Targeting The Autophagy In Bone Marrow Stromal Cells Overcomes Resistance To Vorinostat In Chronic Lymphocytic Leukemia

OBJECTIVE:The aim of this work is to investigate the mechanism of stromal-mediated vorinostat(Vor)drug resistance,to validate the drug-resistance of Vor treatment in chronic lymphocytic leukemia(CLL)patients,and to develop an effective strategy for overcoming this stromal-mediated Vor resistance to improve the clinical treatment effect of CLL patients.METHODS:To address above issues,we used freshly isolated CLL cells from peripheral blood samples of CLL patients,and co-cultured them with bone marrow stromal cell lines to examine autophagy activity and metabolic changes in both CLL cells and stromal cells after vorinostat treatment.Flow cytometry was used to detect the drug sensitivity of CLL cells to Vor treatment,and the production of reactive oxygen species,and the autophagy and mitochondrial autophagy.Western blot was applied to monitor the experission levels of the autophagy and mitochondrial autophagy biomarkers,the changes of glycolysis related proteins and the gene knockdown efficiency of siRNA.The siRNA technology was used for the knockdown of the autophagy-related gene Atg5.RESULTS:The results demonstrated that CLL cells were under intrinsic oxidative stress which was further enhanced by vorinostat treatment,and released hydrogen peroxide outside the cells.The adjacent stromal cells took up hydrogen peroxide and drove autophagy,mitophagy and glycolysis,resulting in the local production of high-energy mitochondrial fuels,which were then taken up by CLL cells to be effectively utilized through mitochondrial oxidative phosphorylation to enable more ATP production.Notably,targeting autophagic stromal cells with autophagy inhibitor remarkably decreased stromal protection against vorinostat treatment in CLL cells.CONCLUSIONS:We demonstrated that vorinostat treatment indirectly promotes autophagy,mitophagy and glycolysis of bone marrow stroma in leukemia microenvironment,and thus promotes the development of drug resistance in CLL cells.This finding broadens our understanding of microenvironment-mediated drug resistance in CLL cells.In addition,we also found that the manipulation of autophagic stromal cells could serve as a novel promising strategy to circumvent stroma-mediated drug resistance in CLL cells,which suggests that directly targeting stroma and associated signaling pathways may be an effective means of leukemia therapy.