P486: CD9 MARKS DIFFERENTIATION AND ANTI-TUMOR IMMUNITY IN PEDIATRIC ACUTE MYELOID LEUKEMIA

Abstract

Background: Despite intensive treatment regimes, the clinical outcomes for children with acute myeloid leukemia (AML) remain suboptimal, underscoring the need to decipher the underlying pathology and translate into therapeutic modalities. Emerging evidence suggest myeloblasts could evolve multiple machineries to evade immune patrol and hinder immunotherapies. Aims: We recently reported the importance of CD9 in pediatric ALL but its role in AML remains unknown. In this disease context, we pursued to characterize its prognostic significance, elucidate its regulation and function, and identify its role in leukemia immunosurveillance. Methods: Patients were stratified based on CD9 status for comparison of long-term survival. Epigenetic control of CD9 was investigated by ChIP-sequencing and confirmed by HDACi treatment. Impact of CD9 on leukemia aggressiveness was measured by competition and colony formation assays. Influence of CD9 on leukemia progression was evaluated in xenograft models, coupled with global transcriptome profiling of AML samples. Immunomodulatory effect of CD9 was dissected by single-cell transcriptomics and validated by flow cytometry. Immune-related CD9 interactors were identified by IP-MS, followed by proof-of-function experiments in an immune-reconstituted mouse model. Results: The expression of CD9 on blasts of AML patients (12.2%, n=82) was significantly lower than those of ALL patients (90.4%, n=219, P<0.001) or stem cells from normal bone marrow donors (48.4%, n=22, P=0.014). Among AML cases, the blasts of 32 patients (39%) were CD9+. The 5-year relapse-free survival rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018). A marked decrease of H3K9/27Ac occupancy in the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P<0.05), and strongly correlated with CD9 repression (r=0.585-0.719, P<0.01). Exposure of CD9- AML cell lines (n=8) or samples (n=9) to panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P<0.05), and restored activating histone acetylation marks (4.1-41.6-fold, P<0.05). Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation (P<0.01) and colony formation (P=0.002). NOD/SCID mice receiving CD9+ AML exhibited a drastic reduction of leukemic load by 70.7-91.8%. Molecular expression analysis revealed decreased stemness (NES: -1.7, P=0.01) and increased monocyte (NES: 1.8, P=0.034) gene signatures in CD9+ patient samples. Concordantly, a profound up-regulation of CD9 (9.4-51.1-fold, P<0.01) was observed in PMA-mediated monocyte/macrophage differentiation but not in ATRA-mediated neutrophil differentiation of myeloblasts. Mechanistically, CD9 promoted basal and IFNγ-induced MHC-I/II expression (P<0.01) through the JAK2/STAT5 axis. Inter-patient comparisons of bone marrow samples (n=31) revealed a higher MHC-I expression in CD9+ AML (P<0.001). Interestingly, CD9 physically bound to MHC-I/II, formed an immune complex and regulated intracellular trafficking. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13), concomitant with a robust bone marrow infiltration of cytotoxic T cells. Summary/Conclusion: Our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining differentiation and immune recognition in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy.