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

项目总结/摘要 前列腺癌（PC）是美国男性中第二常见的癌症，也是第二大致命癌症。选项 用于治疗转移性去势抵抗性PC（mCRPC）的方法有限，需要新的治疗策略。 大约20%的mCRPC患者患有携带DNA修复基因突变的肿瘤，大多数在同源基因中。 重组（HR）途径（例如BRCA 2）。大约5%的mCRPC在CDK 12中有突变，CDK 12是细胞周期蛋白依赖性的。 其缺失导致许多DNA修复基因的异常剪接和mRNA下调的激酶。这些DNA 修复缺陷型肿瘤往往具有很强的侵袭性，但可以治疗，因为已知HR基因（例如BRCA 2）缺失 使其他癌症（例如卵巢癌）对聚（ADP）核糖聚合酶抑制剂（PARPi）和铂敏感 化疗（PLAT）。然而，尚不清楚CDK 12缺失是否会使mCRPC对该治疗敏感。此外，委员会认为， CDK 12突变型mCRPC显示细胞周期基因（例如细胞周期蛋白D1）的反复扩增，很少携带其他基因。 HR突变或ETS家族基因上调（例如TMPRSS 2-ERG融合）。该提案将发展 新的体外和体内模型，以研究DNA修复突变在PC发育中的确切作用， 测试三个假设/特定目标：（1）确定Brca 1，Brca 2或Cdk 12的丢失是否足以驱动或 加速前列腺肿瘤发生并鉴定有助于渗透剂的协同基因组畸变 表型，（2）确定CDK 12突变型PC是否对靶向DNA修复和细胞周期的疗法有反应 检查点，和（3）确定靶向CDK 12在HR缺陷和ETS+前列腺癌中的功效。 我将生成前列腺特异性缺失Brca 1、Brca 2或 cdk 12来测试它们的损失是否足以用于PC开发。小鼠还将与Ptenfl/fl或Pb-MYC小鼠杂交 在已建立的PC小鼠模型中测试DNA修复缺失是否会加速肿瘤的发展。肿瘤将是 通过DNA和RNA测序进行分析，以确定它们是否重现了基因组改变模式，如在 人类肿瘤该项目还将利用经诱导型BRCA 2和CDK 12工程化的PC细胞系 敲除和具有内源性突变的患者来源的异种移植物（PDX），以测试CDK 12的丧失是否致敏 PC至PARPi+PLAT（奥拉帕尼+顺铂）或CDK 4/6抑制剂（ribociclib）。最后，工程PC生产线或PDX 具有内源性BRCA 2缺失或TMPRSS 2-ERG融合的细胞系将用于测试CDK 12敲低或 抑制剂（THZ 531）在HR丧失或ETS家族癌基因上调的细胞中是合成致死的。 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。 CDK 12突变型PC的潜在靶向治疗。此外，这些实验将确定是否瞄准 CDK 12对PC的关键子集有效，这可能会刺激未来的试验和CDK 12的开发 抑制剂，并可能提供新的治疗选择，以延长许多mCRPC患者的生存期。

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.