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Background Oncogenes can alter HLA levels to drive immune escape. However, qualitative changes to the HLA-ligandome remain incompletely understood. We investigated oncogene-mediated editing of the HLA-ligandome during resistance to donor lymphocyte infusion (DLI), a potentially curative immunotherapy for relapse following stem cell transplant.Methods We performed whole exome sequencing and single cell (sc)RNA-seq profiling on relapsed, chronic myeloid leukemia patient samples before/after DLI from the Dana-Farber Cancer Institute and MD Anderson Cancer Center. MuTect2 was used to identify somatic alterations. Harmony was used to batch correct sc-data. K562 and U-937 isogenic cell lines with/without ASXL1 mutations ( ASXL1m) were generated using CRISPR Cas9 mediated gene editing. CD8+ tumor cell line specific T-cells were generated by in vitro priming. Immunopeptidomics was performed and peptides were analyzed using NetMHCpan and MHCflurry.Results We identified ASXL1m in 78%/ 0% of non-responders (NRs)/responders (Rs) (p<0.0001), nominating ASXL1m as a driver of DLI-Resistance. ScRNA-seq revealed enrichment in a leukemic-stem-cell cluster bearing suppressed HLA-I and antigen processing/presentation (APP) genes in NRs and confirmed in additional ASXL1m myeloid malignancies, suggesting ASXL1m may mediate immune escape through HLA-I and APP suppression (p=0.036; p=0.049). To directly test this hypothesis, we CRISPR edited ASXL1m in K562 and U-937 cell lines. Flow cytometry revealed increased HLA-I (p<0.0001) and CD8-killing (p<0.0001) in cell-lines without ASXL1m. Immunopeptidomics demonstrated decreased peptide diversity with ASXL1m, even after treatment with IFNγ (p<0.01). Using isogenic K562 HLA-A*02:01 cell lines to control for HLA-A*02:01 expression, we performed HLA-A*02:01-specific immunopeptidomics (clone BB7.2) and confirmed reduced peptide diversity in ASXL1m cell lines (p=0.026). Peptide binding motif analysis demonstrated altered amino acid usage at key anchor residues (positions 2,9) within the epitope and in the surrounding contexts that shape peptide cleavage (p=0.01; p<0.0001). Computational predictions of peptide binding affinity and HLA-presentation ranks were reduced by ASXL1m (p<0.0001). For mechanistic insight into ASXL1m’s effect on antigen processing, we confirmed altered protein levels of immunoproteasome components, PSMB8, 9, and 10 (p<0.0001).Conclusions In summary, we integrated molecular analyses with immuno-functional studies to define a novel oncogenetic pathway of immune evasion through both quantitative (HLA-I) and qualitative (HLA-ligandome) changes in myeloid cancers. To our knowledge, these findings represent the first report that oncogenes may directly shape antigen processing to alter the HLA-ligandome and likely impact the use of cancer-derived data sets for the rapidly expanding field of antigen discovery.Ethics Approval Written informed consent was obtained from all participating patients in accordance with the ethical standards of the Dana Farber Cancer Institute or MD Anderson Cancer Center institutional review boards.