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MicroRNAs are essential regulators of normal and malignant hematopoiesis. We previously described the function of miR-126 in HSC where it regulates the balance between quiescence and self-renewal (Lechman et al, 2012). 

We aimed at studying the role of miR-126 in induction and maintenance of high-grade B cell malignancies in mouse and humans.

We exploited the following techniques: Transduction of murine bone marrow (BM) cells and human primary ALL blasts with lentiviral vectors (LV); LV integration site (IS) analysis by linear amplification mediated PCR and mapping on the murine genome; RNA sequencing; Immunofluorescence staining

We here report a novel role for miR-126 in the induction and maintenance of high-grade B cell malignancies. By ectopically expressing miR-126 in transplanted BM cells, we observed that up to 60% of mice (n=71) developed B cell tumors. LV IS analysis revealed that all tumors were monoclonal. We then tracked back the leukemic clone to different hematopoietic lineages prospectively purified from the mice 2-6 months before disease onset. IS sharing between normal lineages and the leukemic clone suggests a stem or multipotent progenitor cell origin for most tumors. Importantly, we show that miR-126 is the direct cause for the induction and maintenance of leukemia, since (i) leukemogenesis is abolished when miRNA expression is inhibited by doxycycline (doxy) using a tetracycline-repressible miR-126 cassette, and (ii) established symptomatic leukemia completely regresses when miR-126 is switched off by doxy. We then performed RNA sequencing on blasts purified ex vivo before and 24 to 72h after doxy administration. Gene sets that changed were significantly enriched in migratory functions, cancer pathways and cell cycle regulators. Strikingly, there was a near-complete overlap with pathways that resulted significantly regulated by miR-126 in a second RNAseq dataset obtained from human cord blood (huCB) CD34+ cells following lentiviral miR-126 overexpression or knockdown. Many genes converged on the p53 pathway in both murine B-ALL and huCB CD34+ cells. Indeed, overexpression of miR-126 in huCB CD34+ cells physically displaced p53 from the nucleus and caused a significant reduction of its transcriptional activity. We identified a dozen of genes involved in p53 regulation that were directly targeted by miR-126 or miR-126*, suggesting extensive cooperative regulation of this central tumor suppressor pathway by miR-126. We further studied the function of miR-126 in Philadelphia+ human primary B acute lymphoblastic leukemia (Ph+ B-ALL) where miR-126 expression ranges from low to high in individual patients. We stably knocked down miR-126 in primary blasts from five Ph+ B-ALL patients using a miR-126 sponge LV. Following xenotransplantation, we observed increased apoptosis and impaired engraftment after primary and secondary transplantation in 4 out of 5 diseases (miR-126/KD: n=32 mice; Ctrl: n=37 mice), demonstrating the relevance of miR-126 in human B-ALL.