Epigenetic Silencing of CD9 Shapes a New Mechanism to Render Differentiation Block and Immune Evasion in Pediatric Acute Myeloid Leukemia
Authors: Xu Yaqun , Chan Kathy Yuen Yee , Fok Siu Ping , Wu Yixuan , Li Chi Kong , Leung Kam Tong 
Published: December 2023

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal expansion of myeloid progenitor cells with impaired terminal differentiation. Despite intensive treatment regimes, the clinical outcomes remain suboptimal. Emerging evidence suggest myeloblasts could evolve multiple machineries to evade immune patrol and hinder immunotherapies. Here, we present a new mechanism involving the tetraspanin protein CD9 in orchestrating differentiation arrest and immune escape in pediatric AML.
We first examined CD9 expression and its prognostic impact in patient cohorts of childhood leukemia. 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&lt;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 expression pattern of CD9 was highly associated with cytogenetic/genetic anomalies and FAB subtypes, for instance enrichment of CD9 in M4, M5 and M7 AML. The 5-year relapse-free survival rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018), with its prognostic significance exhibited distinctively in M4/M5 AML.
To elucidate the mechanism governing CD9 silencing, DNA and histone methylation status of CD9 were evaluated by bisulfite and ChIP sequencing for the classical histone marks H3K9/27me3, which indicated methylation may not contribute to CD9 repression. However, a marked decrease of H3K9/27Ac occupancy at the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P&lt;0.05), and strongly correlated with CD9 repression ( r=0.585-0.719, P&lt;0.01). Exposure of CD9- AML cell lines (n=8) or samples (n=9) to the histone deacetylase inhibitor panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P&lt;0.05), restored activating histone acetylation marks (4.1-41.6-fold, P&lt;0.05), and potently suppressed myeloblast proliferation ex vivo (median IC50: 21.4 nM). An epigenetic compound library screen confirmed histone hypoacetylation as the key mechanism driving CD9 silencing.
Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation ( P&lt;0.01) and colony formation ( P=0.002). NOD/SCID mice transplanted with CD9+ cells exhibited a drastically reduced leukemic load in hematopoietic organs by 70.7-91.8% ( P&lt;0.05), a significantly prolonged survival duration ( P&lt;0.001), and a marked regression of extramedullary myeloid sarcoma when compared with animals receiving CD9- cells. Global transcriptome profiling of pediatric AML (n=31) revealed decreased stemness (NES: -1.7, P=0.01) and increased monocyte (NES: 1.8, P=0.034) gene signatures in CD9+ samples. Concordantly, we observed a profound up-regulation of CD9 (9.4-51.1-fold, P&lt;0.01) preceding the appearance of lineage markers (CD14, CD36, CD86, CD115, CD300e, iCD68 and iTNFα) in PMA-mediated monocyte/macrophage differentiation but not ATRA-mediated neutrophil differentiation of myeloblasts. Importantly, the gain of CD9 in THP-1 cells and primary myeloblasts (n=6, P&lt;0.05) consistently augmented the expression of specific monocytic markers, corroborating its cell-intrinsic function in AML differentiation.
Single-cell transcriptomic analyses of bone marrow cells from MV4-11-transplanted mice detected a significant enrichment of differentially regulated genes functioning in antigen processing and presentation. Confirmatory overexpression of CD9 promoted basal and IFNγ-induced MHC-I/II expression ( P&lt;0.01) through the JAK2/STAT5 axis. Inter-patient comparisons (n=31) revealed a higher MHC-I expression in CD9+ AML ( P&lt;0.001). Interestingly, CD9 physically bound to MHC-I/II and formed an immune complex at the cell membrane as revealed by IP-MS, co-IP and confocal microscopy. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13, P&lt;0.05), concomitant with a robust bone marrow infiltration of cytotoxic T cells.
Taken together, our data provided molecular, cellular and clinical evidence showing the plausible function of 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.