PROJECT SUMMARY Cancer and cardiovascular diseases are the two leading causes of death in the US and worldwide. Despite the development of second-generation targeted cancer therapies, including covalent tyrosine kinase inhibitors (TKIs), these chemotherapy agents frequently cause significant cardiovascular toxicities (CTX). Understanding the mechanism of CTX susceptibility and developing mitigation strategies are major current challenges in this field. In Project 2, our team will elucidate the genetic susceptibility of cancer patients to the CTX after treatment with ibrutinib, a covalent Bruton kinase inhibitor. In Aim 1, we will harness multiomics and new multiplexing methodology known as “cell village” to perform population level studies in vitro. In collaboration with Project 1 and Core B, we will recruit ibrutinib-treated patients who developed CTX (“ibrutinib-CTX”) and ibrutinib-treated patients who did not develop CTX (“ibrutinib-nonCTX”) to generate induced pluripotent stem cells (iPSCs). The multiplexing capability of the patient-derived iPSCs will be utilized to form “cell village” pools that can be differentiated into cardiomyocytes (iPSC-CMs). The effect of ibrutinib will be monitored on the transcriptome and epigenome level through single-cell multiomics sequencing technology. In Aim 2, these changes will be examined using expression quantitative trait loci (eQTL) studies to detect variants causal for CTX development, which will be further screened for mitigating pathways in collaboration with Project 3. In Aim 3, we will validate the causality of the top 3 detected genetic variants via genome editing in iPSC lines. The edited lines will then undergo comprehensive functional studies using 3D models of iPSC-derived cardiac organoids (iPSC-COs). In summary, understanding the mechanism and genetic background of ibrutinib-induced cardiotoxicity will contribute significantly to developing patient-specific strategies that can mitigate adverse effects of these drugs. Once established, this versatile and high throughput genetic screening strategy can also be expanded to encompass other covalent TKIs and cancer drugs.