Abstract
Background:Acute myeloid leukemia (AML) accounts for ∼5% of pediatric malignancies with inferior survival rates of 50–70% despite optimal application of risk‐directed, chemotherapy‐based treatment protocols. It is of prime importance to identify novel molecular targets that are amenable to therapeutic interventions, which relies critically on the dissection of key regulatory pathways governing disease progression and/or pathogenesis. CD9, a tetraspanin family protein, has been implicated in cancer progression but its expression, prognostic impact and functions in pediatric AML remain largely unknown.Aims:(1) To characterize the expression and epigenetic regulation of CD9 in a cohort of pediatric AML patients and its association with long‐term survival outcomes; and (2) to elucidate the multi‐faceted functions of CD9 in pediatric AML, focusing on the crucial cellular properties affecting leukemia progression.Methods:Bone marrow (BM) samples were collected from pediatric AML patients consecutively enrolled in the NOPHO‐AML 2004 and NOPHO‐DBH AML 2012 studies. Immunophenotypes of isolated leukemic blasts were characterized with CD9, CD33, CD34 and CD45 antibodies by flow cytometry. Patients were stratified into CD9+ and CD9‐ subgroups for comparison of overall survival (OS) and relapse‐free survival (RFS). Epigenetic control of CD9 in AML cell lines was investigated by bisulfite sequencing of CD9 promoter, and confirmed by decitabine and panobinostat treatment. The impact of CD9 on leukemic cell proliferation, cell division and chemosensitivity was measured by Trypan Blue exclusion, competition, colony formation, SNARF‐1 and Annexin V/7‐AAD assays, subsequent to transduction of a CD9‐expressing lentiviral vector. The influence of CD9 on leukemia progression was evaluated in the NOD/SCID mouse xenograft model.Results:The median CD9 expression on leukemic blasts of AML patients was significantly lower than that on CD34+hematopoietic stem cells of normal BM donors (11.6% vs.46.9%, P < 0.001). Higher abundance of methylated CpG sites in CD9 promoter was detected in AML compared with B‐ALL cell lines (7.3% vs. 1.7%). Treatment of CD9‐ AML cells with the DNA methyltransferase inhibitor decitabine or histone deacetylase inhibitor panobinostat increased CD9 expression by 4.9–64.1‐fold (P < 0.05). Of 58 cases of pediatric AML, blasts of 19 patients (32.8%) were CD9+ (≥20% CD9 expressing cells). Kaplan‐Meier analyses revealed that the 5‐year survival rates of CD9‐ patients were significantly lower than those of CD9+ patients (OS: 48.1% vs.88.2%, P = 0.004; RFS: 35.7% vs.66.7%, P = 0.009). Overexpression of CD9 in the AML cell line MV4–11 reduced proliferation, cell division and clonogenic potential, and enhanced cytarabine‐induced apoptosis (P < 0.05). NOD/SCID mice xenografted with GFP‐tagged, CD9‐overexpressing MV4–11 cells exhibited significantly reduced leukemic load in bone marrow, spleen, blood and liver by ≥71.4% (P < 0.01), and prolonged survival by 1.4‐fold (P = 0.002) when compared with animals transplanted with control, GFP‐only MV4–11 cells.Summary/Conclusion:CD9 was epigenetically silenced in pediatric AML and associated with dismal survival outcomes. Reexpression of CD9 substantially suppressed AML progression and enhanced sensitivity to conventional chemotherapy. Our study collectively established CD9 as a novel prognostic factor and tumor suppressor in pediatric AML, which could potentially be applied for refinement of clinical risk group stratification and derivation of new targeted therapies.
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