HDAC Inhibition Induces Microrna-182, Which Targets RAD51 and Impairs HR Repair to Sensitize Cells to Sapacitabine in Acute Myel
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Published OnlineFirst February 8, 2016; DOI: 10.1158/1078-0432.CCR-15-1063 Cancer Therapy: Preclinical Clinical Cancer Research HDAC Inhibition Induces MicroRNA-182, which Targets RAD51 and Impairs HR Repair to Sensitize Cells to Sapacitabine in Acute Myelogenous Leukemia Tsung-Huei Lai1, Brett Ewald2, Alma Zecevic2, Chaomei Liu2, Melanie Sulda2, Dimitrios Papaioannou1, Ramiro Garzon1, James S. Blachly1, William Plunkett2, and Deepa Sampath1 Abstract Purpose: The double-strand breaks elicited by sapacitabine, a Results: The gene repressors, HDAC1 and HDAC2, became clinically active nucleoside analogue prodrug, are repaired by recruited to the promoter of miR-182 to silence its expression in RAD51 and the homologous recombination repair (HR) path- AML. HDAC inhibition induced miR-182 in AML cell lines and way, which could potentially limit its toxicity. We investigated the primary AML blasts. miR-182 targeted RAD51 protein both in mechanism by which histone deacetylase (HDAC) inhibitors luciferase assays and in AML cells. Overexpression of miR-182, as targeted RAD51 and HR to sensitize acute myelogenous leukemia well as HDAC inhibition–mediated induction of miR-182 were (AML) cells to sapacitabine. linked to time- and dose-dependent decreases in the levels of Experimental Design: Chromatin immunoprecipitation RAD51, an inhibition of HR, increased levels of residual damage, identified the role of HDACs in silencing miR-182 in AML. and decreased survival after exposure to double-strand damage- Immunoblotting, gene expression, overexpression, or inhibi- inducing agents. tion of miR-182 and luciferase assays established that Conclusions: Our findings define the mechanism by which miR-182 directly targeted RAD51. HR reporter assays, apo- HDAC inhibition induces miR-182 to target RAD51 and high- ptotic assays, and colony-forming assays established that the lights a novel pharmacologic strategy that compromises the miR-182, as well as the HDAC inhibition–mediated de- ability of AML cells to conduct HR, thereby sensitizing AML cells creases in RAD51 inhibited HR repair and sensitized cells to to DNA-damaging agents that activate HR as a repair and potential sapacitabine. resistance mechanism. Clin Cancer Res; 22(14); 3537–49. Ó2016 AACR. Introduction intracellular phosphorylation, the resulting triphosphate is incorporated into replicating DNA. In contrast to other nucle- Sapacitabine is an orally available nucleoside analogue pro- oside analogues, this does not terminate replication. Rather, drug that is structurally related to cytarabine. It is active against after additional nascent DNA strand elongation, a b-elimination acute myelogenous leukemia (AML) both in elderly patients and 0 reaction occurs, which causes the analogue to rearrange to a 2 , in relapsed, refractory disease (1, 2). Sapacitabine is metabo- 0 3 -dideoxyconfiguration. This terminates the 3'-end resulting in lized into its active form 20-C-Cyano-20-deoxy-1-b-D-arabino- a single-stranded nick, which becomes converted to a double- pentofuranosylcytosine (CNDAC) by amidases. Following strand break (DSB) upon a subsequent round of DNA replica- tion (3, 4). The DNA DSBs generated by CNDAC (5) or ionizing radiation (6) are partly repaired by the homologous recombi- 1Division of Hematology, Comprehensive Cancer Center, The Ohio 2 nation repair (HR) pathway. HR is governed by multiple State University, Columbus, Ohio. Department of Experimental – – Therapeutics, The University of Texas M.D. Anderson Cancer Center, proteins such as ATM, the MRE11 RAD50 NBS1 complex Houston, Texas. (7), BRCA1/2 and RAD51, a key protein that directs the homol- Note: Supplementary data for this article are available at Clinical Cancer ogy search, and DNA strand exchange that is critical for suc- Research Online (http://clincancerres.aacrjournals.org/). cessful HR repair (8). High levels of RAD51 are linked to resistance to DNA-damaging therapies, relapsing disease, and Corresponding Authors: Deepa Sampath, Division of Hematology, Department of Internal Medicine, Ohio State University, Room 485, 410 W. 12th Ave, poor survival (9, 10). Conversely, cells lacking RAD51 or its Columbus, OH, 43210. Phone: 614-685-3260; Fax: 614-292-3312; E-mail: paralog RAD51D have an increased sensitivity to DNA-damag- [email protected]; and William Plunkett, Department of Experimental ing agents (11) including CNDAC (5). Unlike breast and ovar- Therapeutics, The University of Texas MD Anderson Cancer Center, 1515 ian tumors that often harbor intrinsic defects in HR, AML cells Holcombe Blvd, Houston, TX 77054. Phone: 713-792-3335; Fax: 713-794-4316; usually exhibit a functional HR pathway (12), which may allow E-mail: [email protected] them to limit the toxicity of DNA DSB-inducing agents. doi: 10.1158/1078-0432.CCR-15-1063 The histone deacetylases (HDACs) are a class of chromatin- Ó2016 American Association for Cancer Research. modulating proteins that become recruited to target promoters to www.aacrjournals.org 3537 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst February 8, 2016; DOI: 10.1158/1078-0432.CCR-15-1063 Lai et al. Cell lines Translational Relevance OCI-AML3 and MV4-11 AML cells were obtained from ATCC Sapacitabine is a clinically active nucleoside analogue pro- and maintained in RPMI supplemented with 5% FCS and 1% drug that elicits double-strand DNA breaks. RAD51 is a key glutamine in a 37 C incubator containing 5% CO2. OCI-AML3 protein in the homologous recombination repair (HR) path- cells were authenticated at the Characterized Cell Line Core way that repairs the DNA damage caused by sapacitabine to facility at The University of Texas MD Anderson Cancer Center potentially limit its toxicity. In this study, we identify that miR- (Houston, TX). MV4-11 and HeLa-DR-13 cell lines were authen- 182 targets RAD51 and that miR-182 is silenced by the histone ticated by sequencing at the Ohio State University (OSU; Colum- deacetylases (HDAC) in acute myelogenous leukemia (AML). bus, OH). The HeLa-DR-13 (HeLa-DR) and HEK cells were a gift HDAC inhibition induces miR-182 to cause reciprocal de- from Dr. Parvin (Ohio State University, Columbus, OH) and creases in RAD51 and inhibits HR to sensitize AML cells to maintained as described previously (32). Primary AML cells and sapacitabine. Our findings highlight a novel pharmacologic nucleic acids from normal bone marrow were collected under strategy that targets RAD51 to compromise the ability of AML an Institutional review board–approved protocol and obtained cells to conduct HR, and can be used to sensitize cells to DNA- from the Leukemia tissue bank at OSU. Primary AML blasts were damaging agents that activate HR repair as a potential resis- maintained in RPMI with 20% FCS and 1X Stem Span CC100 tance mechanism. (Stem Cell Technologies). Cytotoxicity assays OCI-AML3 and MV4-11 cells were exposed to 2 mmol/L CNDAC and increasing concentrations of panobinostat or SAHA compact chromatin and silence gene expression. Conversely, for 48 hours following which Annexin assays were conducted by inhibitors of HDACs (HDACi) function by promoting the acet- incubating cells with Annexin V–fluorescein isothiocyanate and ylation of proteins, including histones at gene promoters to propidium iodide (BD Biosciences) for 15 minutes and accumu- induce a transcriptionally active chromatin configuration and lating the fluorescence of at least 10,000 cells on a BD Biosciences reverse HDAC-mediated gene expression (13, 14). HDACis such FACSCalibur flow cytometer to determine the percentage of as vorinostat (SAHA) and belinostat are approved for the treat- apoptotic and viable cells. Drug interactions were analyzed using ment of cutaneous T-cell leukemia (15), whereas panobinostat the CalcuSyn (BioSoft). A combination index (CI) value of 1 is approved for the treatment of refractory multiple myeloma indicated an additive drug interaction, whereas a CI value >1 (16, 17). In AML, HDACi's showed synergy with CNDAC in suggested antagonism and a value <1 denoted synergism (33). xenograft models (18) and were effective in phase I/II trials when combined with demethylating (19) or other chemotherapeutic agents (20). Mechanistically, panobinostat exposure led to miRNA expression array decreases in the levels of ATM, BRCA1, and RAD51 to sensitize miRNA microarray analysis was carried out by LC sciences cells to cytarabine, daunorubicin, or ionizing radiation in solid (http://www.lcsciences.com/) as described in Supplementary tumor cell lines (21, 22). HDACi treatment also leads to the methods. depression an important class of regulatory genes, the miRNAs (23–25). miRNAs bind to complementary sequences in target Immunoblotting RNA to either destabilize it or prevent its transcription in a cell- Immunoblotting was performed with antibodies against and context-specific manner (26, 27). SpecificmiRNAsuch RAD51 (Millipore), H3K9Ac (Millipore), PARP (Cell Signaling as miR-182 are overexpressed in breast cancer and intrinsically Technology), cleaved caspase-3 (Cell Signaling Technology), compromise HR in those cells (28). Other miRNAs, such as g-H2AX and H2AX (Cell Signaling Technology), or GAPDH (Cell miR-103, miR-107, miR-96, and miR-182 target RAD51 and Signaling Technology). other components of the HR pathway in solid tumors (28–31); however, it is not known whether these miRNA target RAD51 in Chromatin immunoprecipitation and protein AML. As RAD51 is well expressed in AML, an investigation of