Supplemental Figure 1. HDAC inhibitors sensitize MV4-11 AML cells to CNDAC. Supplemental Figure 2. Action of CNDAC alone or in combination with 0.5 nM PS over time on Rad51 in MV4-11 cells. Supplemental Figure 3. Action of PS on Rad51 mRNA, Rad50 and Rad51 protein and lack of dependence on caspase-3 cleavage in the HDAC-mediated loss of Rad51 protein Supplemental Figure 4. HDAC inhibition does not decrease levels of the DNA repair proteins ATM or NBS1. Figure 5. HDAC inhibition does not increase levels of miR-182 in response to decitabine (DAC) alone. Figure 6. Recruitment of HDAC1 and HDAC2 at the miR-182 promoter in OCI-AML3 cells before and after exposure to 0.5 nM PS. Figure 7. OCI-.AML3 cells were left untreated, transfected with 100nM of scrambled oligonucleotides (Scr), miR-182 or anti-miR-182 for 48h (n=3) before a portion from each treatment was challenged with 0.5nM PS in combination with 2 μM CNDAC for 48h (n=2), following which the levels of cell death were assayed.
Funding
National Cancer Institute
Department of Health and Human Services
MD Anderson Cancer Center
OSU
ARTICLE ABSTRACT
Purpose: The double-strand breaks elicited by sapacitabine, a clinically active nucleoside analogue prodrug, are repaired by RAD51 and the homologous recombination repair (HR) pathway, which could potentially limit its toxicity. We investigated the mechanism by which histone deacetylase (HDAC) inhibitors targeted RAD51 and HR to sensitize acute myelogenous leukemia (AML) cells to sapacitabine.Experimental Design: Chromatin immunoprecipitation identified the role of HDACs in silencing miR-182 in AML. Immunoblotting, gene expression, overexpression, or inhibition of miR-182 and luciferase assays established that miR-182 directly targeted RAD51. HR reporter assays, apoptotic assays, and colony-forming assays established that the miR-182, as well as the HDAC inhibition–mediated decreases in RAD51 inhibited HR repair and sensitized cells to sapacitabine.Results: The gene repressors, HDAC1 and HDAC2, became recruited to the promoter of miR-182 to silence its expression in AML. HDAC inhibition induced miR-182 in AML cell lines and primary AML blasts. miR-182 targeted RAD51 protein both in luciferase assays and in AML cells. Overexpression of miR-182, as well as HDAC inhibition–mediated induction of miR-182 were linked to time- and dose-dependent decreases in the levels of RAD51, an inhibition of HR, increased levels of residual damage, and decreased survival after exposure to double-strand damage-inducing agents.Conclusions: Our findings define the mechanism by which HDAC inhibition induces miR-182 to target RAD51 and highlights a novel pharmacologic strategy that compromises the ability of AML cells to conduct HR, thereby sensitizing AML cells to DNA-damaging agents that activate HR as a repair and potential resistance mechanism. Clin Cancer Res; 22(14); 3537–49. ©2016 AACR.
