Abstract 1917: Suppression of miR-101a contributes to leukemogenesis by regulating epigenetic and pro-survival pathways in MLL AML stem cells

Abstract

Abstract The occurrence of relapse in acute myeloid leukemia (AML) is attributed to the persistence of leukemic stem cells (LSCs), which possess self-renewal and proliferative ability and contributes to the initiation and maintenance of leukemia (Nature 2001, 414:105-111). LSCs are also inherently drug-resistant (Oncotarget 2010, 1: 552-566). Recently, microRNAs (miRNAs), a class of small noncoding RNAs, have been implicated in regulation of cancer stem cells (CSCs). Several studies have shown that depletion of miRNAs leads to cancer and miRNA overexpression represents a promising strategy for cancer treatment (Nature Rev. 2010, 9: 775-789). Our study aims to develop an LSC-targeted therapeutic strategy by identifying miRNAs regulating gene and epigenetic pathways activated in Mixed-Lineage Leukemia (MLL)-mediated AML stem cells. We performed miRNA microarray analysis of MLL-AF9 mediated pre-leukemic stem cells (pre-LSCs) compared to Hoxa9/Meis1a-derived pre-LSCs and identified a novel tumor suppressor, miR-101a, that is downregulated in MLL-AF9 pre-LSCs (P<0.05, fold change>1.25). Quantitative real-time PCR confirmed the results and further analysis revealed that miR-101a is suppressed in LSCs compared to normal hematopoietic stem cells, suggesting a tumor suppressor role of miR-101a. Serial replating assay showed that overexpressing miR-101a in MLL-AF9 pre-LSCs impaired proliferation as we have observed 60-70% reduction in cell and colony number (p<0.01). Our preliminary in vivo survival studies also showed that primary transplantation of miR-101a overexpressed pre-LSCs in C57BL/6 mice causes a significant delay of MLL AML development. Annexin V and 7-AAD staining followed by flow cytometric analysis revealed a significant increase in apoptosis (p <0.001). To explain the marked reduction in colony forming ability and concomitant increase in apoptosis, we examined the expression of several apoptosis-associated proteins by Western blotting and found a decrease in expression of pro-survival proteins, Myeloid Cell Leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2). We also investigated whether miR-101a regulates epigenetic pathways as epigenetic events in cancer lead to aberrant expression of miRNAs. Strikingly, our result revealed a significant reduction in Enhancer of zeste homolog 2 (Ezh2), a crucial epigenetic regulator in maintenance of MLL AML and a known target of miR-101a. This finding is also accompanied by a global decrease of histone H3K27Me3 mark, implicating that miR-101a may contribute to leukemogenesis via regulating epigenetic pathways in LSCs. Our data show that miR-101a is supressed in MLL AML stem cells and its forced expression impairs LSC functions via reversing the epigenetic landscape and inhibiting pro-survival pathways, leading to apoptotic cell death. Overexpression of miR-101a may provide a means to eradicate drug-resistant LSCs. Citation Format: Estrella Gonzales-Aloy, Murray D. Norris, Jenny Y. Wang. Suppression of miR-101a contributes to leukemogenesis by regulating epigenetic and pro-survival pathways in MLL AML stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1917.