HDAC Inhibition Induces microRNA-182 Which Targets Rad51 Protein and Impairs Homologous Recombination Repair to Sensitize Cells to the Double Strand Break Inducing Nucleoside Analog, Sapacitabine in AML

Abstract Acute myelogenous leukemia (AML) is characterized by multiple genetic and epigenetic abnormalities including a profound dysregulation of microRNA expression. Effective clinical treatment of AML has largely depended on a class of antimetabolites - the nucleoside analogs. Of these, sapacitabine is a nucleoside analog prodrug that is in development for the therapy of AML. It is converted to its active metabolite 2-C-cyano-2-deoxy-1-β(-D-arabino-pentafuranosyl) cytosine (CNDAC), which interferes with DNA synthesis by initially causing a single stranded DNA break that is converted into a double strand break in the subsequent replicative cycle. Such double strand breaks are primarily repaired by the homologous recombination repair (HR) pathway. Consequently, efficient HR may offer a potential resistance mechanism to therapy with sapacitabine. Rad51 is a protein plays a critical role in HR, and high levels of Rad51 are linked to resistance to DNA damaging therapies. Histone deacetylases (HDACs) are chromatin modulating agents that decrease levels of acetylation of histones, repress gene expression. HDAC inhibitors (HDACis) function by modifying chromatin to epigenetically reverse gene silencing of coding and non-coding genes such as the microRNAs (miRs). miRs are endogenous noncoding RNAs 19-25 nucleotides in length that bind to complimentary sequences in target RNA to either destabilize it or prevent its transcription. In this study, we determined that determined primary AML blasts and cell lines express low levels of microRNA-182. Recruitment of HDAC1 and its co-repressors were linked to the epigenetic silencing of miR-182 in AML. Conversely, HDAC inhibition led to accumulation of activating chromatin modifications followed by the upregulation of miR-182 in AML blasts and cell lines. The HDACi-induced increases in miR-182 were linked to decreases in the levels of Rad51, an inhibition in the ability of cells to conduct homologous recombination repair as measured by the Homologous recombination directed repair (HDR) assay, persistent levels of DNA damage as measured by the levels of Ɣ-H2AX and sensitization to sapacitabine. We then mechanistically defined the relation between miR-182 and Rad51. Ectopic expression of miR-182 in AML cell lines identified that Rad51 was a target of miR-182. An assay with luciferase constructs bearing full length or mutated Rad51 3'UTR indentified that Rad51 was a direct target of miR-182. We also determined that ectopic expression of miR-182 attenuated the ability of AML cells to conduct homologus repair as measured by the Homologous recombination directed repair (HDR) assay which resulted in sensitizing AML cells to the cytotoxic action of CNDAC as measured by colony forming assays. In conclusion, our data show that HDAC inhibitors target Rad51 via miR-182 to compromise HR repair to result in higher levels of residual DNA damage and sensitize AML cells to double strand damaging agents such as CNDAC. Disclosures No relevant conflicts of interest to declare.