Nonsense-Mediated RNA Decay Is a Targetable Vulnerability in Splicing Factor Mutant Myeloid Neoplasms by Enhancing R-Loop Accumulation and DNA Damage.

Mutant spliceosome proteins (e.g., U2AF1S34F, SF3B1K700E, or SRSF2P95H) alter RNA splicing in myeloid neoplasms, leading to increased production of nonsense transcripts. Inhibiting the nonsense-mediated RNA decay (NMD) pathway, which is responsible for the degradation of nonsense transcripts, preferentially kills cells expressing mutant spliceosome proteins in vitro. In this study, we used an inhibitor of the kinase SMG1, a key regulator of NMD, to provide in vivo evidence that NMD is a therapeutic vulnerability for splicing factor mutant myeloid neoplasms. Primary mouse acute myeloid leukemia cells and human K562 leukemia cell lines expressing splicing factor mutants were more sensitive than wild-type (WT) cells to in vivo inhibition of SMG1 (SMG1i). Disruption of NMD activity by SMG1i led to increased R-loop levels in spliceosome WT cells, which were further increased in treated U2AF1S34F cells. This R-loop accumulation was accompanied by an increase in DNA damage. Degradation of R-loops with RNase H1 rescued spliceosome mutant cells from NMD inhibition-induced cell death. In U2AF1S34F cells, SMG1i increased NMD transcript isoforms (with reduced but detectable protein levels), which were enriched for DNA repair genes, including ATR and RAD51. Consequently, SMG1i-induced cell death in splicing factor mutant leukemias could be further enhanced by the inhibition of ATR or RAD51. This study shows that in vivo targeting of NMD is a therapeutic strategy to treat myeloid neoplasms with aberrant splicing. Cells with spliceosome mutations are sensitive to SMG1 inhibition in vivo due to R-loop accumulation, suggesting that nonsense-mediated decay targeting, alone or with DNA repair inhibitors, could be effective in splicing-mutant myeloid neoplasms.