MiR-126 Promotes Leukemogenesis in Inv(16) Acute Myeloid Leukemia

MicroRNAs (miRNAs) are short non-coding RNA molecules that inhibit the expression of multiple target proteins. MiRNA expression profiles can predict prognosis and are associated with distinct cytogentic abnormalities in acute myeloid leukemia (AML). Core-binding factor (CBF) AML, including inv(16)(p13q22)/t(16;16)(p13.1;q22) and t(8;21) chromosomal rearrangements, express significantly higher levels of miRNA (miR)-126 than non-CBF AML. We and others have shown that miR-126 is highly expressed in hematopoietic stem cells and leukemic stem cell (LSC)-enriched subpopulations and functions to promote quiescence. We previously reported that miR-126 expression is associated with an AML LSC-gene expression signature and independently predicts poor prognosis in cytogenetically normal AML. However, the role of miR-126 and the mechanism of dysregulation in inv(16) AML remains unknown.

Inv(16) chromosomal rearrangement generates a leukemogenic fusion oncogene CBFB - MYH11 (CM). We utilized a conditional Cbfb-MYH11 knock-in (Cbfb^56M/+ / Mx1-Cre) mouse that can be induced to express CM from the endogenous Cbfb promoter, thus mimicking human inv(16) AML. Time sequential miRNA-seq analysis revealed that miR-126-3p and miR-126-5p expression was significantly up-regulated (log fold change =2.09 and 2.36 respectively; p< 0.0001) early (1 month) and increased progressively throughout leukemia progression (log fold change = 4.11 and 4.45; p< 0.0001). Increased expression of miR-126 was observed in multiple preleukemic progenitor subpopulations, including LSK (Lin-Sca1+ckit+) and pre-megakaryocyte/erythrocyte (Pre-Meg/E; Lin^-ckit⁺Sca1^-CD16/32^-/loCD105^-CD150⁺) in CM mice.

To determine the functional contribution of miR-126 to inv(16) leukemogenesis, we crossed our CM knock-in mice with a conditional miR-126 knockout. Preliminary survival results indicate that CM/miR-126^△/+ (heterozygote) exhibit significantly prolonged survival compared to CM mice, with a median survival of 266 vs. 159 days, respectively (p<0.05). Survival monitoring is ongoing for a cohort of CM/miR-126^△/△(homozygote) mice. To date, this cohort is already demonstrating significantly enhanced survival compared to CM mice (p<0.05).

We hypothesize that high miR-126 expression might contribute to survival and quiescence of CM AML LSK, which is enriched for leukemia-initiating cells. Thus, we performed lentiviral-mediated miR-126 knockdown in CM AML LSK cells and observed increased apoptosis and reduced quiescence in vitro . To assess the role of miR-126 in leukemia progression in vivo, we designed a novel CpG-anti-miR-126 inhibitor. This compound comprises an inhibitory anti-miR-126 RNA component that is linked to a Class A CpG motif for targeted cell delivery. We showed that the CpG-anti-miR-126 is efficiently taken up (60-90%) by AML blasts and LSK cells and can achieve approximately 50-80% knock-down of miR-126 in vitro and in vivo. Mice transplanted with CMAML and treated with CpG-anti-miR-126 (5 mg/kg, 5 times/week for 5 weeks) had a significant delay in leukemia progression and enhanced survival (median survival 92 days vs 35 days; n=9-10, p=0.0339), supporting the notion that high miR-126 contributes to leukemia growth. We further evaluated the effects of combining CpG-anti-miR-126 treatment with chemotherapy, adopting a regimen consisted of Cytarabine (Ara-C; i.p. 100 mg/kg for 5 days) and daunorubicin (DNR; i.v. 3 mg/kg for 3 days). We observed significantly improved survival in mice treated with CpG-anti-miR-126 combined with Ara-C and DNR compared to CpG-scrambled combined with Ara-C and DNR (median survival 183 days vs. 64 days; n=9-10; p=0.0305). These studies demonstrate a key role for miR-126 in promoting leukemogenesis of inv(16) AML. Studies to elucidate the mechanism underlying miR-126 dysregulation and function in inv(16) AML are ongoing.