SUMMARY Splicing is a critical biological process in cancer initiation and progression. In pancreatic cancer, few studies have investigated the role of splicing regulators or their spliced targets in disease progression. Using a high- throughput, genome-wide genetic screen in vivo for novel events promoting KRAS-driven pancreatic ductal adenocarcinoma (PDAC), we recently identified a unique role for the RNA splicing factor RBFOX2 as a latent tumor suppressor driving disease progression. We showed RBFOX2 nuclear abundance is significantly decreased in poorly differentiated human PDAC and that RBFOX2 depletion promotes a highly metastatic disease in orthotopic models. We found RBFOX2 loss promotes exon skipping events associated with stem cell potential and invasion, including ABI1. We hypothesize that RBFOX2-mediated splice-switching in key regulators of cell invasion and differentiation programs is central to its role in promoting PDAC progression. In this proposal, we will examine the oncogenic role of these RBFOX2-mediated splicing events in PDAC progression. In Aim 1, we will investigate the oncogenic role(s) of the ABI1∆Ex9 isoform alternatively spliced by RBFOX2 using proteomics approaches to elucidate novel protein interactions and secondary modifications contributing to ABI1∆Ex9 function in driving invasive phenotypes. In Aim 2, we will investigate the contribution of “ESC-like” exon exclusion events regulated by RBFOX2 to PDAC cell differentiation and disease progression using i) splice- switching Anti-Sense Oligonucleotides (AONs) to mimic exon-skipped isoforms and investigate their cooperativity in driving ex vivo stem cell-like phenotypes and ii) inducible cDNA isoforms to understand their role in promoting disease progression in vivo. In Aim 3, we will develop an RBFOX2 splicing signature using PDXs and PDOs from patients with rapid relapse and progression-free disease using state of the art splicing analysis platforms (bulk and scRNAseq PacBio/isoseq coupled to Illumina short read sequencing) and investigate its association with disease relapse. We expect that our study will reveal the biological relevance of the RBFOX2- promoted oncogenic splicing program in pancreatic cancer progression and identify new molecular vulnerabilities that can be harnessed to improve disease outcomes for pancreatic cancer patients.