN (C. Zhang C. et al, (2016).
  • MS 140 potently and selectively inhibits and degrades CDK4/6 kinases in CDK4/6i-S tumor cells by targeting them to the CRL4-CRBN-E3 ubiquitin complex.
  • Treatment of various Rb-proficient tumor cell lines with MS 140 is found to result in 3 to 30-fold greater suppression of both Rb/E2F signaling and cell growth, compared to PB, in the CDK4/6i-S cells identified in our first screen (FIGS. 4G-H, and FIG. HE).
  • H358 A notable exception is H358, in which MS140 only modestly decreased CDK4 levels or inhibited Rb signaling as compared to PB (FIG. 11F). However, this cell line exhibits very low endogenous levels of CRBN (FIG. IE), consistent with the increased effectiveness of MS 140 being CRBN-dependent.
  • FIG. IE endogenous levels of CRBN
  • FIG. HG All Mantle Cell Lymphomas (MCL) tested show sensitivity to PB and higher sensitivity to MS 140 (FIG. HG), associated with more potent inhibition of Rb/E2F signaling by MS 140 (FIGS. 11H-I), consistent with these cells being predominantly CDK4-driven and expressing low levels of CDK6.
  • CDK4/6i-S phenotype a smaller group of cancer cell lines, mainly of hematopoetic origin and driven by CDK6 (Kim S. et al, (2016) Oncotarget 9, 35226-35240; Ghandi M. et al, (2019) Nature 569, 503-508), show a similar CDK4/6i-S phenotype (FIGS. 4I-J).
  • FIG. 11 J These data on growth response of hematopoetic tumor lines to PB and MS 140 are summarized in FIG. 11 J.
  • MS 140 is more effective than PB in inhibiting Rb/E2F signaling and cell growth of many CDK4/6i-S tumor cell lines.
  • CDK4/6i-S cells are predominantly those expressing CDK4 and low CDK6, but a smaller subset of CDK6-driven tumor cells are identified, that are also sensitive to CDK4/6i and more sensitive to the CDK4/6 degrader.
  • MS 140 was less potent in promoting CDK6 protein degradation and in suppressing cell growth, compared to shRNA-mediated CDK6 knockdown. These results prompted us to directly compare the extent of CDK4/6 degradation by MS 140 in CDK4/6i-S and CDK4/6i-R cell lines. Treatment with MS 140 resulted in potent degradation of CDK4/6 in both CDK4- driven and CDK6-driven CDK4/6i-S cells, but promoted only minimal CDK4/6 degradation in CDK6-driven CDK4/6i-R cells (FIG. 5E).
  • YKL-06-102 was similarly ineffective to the rest of CDK4/6-degraders in degrading CDK6 in the CDK4/6-R cells.
  • failure to degrade CDK4/6 in CDK4/6i-R cells is a general property of CDK4/6 degraders.
  • CDK6-dependent tumor cells can be divided in two groups. In most tumor cells that express CDK6, in which CDK4/6i bind weakly to CDK6 and these cells are therefore resistant to CDK4/6i (and consequently CDK4/6 degraders). In a smaller group of CDK6-dependent cells, CDK6 binds strongly to CDK4/6i (a higher than 1°C shift in Tm as measured by CETSA), and they are thus sensitive to CDK4/6i and degraders.
  • CDK4/6i-resistant cells express CDK6 as a thermostable, weak HSP90 client protein
  • CDK6 immunoprecipitation of CDK6 was carried out followed by mass spectrometry analysis of interacting partners.
  • CDK6 immunoprecipitated by a CDK4/6-S KMS-12-PE
  • CDK4/6i-R Calu6 cell line
  • CDK6 Association of CDK6 with known CDK6 interactors, including members of the cyclin, INK4 or CIP/KIP families correlated well with their relative basal expression in the two cell lines (FIG. 6A and FIG. 13A).
  • a 3-5 fold higher interaction of CDK6 is seen with components of the HSP90/CDC37 chaperoning complex in CDK4/6i-S compared to CDK4/6i-R cells (FIG. 6B).
  • the observation was further confirmed by direct co- immunoprecipitation experiments showing a much stronger interaction of CDK6 with HSP90 and CDC37 in CDK4/6i-S as compared to CDK4/6i-R tumor cells (FIG. 6C).
  • the findings here support a model by which, in CDK6-driven and CDK4/6i-S tumor cells, the CDK6 protein population is enriched in highly active conformations that have strong binding affinity for current CDK4/6i (and consequently for CDK4/6 degraders).
  • CDK4/6i-R cells CDK6 is expressed as thermostable, weak HSP90-client, with lower affinity for the CDK4/6L
  • S178P Selfstaele, L. et al, (2009) Mol Cell Biol 29, 4188-4200
  • CDK6 is also found to be highly thermostable and resistant to degradation by MS 140, similar to the CDK6 state in PB-resistant cells (FIG. 13G and not shown).
  • MS 140 was found to be more potent than PB in CDK4/6i-S tumors, but less potent than PB in CDK4/6i-R tumors, because in the latter cells, CDK6 binds weakly to CDK4/6i. This raises the possibility that MS 140 would also minimally affect normal tissues expressing CDK6 with similar properties, and it would thus exhibit a higher therapeutic index than PB.
  • the effect of treatment of the same daily dose of either MS 140 or PB was assessed on expression of known E2F targets in tumor and normal tissue (kidney and liver). It is found that treatment with MS 140 results in more potent inhibition in the tumor than in normal tissue of several E2F -target transcripts, compared to PB (FIG.
  • CDK4/6-directed degradation may be both a highly effective and well-tolerated therapeutic strategy for patients with CDK4-driven tumors (ER+ breast cancers, Ewing sarcomas, MCL, etc), as well as for patients with tumors dependent on the CDK4/6insensitive CDK6.

Abstract

La présente invention concerne des biomarqueurs, par exemple, dans le diagnostic et le traitement du cancer. L'invention concerne des méthodes de traitement du cancer sur la base des taux de CDK6 et/ou de CDK4 dans les tumeurs. L'invention concerne également des procédés d'identification de composés utiles dans le traitement de cancers, et des Kits pour quantifier les taux de CDK6 et/ou de CDK4.
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US20030039998A1 (en) * 1999-09-30 2003-02-27 National Jewish Medical & Research Center Method for regulating cell growth and assays related thereto
US20070202555A1 (en) * 2006-02-28 2007-08-30 Sysmex Corporation Method for judging feature of malignant tumor
WO2018106870A1 (fr) * 2016-12-08 2018-06-14 Icahn School Of Medicine At Mount Sinai Compositions et méthodes pour le traitement du cancer à médiation par cdk4/6

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US20030039998A1 (en) * 1999-09-30 2003-02-27 National Jewish Medical & Research Center Method for regulating cell growth and assays related thereto
US20070202555A1 (en) * 2006-02-28 2007-08-30 Sysmex Corporation Method for judging feature of malignant tumor
WO2018106870A1 (fr) * 2016-12-08 2018-06-14 Icahn School Of Medicine At Mount Sinai Compositions et méthodes pour le traitement du cancer à médiation par cdk4/6

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