Abstract 1917: Suppression of miR-101a contributes to leukemogenesis by regulating epigenetic and pro-survival pathways in MLL AML stem cells
Authors: Estrella Gonzales-Aloy, Murray D. Norris, Jenny Y. Wang
Journal: Cancer Research (2016)

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&amp;lt;0.05, fold change&amp;gt;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&amp;lt;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 &amp;lt;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.