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Immunofluorescence staining of gH2AX DNA damage marker in human CD34+ hematopoietic progenitor cells.

Heterozygous somatic mutations in spliceosome genes (U2AF1, SF3B1, ZRSR2, or SRSF2) occur in >50% of patients with myelodysplastic syndrome (MDS). These mutations occur early in disease development, suggesting that they contribute to MDS pathogenesis and may represent a unique genetic vulnerability for targeted therapy. Here, we show that RNA splicing perturbation by expression of the U2AF1(S34F) mutant causes accumulation of R loops, a transcription intermediate containing RNA:DNA hybrids and displaced single-stranded DNA, and elicits an ATR response. ATR inhibitors (ATRi) induced DNA damage and cell death in U2AF1(S34F)-expressing cells, and these effects of ATRi were enhanced by splicing modulating compounds. Moreover, ATRi-induced DNA damage was suppressed by overexpression of RNaseH1, an enzyme that specifically removes the RNA in RNA:DNA hybrids, suggesting that the ATRi sensitivity of U2AF1(S34F)-expressing cells arises from R loops. Taken together, our results demonstrate that ATR may represent a novel therapeutic target in patients with MDS carrying the U2AF1(S34F) mutation and potentially other malignancies harboring spliceosome mutations.Significance: This study provides preclinical evidence that patients with MDS or other myeloid malignancies driven by spliceosome mutations may benefit from ATR inhibition to exploit the R loop–associated vulnerability induced by perturbations in splicing. Cancer Res; 78(18); 5363–74. ©2018 AACR.

U2AF1(S34F)-expressing cells induce R loop-associated ATR-mediated RPA32 phosphorylation.

Protein expression levels of R loop regulators in U2AF1(WT)- or U2AF1(S34F)-expressing cells.

RNA splicing perturbation in cells confers sensitivity to ATR inhibitors.

Combination of RNA splicing modulator compounds and ATR inhibition sensitizes U2AF1(S34F)-expressing cells.

List of primers used for RNA splicing assay

Expression of wild type or different mutants of U2AF1.

RNA splicing perturbation by pharmacologic compounds induce R loop and ATR-mediated RPA32 phosphorylation.