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

这 修订后 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，具有长期的生产性合作历史，以研究细胞内的影响 BRCA1介导的DDR上的膀胱癌PD-L1信号及其如何改变对PARP的敏感性 抑制剂和免疫检查点阻断原位/转移性小鼠和人类模型的免疫治疗。 我们测试了可移植的小鼠膀胱癌MB49和MBT-2，以及人膀胱癌RT4， UM-UC3和UM-UC14，代表基础和管腔组织学加上新的PDX、异种移植和类器官 模型和我们的新型GEMM都是根据审阅者的要求 。使用控制询问细胞内PD-L1信号 与基因操纵的肿瘤和其他方法 包括新的非生化方法 审阅者请求 。我们将一起使用这些临床相关的模型来测试我们的总体假设 膀胱癌PD-L1信号促进抑制同源重组DDR通路 PARP抑制剂和免疫检查点阻断免疫治疗的疗效。 目的1：明确PD-L1介导的心率调节机制。我们将使用经过充分验证的基因， 生化和药理学方法加上同源重组的权威细胞报告分析， RNA-Seq，蛋白质组学，RPPA，免疫印迹， CRISPR图书馆屏幕，有机化合物 和图像的数字成像 分析软件，以了解PD-L1如何调节BRCA1功能并测试机制，包括 用于肿瘤的免疫原性。我们确定了FDA批准的药理药物，我们将其重新用于消耗 肿瘤PD-L1和抑制心率，我们对其机制进行了定义。 目的2：明确PD-L1受控同源重组/BRCA1效应的治疗后果。 对照具有BRCA1和相关分子遗传扰动的PD-L1基因耗竭细胞将 通过向小鼠体内挑战工程细胞并用单一试剂或药物治疗来测试体内效应 根据目标1的调查结果，评估治疗效果和具体机制。我们用 可移植的同基因小鼠模型，以及人类异种移植和人源化小鼠模型 我们在这方面有丰富的经验。值得注意的是，我们将评估肿瘤的微环境和免疫 对使用遗传免疫改变的小鼠、过继细胞转移和免疫的治疗的贡献 阻断/中和分子。活体数据是在我们擅长的体外分析中进行机械精炼的。 PDX，有机化合物

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