Implications of PARP1 in myelodysplastic syndromes and targeted therapy

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PROJECT SUMMARY/ABSTRACT Myelodysplastic syndromes (MDS), a heterogenous group of clonal hematopoietic stem cell disorders, are an acquired bone marrow failure syndrome. MDS is characterized by ineffective hematopoiesis resulting in peripheral blood cytopenia and progenitor expansion. Genes encoding for RNA splicing factors (U2AF1, SF3B1, SRSF2, and ZRSR2) are frequently mutated and occur in the founding clones of MDS, representing a unique class of genetic vulnerability for targeted therapy. However, despite the prevalence of spliceosome mutations, how such mutations impact different cellular mechanisms are largely unclear. Recent studies by us and others suggest that R-loops, a group of transcription intermediates containing RNA:DNA hybrids and displaced single- stranded DNA, are a source of genomic instability induced by different spliceosome mutants. In the preliminary studies leading to this application, we find that PARP1 is activated by R-loops and it plays a key role in suppressing R-loop-associated DNA damage. Furthermore, we show that MDS-associated RNA splicing factor mutations promote R-loop accumulation and render cells sensitive to PARP inhibition. These exciting findings lead us to hypothesize that PARP1 is a key sensor of R-loops and a critical suppressor of R-loop-associated DNA damage. Furthermore, aberrant R-loop accumulation represents a new targetable vulnerability in MDS- associated splicing factor mutant cells, making PARP inhibition an attractive way to target R-loop vulnerability in MDS. Finally, since PARP inhibitors achieved limited FDA approval in different diseases, repurposing PARP inhibitors to treat MDS patients harboring RNA splicing factor mutations may provide the fastest route to translate our findings to the clinics. To test these hypotheses, in Aim 1, we will elucidate mechanisms by which PARP1 is activated by R-loops. In Aim 2, we will identify global PARP1 substrates and R-loop distribution landscape in U2AF1-mutant cells, providing a proteomic and genomic view of how PARP1 regulates R-loops. In Aim 3, we will evaluate whether PARP inhibitor, olaparib, can selectively eliminate MDS-associated splicing mutant cells in vitro and in vivo. Together, these studies will mechanistically explain how R-loops are sensed by PARP1 in splicing mutant cells, reveal how PARP1 guards cells against R-loop-associated genomic instability, and address whether R-loop-associated vulnerability in spliceosome-mutant MDS cells can be exploited by PARP inhibitors as targeted MDS therapy. The combined expertise in R-loops, PARP1, DNA damage response and spliceosome mutations in MDS (Nguyen laboratory), PARP regulation by proteomic approach (Leung laboratory, co-I), and MDS GEMM mouse models of U2AF1 and SRSF2 mutations (Lee laboratory, co-I) provides us the unique opportunity to characterize PARP1 function in cells expressing MDS-associated mutations. These studies will not only significantly advance our understanding of R-loop biology and PARP1 signaling, but also repurpose the use of FDA-approved PARP inhibitors in targeted therapy for MDS patients harboring RNA splicing factor mutations by exploiting R-loop-associated vulnerability.