Regulatory Pathways Compromised by PRMT5 Inhibition in Cancers with MTAP Loss - PROJECT SUMMARY The arginine methyltransferase PRMT5 has been identified as a potential target in cancers with loss of the metabolic enzyme MTAP, which occurs in ~15% of all malignancies including several (such as glioblastoma, pancreatic ductal adenocarcinoma, and esophageal cancer) that are refractory to standard therapies. MTAP loss partially impairs PRMT5 activity and sensitizes MTAP-deleted cancer cells to pharmacologic PRMT5 inhibition. As a result, the development of PRMT5 pathway inhibitors has accelerated, including two PRMT5 inhibitors recently reported to have clinical activity in patients with various MTAP-deleted solid tumor malignancies. While PRMT5 is a well-established regulator of multiple cellular pathways including transcription, RNA processing, and DNA repair, how it regulates these pathways and which processes are critical for maintaining cell viability is unclear. To address this knowledge gap, a genome-scale CRISPR activation platform has been leveraged to identify genes that modulate sensitivity to PRMT5 pathway inhibition in MTAP-deleted cancer cell models. Among the top-scoring gene products that reduce sensitivity of MTAP-deleted cells to PRMT5 pathway inhibition are previously unrecognized regulators of DNA damage response (DDR) and potential new PRMT5 substrates. The overall objective of this proposal is to define key components of the PRMT5 axis that impair cell viability upon PRMT5 inhibition and to define pathways that can restore viability. The central hypothesis is that the top- scoring gene products that reduce sensitivity to PRMT5 pathway inhibition represent previously unrecognized regulators of DDR, activate DDR pathways that protect cells from PRMT5 pathway inhibition, and may represent core PRMT5 pathway components. This hypothesis will be tested by pursuing 2 specific aims: 1) Determine how top-scoring proteins mediate responses to DNA damage and 2) Define mechanistic connections between top- scoring proteins and the PRMT5 pathway. The project will examine the impact of these gene products on generation of double-strand DNA breaks, R-loop formation, and homologous recombination (HR) pathways. Proteomic, biochemical, and genetic approaches will be leveraged to gain mechanistic insights about how these proteins intersect with the PRMT5 axis. The proposed work is significant because it will define genes and pathways capable of restoring viability of MTAP-deleted cells exposed to PRMT5 pathway inhibition and will inform a molecular and functional annotation of downstream effects of PRMT5 and compensatory pathways. The approach is innovative and distinct from prior studies because it builds on functional genomic insights to focus squarely on the therapeutically relevant functional roles of PRMT5. Successful completion of these aims will identify processes dysregulated by PRMT5 inhibition that impair cell viability, delineate molecular mediators of these processes, and provide fundamental insights about mechanisms of DDR regulation. The work may also identify additional therapeutic targets in the PRMT5 axis.
