Bladder cancer PD-L1 control of homologous recombination: Basic mechanisms applied to novel treatments

这 修订 MPI提案汇集了膀胱癌，肿瘤生物学，DNA损伤反应（DDR）方面的专家 和临床前模型，具有长期的生产合作历史，以研究细胞内 膀胱癌PD-L1信号对BRCA 1介导的DDR的影响，以及这些信号如何改变对PARP的敏感性 抑制剂和免疫检查点阻断免疫疗法。 我们测试了可移植的小鼠膀胱癌细胞系MB 49和MBT-2，以及人膀胱癌细胞系RT 4， UM-UC 3和UM-UC 14，代表基底和管腔组织学加上新的PDX、异种移植物和类器官 模型和我们的小说GEMM所有根据审查员的要求 .使用对照品询问细胞内PD-L1信号 与基因操作肿瘤和其他方法相比， 包括新的非生物化学方法， 审稿人要求 .我们将一起使用这些临床相关模型来检验我们的总体假设， 膀胱癌PD-L1信号促进同源重组DDR途径， PARP抑制剂和免疫检查点阻断免疫治疗的疗效。 目的1：确定PD-L1介导的HR控制机制。我们将使用经过充分验证的遗传学， 生物化学和药理学方法加上同源重组的确定性细胞报告基因测定， RNA测序、蛋白质组学、RPPA、免疫印迹、 CRISPR文库筛选，类器官 和数字成像 分析软件，以了解PD-L1如何调节BRCA 1功能，并测试机制，包括 对于肿瘤免疫原性。我们确定了FDA批准的药物， 肿瘤PD-L1和抑制HR，我们定义了机制。 目的2：确定PD-L1控制的同源重组/BRCA 1效应的治疗后果。 对照组与基因PD-L1耗竭细胞（BRCA 1和相关分子基因干扰）相比， 通过将工程化细胞激发到小鼠中并用单一药剂治疗来测试体内效应，或 基于目标1的发现，对联合用药进行研究，以评估治疗疗效和具体机制。我们使用 可移植的同基因小鼠模型，以及人异种移植和人源化小鼠模型 我们在这方面有着丰富经验。值得注意的是，我们将评估肿瘤微环境和免疫 使用遗传免疫改变的小鼠、过继细胞转移和免疫治疗的贡献 阻断/中和分子。体内数据在我们擅长的体外试验中进行了机械细化。 PDX，类器官

This revised MPI proposal assembles experts in bladder cancer, tumor biology, DNA damage response (DDR) and pre-clinical models with a long-standing history of productive collaborations to study effects of intracellular bladder cancer PD-L1 signals on BRCA1-mediated DDR, and how such signals alter sensitivity to PARP inhibitors and immune checkpoint blockade immunotherapy in orthotopic/metastatic mouse and human models. We test transplantable mouse bladder cancer lines MB49 and MBT-2, and the human bladder cancer lines RT4, UM-UC3 and UM-UC14, representing basal and luminal histologies plus new PDX, xenograft and organoid models and our novel GEMM all per reviewer request . Intracellular PD-L1 signals are interrogated using control versus genetically manipulated tumors and other approaches including new non-biochemical approaches per reviewer request . We will use these clinically relevant models together to test our overarching hypothesis that bladder cancer PD-L1 signals promote the homologous recombination DDR pathway that suppresses PARP inhibitor and immune checkpoint blockade immunotherapy efficacy. Aim 1: Define mechanisms for PD-L1-mediated control of HR. We will use well-validated genetic, biochemical and pharmacologic approaches plus definitive cell reporter assays for homologous recombination, RNA-seq, proteomics, RPPA, immunoblots, CRISPR library screens, organoids and digital imaging with image analysis software to understand how PD-L1 regulates BRCA1 functionality and to test mechanisms, including for tumor immunogenicity. We identified FDA-approved pharmacologic agents that we repurposed to deplete tumor PD-L1 and inhibit HR, for which we define mechanisms. Aim 2: Define treatment consequences of PD-L1 controlled homologous recombination/BRCA1 effects. Control versus genetically PD-L1 depleted cells with genetic perturbations of BRCA1 and related molecules will be tested for in vivo effects by challenging engineered cells into mice and treating with single agents or combinations, based on findings from Aim 1 to assess treatment efficacy and specific mechanisms. We use transplantable, syngeneic mouse models, and human xenograft and humanized mouse models in which we have extensive experience. Notably, we will assess tumor microenvironmental and immune contributions to treatments using genetically immune altered mice, adoptive cell transfers and immune blocking/neutralizing molecules. In vivo data are mechanistically refined in in vitro assays in which we are expert. PDX, organoids