Histone Hypoacetylation-Driven CD9 Repression Arrests Differentiation and Evades Immunosurveillance in Pediatric Acute Myeloid Leukemia
Authors: Xu Yaqun , Chan Kathy , Fok Siu Ping , Man Toni Ki Fong , Li Chi Kong , Leung Kam Tong 
Published: November 2021

Abstract
Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy characterized by uncontrolled proliferation of myeloid progenitor cells accompanied by impaired terminal differentiation. Despite intensive treatment regimes, the clinical outcomes remain poor, underscoring the need to decipher the underlying pathology and implement therapeutic interventions. Emerging evidence suggest myeloblasts could evolve machineries to evade T cell patrol and hinder immunotherapies. Here, we present a new mechanism driving immune escape in the context of pediatric AML, based on our discoveries of CD9 in hematopoietic stem cells (HSC; Leung et al, Blood, 2011) and acute lymphoblastic leukemia (ALL; Leung et al, Leukemia, 2020).
We first examined CD9 expression and its prognostic impact in patient cohorts of childhood leukemia. The expression of cell surface CD9 on blasts of pediatric AML patients (13.2%, n=81) was significantly lower than that of pediatric ALL patients (90.4%, n=181, P&lt;0.001) or that on CD34+ HSC of normal bone marrow donors (48.4%, n=22, P=0.014). Among pediatric AML cases, the blasts of 32 patients (39.5%) were CD9+. The 5-year RFS rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018).
Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation (P&lt;0.01), Ki-67 expression (P=0.041) and colony formation (P=0.002). NOD/SCID mice transplanted with CD9+ cells exhibited a drastic reduction of leukemic load in the bone marrow, spleen, blood and liver 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 transplanted with CD9- MV4-11 cells.
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&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) and samples (n=9) with a 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 suppressed myeloblast proliferation ex vivo (median IC50: 21.4 nM).
Mechanistically, 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+ patient samples. Moreover, single-cell transcriptomic analyses of total bone marrow cells from MV4-11-tranplanted mice detected a significant enrichment of differentially regulated genes functioning in the antigen processing and presentation pathway. Concordantly, we observed a profound up-regulation of CD9 (9.4-51.1-fold, P&lt;0.001) in PMA-mediated monocyte/macrophage-like AML differentiation cultures preceding the appearance of lineage markers CD14, CD36 and iCD68 as well as the antigen presentation molecule MHC-I. In the overexpression system, CD9 not only elevated the expression of monocytic markers, but also promoted basal and IFNγ-induced MHC-I/II expression (P&lt;0.01) through the JAK/STAT axis. Inter-patient comparisons of bone marrow samples (n=27) revealed a higher MHC-I expression in CD9+ than CD9- AML (MFI: 61820 vs. 18601, P&lt;0.001). Interestingly, tetraspanin CD9 physically bound to MHC-I/II and formed an immune complex as revealed by co-immunoprecipitation. 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. Syngeneic transplantation in immunocompetent mice, AML/T cell co-cultures, antigen-specific assays and panobinostat priming/T cell adoptive transfer are currently underway to fully dissect the role of CD9 in leukemia immunity.

Taken together, our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining differentiation and immunosurveillance in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy.

Disclosures
No relevant conflicts of interest to declare.