The RAS/RAF/MEK/ERK pathway is frequently hyperactivated in several tumors. In leukemia, this activation can arise, among other mechanisms, from point mutations in the RAS genes, which are important in acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML), or from chromosomal translocations such as the BCR-ABL gene, which is a driver mutation in chronic myeloid leukemia (CML) and some cases of ALL. The hyperactivation of this pathway stimulates cell proliferation and, consequently, the production of reactive oxygen species (ROS), which is one of the main mechanisms involved with induction of cellular senescence in tumors. Thus, tumor cells that harbor the mutated RAS gene are critically dependent on feedback mechanisms to regulate pathway activation. Jang et al. demonstrated that the ubiquitinligase HUWE1 acts on a negative feedback mechanism that controls the activation of ERK1/2. Although widely studied in the context of tumorigenesis, the role of this molecule in events related to leukemogenesis has not yet been described. In this study, leukemia cell lines and human hematopoietic stem and progenitors cells (HSPCs) with KRASG12V mutation were transduced with miR-E lentiviral particles for HUWE1 knockdown. Cell proliferation, apoptosis, cell cycle analysis, ROS production and analysis of gene and protein expression were performed in cell lines; cumulative growth analysis, cobblestones area formations, clonogenic capacity and differentiation profile analysis were performed in HSPCs. In cell lines, it was observed that HUWE1 knockdown reduced the proliferative capacity of Nalm-6, K562 and THP-1, but not of HL-60. Besides that, it caused a reduction in ROS production (p<0,05), associated with reduction of apoptosis rates (p<0,01), especially in K562 in which it also promoted activation of ERK1/2. In HSPCs, a reduction of the proliferative capacity was observed in cultures expressing KRASG12V in combination with HUWE1 knockdown. In the same conditions, a drastic reduction of clonogenic capacity (p<0,001), especially of BFU-E colonies, was observed. HUWE1 knockdown also changed differentiation profile to the monocytic lineage. Results suggest that HUWE1 might play a role in leukemogenesis process and differentiation of human HSPCs, acting in the modulation of RAS/RAF/MEK/ERK. (AU) |
