U2AF1 Mutant Myeloid Neoplasms are Preferentially Sensitive to In Vivo Inhibition of Nonsense-Mediated RNA Decay

Expression of mutant spliceosome proteins (e.g., U2AF1S34F, SF3B1K700E, or SRSF2P95H) alters RNA splicing in myeloid neoplasms, leading to increased production of nonsense transcripts. We have previously shown that inhibiting the nonsense–mediated RNA decay (NMD) pathway, which is responsible for degradation of nonsense transcripts, preferentially kills cells expressing mutant spliceosome proteins. In this study, we used a novel inhibitor (SMG1i-63) of the kinase SMG1, a key regulator of NMD, to provide in vivo evidence that NMD is also a therapeutic vulnerability for splicing factor mutant myeloid neoplasms. We show that primary mouse acute myeloid leukemia cells and human K562 leukemia cell lines expressing splicing factor mutants were more sensitive than wild-type cells to in vivo inhibition of SMG1 (SMG1i). Disruption of NMD activity by SMG1i led to increased R-loops levels in spliceosome wild-type cells, which are further increased in U2AF1S34F treated 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, indicating that R-loop formation is a primary mechanism of drug sensitivity. In U2AF1S34F cells, SMG1i led to increased detection of NMD transcript isoforms (with reduced but detectable protein levels) for DNA repair genes, including ATR and RAD51. Consequently, SMG1i-induced cell death in splicing factor mutant leukemias could be further enhanced by inhibition of the DNA damage response proteins ATR or RAD51. This study shows that in vivo targeting of NMD is a therapeutic strategy to treat myeloid neoplasms with aberrant splicing.