Overexpression and Knockout of Mir-126 Both Promote Leukemogenesis through Targeting Distinct Gene Signaling
Authors: Su Rui , Li Zejuan , Chen Ping , Li Yuanyuan , Chao Hu , Wang Yungui , Arnovitz Stephen , He Miao , Gurbuxani Sandeep , Zuo Zhixiang , Elkahloun Abdel G. , Li Shenglai , Weng Hengyou , Huang Hao , Neilly Mary Beth , Wang Shusheng , Larson Richard A. , Zhang Jiwang , Jiang Xi , Wei Minjie , Jin Jie , Liu Paul P. , Chen Jianjun 
Published: October 2019

Abstract
MicroRNAs (miRNAs) have been implicated in the pathogenesis of various types of cancers. We reported previously that miR-126 likely functioned as an oncogene in acute myeloid leukemia (AML) (Li Z, et al. PNAS. 2008 Oct 7; 105:15535-40), which was confirmed by others (de Leeuw DC, et al. Cancer Research. 2014 Apr 1,74:2094-105; Dorrance AM, et al. Leukemia. 2015). However, it was also reported that miR-126 knockdown in normal hematopoietic stem/progenitor cells (HSPCs) resulted in expansion of long-term HSCs (Lechman ER, et al. Cell Stem Cell. 2012 Dec 7,11:799-811). Thus, the role of miR-126 in normal and malignant hematopoiesis warrants further investigation.
Here we show that miR-126 is particularly overexpressed in t(8;21) AML and increased expression of miR-126 is associated with poor prognosis in patients with t(8;21) AML. To determine the function of miR-126 in leukemogenesis, we conducted both gain- and loss-of-function in vivo studies of miR-126 in t(8;21) AML models via mouse bone marrow transplantation (BMT). Surprisingly, we found that both overexpression and knockout of miR-126 accelerated leukemogenesis by the t(8;21) fusion genes, AML1-ETO (AE) or AML1-ETO9a (AE9a; a potent oncogenic isoform of AE). For example, forced expression of miR-126 (by retroviral transduction?) significantly accelerated AE9a-mediated leukemogenesis (median survival of AE9a+miR-126 vs. AE9a: 234 days vs. 317 days, p <0.01), and AE9a -transduced miR-126-/- HSPCs (miR-126KO+AE9a) caused leukemia significantly faster than did AE9a -transduced wild-type HSPCs (AE9a) in recipients (median survival: 130 days vs. 317 days, p <0.0001).
To assess the impact of miR-126 overexpression and depletion on long-term self-renewal of t(8;21) leukemia stem/initiating cells (LSCs/LICs), we performed serial mouse BMT assays with leukemic BM cells collected from the prior generation of BMT recipients as donor cells. In the secondary mouse BMT assay, we found that the AE9a+miR-126 group showed similar leukemia development to the miR-126KO+AE9a group (median survival: 79 days vs. 81 days, p =0.22), and both groups had significantly shorter survival (p <0.01) than did the AE9a group (median survival: 116 days). Strikingly, the AE9a +miR-126 group developed leukemia significantly faster than did the miR-126KO+AE9a group in both tertiary and quaternary BMT (median survival: 28 days vs. 43 days, p =0.0001 in tertiary; 35 days vs. 49 days, p <0.0001 in quaternary), and both groups developed leukemia significantly faster (p <0.0001) than did the AE9a group. Our limiting dilution assays with mouse BM leukemic cells collected from secondary BMT recipients as donor cells showed that the estimated LSCs/LICs frequency of the AE9a+miR-126 group is significantly greater (p <0.001) than that of the miR-126KO+AE9a group (1/2,476 vs. 1/27,399; >10 fold), and both were significantly higher (p <0.01) than that (1/166,619) of the AE9a group. Furthermore, consistent with the association of increased expression of miR-126 with poor survival of t(8;21) AML patients, our in vivo and in vitro studies demonstrated that depletion or inhibition of miR-126 significantly sensitized t(8;21)-fusion-induced mouse leukemia or primary human leukemia cells to standard chemotherapy.
Our mechanistic studies indicate that miR-126 overexpression activates genes that are highly expressed in LSCs/LICs and/or primitive HSPCs through directly targeting ERRFI1 and SPRED 1, which in turn activate the MAPK signaling pathway. On the other hand, miR-126 knockout activates genes that are highly expressed in committed, more differentiated hematopoietic cells and triggers the WNT/β-catenin signaling pathway by inducing FZD7 expression. We show that ERRFI1, SPRED 1 and FZD7 are bona fide targets of miR-126, and their increased expression is associated favorable survival of t(8;21) AML patients. Overall, our data show that both gain- and loss-of-function of a single miRNA (e.g., miR-126) can promote tumorigenesis and enhance long-term self-renewal/progression of LSCs/LICs, through targeting distinct gene signaling and thus being associated with different biological consequences. As miR-126 depletion can sensitize AML cells to standard chemotherapy, our data also suggest that miR-126 represents a promising therapeutic target.

Disclosures
No relevant conflicts of interest to declare.