H3K79me2 Profiling Reveals DOT1L Regulated Splicing in MLL-rearranged AML [ChIP-Seq]

Aberrant H3K79 dimethylation by DOT1L is a defining feature of MLL-rearranged (MLLr) acute myeloid leukemia (AML), but whether this modification influences alternative splicing remains unclear. Here, we performed H3K79me2 ChIP-seq and RNA-seq on primary samples from 24 MLLr AML patients, 4 wild-type MLL AML patients, and 4 healthy bone marrow donors. We found that a subset of exon skipping (SE) events was enriched at H3K79me2-occupied loci in MLLr samples. DOT1L inhibition with EPZ5676 led to broad changes in SE patterns, and many switched events showed concurrent reduction in local H3K79me2 signal. Pathway analysis of genes harboring these events revealed enrichment for RNA processing and splicing-related functions, with additional involvement of DNA repair and apoptosis-associated pathways. To identify potential mediators, we performed rapid immunoprecipitation mass spectrometry (RIME) in MV4-11 and MOLM-14 cells and detected SRSF2 and hnRNP family proteins in complex with DOT1L; these interactions were confirmed by co-immunoprecipitation. Combined inhibition of DOT1L and selected splicing factors reduced cell proliferation more effectively than single-agent treatment, both in MLLr cell lines and in xenograft models. These results indicate that H3K79me2 contributes to alternative splicing regulation through splicing factor recruitment in MLLr AML and provide a rationale for coordinated epigenetic–splicing therapeutic strategies.