Genome-Wide Identification of a Chromatin-Splicing Regulatory Axis Driven by DOT1L in MLL-Rearranged Acute Myeloid Leukemia

Background/Objectives: Aberrant H3K79 dimethylation (H3K79me2) by DOT1L is a defining feature of MLL-rearranged (MLLr) acute myeloid leukemia (AML), but whether this modification influences alternative splicing is unclear. We examined the relationship between H3K79me2 and exon skipping in primary MLLr AML. Methods: 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, with matched profiling before and after DOT1L inhibition using EPZ5676. DOT1L co-immunoprecipitation with mass spectrometry (IP-MS) was performed in MV-4-11 and MOLM-14 cells, and an aggregate splicing score was evaluated in the TCGA-AML cohort. Results: A subset of exon skipping (SE) events was enriched at H3K79me2-occupied loci in MLLr samples. EPZ5676 remodeled SE patterns, and many switched events showed concurrent loss of local H3K79me2. Genes harboring these events were enriched for RNA processing, DNA repair, and apoptosis functions. Core spliceosomal and hnRNP proteins were prominent in the DOT1L interactome and were reduced after EPZ5676, and SRSF2 and hnRNPA1 binding motifs were enriched near the regulated exons. About two-thirds of the common switched events overlapped a local H3K79me2 peak, and an aggregate splicing score derived from these events stratified patients by overall survival in the TCGA-AML cohort, with higher scores associated with shorter survival. Conclusions: These data support a model in which H3K79me2 contributes to alternative splicing regulation in MLLr AML, linking the core epigenetic lesion of this disease to aberrant RNA processing with potential prognostic relevance.