R-loop accumulation induced by splicing perturbation triggers PARP1/2 response

Abstract

Mutation in genes encoding for U2AF1, SRSF2, and SF3B1 are commonly mutated in myelodysplastic syndromes (MDS) and other hematological malignancies, highlighting a unique genetic vulnerability for targeted therapies. Recent studies showed that perturbing RNA splicing by pharmacological modulators selectively sensitizes RNA splicing mutant cancers in vitro and in vivo models. However, the use of pharmacological modulators in clinical trials did not show improved efficacy in patients carrying RNA splicing factor mutations, pointing to a need for drug combination strategies. We previously reported that RNA splicing perturbation caused by pharmacological splicing modulators trigger aberrant accumulation of R-loops, a three-stranded nucleic acid structure consisting of an RNA:DNA hybrid and a displaced single-stranded DNA and depend on R-loop response signaling for survival. Based on these findings, we hypothesize that targeting different R-loop response pathways enhances cancer cell killing when combined with RNA splicing modulators. Here, we identified that RNA splicing modulator, pladienolide B (Plad-B) elicited a poly ADP-ribosylation (PARP) response in an R-loop dependent manner. PARP inhibitor, olaparib, induced more DNA damage in cells treated Plad-B, suggesting a new therapeutic drug combination that may enhance cell death. Surprisingly, we found that the PARP response in Plad-B treated cells requires both PARP1 and PARP2 function. This is in stark contrast to their known functions in DNA repair where PARP1 plays the dominant role. Taken together, we propose a two-step model where PARP1 initiates and PARP2 amplifies the ADP-ribosylation signaling to prevent R-loop-associated genomic instability. Importantly, our results highlight a new R-loop regulatory signaling mediated by PARP1/2 and a potential of repurposing FDA-approved PARP inhibitors to treat MDS patients harboring splicing factor mutations either as a monotherapy or in combination with RNA splicing modulators.