A unifying mechanism for shared splicing aberrations in splicing factor mutant cancers

Cancer-associated mutations in splicing factors (SFs) such as SF3B1, U2AF1, and SRSF2 generate well-recognized cis-acting alterations in splice site choice, specific to each mutational subtype. However, there is substantial overlap in alternative splicing (AS) defects across these genetically distinct mutation groups which has remained unexplained. We hypothesized that this convergence reflects a shared trans-regulatory mechanism. To answer this, we profiled global transcriptomes to delineate AS programs across SF-mutant states using a cohort of 395 patients with SF-mutant clonal myeloid disorders and 64 healthy donors. While the majority of AS alterations were mutation-specific, we identified a robust subset of shared events, strongly enriched for retained intron (RI) signatures. These RI defects were bidirectional (both increase and decrease in RI) yet strikingly concordant across SF3B1, U2AF1, and SRSF2 mutants. Given that RNA binding proteins (RBPs) are the primary regulators of AS, we compared these SF-mutant RI to those in cells with loss of 356 individual RBPs (ENCODE). SF-mutant RI closely mirrored RI in resulting from SRSF1 loss of function. Phosphoproteomics revealed that SF-mutant cells have hypophosphorylation of RS domains in SRSF1, impairing its splicing activity. This reduction stemmed from a shift in the AMPKalpha-AKT signaling balance, which suppressed the AKT-SRPK1-SRSF1 phosphorylation axis. We next traced this signaling imbalance to an upstream trigger: R-loop-associated transcriptional stress, which activates a DNA damage response (DDR). DDR activation enhanced AMPKalpha signaling while diminishing AKT activity, thereby reducing SRPK1-mediated phosphorylation of SRSF1. Pharmacologic DDR activation recapitulated SRSF1 hypophosphorylation, decreased AKT/SRPK1 activity, and induced RI defects resembling those in SF-mutant cells. Conversely, relief of DDR signaling restored AKT/SRPK1 activity, normalized SRSF1 phosphorylation, and corrected RI abnormalities. These results were confirmed in SF-mutant clonal myeloid cells from patients using RNAseH over-expression to reverse R-loops and quantifying effect on colony formation by CFU assays. Together, our findings demonstrate that, beyond distinct cis-acting splice site changes, SF-mutant cancers share a stress-driven, trans-acting splicing program coordinated through DDR-mediated rewiring of SRSF1 activity. This unified mechanism links replication stress, kinase signaling, and RNA processing across diverse clonal states, and highlights novel actionable nodes (DDR, AMPK/AKT balance, and SRPK1-SRSF1 coupling) with therapeutic potential.