Abstract 5235: Identification of microRNA<i>-3662</i> as a novel tumor suppressor miR
Authors: Sophia Maharry, Sujay Mehta, Sandya Liyanarachchi, Guido Marcucci, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld
Journal: Cancer Research (2014)

Abstract Background: Despite all progress, the prognosis of many acute myeloid leukemia (AML) patients remains poor. Identification of candidate genes for targeted treatment strategies may be beneficial for patient survival. Using online prediction programs, we identified miR-3662 as a novel tumor suppressor candidate in AML. Its predicted targets include numerous genes involved in hematopoietic differentiation and leukemogenesis, such as the oncogene BAALC. So far, miR-3662 has not been implicated in cancer biology. Aims: First, we strived to elucidate the role of miR-3662 in hematopoietic differentiation and leukemogenesis. Second, we aimed to experimentally validate BAALC as a miR-3662 target to explore the downstream biology of the tumor suppressor candidate. Methods &amp; Results: As tumor suppressor genes are often downregulated in cancer, we determined the expression of miR-3662 in peripheral blood blasts from cytogenetically normal (CN-) AML patients (n=10) and peripheral blood samples from non-cancer controls (n=20). Indeed, the non-cancer controls had an average 97-fold (P≤.001) higher miR-3662 expression compared to CN-AML patients, suggesting a downregulation of miR-3662 in leukemic cells. Next, we assessed the effects of forced miR-3662 expression and knock-down of miR-3662 on the growth and colony formation of hematopoietic progenitor cells (CD34+) from three healthy donors. While miR-3662 increased cell growth and colony formation, antagomiR-3662 reduced both parameters (miR-3662 vs. scramble: P≤.001; antagomiR-3662 vs. scramble: P≤.001). In contrast, colony forming assays with leukemia cell lines (OCI-AML3, KG1a) after stable infection with miR-3662 versus scramble reduced the cell growth and colony formation (miR-3662 vs. scramble: both P≤.001). To assess the effect of miR-3662 on leukemia cell survival, we performed caspase-3/7 assays with the leukemic blasts of five CN-AML patients infected with miR-3662 or scramble. miR-3662 increased caspase-3/7 activity (miR-3662 vs. scramble, P=.004), indicating miR-3662-mediated increased apoptosis. Thus, miR-3662 has tumor suppressive potential. Next we tested the effects of miR-3662 on the expression of its predicted target gene BAALC by overexpressing miR-3662 in two AML cell lines (KG1a, MV4-11) and blasts of CN-AML patients (n=10). miR-3662 reduced BAALC expression in both AML cell lines (KG1a: 70% reduction, MV4-11: 75% reduction, both P≤.001) and in the CN-AML patients (average reduction: 38.6%, P=.007). Finally, we performed luciferase assays with a BAALC 3′-UTR construct. Co-transfection with miR-3662 led to a 38% reduction of luciferase activity (miR-3662 vs. scramble, P≤.001), indicating that miR-3662 directly targets BAALC. Conclusion: miR-3662 is a novel tumor suppressor miR that has potential implications for leukemogenesis, as it increased colony formation and differentiation in CD34+ cells and decreased growth of leukemic cells. Citation Format: Sophia Maharry, Sujay Mehta, Sandya Liyanarachchi, Guido Marcucci, Clara D. Bloomfield, Albert de la Chapelle, Ann-Kathrin Eisfeld. Identification of microRNA-3662 as a novel tumor suppressor miR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5235. doi:10.1158/1538-7445.AM2014-5235