Abstract 4225: Targeting RNA splicing sensitizes MTAP-deleted cancers to MTA-cooperative PRMT5 inhibitors

Abstract AMG 193, currently in Phase 1/2 trials, is an MTA-cooperative PRMT5 inhibitor that is showing promise as a targeted therapy in MTAP-deleted tumors. MTAP deletion, which occurs in ∼10-15% of cancers, results in the accumulation of its substrate MTA leading to the partial inhibition of the arginine methyltransferase PRMT5. AMG 193, and its representative analog AM-9747, selectively bind PRMT5 in the presence of MTA resulting in preferential growth inhibition of MTAP-deleted tumor cells while sparing normal WT cells. Mechanistically, AMG 193 treatment induces DNA damage, cell cycle arrest, and a change in alternative splicing. We previously showed that in a large tumor cell line panel, elevated baseline levels of intron retention positively correlated with sensitivity to AMG 193 suggesting alternative splicing could potentially be utilized as a biomarker (Belmontes & Slemmons et al, 2024). As a result, we hypothesized that co-targeting pre-mRNA splicing could sensitize MTAP-deleted cells to PRMT5 inhibition. To identify modulators of response or resistance to MTA-cooperative PRMT5 inhibitors, we performed a series of drug anchored genome-wide CRISPR knockout screens in engineered MTAP-deleted cell lines (HAP1, HCT116) and endogenous MTAP-deleted cell lines (A549, MIAPACA2). In line with our previous data that PRMT5 inhibition induces alternative splicing and intron retention is associated with AMG 193 sensitivity, these screens identified components of pre-mRNA splicing and RNA processing as modulators of AMG 193 response. One exemplary hit, AKAP17A, is involved in the regulation of alternative splicing. Here we demonstrate that AKAP17A knockout drives sensitivity to MTA-cooperative PRMT5 inhibition resulting in a 3-4X IC50 decrease in HAP1 MTAP-deleted cells and a 30% increase in max effect in A549 and MIAPACA2 cells. In the MIAPACA2 xenograft model, which has moderate sensitivity to PRMT5 inhibition (average tumor growth inhibition of ∼50%), the addition of AKAP17A knockout drives tumor regressions. Knockout of other components of the spliceosome or splicing regulators similarly sensitizes MTAP-deleted cells to AM-9747 treatment in vitro. In a complementary pharmacologic approach, treatment with spliceosome inhibitors synergizes with PRMT5 inhibition in MTAP-deleted cell lines but not in MTAP WT cells. In summary, our data suggests that alternative splicing is a vulnerability in MTAP-deleted cell lines that can be perturbed to augment the activity of MTA-cooperative PRMT5 inhibitors. Additional resistance and sensitization pathways identified from the CRISPR screens are currently being evaluated using in vitro and in vivo assays. Furthermore, clinical biomarker datasets from the ongoing early phase trials will be utilized to validate these preclinical findings. Citation Format: Katherine K. Slemmons, Chun Su, Siyuan Liu, Po-Wen Yu, Michele McEntee, Eric Yuan, Jasper Lee, Jiamiao Lu, Aaron Horning, Antreas Hindoyan, Brian Belmontes, Paul E. Hughes. Targeting RNA splicing sensitizes MTAP-deleted cancers to MTA-cooperative PRMT5 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4225.