Therapeutic Targeting of Spliceosome Mutant Myeloid Neoplasms Via PARP1 Inhibition

Somatic mutations in splicing factors (SF) SF3B1, SRSF2, U2AF1 are observed across a spectrum of clonal myeloid disorders including myelodysplastic syndromes (MDS). Previous studies identified increased R-loops in SF-mutant cells. Abnormal R-loop accumulation is associated with increased genomic instability and preferential sensitivity to ATR kinase inhibitor. Motivated by these observations, we set out to identify novel therapeutic targets in SF-mutant myeloid malignancies. Using MLLAF9 Srsf2WT and Srsf2P95Hcells, we performed a focused drug screen using inhibitors targeting different DNA damage response pathways and drugs that inhibit DNA replication or DNA metabolic processes. Unexpectedly, Srsf2P95H cells displayed increased sensitivity to inhibitors targeting PARP1/2 (Fig. 1A-C). Olaparib (PARPi) treatment induced higher apoptosis in Srsf2P95Hcells than in Srsf2WTcells (Fig. 1D). To ensure this phenomenon extends beyond Srsf2 mutation, we generated MLLAF9 leukemia on U2af1WT and U2af1S34Fbackgrounds. PARPi treatment in U2af1S34F cells resulted in reduced cell viability and increased apoptosis compared to U2af1WT cells (Fig. 1E-G). Next, we genetically inactivated Parp1 in Srsf2WT and Srsf2P95H cells. The absence of Parp1 did not affect the growth of Srsf2WT cells (Fig. 1H). In contrast, Parp1 deletion significantly reduced proliferation of Srsf2P95H cells (Fig. 1I).Finally, to confirm this phenotype in vivo, we transplanted established MLLAF9 Srsf2WTor Srsf2P95H leukemia into syngeneic mice. Eleven days post-transplant, mice received either vehicle or PARPi. Mice transplanted with Srsf2WT cells showed no survival advantage when treated with PARPi (Fig. 1J). In contrast, PARPi treatment significantly prolonged survival of mice transplanted with Srsf2P95H cells compared to vehicle-treated mice (Fig. 1K). To examine whether PARPi sensitivity is conserved in humans, we treated K562 cells expressing either SRSF2P95H or SF3B1K700E mutation. SRSF2P95H and SF3B1K700E cells showed increased sensitivity to olaparib and rucaparib (Fig. 1L-M). This observation was further validated in a panel of primary AML blasts carrying SRSF2 and U2AF1 mutations(Fig. 1N). Taken together, our results identify PARP1 as a novel therapeutic target in SF-mutant myeloid neoplasms.

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.

Graubert:Janssen: Research Funding; Juno Therapeutics: Honoraria; H3 Biomedicine: Honoraria.