Abstract 3748: U2AF1 mutations rescue deleterious exon skipping induced by KRAS mutations

Abstract U2AF1S34F is one of the most common somatic amino acid substitutions in lung adenocarcinoma; however, the selective pressure behind this splicing factor mutation is poorly understood. To assess the function of U2AF1S34F that results in its selection in lung cancer, we used prime editing to modify the endogenous U2AF1 gene in lung adenocarcinoma cells. Analysis of the resulting changes in alternative splicing identified KRAS as a key target modulated by U2AF1S34F in KRASG12S-mutant A549 cells, but not KRASG12V-mutant NCI-H441 cells. A broad analysis of cancer cell lines identified frequent skipping of KRAS exon 2 in KRASG12S-mutant cells specifically, and this exon skipping is in turn reduced by the acquisition of U2AF1S34F mutations. We observed that the RNA sequence of the KRASG12S mutation resembles a U2AF1 binding site, and KRAS exon 2 skipping is completely abolished when this site is mutated to an alternative, dissimilar codon for serine. We found that KRAS exon 2 skipping is detrimental to KRAS signaling and cell growth. As such, U2AF1S34F mutations are highly enriched in KRASG12S-mutant primary lung adenocarcinomas based on data from AACR Project GENIE and Foundation Medicine Inc. (Odds Ratio = 7.4, p = 4.78 x 10-73). While U2AF1 mutations have previously been reported to be truncal, variant allele frequency analysis suggests that U2AF1S34F occurs as a secondary mutation in KRASG12S-mutant cancers, likely as a means of rescuing full length KRAS expression. Furthermore, analysis of the MSK-CHORD dataset found that patients with KRASG12S-mutant solid tumors have worse overall survival in the presence of secondary U2AF1S34F mutations (p = 0.0169). To identify additional splicing factor/oncogene relationships, we examined mutations which caused exon skipping within the mutated gene and which had a significant co-occurrence with U2AF1, SRSF2 or SF3B1 mutations. An analysis of the AACR Project GENIE and Foundation Medicine Inc. datasets revealed a strong co-occurrence of KRASQ61R and U2AF1I24T mutations, predominantly in pancreatic cancers (Odds Ratio = 46.6, p = 4.90 x 10-30). We found that KRASQ61R leads to KRAS exon 3 skipping, which in turn limits cell growth. However, expression of U2AF1I24T in KRASQ61R-mutant cells rescues KRAS exon 3 inclusion. While U2AF1I24T mutations are virtually unstudied, variant allele frequency analysis indicates that U2AF1I24T occurs as a secondary mutation in KRASQ61R-mutant pancreatic cancers as a means of rescuing KRAS exon 3 inclusion. Additionally, patients with pancreatic cancers harboring both KRASQ61R and U2AF1I24T mutations have worse survival outcomes than those with KRASQ61R mutations alone (p = 0.0003). Our findings describe a novel mechanism for splicing factor mutations in cancer, whereby secondary mutations in splicing factors are positively selected for to rescue splicing defects arising from oncogenic mutations. Citation Format: David M. Walter, Katherine Cho, Smruthy Sivakumar, Iris T.-H. Lee, Anders B. Dohlman, Ethan Shurberg, Kevin X. Jiang, Akansha A. Gupta, Garrett M. Frampton, Matthew Meyerson. U2AF1 mutations rescue deleterious exon skipping induced by KRAS mutations [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 3748.