Abstract 263: Histone hypoacetylation of CD9 mediates differentiation arrest and immune evasion in pediatric acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by impaired terminal differentiation. Despite intensive treatment regimes, the clinical outcomes remain suboptimal. Here, we report a new mechanism involving the tetraspanin protein CD9 in orchestrating differentiation arrest and immune escape in pediatric AML. The expression of CD9 on blasts of AML patients (12.5%, n=115) was significantly lower than those of ALL patients (93.6%, n=343, P<0.0001) or hematopoietic stem cells from normal bone marrow donors (48.4%, n=22, P=0.004). Among AML cases, 43 patients (37.4%) were CD9+, with 5-year event-free survival rate significantly higher than CD9- patients (58.1% vs. 38.3%, P=0.040). A marked decrease of H3K9/27Ac occupancy in the CD9 locus was observed in AML than in ALL cells, which strongly correlated with CD9 repression (r=0.585-0.719, P<0.01). Exposure of CD9- AML to the HDAC inhibitor panobinostat significantly elevated CD9 mRNA and protein level, restored histone acetylation marks, and suppressed myeloblast proliferation ex vivo (median IC50: 21.4 nM). Compound library screen with a collection of 896 epigenetic agents confirmed focal hypoacetylation as the key driver of CD9 silencing. Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation and colony formation. NOD/SCID mice transplanted with CD9+ cells exhibited reduced leukemic load, prolonged survival duration (P<0.001), and a marked regression of extramedullary sarcoma. RNA-seq of 114 pediatric AML revealed decreased stemness and increased monocyte gene signatures in CD9+ samples. Concordantly, we observed a profound up-regulation of CD9 with the appearance of lineage markers (CD14, CD36, CD86, CD115, CD300e) in monocytic-supportive ex vivo culture. The gain of CD9 in primary myeloblasts (n=13) consistently augmented monocytic markers. Transcriptional network analysis revealed CD9+ AML exhibited enhanced antigen processing and presentation. Confirmatory overexpression of CD9 promoted basal and IFNγ-induced MHC-I/II expression through JAK2/STAT5 axis. Interestingly, CD9 physically bound to MHC-I/II and formed an immune complex on the membrane. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML, concomitant with a robust BM infiltration of GZMB+ cytotoxic T cells as revealed by single-cell transcriptomics and CyTOF deep T cell profiling. Importantly, panobinostat priming rejuvenates immune sensitization via CD9 transcriptional activation in humanized mice, resulting in robust leukemia clearance. Taken together, our data provided molecular, cellular and clinical evidence showing CD9 as a key driver intertwining monocytic differentiation and immune recognition in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy. Citation Format: Histone hypoacetylation of CD9 mediates differentiation arrest and immune evasion in pediatric acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 263.