SUMO inhibition reveals temporally distinct intron retention programs shaping the immunopeptidome in acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) exhibits RNA-processing defects that can expose antigens encoded by non-canonical genomic regions. Here, we combine time-resolved RNA sequencing with discovery and targeted HLA-I immunopeptidomics to determine how SUMOylation controls intron retention and antigen presentation. In three AML cell lines, SUMO inhibition with TAK-981 induced an interferon-associated antigen-processing response and extensively remodeled the immunopeptidome. In MOLM-13 cells, 7,741 dynamically retained introns formed early- and late-rising programs distinguished by intron architecture, splice-site strength and RNA-binding-protein motifs. Among 16,965 HLA-I ligands detected over time, 438 arose from retained introns and 167 mapped to dynamically regulated events, revealing heterogeneous coupling between intron retention and peptide display. TAK-981 further exposed 453 treatment-induced antigens, nearly 60% of which were intron-derived. Targeted mass spectrometry identified 68 sequence-validated intronic ligands from 39 previously unannotated peptideins, with individual retained introns generating multiple, HLA-dependent ligands across AML models. Their source events recurred in untreated primary AML, with subsets enriched relative to healthy hematopoietic samples. These findings establish SUMO-dependent RNA processing as a determinant of non-canonical HLA-I presentation and identify recurrent intron-derived antigens for therapeutic evaluation.