The role of CYBB in maintenance of MLL-rearranged leukemia

Abstract

BACKGROUND: MLL (mixed lineage leukemia)-rearranged leukemia is caused by the chromosomal translocation of the MLL and partner genes. Previous studies demonstrated that the Cytochrome b beta chain (Cybb) gene is severely down-regulated in Mll-rearranged leukemia. Cybb encodes the catalytic subunit of NAPDH oxidase 2 (Nox2) complex and its expression is closely associated with the reactive oxygen species (ROS) level. ROS has been shown to influence stem cells maintenance and progenitors differentiation in hematopoietic system. The down-regulation of Cybb may suppress intracellular ROS level and therefore enhance stemness of Mll-rearranged leukemia. AIMS: To investigate the function of Cybb and Nox2-generated ROS in the maintenance of MLL-rearranged leukemic cells. METHODS: We have established a novel Mll-Een cell line from an Mll-Een knock-in mouse model. We compared the expression level of Cybb in the Mll-Een cells and wild-type bone marrow cells isolated from C57BL/6 mice by qPCR analysis. Intracellular ROS level was quantified by CellRox staining. Overexpression of Cybb in Mll-Een cells was done by lentiviral transduction. The ROS level and leukemic features of Cybb overexpressing Mll-Een cells were assessed by CellRox staining, colony forming assay and proliferation assay. RESULTS: We found that Cybb expression was severe down-regulated in the Mll-Een cell line when compared with the wild-type bone marrow cells. Besides, the intracellular ROS level of Mll-Een cells was also reduced. Overexpression of Cybb in Mll-Een leukemia cell increased the intracellular ROS level by over 30%. The colony forming number of Cybb overexpressing cells was sharping decreased by 89%, indicating that Cybb expression attenuates the colony forming potential of Mll-Een cells. Importantly, the colony morphology of Cybb overexpressing cells consists of mainly terminally differentiated cell colonies, which are 5 times higher in number than the control sample (57% vs 11%). These results suggested Cybb reduces self-renewal potential and promotes myeloid differentiation of Mll-Een leukemia. Furthermore, proliferation ability of Cybb overexpressing Mll-Een cells was greatly reduced by 87%, implying an impaired proliferative potential. SUMMARY: Overexpression of Cybb in Mll-Een cells increases ROS level and attenuates leukemic feature, such as the self-renewal potential and proliferative ability. Cybb also promotes myeloid differentiation in Mll-rearranged leukemia cells.