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