We recently identified the importance of CD9 on disease progression of acute lymphoblastic leukemia (ALL), but its role in acute myeloid leukemia (AML) remains unknown. This study aims to: (1) characterize CD9 expression in pediatric AML and its association with clinical outcomes; and (2) elucidate its regulations, functions and mechanisms.
Immunophenotypes of diagnostic BM were characterized by flow cytometry. Patients were stratified into CD9+ and CD9- subgroups for comparison of long-term survival. Epigenetic control of CD9 was investigated by bisulfite sequencing, and confirmed by decitabine treatment. Impact of CD9 on cell proliferation, division and chemosensitivity was measured by competition, colony and apoptosis assays. Influence of CD9 on leukemia progression was evaluated in xenograft models, coupled with single-cell transcriptomics to dissect the underlying mechanisms.
CD9 expression on blasts of AML patients was significantly lower than that on stem cells of BM donors (12.2% vs. 48.4%). Among 64 AML cases, blasts of 24 patients (37.5%) were CD9+. The 5-year survival rates of CD9- patients were significantly lower than those of CD9+ patients (OS: 52.3% vs. 75.9%, RFS: 35.0% vs. 63.3%). Methylated CpG sites on CD9 promoter were more abundant in AML compared with ALL cell lines (14.5% vs. 4.7%). Decitabine treatment increased CD9 expression by 4.7-65.2-fold. Overexpression of CD9 reduced proliferation, division and clonogenicity of MV4-11 cells, and enhanced cytarabine-induced apoptosis. NOD/SCID mice transplanted with CD9+ cells exhibited a drastic reduction of leukemic load by 70.7-91.8%, concomitant with an increased expression of MHC molecules on leukemic cells, and infiltration of T cells and NK cells into the BM.
CD9 was epigenetically silenced in pediatric AML and conferred a dismal survival. Reexpression of CD9 substantially suppressed leukemia progression via immune sensitization. Our data established CD9 as a novel tumor suppressor in pediatric AML, which endow a huge translational potential.