Investigating the Role of DNA Repair Mutations in Prostate Cancer Initiation, Progression, and Susceptibility to Targeted Therapies

项目摘要/摘要 前列腺癌(PC)是美国男性第二常见的癌症，也是第二致命的癌症。选项 对于转移性去势耐药PC(MCRPC)的治疗是有限的，需要新的治疗策略。 大约20%的mCRPC患者的肿瘤含有dna修复基因突变，主要位于同源 重组(HR)途径(如BRCA2)。大约5%的mCRPC有CDK12的突变，CDK12是一种细胞周期蛋白依赖性的基因 该蛋白的缺失导致许多DNA修复基因的异常剪接和mRNA下调。这些DNA 修复缺陷的肿瘤往往非常具有侵袭性，但可以治疗，因为HR基因(例如BRCA2)的丢失是已知的 使其他癌症(如卵巢癌)对聚(ADP)核糖聚合酶抑制剂(PARPI)和铂敏感 化疗(Plat)。然而，目前尚不清楚CDK12缺失是否会使mCRPC对这种疗法敏感。此外， CDK12突变的mCRPC在细胞周期基因(如细胞周期蛋白D1)中反复扩增，很少携带其他 Ets家族基因的HR突变或上调(例如TMPRSS2-ERG融合)。这项提议将得到发展。 新的体外和体内模型研究DNA修复突变在PC发育中的确切作用 检验三个假设/具体目标：(1)确定BRCA1、BRCA2或CDK12的丢失是否足以驱动或 加速前列腺癌的发生并确定导致穿透性病变的协同基因组异常 表型，(2)确定CDK12突变的PC是否对针对DNA修复和细胞周期的治疗有反应 以及(3)确定靶向CDK12在HR缺陷和ETS+前列腺癌中的疗效。 我将生成具有前列腺特异性BRCA1、BRCA2或 CDK12来测试它们的损失是否足以进行PC开发。小鼠也将与Ptenfl/fl或PbMYC小鼠杂交 在已建立的PC小鼠模型上测试DNA修复缺失是否可以加速肿瘤的发展。肿瘤将会是 通过DNA和RNA测序进行分析，以确定它们是否重现了如 人类肿瘤。该项目还将利用可诱导的BRCA2和CDK12基因工程的PC细胞系 带有内源性突变的基因敲除和患者来源的异种移植(PDX)测试CDK12的缺失是否会增敏 PC至PARPI+Plat(奥拉帕利布+顺铂)或CDK4/6抑制剂(核环素)。最后，设计PC生产线或PDX 具有内源性BRCA2缺失或TMPRSS2-ERG融合的品系将用于测试CDK12基因敲除或 抑制(THZ531)在HR丢失或Ets家族癌基因上调的细胞中是综合致命的。 总之，这些研究将解决有关DNA修复丢失在PC和测试中的作用的关键问题 CDK12突变PC的潜在靶向治疗。此外，这些实验将确定目标是否 CDK12可以有效地治疗PC的关键亚群，这可能会刺激CDK12的未来试验和开发 抑制剂，并有可能提供新的治疗选择，以延长许多mCRPC患者的生存。

PROJECT SUMMARY/ABSTRACT Prostate cancer (PC) is the second most common cancer in American men and second most lethal. Options for treating metastatic castration-resistant PC (mCRPC) are limited and new therapeutic strategies are needed. About 20% of mCRPC patients have tumors harboring mutations in DNA repair genes, mostly in the homologous recombination (HR) pathway (e.g. BRCA2). About 5% of mCRPCs have mutations in CDK12, a cyclin-dependent kinase whose loss leads to aberrant splicing and mRNA downregulation of many DNA repair genes. These DNA repair-deficient tumors tend to be very aggressive but can be treatable, as loss of HR genes (e.g. BRCA2) is known to sensitize other cancers (e.g. ovarian) to poly(ADP) ribose polymerase inhibitors (PARPi) and platinum chemotherapy (PLAT). However, it is unknown if CDK12 loss sensitizes mCRPC to this therapy. Furthermore, CDK12-mutant mCRPC show recurrent amplifications in cell cycle genes (e.g. cyclin D1) and rarely carry other HR mutations or upregulation of ETS-family genes (e.g. TMPRSS2-ERG fusions). This proposal will develop new in vitro and in vivo models to investigate the precise role of DNA repair mutations in PC development by testing three hypotheses/specific aims: (1) determine if loss of Brca1, Brca2, or Cdk12 is sufficient to drive or accelerate prostate tumorigenesis and identify cooperating genomic aberrations that contribute to a penetrant phenotype, (2) determine if CDK12-mutant PCs respond to therapies targeting DNA repair and cell cycle checkpoints, and (3) determine the efficacy of targeting CDK12 in HR-deficient and ETS+ prostate cancers. I will generate conditional genetic mouse models with prostate-specific deletion of Brca1, Brca2, or Cdk12 to test if their loss is sufficient for PC development. Mice will also be crossed to Ptenfl/fl or Pb-MYC mice to test if DNA repair loss can accelerate tumor development in established mouse models of PC. Tumors will be analyzed by DNA and RNA sequencing to determine if they recapitulate genomic alteration patterns as seen in human tumors. This project will also utilize PC cell lines engineered with inducible BRCA2 and CDK12 knockdown and patient derived xenografts (PDX) with endogenous mutations to test if loss of CDK12 sensitizes PC to PARPi+PLAT (olaparib+cisplatin) or CDK4/6 inhibitor (ribociclib). Lastly, engineered PC lines or PDX lines with endogenous BRCA2 loss or TMPRSS2-ERG fusion will be used to test if CDK12 knockdown or inhibition (THZ531) is synthetically lethal in cells with HR loss or ETS-family oncogene upregulation. Together, these studies will address key questions concerning the role of DNA repair loss in PC and test potential targeted therapies for CDK12-mutant PC. Furthermore, these experiments will determine if targeting CDK12 can be effective for key subsets of PC, which could spur future trials and development of CDK12 inhibitors and potentially offer new therapeutic options to extend survival for many mCRPC patients.