P736: BCOR MUTATION REGULATES MYELODYSPLASTIC SYNDROMES PROGRESSION THROUGH REPRESSING AUTOPHAGY FLUX MEDIATED BY HDAC6 ACTIVATION

Abstract

Background: Past studies have revealed that mutations in bcor are involved in various human cancers including MDS and AML. Autophagy plays a critical role in cancer progression. However, the role of bcor mutations in autophagy and MDS progression remains subject to debate. Aims: This study investigated the role of bcor mutations in autophagy and MDS progression. Methods: Cell lines with bcorp483L stable overexpression were established with a recombinant lentiviral vector. The effects of bcor mutations on cell proliferation, cell cycle progression, and cell apoptosis were detected by CCK8 assay, colony formation assay, and flow cytometry. We detected changes in autophagy levels by Western blot (WB), quantitative polymerase chain reaction (qPCR), and most importantly, transmission electron microscopy. The function of histone deacetylase 6 (HDAC6) was inhibited by Tubasatin A （TBA）, and We detected the consequent changes in the functional phenotype of the cells by WB and qPCR. The interactions between related proteins were analyzed by a co-immunoprecipitation assay. To test cell lines for mitochondrial dysfunction, bcorp483L mutant cells, negative control cells, and wild-type cells were double-stained with MitoTracker Green and MitoTracker Red, or single-stained with MitoSOX followed by fluorescence-activated cell sorting (FACS) analysis. The changes in pyroptosis level caused by bcorp483L mutation were detected by WB and qPCR. Results: The cell proliferative ability of bcorp483L mutant cells was repressed compared to the negative control cells. Besides, bcorp483L mutation could promote cell apoptosis and block the cell cycle progression to the G2 phase. Notably, bcorp483L mutation repressed autophagic flux in cultured cells, as corroborated by decreased expression of autophagy marker genes (e.g., ATG7, ATG10) and reduced numbers of autophagosomes or autolysosomes, which were accompanied by upregulation of HDAC6, downregulation of FoxO1 and ac-FOXO1. HDAC6 was required for inhibiting the deacetylation of FoxO1. Enhanced HDAC6 expression and repression of FOXO1 were required for bcorp483L -mutation-deduced autophagy. Depression of HDAC6 by TBA was sufficient for increasing the expression of FOXO1, ac-FOXO1, and inducing autophagic flux. Notably, we examined the direct interaction between HDAC6 and FOXO1, FOXO1 and ATG7. Bcorp483L mutated cells accumulated dysfunctional, non-respiring mitochondria and increased mitochondrial ROS production compared to negative control cells. And we found upward adjustment of pyroptosis level and DNA damage. Image:Summary/Conclusion:Bcor p.P483L mutation could disrupt BCOR protein structure and function, therefore reducing the transcriptional repressive effect of BCOR on HDAC6. HDAC6 was upregulated and decreases the acetylation of FOXO1, further repressing autophagy. We further verified the protein: protein interaction between HDAC6 and FOXO1, FOXO1 and ATG7. Defects in the clearance of damaged mitochondria by bcorp483L-mutation-regulated autophagy contribute to the accumulation of cellular reactive oxygen species（ROS）, thus promoting cell apoptosis and repressing cell proliferation. The escalation in pyroptosis levels was found after bcor mutations and may be partly due to increased ROS levels. We also found increased gene damage and increased likelihood of gene mutations in bcorp483L mutant cells, which would promote MDS transformation into AML. These findings indicate a completely novel BCOR-HDAC6-FOXO1-ATG7 axis in the regulation of autophagy and MDS progression, providing potential possibilities for MDS treatment.