Session: 636. Myelodysplastic Syndromes—Basic and Translational: Clonal Trajectories and Novel Therapies
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acquired Marrow Failure Syndromes, Acute Myeloid Malignancies, AML, Translational Research, Bone Marrow Failure Syndromes, Combination therapy, Diseases, Myeloid Malignancies, Biological Processes, molecular biology, pathogenesis
PARP inhibitors preferentially kill cells with defective homologous recombination (HR)-mediated DNA repair. We asked if the observed PARPi sensitivity in SF-mutant cells was due to defects in HR. To measure HR activity, we performed a CRISPR-Cas9/mClover assay. The assay measures HR-dependent insertion of an mClover-containing donor cassette into Cas9-mediated double-stranded breaks in the LMNA gene, which results in the expression of a green fluorescent Lamin A/C protein. There was no significant difference in the relative percentage of mClover positivity in SRSF2P95H or U2AF1S34F cells compared to control (Fig. 1O), suggesting that these cells are HR-proficient. Interestingly, SF3B1K700E cells showed significantly reduced HR efficiency, consistent with a published study. We next investigated cellular PARP1 activity in SRSF2- and U2AF1-mutant cells by monitoring Poly-ADP-ribosylated chain (PAR) levels. SRSF2P95H cells had elevated PAR level compared to SRSF2WT cells (Fig. 1P). Since PARP1 is known to associate with R-loops, we wanted to test whether increased PARP1 activity and PARPi sensitivity are R-loop dependent. We generated SRSF2-mutant cells that inducibly express RNaseH1, an enzyme that specifically cleaves the RNA moiety within the RNA:DNA hybrids. Induction of RNaseH1 reduced PAR level and PARPi sensitivity in SRSF2P95H cells, suggesting that SRSF2-mutant cells elicit a PARP1 response to promote survival in an R-loop-dependent manner (Fig. 1Q). Lastly, since SF-mutant cells are also sensitive to ATRi, we tested if combining PARPi and ATRi can further sensitize SF-mutant cells. Combined PARPi + ATRi treatment induced synergistic killing of Srsf2- and U2af1-mutant cells compared to wildtype cells in cell viability and colony forming assays (Fig. 1R-S). In summary, this study provides a pre-clinical rationale for therapeutic targeting of PARP1 in SF-mutant leukemia. Moreover, PARP and ATR inhibitor combination could emerge as a new therapeutic strategy in this genetically distinct disease subtype.
Disclosures: Graubert: Janssen: Research Funding; Juno Therapeutics: Honoraria; H3 Biomedicine: Honoraria.
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