Elucidating molecular mechanisms of PARP2 at R loops in myelodysplastic syndromes

ABSTRACT Myelodysplastic syndromes (MDS), a group of blood disorders diagnosed in nearly 15,000 Americans annually, are characterized by patients with high-risk MDS having a median survival of less than two years. Roughly half of all MDS patients possess a mutation in an RNA splicing factor (SF) gene: most commonly U2AF1, SRSF2, or SF3B1. Mutations in U2AF1 and SRSF2 are associated with faster disease progression to secondary acute myeloid leukemia and with poorer patient outcomes. Complicating treatment, MDS-associated SF mutations are mutually exclusive and cause unique patterns of alternative splicing events with non-overlapping outcomes, based on the individual mutations. Overcoming this, previous work in the Nguyen lab identified R loops, three- stranded RNA:DNA hybrid transcription intermediates, as a unifying mechanism of disease pathogenesis induced by MDS-associated SF mutations. Recently, the lab further found that SF mutations sensitize cells to inhibitors of poly(ADP-ribose) polymerase (PARP) enzymes, and that PARP1 plays a vital role in regulating R loops and maintaining genomic stability in SF-mutant cells. The proposed project aims to expand on this prior work by elucidating the mechanism by which PARP2 regulates R loops in cells possessing MDS-associated SF mutations. Preliminary results show that PARP2 associates with R loops, and that loss of PARP2 leads to both a reduction in ADP-ribosylation signaling in SF-mutant cells, and R-loop accumulation. In contrast, loss of PARP2 had no effect on ADP-ribosylation levels following direct DNA damage alone. PARP2 was also found to interact with the R-loop-resolving helicase DDX41. The central hypothesis of this project is that PARP2 plays a unique role at R loops to prevent R-loop-associated genomic instability in SF-mutant cells. This hypothesis will be tested through two specific aims evaluating PARP2 function at R loops: 1) elucidate how PARP2 senses R loops in U2AF1S34F cells, and 2) determine the impact of DDX41 ADP-ribosylation at R loops in SF-mutant cells. Results obtained through completion of these aims will provide new insights into how cells maintain genomic stability in response to R-loop accumulation, explicate a mechanism by which PARP inhibitors sensitize SF-mutant cells, and potentially identify novel biomarkers to predict this sensitivity. This proposal will be completed at the University of Minnesota, under the co-mentorship of Dr. Hai Dang Nguyen, an expert in the DNA damage response, R-loop regulation, and hematologic disorders, and Dr. David Largaespada (co-sponsor), an expert in molecular-genetic mechanisms of pathogenesis. To complement this expertise, I will receive further guidance from my collaborators: Dr. Stanley Lee, who specializes in mouse models of splicing-factor mutant MDS, and Dr. Anthony Leung, an expert in ADP-ribosylation signaling. This mentorship team, paired with the collaborative research environment, provides a unique opportunity for the candidate to complete the research aims described in this proposal and achieve his goal of becoming a molecular translational scientist working to uncover mechanisms of pathogenesis in hematologic disorders.