Project Summary Cardiotoxicities associated with small molecule cancer therapeutics are well documented. However, emerging reports of adverse cardiac reactions due to monoclonal antibody-based immune checkpoint inhibitors (ICIs) is a growing concern. These ICIs act on programmed cell death protein 1 (PD-1) receptor or its ligand programmed cell death-ligand 1 (PD-L1) enhancing antitumor response, and these often lead to such immune-related adverse events (iRAEs). Several studies point toward the homology between tumor antigens and cardiac proteins as one of the reasons for outcomes such as myocarditis, changes in cardiac rhythm, and vasculitis. Due to the rare, early detection iRAEs and scarcity of patient samples. The mechanism of ICI-induced cardiac disease remains elusive. Hence, utilizing a unique multi-disciplinary approach and expertise in induced pluripotent stem cell (iPSC) technology, immunology and bioengineering strategies, I hypothesize that, iPSC-derived 3-D cardiac organoids (COs) with immune cells can be used to model ICI-induced cardiac injury. Unraveling the disease mechanism of cardiac injury due to ICI treatment using a reductionist approach may lead to discovery of new targets that can confer cardioprotection for immunotherapies. In order to accomplish this goal, I have defined three specific aims: (1) I will generate COs through self-assembly of iPSC-cardiomyocytes, iPSC-endothelial cells and iPSC-cardiac fibroblasts and test two commonly used ICIs Pembrolizumab (PD-1 inhibitor) and Atezolizumab (PD-L1 inhibitor). I will for assess for changes in CO function in the presence and absence of peripheral blood mononuclear cells (PBMCs) used for iPSCs derivation (2) I will conduct in-depth molecular analyses of the COs at a single cell level and identify potential targets responsible to hyper immune response can cardiac dysfunction (3) Finally, I will use cutting-edge genome editing techniques to knockdown the gene targets that help rescue the CO function after ICI treatment. The targets identified in Aim 2 will be validated again in mouse models by injecting ICIs into mice with diverse immune backgrounds followed by evaluation of heart function and molecular changes within the tissue. In this multidisciplinary project, with the support from my esteemed panel of mentors, advisors and collaborators, I am confident that I will receive par excellence training to accomplish both scientific and career development goals. Given my scientific track record to date, and complementary training sought through this K99/R00 award, together will help me develop and lead several research areas building toward a successful independent career.

Project Narrative The mechanism of cardiotoxicity due to immune checkpoint inhibitors (ICI) remains poorly understood due varied clinical symptoms cause due to inflammation. The goal of this project is to unravel the underlying mechanisms by systematically studying the effect of ICIs using multicellular cardiac organoids (CO) derived from patient-specific induced pluripotent stem cells (iPSCs). The results of the study may lead to development of drugs to preserve heart function in cancer patients.