Crispr-mediated genome editing reveals a preponderance of non-oncogene addictions as targetable vulnerabilities in pleural mesothelioma

•A comprehensive genome-wide CRISPR knockout screen identified novel susceptibilities in PM cells.•Non-oncogenic stress-responsive pathways, such as DNA damage are critical gene dependencies.•Integrated analysis prioritizes CDK7, CHK1, HDAC3, RAD51, TPX2, and UBA1 as targetable non-oncogene dependencies.•The results reveal overlooked aspects of PM biology and provide a blueprint for developing novel targeted therapies. Pleural mesothelioma (PM) is an aggressive cancer with limited treatment options. In particular, the frequent loss of tumor suppressors, a key oncogenic driver of the disease that is therapeutically intractable, has hampered the development of targeted cancer therapies. Here, we interrogate the PM genome using CRISPR-mediated gene editing to systematically uncover PM cell susceptibilities and provide an evidence-based rationale for targeted cancer drug discovery. This analysis has allowed us to identify with high confidence numerous known and novel gene dependencies that are surprisingly highly enriched for non-oncogenic pathways involved in response to various stress stimuli, in particular DNA damage and transcriptional dysregulation. By integrating genomic analysis with a series of in vitro and in vivo functional studies, we validate and prioritize several non-oncogene addictions conferred by CDK7, CHK1, HDAC3, RAD51, TPX2, and UBA1 as targetable vulnerabilities, revealing previously unappreciated aspects of PM biology. Our findings support the growing consensus that stress-responsive non-oncogenic signaling plays a key role in the initiation and progression of PM and provide a functional blueprint for the development of unprecedented targeted therapies to combat this formidable disease.