Abstract
PurposeWe recently found that the tetraspanin family member, CD82, which is aberrantly expressed in chemotherapy-resistant CD34+/CD38− acute myelogenous leukemia (AML) cells, negatively regulates matrix metalloproteinase 9, and plays an important role in enabling CD34+/CD38− AML cells to adhere to the bone marrow microenvironment. This study explored novel functions of CD82 that contribute to AML progression.Materials and MethodsWe employed microarray analysis comparing the gene expression profiles between CD34+/CD38− AML cells transduced with CD82 shRNA and CD34+/CD38− AML cells transduced with control shRNA. Real-time RT-PCR and western blot analysis were performed to examine the effect of CD82 knockdown on the expression of the polycomb group member, enhancer of zeste homolog 2 (EZH2), in leukemia cells. A chromatin immunoprecipitation assay was performed to examine the effect of CD82 expression on the amount of EZH2 bound to the promoter regions of tumor suppressor genes in leukemia cells. We also utilized methylation-specific PCR to examine whether CD82 expression influences the methylation status of the tumor suppressor gene promoter regions in leukemia cells.ResultsMicroarray analysis revealed that levels of EZH2 decreased after shRNA-mediated depletion of CD82 in CD34+/CD38− AML cells. Moreover, the antibody-mediated blockade of CD82 in leukemia cells lowered EZH2 expression via activation of p38 MAPK signaling, decreased the amount of EZH2 bound to the promoter regions of the tumor suppressor genes, and inhibited histone H3 lysine 27 trimethylation in these promoter regions, resulting in upregulation of the tumor suppressors at both the mRNA and protein levels.
Highlights
Acute myelogenous leukemia (AML) originates from hematopoietic stem/progenitor cells and is maintained by a subset of leukemia stem cells (LSCs), which are assumed to be enriched for the CD34+/CD38− fraction [1,2,3]
A chromatin immunoprecipitation assay was performed to examine the effect of CD82 expression on the amount of enhancer of zeste homolog 2 (EZH2) bound to the promoter regions of tumor suppressor genes in leukemia cells
The antibody-mediated blockade of CD82 in leukemia cells lowered EZH2 expression via activation of p38 MAPK signaling, decreased the amount of EZH2 bound to the promoter regions of the tumor suppressor genes, and inhibited histone H3 lysine 27 trimethylation in these promoter regions, resulting in upregulation of the tumor suppressors at both the mRNA and protein levels
Summary
Acute myelogenous leukemia (AML) originates from hematopoietic stem/progenitor cells and is maintained by a subset of leukemia stem cells (LSCs), which are assumed to be enriched for the CD34+/CD38− fraction [1,2,3]. It is noteworthy that p38 is less phosphorylated in CD34+/CD38− AML cells than in normal hematopoietic stem cells (HSCs) and H2O2-induced senescent HSCs [9]. EZH2 mutations were found in 7% of follicular lymphomas and 22% of diffuse large cell B-cell lymphomas [13]. These mutations cause reduction of p16 expression and are recognized as gain of function mutations [14]. EZH2 mutations were identified in 10–13% of poor-prognosis myelodysplastic syndromes-myeloproliferative neoplasms, 6% of myelodysplastic syndromes, 6% of chronic myelomonocytic leukemia, and 1.7% of AML [13, 15,16,17]. EZH2 mutations in myeloid neoplasia were characterized by decreased H3K27me and increased chromatin relaxation at specific gene loci accompanied by higher transcriptional activity [18]
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