Single-cell genomics reveals the genetic and molecular bases for escape from mutational epistasis in myeloid neoplasms.

Large-scale sequencing studies of hematologic malignancies have revealed notable epistasis among high-frequency mutations. One of the most striking examples of epistasis occurs for mutations in RNA splicing factors. These lesions are amongst the most common alterations in myeloid neoplasms and generally occur in a mutually exclusive manner, a finding attributed to their synthetic lethal interactions and/or convergent effects. Curiously, however, patients with multiple concomitant splicing factor mutations have been observed, challenging our understanding of one of the most common examples of epistasis in hematologic malignancies. Here we performed bulk and single cell analyses of myeloid malignancy patients harboring >2 splicing factor mutations to understand the frequency and basis for the co-existence of these mutations. Although mutations in splicing factors were strongly mutually exclusive across 4,231 patients (q<0.001), 0.85% harbored two concomitant bona fide splicing factor mutations, ~50% of which were present in the same individual cells. However, the distribution of mutations in double mutants deviated from those in single mutants with selection against the most common alleles, SF3B1K700E and SRSF2P95H/L/R, and selection for less common alleles, such as SF3B1 non-K700E mutations, rare amino acid substitutions at SRSF2P95, and combined U2AF1S34/Q157 mutations. SF3B1 and SRSF2 alleles enriched in double mutants had reduced effects on RNA splicing and/or binding compared to the most common alleles. Moreover, dual U2AF1 mutations occurred in cis with preservation of the wild-type allele. These data highlight allele-specific differences as critical in regulating molecular effects of splicing factor mutations as well as their co-occurrences/exclusivities with one another.

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