CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application Serial No. 63/407,870, filed on September 19, 2022. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named “07039-2154W01.xml.” The XML file, created on August 17, 2023, is 40000 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This document relates to methods and materials for assessing and/or treating mammals (e.g., humans) having prostate cancer. For example, the methods and materials provided herein can be used to determine whether or not a prostate cancer (e.g., a castrationresistant prostate cancer (CRPC)) is likely to respond to a particular cancer treatment (e.g., treatment with one or more inhibitors of a poly (ADP-ribose) polymerase (PARP) polypeptide). Also provided are methods and materials for using one or more cancer treatments to treat a mammal (e.g., a human) identified as likely to respond to a particular cancer treatment.

BACKGROUND INFORMATION

Prostate cancer (PCa) is the second leading cause of cancer death in American men and androgen deprivation therapy (ADT) is the mainstay treatment of advanced/metastatic PCa. Despite initial response to ADT, a majority of these patients eventually develop CRPC associated with a reduced survival rate (Garcia, Cancer, 118(10):2583-2593 (2012); Petrylak, Am. J. Manag. Care, 19:8366-8375 (2013); and Huang etal., J. Hematol. Oncol., 5:35 (2012)). SUMMARY

Alternative splicing of the androgen receptor (AR) gene, driven by usage of intronic polyadenylation (IPA) sites within the AR gene, can result in expression of androgen receptor variants (AR-Vs) that have been implicated in the development of CRPC. This document is based, at least in part, on the discoveries that a complex including (i) a CYCLIN K polypeptide and (ii) a cyclin-dependent kinase 12 (CDK12) polypeptide (e.g., a CYCLIN K- CDK12 complex) can regulate IPA usage in the AR gene leading to AR-V production and hormonal therapy resistance in CRPC, and that (a) the presence of a CDK12 polypeptide deficiency and/or (b) the presence of a reduced level of expression of a CCNK nucleic acid (e.g., resulting in a reduced level of a CYCLIN K polypeptide) can be used to determine that a prostate cancer (e.g., CRPC) is likely to be responsive to one or more inhibitors of a PARP polypeptide.

This document provides methods and materials for assessing and/or treating prostate cancer. In some cases, this document provides methods and materials for determining whether or not a mammal (e.g., a human) having prostate cancer (e.g., CRPC) is likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PARP polypeptide), and, optionally, administering to the mammal one or more cancer treatments selected based, at least in part, on whether or not the mammal is likely to respond to a particular cancer treatment. For example, a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal having prostate cancer can be assessed to determine if the mammal is likely to respond or is unlikely to respond to a particular cancer treatment based, at least in part, on (a) the presence or absence of a CDK12 polypeptide deficiency and/or (b) the presence or absence of a reduced level of expression of CCNK nucleic acid in the sample.

As demonstrated herein, the (a) the presence or absence of a CDK12 polypeptide deficiency and/or (b) presence or absence of a reduced level of expression of a CCNK nucleic add in a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal (e.g., a human) having prostate cancer can be used to determine whether or not that prostate cancer is likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PARP polypeptide). For example, a mammal (e.g., a human) having prostate cancer (e g., CRPC) can be identified as being likely to respond to one or more inhibitors of a PARP polypeptide based, at least in part, on (a) the presence of a CDK12 polypeptide deficiency and/or (b) the presence of a reduced level of expression of a CCNK nucleic acid (e.g., resulting in a reduced level of a CYCLIN K polypeptide) in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal, and, optionally, one or more inhibitors of a PARP polypeptide can be administered to the mammal (e.g., to treat the mammal). Also as demonstrated herein, inhibition of a CYCLIN K/CDK12 complex can sensitize a prostate cancer (e.g., CRPC) to one or more inhibitors of a PARP polypeptide. For example, one or more inhibitors of a CYCLIN K/CDK12 complex can be administered to a mammal (e.g., a human) having prostate cancer (e.g., CRPC) to sensitize the prostate cancer to one or more inhibitors of a PARP polypeptide, and, optionally, one or more inhibitors of a PARP polypeptide can be administered to the mammal to treat the mammal.

Having the ability to identify a mammal having prostate cancer as being likely to respond to a particular cancer treatment based, at least in part, on (a) the presence or absence of a CDK12 polypeptide deficiency and/or (b) the presence or absence of a reduced level of expression of a CCNK nucleic acid provides a unique and unrealized opportunity to provide an individualized approach in selecting effective prostate cancer therapies. Also, having the ability to identify a mammal having prostate cancer as being likely to respond to an inhibitor of a PARP polypeptide based, at least in part, on (a) the presence of a CDK12 polypeptide deficiency and/or (b) the presence of a reduced level of expression of a CCNK nucleic acid (e.g., resulting in a reduced level of a CYCLIN K polypeptide) provides a unique and unrealized opportunity to provide a safe and effective use of inhibitors of a PARP polypeptide to treat prostate cancer in this identified patient population.

In general, one aspect of this document features methods for assessing a mammal having a prostate cancer. The methods can include, or consist essentially of, (a) determining if a sample from a mammal having a prostate cancer contains the presence or absence of at least one of (i) a CDK12 polypeptide deficiency, and (ii) a reduced level of a CYCLIN K polypeptide; (b) classifying the prostate cancer as being likely to respond to an inhibitor of a PARP polypeptide if the presence of at least one of the (aXi) and the (aXii) is determined; and (c) classifying the prostate cancer as not being likely to respond to an inhibitor of a PARP polypeptide if the absence of each of the (a)(i) and the (a)(ii) is determined. The mammal can be a human. The prostate cancer can be a CRPC. The presence or absence of the CDK12 polypeptide deficiency can be determined. The sample can include the presence of the CDK12 polypeptide deficiency. The presence can be determined by detecting one or more mutations within the CDK12 polypeptide. The one or more mutations within the CDK12 polypeptide can include an amino acid substitution at residue 909 of a human CDK12 polypeptide having SEQ ID NO:43. The one or more mutations within the CDK12 polypeptide can include a G909R substitution, a G909E substitution a G909D substitution, a G909K substitution, or a G909H substitution. The presence or absence of the reduced level of said CYCLIN K polypeptide can be determined. The sample can include the presence of the reduced level of the CYCLIN K polypeptide. The method can include determining the presence of the CDK12 polypeptide deficiency and the presence of the reduced level of a CYCLIN K polypeptide. The method can include determining the absence of the CDK12 polypeptide deficiency and the absence of the reduced level of a CYCLIN K polypeptide. The sample can be a tissue sample comprising a prostate cancer cell. The inhibitor of the PARP polypeptide can be olaparib, rucaparib, niraparib, or talazoparib.

In another aspect, this document features methods for selecting a treatment for a mammal having a prostate cancer. The methods can include, or consist essentially of, (a) determining that a sample from a mammal having a prostate cancer contains at least one of (i) the presence of a CDK12 polypeptide deficiency and (ii) the presence of a reduced level of a CYCLIN K polypeptide, thereby identifying the mammal as being an identified mammal for treatment with an inhibitor of a PART polypeptide; and (b) selecting an inhibitor of a PARP polypeptide to be a treatment for the identified mammal. The mammal can be a human. The prostate cancer can be a CRPC. The sample can be a tissue sample comprising a prostate cancer cell. The method can include determining the presence of the CDK12 polypeptide deficiency and the presence of the reduced level of a CYCLIN K polypeptide. The inhibitor of the PARP polypeptide can be olaparib, rucaparib, niraparib, or talazoparib. The method can include administering the inhibitor of the PARP polypeptide to the mammal. The mammal can be a human. The prostate cancer can be a CRPC. The sample can be a tissue sample comprising a prostate cancer cell. The inhibitor of the PARP polypeptide can be olaparib, rucaparib, niraparib, or talazoparib. In another aspect, this document features methods for selecting a treatment for a mammal having a prostate cancer. The methods can include, or consist essentially of, (a) determining that a sample from a mammal having a prostate cancer contains (i) the absence of a CDK12 polypeptide deficiency and (ii) the absence of a reduced level of a CYCLIN K polypeptide, thereby identifying the mammal as being an identified mammal for treatment with either (1) a treatment that does not comprise an inhibitor of a PARP polypeptide or (2) a treatment that comprises an agent that sensitizes prostate cancer to an inhibitor of a PARP polypeptide and an inhibitor of a PARP polypeptide; and (b) selecting either (1) the treatment that does not comprise the inhibitor of a PARP polypeptide or (2) the treatment that comprises the agent and the inhibitor of said PARP polypeptide to be a treatment for the identified mammal. The method can include selecting the treatment that does not comprise the inhibitor of the PARP polypeptide. The treatment that does not include the inhibitor of the PARP polypeptide can include treatment with an anti-cancer agent. The anti-cancer agent can be docetaxel, cabazitaxel, mitoxantrone, estramustine, leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, darolutamide, or rezvilutamide. The method can include administering the anti-cancer agent to the mammal. The treatment that does not include the inhibitor of the PARP polypeptide can include a surgery to remove the prostate cancer, radiation treatment, and/or prostate tissue ablation. The method can include performing the surgery, the radiation treatment, and/or the prostate tissue ablation on the mammal. The mammal can be a human. The prostate cancer can be a CRPC. The sample can be a tissue sample comprising a prostate cancer cell. The inhibitor of the PARP polypeptide can be olaparib, rucaparib, niraparib, or talazoparib.

In another aspect, this document features methods for treating a mammal having a prostate cancer. The methods can include, or consist essentially of (a) determining that a sample from a mammal having a prostate cancer contains at least one of (i) the presence of a CDK12 polypeptide deficiency, and (ii) the presence of a reduced level of a CYCLIN K polypeptide; and (b) administering, to the mammal, an inhibitor of a PARP polypeptide. The mammal can be a human. The prostate cancer can be a CRPC. The sample can be a tissue sample comprising a prostate cancer cell. The method can include determining the presence of the CDK12 polypeptide deficiency and the presence of the reduced level of a CYCLIN K polypeptide. The inhibitor of the PARP polypeptide can be olaparib, rucaparib, niraparib, or talazoparib.

In another aspect, this document features methods for treating a prostate cancer. The methods can include, or consist essentially of, administering an inhibitor of a PARP polypeptide to a mammal that was identified as having at least one of (i) the presence of a CDK12 polypeptide deficiency, and (ii) the presence of a reduced level of a CYCLIN K polypeptide in a sample obtained from the mammal. The mammal can be a human. The prostate cancer can be a CRPC. The sample can be a tissue sample comprising a prostate cancer cell. The method can include determining the presence of the CDK12 polypeptide deficiency and the presence of the reduced level of a CYCLIN K polypeptide. The inhibitor of the PARP polypeptide can be olaparib, rucaparib, niraparib, or talazoparib.

In another aspect, this document features methods for treating a mammal having a prostate cancer. The methods can include, or consist essentially of (a) determining that a sample from a mammal having a prostate cancer contains (i) the absence of a CDK12 polypeptide deficiency and (ii) the absence of a reduced level of a CYCLIN K polypeptide; and (b) performing a treatment on the mammal that is either (1) a treatment that does not comprise an inhibitor of a PARP polypeptide or (2) a treatment that comprises an agent that sensitizes prostate cancer to an inhibitor of a PARP polypeptide and an inhibitor of a PARP polypeptide. The method can include performing the treatment that does not comprise the inhibitor of the PARP polypeptide. The treatment that does not include the inhibitor of the PARP polypeptide can include treatment with an anti-cancer agent. The anti-cancer agent can bedocetaxel, cabazitaxel, mitoxantrone, estramustine, leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, darolutamide or rezvilutamide. The treatment that does not include the inhibitor of the PARP polypeptide can include a surgery to remove the prostate cancer, radiation treatment, and/or prostate tissue ablation. The mammal can be a human. The prostate cancer can be a CRPC. The sample can be a tissue sample including a prostate cancer cell. The inhibitor of the PARP polypeptide can be olaparib, rucaparib, niraparib, or talazoparib. In another aspect, this document features methods for treating a prostate cancer. The methods can include, or consist essentially of, performing a treatment on a mammal identified as having (i) the absence of a CDK12 polypeptide deficiency and (ii) the absence of a reduced level of a CYCLIN K polypeptide in a sample obtained from the mammal, where the treatment is either (1) a treatment that does not comprise an inhibitor of a PARP polypeptide or (2) a treatment that comprises an agent that sensitizes prostate cancer to an inhibitor of a PARP polypeptide and an inhibitor of a PARP polypeptide. The method can include performing the treatment that does not comprise the inhibitor of the PARP polypeptide. The treatment that does not include the inhibitor of the PARP polypeptide can include treatment with an anti-cancer agent. The anti-cancer agent can be docetaxel, cabazitaxel, mitoxantrone, estramustine, leuprolide, goserelin, triptorelin, histrelin, degarelix, abiraterone, ketoconazole, flutamide, bicalutamide, nilutamide, enzalutamide, apalutamide, darolutamide or rezvilutamide. The treatment that does not include the inhibitor of a PARP polypeptide can include a surgery to remove the prostate cancer, radiation treatment, and/or prostate tissue ablation. The mammal can be a human. The prostate cancer can be a CRPC. The sample can be a tissue sample including a prostate cancer cell. The inhibitor of the PARP polypeptide can be olaparib, rucaparib, niraparib, or talazoparib.

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

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

FIGS. 1 A-1E show that CDK12 inhibition confers AR antagonist resistance in LAPC4 PCa cells. FIGS. 1 A LAPC4 cells were cultured in regular medium (containing fetal bovine serum without charcoal stripping), and were treated with vehicle (mock) or enzalutamide (ENZ) in the presence or absence of indicated drugs for 72 hours and followed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 -sulfophenyl)-2H- tetrazolium (MTS) assay. Three biological replicates were analyzed. **P < 0.01; ***p < 0.001. Dimethyl sulfoxide (DMSO), suberoylanilide hydroxamic acid (SAHA), trichostatin A (TSA), camptothecin (CPT), cycloheximide (CHX). FIG. IB) LAPC4 cells were treated with ENZ in combination with or without indicated drugs for one week and subjected to sulforhodamine B (SRB) assay. FIGS. 1C-1E) LAPC4 cells were stably infected with lentivirus expressing non-specific control small hairpin RNA sequences (shNS) or CDK12- specific small hairpin RNA sequences (shRNAs) (shCDK12 #1 and #2). Transfected cells were subjected to Western Blot (WB) analysis (FIG. 1C) and to a colony formation assay in the presence or absence of ENZ treatment (5 pM) for 11 days (FIG. ID and IE). Representative images showing colonies of the indicated cells are shown in FIG. ID and quantitative data are shown in FIG. IE. Mitogen-activated protein kinase 1 (ERK2), a loading control for WB. Three biological replicates were analyzed. ***P < 0.001.

FIGS. 2A-2K show that CDK12 alteration promotes AR IP A usage and androgen receptor splice variant 7 (AR-V7) expression. FIG. 2A) The frequency of the usage of polyadenylation sites (PASs) between exons 2 and 5 of AR. chromosome X (X). FIG. 2B). An analysis of 3' region extraction and deep sequencing (3’-READS) shows an APA peak in AR intron 3 region. FIG. 2C) A real-time polymerase chain reaction (RT-qPCR) analysis of AR-V7 and AR-FL mRNA using the indicated transcript-specific primers in LNCaP cells cultured in regular medium and treated with vehicle (DMSO) or indicated drugs. Three biological replicates were analyzed. FIGS. 2D-2E) LNCaP and C4-2 cells were treated with vehicle or THZ531 for 18 hours and subjected to the RT-qPCR analysis with indicated primers. Three biological replicates were analyzed. *P < 0.05; **P < 0.01; ***P < 0.001. FIGS. 2F-2G) LNCaP and C4-2 cells were transfected with indicated siRNAs for 72 hours and subjected to WB (FIG. 2F) and RT-qPCR analysis using indicated primers (FIG. 2G). ERK2 was used as a loading control for WB. Three biological replicates were analyzed. *P < 0.05; **P < 0.01; ***P < 0.001. FIGS. 2H-2K) 22Rvl and LAPC4 cells were stably infected with lentivirus expressing indicated shRNAs, and were subjected to WB (FIG. 2H and FIG. 2J). The cells of FIGS. 2H and 2J were cultured in medium containing charcoal-stripped serum (CSS) in the presence or absence of dihydrotestosterone (DHT; 1 nM) for 6 hours followed by RT-qPCR analysis using KLK3 gene primers (FIGS. 21 and 2K). Three biological replicates were analyzed. ***P < 0.001.

FIGS. 3 A-3I show that CDK12 alteration promotes AR IP A and ENZ resistance. FIG. 3 A) WB analysis of AR protein expression in the indicated PCa cell lines. ERK2 was used asa loading control for WB. FIG. 3B) A screenshot from the University of California, Santa Cruz (UCSC) Genome Browser of RNA-seq in the AR locus in the indicated PCa cell lines. FIG. 3C) A spectrum of CDK12 gene mutations detected in TCGA PCa patient samples (upper panel), and mutations in CDK12 G909 residue in 22Rvl cell line and patient samples of indicated cancer types (lower panel). FIG. 3D) A UCSC screenshot of RNA-seq in the AR locus in 22Rvl cell line and the TCGA-HC-A6HX patient sample. FIGS. 3E-3F) A WB (FIG. 3E) and a RT-qPCR analysis (FIG. 3F) in stable 22Rvl cell lines with or without CDK12 knockout and/or rescued with wild type (WT) or mutant CDK12. Three biological replicates were analyzed. ***P < 0.001. FIG. 3) Schematic diagram showing the genomic duplication in the region between exons 2 and 4 of the AR gene and the transcripts of AR-FL and AR-F7 in 22Rvl cells. FIGS. 3H-3I) CDK12 KO and rescued 22Rvl cells were treated with ENZ and subjected to colony formation assay. At 11 days after treatment, cell colonies were photographed (FIG. 3H) and quantified (FIG. 31). Three biological replicates were analyzed. ***P < 0.001.

FIGS. 4A-4H show that CDK12 alteration associates with hyperactivation of AR signaling pathway and IP A usage in AR gene locus in patient samples. FIGS. 4A-4B) A comparison of RNA-seq reads in the IP A sites in intron 3 of the CSTF3 (FIG. 4A) and AR (FIG. 4B) gene between CDK12 WT (n= 487) and deficient (n = 12) PCa patient samples in the TCGA cohort. ***P < 0.001. FIG. 4C) A comparison of AR score between CDK12 WT (n= 487) and deficient (n = 12) PCa patient samples from the TCGA cohort. *P < 0.05. FIG 4D) A PFS analysis between CDK12 WT (n= 487) and deficient (n = 12) PCa patient samples from the TCGA cohort. FIGS. 4E-4F). An overall survival (OS) analysis between CDK12 WT and deficient patient samples of the Stand Up to Cancer (SU2C) (FIG. 4E, n = 310 versus 15) or West Coast Dream Team (WCDT) cohort (FIG. 4F, n= 95 versus 3). FIGS. 4G-4H) A comparison of AR score between CDK12 WT and deficient patient samples of the SU2C (FIG. 4G, n = 310 versus 15) or WCDT cohort (FIG. 4H, n= 95 versus 3). *P < 0.05; **P < 0.01.

FIGS. 5A-5Q show that ADT promotes global IP A usage including AR IP A usage through repressing CCNK transcription. FIG 5 A) Measurement of IP A usage in C4-2 cells cultured in CSS medium supplemented with or without DHT (10 nM) by analyzing RNA-seq data (GSM2432781 versus GSM2432783). FIG. 5) WB analysis of indicated proteins in C4-2 and LNCaP cells cultured in CSS medium supplemented with or without DHT (10 nM) for 48 hours. ERK2 was used as a loading control for WB. FIG. 5C) A UCSC screenshot of RNA-seq data in the CCNK locus in C4-2 cells cultured in CSS medium supplemented with vehicle (mock) or DHT (10 nM) for 24 hours. FIG. 5D) A RT-qPCR analysis of CCNK mRNA expression in C4-2 and LNCaP cells cultured in CSS medium supplemented with vehicle (mock) or DHT (10 nM) for 48 hours. Three biological replicates were analyzed. ***P < 0.001. FIGS. 5E-5F) A WB (FIG. 5E) and a RT-qPCR analysis (FIG. 5F) in C4-2 and LNCaP cells cultured in regular medium treated with vehicle (mock) or ENZ (10 pM) for 72 hours. Three biological replicates were analyzed. ***P < 0.001. FIG. 5G-5H) A RT- qPCR (FIG. 5G) and a WB (FIG. 5H) analysis of CYCLIN K in LuCaP35 xenograft tumors from male mice treated with sham castration (control) or castration for a week. FIG. 51) Analysis of microarray data (GDS4120) for CCNK mRNA expression in LuCaP35 xenograft tumors in mice with or without castration (control versus castration). FIG. 5J) Analysis of RNA-seq for the expression of CCNK mRNA in paired PCa tissues from 36 patients before and after ADT and ENZ treatment. FIG. 5K) Meta-analysis of CMP data of transcription factors using the cistrome software to define possible transcription factors that could bind to the CCNK gene locus. FIG. 5L) A UCSC screenshot of AR ChlP-seq data in the CCNK gene locus in C4-2 and LNCaP cells cultured in CSS medium supplemented with vehicle or DHT (10 nM) for 24 hours. FIG. 5M) ChlP-qPCR analysis of AR occupancy in the CCNK gene promoter in C4-2 and LNCaP cells cultured in CSS medium supplemented with vehicle (mock) or DHT (10 nM) for 24 hours. Three biological replicates were analyzed. ***P < 0.001; n.s., not significant. FIG. 5N) A UCSC screenshot of AR ChlP-seq data in the CCNK gene locus in C4-2 cells treated with vehicle (mock) or ENZ (10 pM) for 24 hours. FIG. 50) A ChlP-qPCR analysis of AR occupancy in the CCNK gene promoter in C4-2 and LNCaP cells treated with vehicle or ENZ (10 pM) for 24 hours. Three biological replicates were analyzed. ***P < 0.001; n.s., not significant. FIGS. 5P-5Q) AWB (FIG. 5P) and aRT-qPCR analysis (FIG. 5Q) in C4-2 and LNCaP cells were transfected with control or AR-specific siRNAs for 72 hours. Three biological replicates were analyzed. ***P < 0.001.

FIGS. 6A-6K show that PARP1 inhibitor induces synthetic lethality in PCa cells treated with ENZ. FIGS. 6A-6C) AWB (FIG. 6A) and RT-qPCR analyses (FIG. 6B and 6C) in C4-2 cells treated with indicated drugs for three days. ERK2 was used as a loading control for WB. Three biological replicates were analyzed. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. FIG. 6D) MTS assay in C4-2 cells treated with indicated drugs. Three biological replicates were analyzed. **P < 0.01; ***P < 0.001. FIGS. 6E-6F) A colony formation assay in C4-2 cells treated with indicated drugs. At 11 days after treatment, colonies were photographed (FIG. 6E) and quantified (FIG. 6F). Three biological replicates were analyzed. ***P < 0.001. FIGS. 6G-6I) A WB (FIG. 6G) and a RT-qPCR analysis (FIGS. 6H and 61) in C4-2 cells transfected with indicated plasmids and treated with vehicle or ENZ. iPrimer and dPrimer represent the pair of primers for amplification of transcripts generated by intronic PAS and distal PAS usage, respectively. Three biological replicates were analyzed. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant. FIGS. 6J-6K) A colony formation assay in C4-2 cells transfected with indicated plasmids and treated with vehicle or ENZ. At 11 days after treatment, colonies were photographed (FIG. 61) and quantified (FIG. 6K). Three biological replicates were analyzed. ***P < 0.001.

FIG. 7 is a schematic of an exemplary mechanism underlying androgen regulation of CCNK expression. The left panel shows an activation of the AR by androgens leads to transcriptional upregulation of CCNK mRNA, which in turn results in the activation of the CYCLIN K/CDK12 complex and subsequent suppression of the IP A usage in the AR gene locus and inhibition of AR variant expression. The right panel shows an inactivation of the CYCLIN K/CDK12 complex due to deletion or mutation of the CDK12 gene or downregulation of CCNK caused by ADT or ENZ treatment. This leads to the APA activation in the AR and homologous recombination (HR) repair gene loci (BRCAness), which promotes the IP A usage in the AR locus and AR variant expression, thereby contributing to endocrine therapy resistance and castration-resistant progression of PCa. The CYCLIN K/CDK12 inactivation also makes cancer cells susceptible to PARP inhibitor.

FIGS. 8A-8B show viability measurements in LAPC4 cells following drug treatment and IP A usage in intron 3 of the AR gene in normal tissues of humans. Fig. 8 A) LAPC4 cells cultured in regular medium (containing fetal bovine serum without charcoal stripping) were treated with indicated drugs for 72 hours and subjected to MTS assay. Three biological replicates were analyzed. n.s., not significant. FIG. 2B) A UCSC screenshot of RNA-seq showing the usage of IP A site in intron 3 of the AR gene (highlighted) in various normal human tissues using SAP AS (strategy of sequencing APA sites) through the PolyASite website (polyasite.unibas.ch).

FIGS. 9A-9B show an analysis o£AR-V7 mRNA expression in PCa cell lines treated with CYCLIN K inhibitor. RT-qPCR analysis of expression of AR-V7 and AR-FL mRNA in LNCaP (FIG. 9 A) and C4-2 (FIG. 9B) cells treated with different doses of the CYCLIN K inhibitor CR8 for 18 hours. Three biological replicates were analyzed. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant.

FIGS. 10A-10D show CDK12 gene mutation analysis in 22Rvl cell line and metaanalysis of RNA-seq data for IP A usage in intron 3 of the AR gene in PCa patient samples. FIGS. 10A and 10B) Sanger sequencing (FIG. 10A) and sequence alignment (FIG. 10B) show a G909R missense mutation in the kinase domain of CDK12 in 22Rvl cells. Sanger sequencing (FIG. 10A) contains a portion of a human CDK12 gene sequence containing a mutation missense mutation (SEQ ID NO:1) that can encode a CDK12 G909R polypeptide. The sequence alignment (FIG. 10B) contains a portion of a wild type human CDK12 gene sequence (SEQ ID NO:2), a portion of the encoded wild type CDK12 polypeptide (SEQ ID NO:3), a portion of a human CDK12 gene sequence containing a mutation missense mutation (SEQ ID NO: 1) that can encode a CDK12 G909R polypeptide, and a portion of the encoded CDK12 G909R polypeptide (SEQ ID NO:4). FIG. 10C shows a UCSC screenshot of RNA- seq in the AR exon 3/intron 3 region in 22Rvl cell line and the TCGA-HC-A6HX patient sample from TCGA (SEQ ID NO: 5). A putative alternative polyadenylation (APA) site in intron 3 of the AR gene is highlighted in bold (black). FIG. 10D) A UCSC screenshot of RNA-seq data in the ATM and FANCD2 gene loci in CDK12 deleted/mutated patient samples and randomly selected CDK12 WT samples in the TCGA cohort. Data from LNCaP and 22Rvl cell lines were used as a negative and positive control, respectively.

FIGS. 11A-11C show an analysis of IP A usage in C4-2 cells treated with DHT, AR- V7 mRNA expression in C4-2 and LNCaP cells treated with ENZ. FIG. 11 A) Meta-analysis of the correlation between P value (gene cutoff stringency) and IP A ratio (suppressed IPA/activated IP A) using RNA-seq data generated from C4-2 cells cultured in CSS medium supplemented with or without DHT (10 nM) (GSM2432781 versus GSM2432783). FIG. 1 IB) A RT-qPCR analysis of AR-V7 and AR-FL mRNA expression in C4-2 and LNCaP cell lines treated with vehicle (mock) or ENZ (10 pM) for 72 hours. Three biological replicates were analyzed. ***P < 0.001; n.s., not significant. FIG. 11C) Meta-analysis of the correlation between AR and CCNK mRNA expression levels in PCa patient samples of the TCGA cohort.

DETAILED DESCRIPTION

This document provides methods and materials for assessing and/or treating mammals (e.g., humans) having prostate cancer (e.g., CRPC). In some cases, the methods and materials provided herein can be used to determine whether or not a mammal having prostate cancer (e.g., CRPC) is likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PARP polypeptide). For example, a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal having prostate cancer (e.g., CRPC) can be assessed for (a) the presence or absence of a CDK12 polypeptide deficiency and/or (b) the presence or absence of a reduced level of expression of CYCL1N K polypeptides to determine whether or not the mammal is likely to respond to one or more inhibitors of a PARP polypeptide (e.g., olaparib). In some cases, the methods and materials provided herein also can include administering one or more cancer treatments to a mammal having prostate cancer (e.g., CRPC) to treat the mammal (e.g., one or more cancer treatments selected based, at least in part, on whether or not the mammal is likely to respond to a particular cancer treatment such as one or more inhibitors of a PARP polypeptide).

A mammal (e.g., a human) having prostate cancer (e.g., CRPC) can be assessed to determine whether or not the cancer is likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PARP polypeptide) by detecting (a) the presence or absence of a CDK12 polypeptide deficiency and/or (b) the presence or absence of a reduced level of expression of a CYCLIN K polypeptide in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal. As described herein, the presence of a CDK12 polypeptide deficiency and/or the presence of a reduced level of expression of a CYCLIN K polypeptide in a sample obtained from the mammal having prostate cancer (e.g., CRPC) can be used to determine that mammal is likely to respond to one or more inhibitors of a PARP polypeptide. For example, the presence of a CDK12 polypeptide deficiency (e.g., the presence of a reduced level of a CDK12 polypeptide or the presence of one or more mutations within a CDK12 polypeptide such as one or more mutations that can increase AR IPA usage) in a sample obtained from a mammal having prostate cancer (e.g., CRPC) can be used to identify that mammal as being likely to respond to one or more inhibitors of a PARP polypeptide (e.g., olaparib). In another example, the presence of a reduced level of expression of a CYCLIN K polypeptide in a sample obtained from a mammal having prostate cancer (e g., CRPC) can be used to identify that mammal as being likely to respond to one or more inhibitors of a PARP polypeptide (e.g., olaparib). Also as demonstrated herein, one or more inhibitors of a CYCLIN K/CDK12 complex can be used to sensitize prostate cancers to one or more inhibitors of a PARP polypeptide. For example, one or more inhibitors of a CYCLIN K/CDK12 complex can be administered to a mammal having prostate cancer (e.g., CRPC) to sensitize the mammal to one or more inhibitors of a PARP polypeptide, and optionally, the mammal can be administered one or more inhibitors of a PARP polypeptide (e.g., to treat the mammal).

Any appropriate mammal having prostate cancer (e.g., CRPC) can be assessed and/or treated as described herein. Examples of mammals that can have prostate cancer and can be assessed and/or treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. In some cases, a mammal can be a mammal that was treated with one or more (e.g., one, two, three, four, five, or more) anti-androgen agents. Examples of anti-androgen agents include, without limitation, leuprolide (e.g, LUPRON DEPOT® and ELIGARD®), goserelin (e.g, ZOLADEX®), triptorelin (e.g., TRELSTAR®), histrelin (e.g., VANTAS®), degarelix (e.g., FIRMAGON®), abiraterone (e.g., ZYTIGA®), ketoconazole (e.g., NIZORAL®), flutamide (e.g, EULEXIN®), bicalutamide (e.g., CASODEX®), nilutamide (e.g, NILANDRON®), enzalutamide (e.g., XTANDI®), apalutamide (e.g., ERLEADA®), darolutamide (e.g., NUBEQA®), and rezvilutamide. In some cases, a mammal can be a male mammal. For example, a male human having prostate cancer (e.g., CRPC) can be assessed and/or treated as described herein.

When assessing and/or treating a mammal (e.g., a human) having prostate cancer as described herein, the prostate cancer can be any type of prostate cancer. A prostate cancer assessed and/or treated as described herein can be any stage of prostate cancer (e.g., stage I, stage II, stage in, or stage IV). A prostate cancer assessed and/or treated as described herein can be any grade of prostate cancer (e.g., grade 1, grade 2, or grade 3). A prostate cancer assessed and/or treated as described herein can have any Gleason score. In some cases, a prostate cancer assessed and/or treated as described herein can be a primary cancer (e.g., a localized primary cancer). In some cases, a prostate cancer assessed and/or treated as described herein can have metastasized. In some cases, a prostate cancer assessed and/or treated as described herein can be castration-sensitive prostate cancer (CSPC). In some cases, a prostate cancer assessed and/or treated as described herein can be CRPC. In some cases, a prostate cancer assessed and/or treated as described herein can be hormone-refractory prostate cancer (HRPC).

In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having prostate cancer (e.g., CRPC). Any appropriate method can be used to identify a mammal as having prostate cancer. For example, physical examination (e.g., a digital rectal examination (DRE)), laboratory testing (e.g., blood tests for prostate-specific antigen (PSA) test), imaging techniques (e.g., ultrasound, magnetic resonance imaging (MRI), bone scann, computerized tomography (CT) scan, and positron emission tomography (PET) scan), and biopsy techniques can be used to identify a mammal (e.g., a human) as having prostate cancer.

In some cases, a prostate cancer (e.g., CRPC) can be identified as likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PART polypeptide) based, at least in part, on the presence or absence of a CDK12 polypeptide deficiency. The term “CDK12 polypeptide deficiency” as used herein can be a reduced level of a CDK12 polypeptide. In some cases, a CDK12 polypeptide deficiency can be present within cancer cells because of one or more mutations in the nucleic acid sequence that encodes a CDK12 polypeptide and/or one or more mutations in the nucleic acid sequence that controls the expression of a CDK12 polypeptide. For example, frame-shift mutations within the nucleic acid sequence that encodes a CDK12 polypeptide can result in a CDK12 polypeptide deficiency in that less full-length, wild-type CDK12 polypeptides are present within the cancer cell. In another example, one or more mutations (e.g., one or more substitutions) within the nucleic acid that encodes a CDK12 polypeptide can result in a CDK12 polypeptide deficiency in that less functional CDK12 polypeptides are present within the cancer cell. In another example, one or more mutations in a promotor region that controls the expression of a CDK12 polypeptide can result in a CDK12 polypeptide deficiency in that less CDK12 polypeptides are present within the cancer cell. In some cases, the presence of a CDK12 polypeptide deficiency can increase AR IPA usage.

In some cases, a prostate cancer (e.g., CRPC) can be identified as likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PARP polypeptide) based, at least in part, on the presence or absence of a CDK12 polypeptide deficiency. For example, a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal having prostate cancer (e.g., CRPC) can be assessed for the presence or absence of a reduced level of a CDK12 polypeptide in the sample. For example, a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal having prostate cancer (e.g., CRPC) can be assessed for the presence or absence of a reduced level of mRNA encoding a CDK12 polypeptide in the sample and/or a reduced level of CDK12 polypeptides in the sample. The term “reduced level” as used herein with respect to a level of a CDK12 polypeptide (or mRNA) refers to any level that is lower than a reference level of the CDK12 polypeptide (or mRNA). The term “reference level” as used herein with respect to a CDK12 polypeptide (or mRNA) refers to the level of the CDK12 polypeptide (or mRNA) typically observed in a comparable sample (e.g, a comparable control sample) from one or more mammals (e.g., humans) without prostate cancer. Comparable control samples can include, without limitation, prostate cell samples from normal (e.g., healthy) mammals and non-cancerous prostate cells from a mammal having prostate cancer. In some cases, a reduced level of a CDK12 polypeptide (or mRNA) can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold less relative to a reference level of the CDK12 polypeptide (or mRNA). In some cases, a reduced level of a CDK12 polypeptide can be an undetectable level. In some cases, a reduced level of an mRNA encoding a CDK12 polypeptide can be an undetectable level. It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is a reduced level.

Any appropriate method can be used to detect the presence or absence of a reduced level of expression of a CDK12 polypeptide within a sample (e.g., a sample containing one or more prostate cancer cells) obtained from a mammal (e.g., a human). In some cases, a level of CDK12 polypeptide expression within a sample can be determined by detecting the presence, absence, or level of the CDK12 polypeptide in the sample. For example, immunoassays (e.g., immunohistochemistry (IHC) techniques and western blotting techniques), mass spectrometry techniques (e.g., proteomics-based mass spectrometry assays or targeted quantification-based mass spectrometry assays), enzyme-linked immunosorbent assays (ELISAs), and radio-immunoassays can be used to determine the presence, absence, or level of a CDK12 polypeptide in a sample. In some cases, a level of CDK12 polypeptide expression within a sample can be determined by detecting the presence, absence, or level of mRNA encoding the CDK12 polypeptide in the sample. For example, polymerase chain reaction (PCR)-based techniques such as quantitative RT-PCR techniques, gene expression panel (e.g., next generation sequencing (NGS) such as RNA-seq), and in situ hybridization can be used to determine the presence, absence, or level of mRNA encoding a CDK12 polypeptide in the sample. In some cases, the presence or absence of a reduced level of expression of a CDK12 polypeptide within a sample from a mammal having prostate cancer can be determined as described in Example 1.

In some cases, a prostate cancer (e.g., CRPC) can be identified as likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PART polypeptide) based, at least in part, on the presence or absence of (a) one or more mutations in nucleic acid encoding a CDK12 polypeptide that create a CDK12 polypeptide deficiency and/or (b) one or more mutations in nucleic acid that controls expression of a CDK12 polypeptide and creates a CDK12 polypeptide deficiency. For example, a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal having prostate cancer (e.g., CRPC) can be assessed for the presence or absence of one or more mutations in nucleic acid encoding a CDK12 polypeptide and/or in nucleic acid that controls expression of a CDK12 polypeptide.

Examples of mutations of a CDK12 polypeptide that can create a CDK12 polypeptide deficiency within prostate cancer cells and can be used to assess a mammal (e.g., a human) as described herein include, without limitation, nucleic add mutations that result in an amino acid substitution at residue 909 of a CDK12 polypeptide (e.g, a CDK12 G909 amino acid substitution) of SEQ ID NO:43 (see, e.g., Example 2). For example, a CDK12 polypeptide having a substitution of the glycine (G) at CDK12 G909 with another charged amino acid can create a CDK12 polypeptide deficiency. Examples of amino acids that can be substituted for the G at CDK12909 create a CDK12 polypeptide deficiency within prostate cancer cells as described herein include, without limitation, arginine (R), glutamate (E), aspartic acid (D), lysine (K), and histidine (H). In some cases, a CDK12 polypeptide having a G909R mutation in a CDK12 polypeptide (e.g, a CDK12 G909R polypeptide) can create a CDK12 polypeptide deficiency. In some cases, a CDK12 polypeptide having a G909E mutation in a CDK12 polypeptide (e.g., a CDK12 G909E polypeptide) can create a CDK12 polypeptide deficiency.

Any appropriate method can be used to detect the presence or absence of one or more mutations in nucleic acid encoding a CDK12 polypeptide that create a CDK12 polypeptide deficiency and/or one or more mutations in nucleic acid that controls expression of a CDK12 polypeptide and creates a CDK12 polypeptide deficiency within a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal (e.g., a human) having prostate cancer (e.g., CRPC). For example, mass spectrometry, and Edman degradation can be used to identify the presence or absence of one or more mutations in a CDK12 polypeptide, and nucleic acid sequencing techniques (e.g., Sanger sequencing and nextgeneration sequencing) can be used to identify the presence or absence of one or more mutations in nucleic acid encoding a CDK12 polypeptide and/or controlling the expression of a CDK12 polypeptide. For example, nucleic acid sequencing techniques can be used to detect the presence or absence of one or more mutations in nucleic acid encoding a CDK12 polypeptide and/or controlling the expression of a CDK12 polypeptide.

A sample (e.g., a sample containing one or more cancer cells) obtained from a mammal (e.g., a human) having prostate cancer (e.g., CRPC) can be assessed for a CDK12 polypeptide deficiency in any appropriate CDK12 polypeptide. Examples of CDK12 polypeptides that can be assessed for a CDK12 polypeptide deficiency in a sample obtained from a mammal having prostate cancer (e.g., CRPC) include, without limitation, those set forth in the National Center for Biotechnology Information (NCBI) databases at, for example, accession no. Q9NYV4 (version Q9NYV4.2), accession no. NP_057591 (version NP_057591.2), and accession no. NP_055898 (version NP_055898.1).

In some cases, a prostate cancer (e.g., CRPC) can be identified as likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PARP polypeptide) based, at least in part, on the presence or absence of a reduced level of expression of a CCNK nucleic add (e.g., resulting in a reduced level of a CYCLIN K polypeptide). For example, a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal having prostate cancer (e.g., CRPC) can be assessed for the presence or absence of a reduced level of a CYCLIN K polypeptide in the sample. For example, a sample (e.g., a sample containing one or more cancer cells) obtained from a mammal having prostate cancer (e.g., CRPC) can be assessed for the presence or absence of a reduced level of mRNA encoding a CYCLIN K polypeptide in the sample. The term “reduced level” as used herein with respect to a level of a CYCLIN K polypeptide (or mRNA) refers to any level that is lower than a reference level of the CYCLIN K polypeptide (or mRNA). The term “reference level” as used herein with respect to a CYCLIN K polypeptide (or mRNA) refers to the level of the CYCLIN K polypeptide (or mRNA) typically observed in a comparable sample (e.g., a comparable control sample) from one or more mammals (e.g., humans) without prostate cancer. Comparable control samples can include, without limitation, prostate cell samples from normal (e.g., healthy) mammals and non-cancerous prostate cells from a mammal having prostate cancer. In some cases, a reduced level of a CYCLIN K polypeptide (or mRNA) can be a level that is at least 2 (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 35, or at least 50) fold less relative to a reference level of the CYCLIN K polypeptide (or mRNA). In some cases, a reduced level of a CYCLIN K polypeptide can be an undetectable level. In some cases, a reduced level of an mRNA encoding a CYCLIN K polypeptide can be an undetectable level. It will be appreciated that levels from comparable samples are used when determining whether or not a particular level is a reduced level.

Any appropriate method can be used to detect the presence or absence of a reduced level of expression of a CYCLIN K polypeptide within a sample (e.g., a sample containing one or more prostate cancer cells) obtained from a mammal (e.g., a human). In some cases, a level of CYCLIN K polypeptide expression within a sample can be determined by detecting the presence, absence, or level of the CYCLIN K polypeptide in the sample. For example, immunoassays (e.g., IHC techniques and western blotting techniques), mass spectrometry techniques (e.g., proteomics-based mass spectrometry assays or targeted quantification-based mass spectrometry assays), ELISAs, and radio-immunoassays can be used to determine the presence, absence, or level of a CYCLIN K polypeptide in a sample. In some cases, a level of CYCLIN K polypeptide expression within a sample can be determined by detecting the presence, absence, or level of mRNA encoding the CYCLIN K polypeptide in the sample. For example, PCR-based techniques such as quantitative RT-PCR techniques, gene expression panel (e.g., NGS such as RNA-seq), and in situ hybridization can be used to determine the presence, absence, or level of mRNA encoding a CYCLIN K polypeptide in the sample. In some cases, the presence or absence of a reduced level of expression of a CYCLIN K polypeptide within a sample from a mammal having prostate cancer can be determined as described in Example 1.

A sample (e.g., a sample containing one or more cancer cells) obtained from a mammal (e.g., a human) having prostate cancer (e.g., CRPC) can be assessed for any appropriate CYCLIN K polypeptide. Examples of CYCLIN K polypeptides that can be detected in a sample obtained from a mammal having prostate cancer (e.g., CRPC) include, without limitation, those set forth in the NCBI databases at, for example, accession no. NP_001092872 (version NP_001092872.1) and accession no. XP_005268211 (version XP_005268211.1).

In some cases, a sample from a mammal (e.g., a human) having prostate cancer (e.g., CRPC) and assessed as described herein (e.g., for (a) the presence or absence of a CDK12 polypeptide deficiency and/or (b) the presence or absence of a reduced level of expression of a CYCLIN K polypeptide) also can be assessed for the presence or absence of one or more short-form mutations of one or more HR genes. For example, the presence of a short-form mutations in an HR gene can result in homologous recombination deficiency (HRD). For example, the presence of a short-form mutations in an HR gene can result in increased IP A usage in the HR gene. Examples of HR genes in which a short-form mutation can be detected in a sample obtained from a mammal having prostate cancer (e.g., CRPC) include, without limitation, breast cancer gene (BRC A) 1 genes, BRCA2 genes, ataxia-telangiesctasia mutated (ATM) genes, FA complementation group D2 (FANCD2) genes, Werner syndrome (WRN) genes, and ATR Serine/Threonine Kinase (ATR) genes.

Any appropriate sample from a mammal (e.g., a human) having prostate cancer (e.g., CRPC) can be assessed as described herein (e.g., for (a) the presence or absence of a CDK12 polypeptide deficiency and/or (b) the presence or absence of a reduced level of expression of a CYCLIN K polypeptide). In some cases, a sample can be a biological sample. In some cases, a sample can contain one or more cancer cells. In some cases, a sample can contain one or more biological molecules (e.g., nucleic acids such as DNA and RNA, polypeptides, carbohydrates, lipids, hormones, and/or metabolites). Examples of samples that can be assessed as described herein include, without limitation, tissue samples (e.g., prostate tissue samples or prostate cancer tissue biopsies), fluid samples (e.g., whole blood, serum, plasma, urine, and saliva), and cellular samples. A sample can be a fresh sample or a fixed sample (e.g., a formaldehyde-fixed sample or a formalin-fixed sample). In some cases, a sample can be a processed sample (e.g., an embedded sample such as a paraffin or OCT embedded sample). In some cases, one or more biological molecules can be isolated from a sample. For example, nucleic acid (e.g., DNA and RNA such as messenger RNA (mRNA)) can be isolated from a sample and can be assessed as described herein. In some cases, one or more polypeptides can be isolated from a sample and can be assessed as described herein.

In some cases, a mammal (e.g., a human) having prostate cancer (e.g., CRPC) can be identified as being likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PARP polypeptide) based, at least in part, on (a) the presence or absence of a CDK12 polypeptide deficiency and/or (b) the presence of a reduced level of expression of a CYCLIN K polypeptide in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal. For example, a mammal having prostate cancer (e.g., CRPC) can be identified as being likely to respond to one or more inhibitors of a PARP polypeptide (e.g., olaparib) based, at least in part, on a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal having the presence of a CDK12 polypeptide deficiency (e.g., having a reduced level of a CDK12 polypeptide and/or having the presence of one or more mutations that create a CDK12 polypeptide deficiency). In another example, a mammal having prostate cancer (e.g., CRPC) can be identified as being likely to respond to one or more inhibitors of a PARP polypeptide (e.g., olaparib) based, at least in part, on a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal having a presence of a reduced level of expression of a CYCLIN K polypeptide.

In some cases, a mammal (e.g., a human) having prostate cancer (e.g., CRPC) and identified as being likely to respond to a particular cancer treatment (e.g., one or more inhibitors of a PARP polypeptide) as described herein (e.g., based, at least in part, on (a) the presence of a CDK12 polypeptide deficiency and/or (b) the presence of a reduced level of expression of a CYCLIN K polypeptide in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal) can be administered or instructed to self-administer one or more (e.g., one, two, three, four, five, or more) inhibitors of a PARP polypeptide. For example, a mammal having prostate cancer (e.g., CRPC) and identified as having (a) a presence of a CDK12 polypeptide deficiency and/or (b) a presence of a reduced level of a CYCLIN K polypeptide in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal can be administered or instructed to self-administer one or more inhibitors of a PARP polypeptide. An inhibitor of a PARP polypeptide can be any appropriate inhibitor of PARP polypeptide activity (e.g., anti-PARP antibodies such as neutralizing anti- PARP antibodies and small molecules that target a PARP polypeptide) or any appropriate inhibitor of PARP polypeptide expression (e.g., nucleic acid molecules designed to induce RNA interference of PARP polypeptide expression such as siRNA molecules and shRNA molecules). Examples of inhibitors of a PARP polypeptide that can be used as described herein include, without limitation, olaparib, rucaparib, niraparib, and talazoparib.

In some cases, a mammal (e.g., a human) having prostate cancer (e.g., CRPC) can be identified as being unlikely to respond to one or more inhibitors of a PARP polypeptide based, at least in part, on (a) the absence of a CDK12 polypeptide deficiency and (b) the absence of a reduced level of expression of a CYCLIN K polypeptide in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal. For example, a mammal having prostate cancer (e.g., CRPC) can be identified as being unlikely to respond to one or more inhibitors of a PARP polypeptide (e.g., olaparib) based, at least in part, on a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal (a) lacking a CDK12 polypeptide deficiency and (b) lacking a reduced level of expression of a CYCLIN K polypeptide.

In some cases, a mammal (e.g., a human) having prostate cancer (e.g., CRPC) and identified as being unlikely to respond to one or more inhibitors of a PARP polypeptide as described herein (e.g., based, at least in part, on (a) the absence of a CDK12 polypeptide deficiency and (b) the absence of a reduced level of expression of a CYCLIN K polypeptide in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal) can be administered or instructed to self-administer one or more (e.g., one, two, three, four, five, or more) alternative cancer treatments (e.g., one or more cancer treatments that are not an inhibitor of a PARP polypeptide). For example, a mammal having prostate cancer (e.g., CRPC) and identified as lacking (a) a CDK12 polypeptide deficiency and (b) a reduced level of a CYCLIN K polypeptide in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal can be administered or instructed to self-administer one or more alternative cancer treatments (e.g., one or more cancer treatments that are not an inhibitor of a PARP polypeptide). In some cases, an alternative cancer treatment that can be used as described herein can include administering one or more cancer drugs (e.g., chemotherapeutic agents, targeted cancer drugs, and immunotherapy drugs) other than an inhibitor of a PARP polypeptide to a mammal in need thereof. Examples of cancer drugs that are not an inhibitor of a PARP polypeptide and that can be administered to a mammal having prostate cancer (e.g., CRPC) and identified as being unlikely to respond to an inhibitor of a PARP polypeptide as described herein include, without limitation, docetaxel (e.g., TAXOTERE®), cabazitaxel (e.g., JEVTANA®), mitoxantrone (e.g., NOVANTRONE®), estramustine (e.g., EMCYT®), leuprolide (e.g, LUPRON DEPOT® and ELIGARD®), goserelin (e.g, ZOLADEX®), triptorelin (e.g., TRELSTAR®), histrelin (e.g., VANTAS®), degarelix (e.g., FIRMAGON®), abiraterone (e.g., ZYTIGA®), ketoconazole (e.g., NIZORAL®), flutamide (e.g, EULEXIN®), bicalutamide (e.g, CASODEX®), nilutamide (e.g, NILANDRON®), enzalutamide (e.g., XT ANDI®), apalutamide (e.g., ERLEADA®), darolutamide (e.g., NUBEQ A®), and rezvilutamide .. In some cases, an alternative cancer treatment that can be used as described herein can include surgery. Examples of surgeries that can be performed on a mammal having prostate cancer (e.g., CRPC) include, without limitation, and radical prostatectomy (removal of the prostate gland). In some cases, an alternative cancer treatment that can be used as described herein can include radiation treatment. In some cases, an alternative cancer treatment that can be used as described herein can include prostate tissue ablation. Examples of ablative therapies that can be performed on a mammal having prostate cancer to treat the mammal as described herein include, without limitation, freezing prostate tissue (e.g., cryoablation or cryotherapy) and heating prostate tissue.

In some cases, a mammal (e.g., a human) having prostate cancer (e.g., CRPC) and identified as being unlikely to respond to one or more inhibitors of a PARP polypeptide as described herein (e.g., based, at least in part, on (a) the absence of a CDK12 polypeptide deficiency and (b) the absence of a reduced level of expression of a CYCLIN K polypeptide in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal) can be administered or instructed to self-administer (a) one or more (e.g., one, two, three, four, five, or more) agents that can sensitize prostate cancer to one or more inhibitors of a PARP polypeptide, and (b) one or more (e.g., one, two, three, four, five, or more) one or more inhibitors of a PARP polypeptide. For example, a mammal having prostate cancer (e.g., CRPC) and identified as (i) lacking a CDK12 polypeptide deficiency and (ii) lacking a reduced level of a CYCLIN K polypeptide in a sample (e.g., a sample containing one or more cancer cells) obtained from the mammal can be administered or instructed to self-administer (a) one or more agents that can sensitize prostate cancer to one or more inhibitors of a PARP polypeptide and (b) one or more one or more inhibitors of a PARP polypeptide. An agent that can sensitize a prostate cancer to one or more inhibitors of a PARP polypeptide can be an inhibitor of a CYCLIN K/CDK12 complex. In some cases, an inhibitor of a CYCLIN K/CDK12 complex can be a molecule that reduces the activity of the CYCLIN K/CDK12 complex. In some cases, an inhibitor of a CYCLIN K/CDK12 complex can be a molecule that reduces formation of a complex between a CYCLIN K polypeptide and a CDK12 polypeptide. In some cases, an inhibitor of a CYCLIN K/CDK12 complex can be an inhibitor of a CYCLIN K polypeptide (e.g., an inhibitor of CYCLIN K polypeptide expression or an inhibitor of CYCLIN K polypeptide activity). In some cases, an inhibitor of a CYCLIN K/CDK12 complex can be an inhibitor of a CDK12 polypeptide (e.g., an inhibitor of CDK12 polypeptide expression or an inhibitor of CDK12 polypeptide activity). Examples of compounds that can reduce polypeptide activity include, without limitation, antibodies (e.g., neutralizing antibodies) and small molecules that target (e.g., target and bind) to the polypeptide. Examples of compounds that can reduce polypeptide expression include, without limitation, nucleic acid molecules designed to induce RNA interference of polypeptide expression (e.g., a siRNA molecule or a shRNA molecule), antisense molecules, and miRNAs. Examples of agents that can sensitize a prostate cancer to one or more inhibitors of a PARP polypeptide (e.g., olaparib) are set forth in Table A below.

In some cases, the one or more inhibitors of a PARP polypeptide can be administered together with the one or more agents that can sensitize a prostate cancer to one or more inhibitors of a PARP polypeptide. In some cases, the one or more inhibitors of a PARP polypeptide can be administered independent of the one or more agents that can sensitize a prostate cancer to one or more inhibitors of a PARP polypeptide. When the one or more inhibitors of a PARP polypeptide are administered independent of the one or more agents that can sensitize a prostate cancer to one or more inhibitors of a PARP polypeptide, the one or more agents that can sensitize a prostate cancer to one or more inhibitors of a PARP polypeptide can be administered first, and the one or more anti-androgen agents administered second, or vice versa.

In some cases, when treating a mammal (e.g., a human) having prostate cancer (e.g., CRPC) as described herein, the treatment can be effective to reduce the size of a cancer (e.g., to reduce the number of cancer cells in the mammal and/or to reduce the volume of one or more tumors in the mammal) within a mammal (e.g., a human). For example, the methods and materials described herein can be used to reduce the size of the cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the methods and materials described herein can be used to reduce the size of the cancer by at least 2-fold (e.g., by 2-fold, 3-fold, 4-fold, 5-fold, or more). In some cases, the methods and materials described herein can be used such that the size (e.g., the number of cancer cells and/or the volume) of one or more tumors present within a mammal does not increase.

In some cases, when treating a mammal (e.g., a human) having prostate cancer (e.g., CRPC) as described herein, the treatment can be effective to improve survival of the mammal. For example, the methods and materials described herein can be used to improve disease-free survival (e.g., relapse-free survival). For example, the methods and materials described herein can be used to improve progression-fiee survival. For example, the methods and materials described herein can be used to improve the survival of a mammal having prostate cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, the materials and methods described herein can be used to improve the survival of a mammal having prostate cancer by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, or about 3 years).

In some cases, when treating a mammal (e.g., a human) having prostate cancer (e.g., CRPC) as described herein, the treatment can be effective to reduce one or more symptoms of the cancer. Examples of symptoms of prostate cancer that can be reduced using the methods and materials described herein include, without limitation, trouble urinating, decreased force in the stream of urine, blood in the urine, blood in the semen, bone pain, losing weight without trying, and erectile dysfunction. For example, the materials and methods described herein can be used to reduce one or more symptoms within a mammal having prostate cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.

In some cases, when treating a mammal (e.g., a human) having prostate cancer (e.g., CRPC) as described herein (e.g., by administering one or more inhibitors of a PARP polypeptide and/or one or more therapies that can sensitize prostate cancer to one or more inhibitors of a PARP polypeptide and, optionally, one or more inhibitors of a PARP polypeptide), the one or more inhibitors of a PARP polypeptide and/or one or more therapies that can sensitize prostate cancer to one or more inhibitors of a PARP polypeptide and, optionally, one or more inhibitors of a PARP polypeptide can be the sole cancer treatment(s) administered to the mammal.

In some cases, when treating a mammal (e.g., a human) having prostate cancer (e.g., CRPC) as described herein (e.g., by administering one or more inhibitors of a PARP polypeptide and/or one or more therapies that can sensitize prostate cancer to one or more inhibitors of a PARP polypeptide and, optionally, one or more inhibitors of a PARP polypeptide), the mammal also can be treated with one or more additional agents/therapies used to treat cancer. Examples of additional agents/therapies that can be used to treat cancer as described herein include, without limitation, surgery, chemotherapies, targeted therapies (e.g., monoclonal antibody therapies), angiogenesis inhibitors, immunosuppressants, and checkpoint blockade therapies. In cases where one or more inhibitors of a PARP polypeptide and/or one or more therapies that can sensitize prostate cancer to one or more inhibitors of a PARP polypeptide and, optionally, one or more inhibitors of a PARP polypeptide are used in combination with one or more additional agents/therapies, the one or more additional agents/therapies can be administered at the same time or independently. For example, the one or more inhibitors of a PARP polypeptide and/or one or more therapies that can sensitize prostate cancer to one or more inhibitors of a PARP polypeptide and, optionally, one or more inhibitors of a PARP polypeptide can be administered first, and the one or more additional agents/therapies can be administered second, or vice versa.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1: CYCLINK downregulation induces androgen receptor gene intronic polyadenylation and castration resistance in prostate cancer

Materials and Methods

Cell culture and chemicals

LNCaP, 22Rvl, RWPE1, DU145, VCaP, PC-3, LAPC4 and 293T cell lines were purchased from the American Type Culture Collection (ATCC). The C4-2 cell line was purchased from Uro Corporation. The C4-2B cell line was obtained from ViroMed Laboratories (Minneapolis, MN). LNCaP, C4-2, C4-2B, PC-3, DU145, BPH1, and 22Rvl cell lines were cultured in RPMI-1640 cell culture medium (Coming) containing 10% fetal bovine serum (FBS), together with 100 pg/mL streptomycin, and 100 U/mL penicillin. VCaP and 293T cells were cultured in DMEM cell culture medium (Coming) containing 10% FBS, with 100 pg/mL streptomycin, and 100 U/mL penicillin. RWPE1 cells were maintained in keratinocyte serum free medium (K-SFM). LAPC4 cells were maintained in Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies, Inc.) containing 10% FBS, 100 pg/mL streptomycin, and 100 U/mL penicillin. LNCaP-RF cells were established by longterm culture of LNCaP cells (approximately > 10 weeks) in RPMI 1640 containing 10% charcoal-stripped serum (CSS). All cell lines were authenticated by STR profiling and cultured in incubator with 37°C and 5% CO2. Information of chemicals used in this study is provided in Table 1.

Stable cell line generation

A lentivirus transduction system was utilized to generate stable cell lines with specific gene knockdown and knockout. Polyethylenimine (PEI) was used to transfect shRNA or sgRNA plasmids together with lentivirus package plasmids (PSPAX2 and PMD2.G) into 293 T cells. 48 hours after transfection, supernatant containing viruses was collected, filtered, and utilized to infect indicated cells. Polybrene (8 pg/mL) was added to the viral supernatant to increase the infection efficiency. 48 hours after infection, the culture medium was replaced with fresh medium, and puromycin (1 pg/mL) was administrated for cell selection. Sequence information of shRNA and sgRNA is provided in Table 2.

Table 2. Information of sequences for shRNAs, siRNAs and sgRNAs

MTS cell proliferation assay

Cell proliferation was measured utilizing the MTS assay (Promega, USA) according to manufacturer’s instruction. Briefly, cells were seeded in a 96-well plate with a density of 2,000 cells per well. At the indicated time points, 10 pL CellTiter 96® Aqueous One Solution reagent (Promega, USA) was added to cells. After incubating at 37°C incubator for 3 hours, cell growth was measured in a microplate reader with absorbance at 490 nm.

Sulforhodamine B (SRB) assay

Cells were re-suspended in fresh culture medium, seeded in triplicate with a density of 2,000 cells/well in 96-well plates, and cultured with indicated drugs for one week. Colonies were fixed by adding 10% TCA buffer for 1 hour. The colonies were further stained by 0.4% (W/V) SRB solution in 1% acetic acid for 5 minutes, and then washed with 1% acetic acid twice to remove the SRB. Then add Tris-base (pH 10.5) to solubilize the bound dye.

Colony formation assay

Cells were re-suspended in fresh culture medium, seeded in triplicate with a density of 2,000 cells/well in 6-well plates, and cultured with indicated drugs for various days depending on cell lines used. Colonies were washed 3 times in PBS and were fixed with 4% paraformaldehyde for 0.5 hours. Colonies were further stained by 0.5% crystal violet for 2 hours, and then washed with water twice to remove the crystal violet. The number of colonies in each well was counted.

Reverse transcription and real-time polymerase chain reaction (RT-qPCR)

Total RNA was extracted from the cells by utilizing Trizol reagent (Ambion, USA) and reversely transcribed into cDNA by utilizing the GoScript kit (Promega, USA). The

SYBR-green Mix (Bio-Rad, USA) and CFX96 Real-Time System (Bio-Rad, USA) were utilized to conduct the real-time PCR according to manufacturer’s instruction. Expression of β-Actin housekeeping gene was used as an inner control, and data were present as mean ±

SD. Sequence information for primers used for qPCR is provided in Table 3.

Table 3. Primers used for RT-qPCR analysis

Antibodies and immunoblotting

For immunoblotting analysis, cells were lysed in modified RIP A buffer (50 mM Tris-

HC1 pH=7.4, 1% Nonidet™ P-40, 0.25% sodium deoxycholate, 150 mMNaCl, 0.1% SDS, and 1 mM EDTA) supplemented with 1% protease inhibitor cocktail. Protein concentration was determined using DC™ protein assay reagent (Bio-Rad, USA). Cell extracts were supplemented with 10% DTT (Thermo Fisher Scientific, USA) and boiled at 95°C for 3 minutes. Samples were subjected to SDS-polyacrylamide gel (Bio-Rad, USA) separation, and the gels were further transferred to nitrocellulose (NC) membranes (Thermo Fisher

Scientific, USA). After transferring, the NC membranes were blocked in 5% non-fat milk

(Bio-Rad, USA) for 1 hour at room temperature and incubated with the indicated primary antibodies at 4°C overnight. The next day, the NC membranes were washed with 1 x TBST for 10 minutes three times and incubated with matched secondary antibody for 1 hour at room temperature. The membranes were washed with 1 x TBST for 10 minutes three times.

Lastly, the signals were developed with SuperSignal West Pico Luminal Enhancer Solution

(Thermo Fisher Scientific, USA) on autoradiography films (HyBlot CL, USA). Detailed information for primary antibodies is provided in Table 4.

Table 4. Information for primary antibodies

Chromatin immunoprecipitation quantitative PCR (ChlP-qPCR)

Chromatin immunoprecipitation experiments were performed. In brief, chromatin was cross-linked for 10 minutes at room temperature with 11% formaldehyde/PBS solution added to cell culture medium. Cross-linked chromatin was then sonicated, diluted and immunoprecipitated with Protein G-plus Agarose beads (Bio-Rad) prebound with antibody at

4°C overnight. Precipitated protein-DNA complexes were eluted, and cross-linking was reversed at 65°C for 16 hours. DNA fragments were purified and analyzed by qPCR. ChlP- qPCR data were analyzed as % input after normalizing each ChIP DNA fraction’s Ct value to the Input DNA fraction’s Ct value.

AR activity score

AR activity score was calculated based on the 20 AR target genes including ABCC4,

ACSL3, ADAM7, C1ORF116, CENPN, EAF2, ELL2, FKBP5, GNMT, HERC3, KLK2, KLK3,

MAP, MED28, MPHOSPH9, NKX3.1, NNMT, PMEPA1, PTGER4 and ZBTB10 as described elsewhere (Hieronymus et al., Cancer Cell 10:321-330 (2006)). In brief, gene expression values (log2 (FPKM)) of each sample were converted to Z-score by Z = (x - p)/o, where p is the average log2 (FPKM) across all samples of a gene and a is the standard derivation of the log2 (FPKM). The Z-scores were then summed across all genes for each sample. Quantification and statistical analysis

Analysis of RNA-seq for the expression of CCNK mRNA in paired PCa tissues from 36 patients before and after ADT and ENZ treatment. Data from RNA-seq (GEO accession number GSE183100) were downloaded and used for analysis in this study. GraphPad Prism 7 was used for statistical analyses of results from RT-qPCR, ChlP-qPCR and cell proliferation assays. The Student’s /-test was used to compare data between two groups. Survival curves were obtained using the Kaplan-Meier method, and the log-rank test was used to test the difference in survival curves. P value < 0.05 was considered statistically significant.

Results

CDK12 inhibition confers AR antagonist resistance in PCa cells

This study sought to identify signaling pathways that regulate ENZ resistance in PCa. The sensitivity of AR-expressing LAPC4 PCa cells derived from the lymph node metastasis of a male patient with CRPC, to an array of signaling pathway inhibitors following the treatment with ENZ was surveyed. In cell viability assays, it was demonstrated that LAPC4 cells responded in various degrees to the treatment of different drugs/chemicals that were tested in the absence of ENZ. However, the effect of CDK12 inhibitor THZ531 showed the opposite (FIG. 8A). Further, treatment of LAPC4 cells with all the drugs except THZ531 substantially enhanced ENZ-induced cell growth inhibition (FIG. 1 A). Similar results were obtained in LAPC4 cells using the independent sulforhodamine B (SRB) assay, which measured cell density and cellular protein content (FIG. IB). To confirm this phenomenon, a genetic approach to generate CDK12 knockdown stable LAPC4 cells using two independent CDK12 gene-specific small hairpin RNAs (shRNAs) was employed. Colony formation assays were then performed using both control and CDK12 knockdown LAPC4 cells. CDK12 depletion decreased expression of its partner protein CYCLIN K (FIG. 1C). It was demonstrated that CDK12 knockdown conferred ENZ-resistant growth on LAPC4 cells (FIGS. ID and IE). Thus, data obtained from both pharmacological and genetic approaches showed that CDK12 inactivation confers ENZ resistance in PCa cells. CDK12 deficiency induces expression of AR-Vs in PCa cells

It was observed that IP A usage was detectable in the intron 3 of the AR gene in several normal tissues including prostate, testis, uterus, and liver, although the level was much lower compared to the usage of the distal PAS (FIG. 8B). Meta-analysis of the messenger RNA (mRNA) 3’ region extraction and deep sequencing (3’-READS) dataset generated from LNCaP cells was also performed. LNCaP cells are a cell line which predominantly expresses the frill length AR (AR-FL). Three putative AR IP A sites in AR introns 2, 3, and 4 were determined (FIG. 2A). Notably, the second peak (X: 67696070 - 67696093), located between exons 3 and 4, exhibited the highest transcripts per million (TPM) value and usage rate (FIGS. 2A and 2B). Two pairs of primers unique for intronic PAS (within the third intron) and distal PAS sites were designed. The two primers correspond to AR-V7 and AR-FL, respectively (FIG. 2C, upper) to define the IP A usage in LNCaP cells after treated with various pathway inhibitors (FIG. 2C, lower). Among the drugs/compounds that were tested, THZ531 induced the highest IP A usage in the intron 3 of AR but had limited effect on distal PAS usage. Accordingly, treatment with THZ531 or CR8, a CYCLIN K inhibitor increased AR-V7 mRNA expression in LNCaP and its castrationresistant derivative cell line C4-2 (FIGS. 2D, 2E, 9A and 9B). Consistent with the effect of CDK12 inhibitor, it was demonstrated that knockdown of CDK12 by two independent small interference RNAs (siRNAs) increased IP A usage in AR intron 3 in both cell lines (FIGS. 2F and 2G). Similarly, stable depletion of CDK12 by gene-specific shRNAs also increased expression of AR-Vs and KLK3 (PSA). KLK3 is a well-known AR target gene in LAPC4 and 22Rvl, a cell line that constitutively expresses AR-Vs (FIGS. 2H-2K). Together, the data reveal that pharmacologic inhibition or genetic depletion of CDK12 induces expression of AR variants in various PCa cell lines.

PCa-associated CDK12 mutations enhance AR IP A usage, AR activity and PCa cell growth Among the six AR-expressing PCa cell lines examined, it was noticed that expression of AR-Vs was much higher in 22Rvl compared to VCaP, LNCaP, and C4-2; and that AR variant expression was almost undetectable in LAPC4 and C4-2B (FIG. 3 A). Meta-analysis of RNA-seq data showed that 22Rvl cells had much higher IP A usage in the intron 3 of AR gene compared to LNCaP and C4-2 cell lines (FIG. 3B). Further analysis showed that, similar to the situation in TCGA PCa patient samples, there is a missense mutation (G909R) in CDK12 gene in 22Rvl cell line (FIGS. 3C, 10A and 10B). The same mutation was also detected in patient samples of other cancer types (FIG. 3C, lower panel). Notably, a missense mutation (G909E, another mutation substituted with a charged amino acid) was also observed in the TCGA PCa patient sample TCGA-HC-A6HX (FIG. 3C, lower panel). Analysis of RNA-seq data revealed that the IP A signaling in the AR intron 3 was highly detectable in both 22Rvl cell line and this patient’s tumor (FIGS. 3D and 10C). Similarly, much higher IP A usage in DNA repair genes such as ATM and FANCD2 was also observed in CDK12-deficient patient samples compared to CDK12 wild-type (WT) counterparts (FIG. 10D).

The functional impact of these CDK12 mutations was also studied. The study knocked out endogenous CDK12 in 22Rvl cells and rescued with WT CDK12, G909R or G909E mutant. Restored expression of mutant, but not wild-type CDK12, enhanced expression of AR variants and KLK3 mRNA, a surrogate of AR activation in 22Rvl cells (FIGS. 3E and 3F). Furthermore, it was determined that the molecular mass of AR-FL in 22Rvl cells is larger (lower mobility on PAGE gel) than the wild-type AR-FL in VCaP, LNCaP, and C4-2 cell lines (FIG. 3 A). This is the result of an additional zinc finger in the DBD caused by splicing of the extra copy of exon 3 (FIG. 3G). However, the additional zinc finger encoded by the extra exon 3 does not exist in the AR-Vs in 22Rvl such that they migrate similarly to those in VCaP, LNCaP, and C4-2 cell lines (FIG. 3 A). A plausible explanation is that independent IP A sites are present in the first intron 3 and the duplicated intron 3 of the AR gene; however, the pre-mRNA of the duplicated intron 3 should not be transcribed when the first IP A site was used in 22Rvl cells (FIG. 3G, top) given that CDK12 is inactivated by the gene mutation. As a result, only the first exon 3, but not the additional exon 3, is expressed along with one copy of those cryptic exons in AR variants (FIG. 3G, bottom). The study examined the effect of CDK12 mutations on AR antagonist resistance. It was demonstrated that CDK12 knockout (KO) 22Rvl cells with restored expression of G909R and G909E mutant CDK12 grew much faster than cells rescued with WT CDK12 when treated with ENZ (FIGS. 3H and 31). Collectively, the data indicate that CDK12 mutations enhance AT? IP A usage, AR activity, and enzalutamide-resistant growth ofPCa cells.

CDK12 deficiency associates "with increased IP A usage, AR signaling activation and poor survival ofPCa patients

To determine the potential impact of CDK12 deficiency on AR signaling and overall survival in patients, the PCa patient data of the TCGA cohort was studied. It was observed that patients with CDK12 defects (mutations and/or deletions) in prostate tumors had much higher CSTF3 IP A usage than patients with wild-type CDK12 (FIG. 4A). This implied that there was enhanced overall APA activities in the CDK12-deficient patient samples. It was found that the IP A usage in intron 3 of the AR gene locus was also higher in CDK12- deficient samples compared to CDK12 WT counterparts (FIG. 4B). It was determined that CDK12-deficient primary PCa in the TCGA cohort had higher AR activity than control samples (FIG. 4C), indicating the aberrant activation of the AR signaling in CDK12-deficient samples. The impact on IP A usage and AR score can be detected in CDK12-deficient tumors, and the number of those cases (12 out of 499 cases, 2.4%) was small in the TCGA cohort. Additionally, the study found that CDK12 defects correlate with shorter progression-free survival of these patients (FIG. 4D). It was demonstrated that CDK12 defects also correlate with inferior overall survival of mCRPC patients in both SU2C and West Coast Dream Team (WCDT) datasets (FIGS. 4E and 4F). CDK12-defi cient patients had a higher AR score compared to CDK12 WT patients in those two cohorts (FIGS. 4G and 4H). Together, these data stress that AR signaling is hyperactivated in PCa harboring CDK12 gene deletions and/or mutations.

Androgen depletion and antiandrogen treatment represses CCNKgene expression

The study examined whether androgen manipulation affects IP A usage globally and at the AR gene locus to induce AR-V expression. APAlyzer software was used to analyze RNA-seq data generated from C4-2 cells cultured in charcoal-stripped medium supplemented with or without dihydrotestosterone (DHT). The study demonstrated that DHT treatment suppressed IP A peaks more than two-fold versus activated peaks (FIGS. 5 A and 11 A), suggesting that DHT treatment induces net suppression of IP A usage. Because the data (FIG. 3) indicated that CDK12 acts as an upstream effector regulating AR-V genesis, the study examined whether DHT treatment affects CDK12 expression. The study showed that DHT had little or no effect on CDK12 protein expression in C4-2 cells (FIG. 5B). In contrast, the study demonstrated that DHT treatment of C4-2 cells substantially increased the expression of CYCLIN K, the regulatory subunit of the CDK12/CYCLIN K kinase complex (FIG. 5B). Similar results were obtained at mRNA level as revealed by RNA-seq data in C4-2 cells and RT-qPCR analysis in C4-2 and LNCaP cells (FIGS. 5C and 5D). The data showed that while ENZ treatment had no obvious effect on CDK12 expression, it substantially suppressed CYCLIN K expression at both protein and mRNA levels and increased AR-V7 expression (FIGS. 5E, 5F and 1 IB). This study examined CCNK mRNA and CYCLIN K protein expression in LuCaP35 PDX tumors grown in mice with or without castration. The study demonstrated that castration decreased expression of CCNK mRNA and CYCLIN K protein in LuCaP35 tumors (FIGS. 5G and 5H). These observations are consistent with microarray data in LuCaP35 PDX tumors showing that CCNK mRNA level was much lower in tumors in castrated mice compared to intact counterparts (FIG. 51). Meta-analysis was performed of RNA-seq data from 36 paired PCa patient samples before and after ADT and ENZ treatments. The study showed that, similar to the effect of KLK3 mRNA, ADT and ENZ treatment substantially decreased CCNK mRNA expression in these patient samples (FIG. 5J).

To determine the molecular mechanism underlying androgen regulation of CCNK expression, the study performed meta-analysis of chromatin immunoprecipitation sequencing (ChlP-seq) data of transcription factors including those that are highly relevant in PCa such as AR, ERG and FOXA1. The enrichment of these factors in the CCNK gene locus was examined. The study demonstrated that AR is one of the top 10 transcript factors highly enriched in the CCNK locus (FIGS. 5K), suggesting that AR might regulate CCNK gene expression by binding to this genomic region. Indeed, analysis of AR ChlP-seq data in C4-2 and LNCaP cell lines showed that DHT treatment largely increased AR occupancy in the proximity of the CCNK gene promoter (FIG. 5L). These results were further confirmed by AR ChlP-qPCR analysis (FIG. 5M). In contrast, ChlP-seq data showed that ENZ treatment inhibited AR occupancy at the CCNK gene locus in C4-2 cells, and these results were further confirmed by ChlP-qPCR analysis in both C4-2 and LNCaP cell lines (FIGS. 5N and 50). In agreement with these observations, the study showed that AR knockdown by two independent siRNAs decreased CYCLIN K expression at both mRNA and protein levels in C4-2 and LNCaP cell lines (FIGS. 5P and 5Q). The study fiirther showed that AR mRNA level was positively correlated with CCNK mRNA expression in PCa specimens in the TCGA cohort (FIG. 11C). Together, these data indicate that CCNK expression is positively regulated by androgens, but negatively regulated by androgen deprivation and antiandrogens in PCa cells in culture, PDX tumors, and patient samples.

Antiandrogen induces synthetic lethality in PCa cells upon PARP1 inhibition

This study treated C4-2 cells with ENZ and the PARP inhibitor olaparib alone or together. The study showed that ENZ treatment decreased expression of HR regulators such as BRCA1, ATM, FANCD2 and WRN at both mRNA and protein levels (FIGS. 6A and 6B). The study fiirther showed that ENZ treatment induced IP A usage in introns of AR and HR genes (FIGS. 6B and 6C). MTS assays showed that co-treatment of C4-2 cells with ENZ and olaparib resulted in greater cell growth inhibition compared to each agent treatment alone (FIG. 6D). These results were fiirther confirmed by colony formation assays (FIGS. 6E and 6F). Rescue experiments showed that forced expression of CYCLIN K not only abolished ENZ-induced IP A usage in both AR and HR repair gene loci, but also prevented ENZ- mediated sensitization of PCa cells to olaparib (FIGS. 6G-6K). Collectively, this data indicate that antiandrogen induces synthetic lethality in PCa cells upon PARP1 inhibition and that this process is mediated by downregulation of CYCLIN K.

Example 2: CDK12 polypeptide sequence

Amino acid sequence of an exemplary CDK12 polypeptide (SEQ ID NO:43):

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.