PROJECT SUMMARY Breast cancer is the most common cancer among women worldwide, affecting one in eight women in the United States during their lifetime. Existing patient-derived models for breast cancer, including cell and organoid cultures and patient-derived xenografts, fail to recapitulate important aspects of breast tumorigenesis. Notably, these models are derived from late-stage tumors, undergo genetic drift and selection in culture, and are incompatible with high-throughput genetic and chemical screening. There is a clear need for more robust preclinical models of breast cancer that enhance translation of basic cancer research into meaningful improvements in patient outcomes. Human induced pluripotent stem cells (hiPSCs) are an appealing tool for modeling breast cancer because they provide a scalable source of patient-derived cells that retain molecular features of underlying cancer cells, including mutational burdens and drug sensitives. Additionally, as hiPSCs are clonally derived from single cells, multiple hiPSC lines from an individual tumor provide a tool to examine subclone-specific contributions to tumorigenesis and therapy response. However, no study to-date has examined the potential of hiPSCs to model breast tumorigenesis. This study aims to develop a novel preclinical model of breast cancer by generating hiPSC lines from human breast tumors and to use this model to examine patient and subclone-specific responses to poly (ADP-ribose) polymerase (PARP) inhibitors. PARP inhibitors target intrinsic DNA damage repair deficiencies in tumor cells and are a promising therapeutic for subsets of highly aggressive, BRCA-mutant triple negative breast cancers. In Aim 1, we will reprogram primary breast cancer cells to an hiPSC state, followed by differentiation into mammary epithelial cells (MECs) for in vitro disease modeling. We will use in-depth sequencing to determine the extent to which the genetic heterogeneity of hiPSC lines recapitulates the clonal heterogeneity of primary tumors. In Aim 2, we will use genetically distinct hiPSC-MEC lines derived from a BRCA mutant breast tumor to examine subclonal differences in DNA damage repair proficiency, intrinsic sensitivity to PARP inhibitors, and propensity to acquire PARP inhibitor resistance. Subclone-specific mechanisms of PARP inhibitor resistance will be mechanistically validated using gene editing in this model. Completion of these aims will fill a critical need for novel in vitro models of breast cancer that accurately capture the genomic heterogeneity of human breast tumors and can be used to model how patient- specific subclonal tumor architecture influences response to therapy. Additionally, the proposed project provides a platform for the applicant’s predoctoral training, with a focus on developing expertise in cancer disease modeling and precision oncology, skills related to experimental design and analysis, proficiency in computational pharmacogenomics, and the professional skil...