Anti-MiR-126 Therapy for Inv(16) Acute Myeloid Leukemia

Inv(16)(p13q22) or t(16;16)(p13.1;q22) [hereafter inv(16)], a chromosomal rearrangement occurred in 5-12% acute myeloid leukemia (AML) patients, disrupts the core-binding factor (CBF) transcription complex and creates a leukemogenic fusion gene CBFb-MYH11 (CM). Only about 50% inv(16) patients achieve long-term survival with standard chemotherapy. Therefore, more effective therapies are necessary to improve outcomes. MicroRNAs (miRNAs) are small non-coding RNA molecules that inhibit multiple target gene expression. High miR-126 expression is a miRNA hallmark of inv(16) AML. However, the function and mechanism of miR-126 dysregulation in inv(16) AML remains elusive. Our results indicate that miR-126 levels are significantly higher in inv(16) AML CD34⁺ and CD34^- blasts compared to normal counterparts. We have previously generated a conditional Cbfb-MYH11 (CM) knock-in (KI) mouse model (Cbfb^+/56M/Mx1-Cre), in which lethal AML develops in 4-6 months after CM induction. We observed an overtime-increased miR-126 in the peripheral blood relative to control (log fold change = 4.11; p< 0.0001) as well as in multiple progenitor subpopulations, including LSK, pre-megakaryocyte/erythrocyte (Pre-Meg/E), pre-granulocyte-macrophage (Pre-GM), and granulocyte-macrophage progenitors (GMP) in CM preleukemic and leukemic mice. Expression of CM in 32D cells significantly upregulated miR-126, pri-miR-126, pre-miR-26 and the host gene Egfl7 compared to vector control or WT CBFb, suggesting that transcriptional activation of miR-126 is induced by CM.

But, is the aberrant expression of miR-126 necessary for leukemia growth in CM AML? To address this question, we crossed the conditional CM KI mice with a miR-126-floxed model (miR-126^f/f). CM/miR-126^△/△ mice showed significantly reduced AML incidence (3 out of 10) and prolonged disease-free survival compared to CM mice (p < 0.0001), indicating that high miR-126 promotes AML growth. Deletion of miR-126 significantly reduced the expansion of preleukemic (6 weeks after induction) stem/progenitor populations (LSK, Pre-GM, and Pre-Meg/E). In addition, we observed significantly increased radiation-induced apoptosis in LSK (CM 11.61±2.277% vs. CM/miR-126^△/△16±1.386%, p=0.03) and Pre-Meg/E (CM 8.885±1.607% vs. CM/miR-126^△/△25.5±3.961%, p=0.0081), which represent leukemia-initiating populations in CM mice (Cai et al, Blood 2016). Furthermore, miR-126 knockdown in human inv(16) AML patient samples led to significantly increased apoptosis in all AML stem/progenitor cell subsets (shCtrl 7.469±1.085% vs. shmiR-126 13.8±1.585% in CD34⁺, p=0.0045; shCtrl 11.59±1.723% vs. shmiR-126 17.97±1.461% in CD34^-, p=0.0122) and reduced quiescence/increased cycling in more primitive subsets (e.g, CD34⁺CD38^-, CD34⁺CD38⁺). These results indicate that high miR-126 contributes to CM leukemia initiation and maintenance by antagonizing stress-induced apoptosis and maintaining the quiescence of stem/progenitor cells. Thus, miR-126 may be a novel therapeutic target in this subtype AML.

To target aberrantly expressed miR-126 in CM-AML, we designed a CpG-anti-miR-126 oligonucleotide (ODN) inhibitor (named miRisten, Bin Zhang et al, Nat Med 2018) that is efficiently (60-90%) taken up and achieved 50-80% reduction of miR-126 in AML LSK and blasts. Compared with a scramble ODN control (Ctrl), in vivo miRisten treatment (20 mg/kg i.v. daily for 3 weeks) significantly reduced CM-AML burden in spleen (Ctrl 71.23±3.756% vs. miRisten 51.91±5.788%, p=0.0103) and bone morrow (Ctrl 66.07±3.203% vs. miRisten 50.9±2.999%, p=0.0038), and reduced the frequency of LSK (Ctrl 1.72±0.6176% vs. miRisten 0.2316±0.03894%, p=0.0482). Importantly, miRisten treatment significantly reduced the leukemia-initiating capacity with prolonged survival in secondary transplants compared to Ctrl (median survival 95.5 days vs. 84.5 days; n=10, p=0.0004). When we combined miRisten with chemotherapy, adopting a regimen consisted of Cytarabine (Ara-C; 50 mg/kg i.p. daily for 5 days) and daunorubicin (DNR; 1.5 mg/kg i.v. every other day for 3 days), we observed a further reduction of AML burden and prolonged survival compared to chemotherapy alone (median survival 102 days vs. 84 days, n=10-11, p<0.0001). Therefore, miRisten is a novel targeted therapeutics that effectively targets miR-126 and mediates elimination of AML blasts and leukemia-initiating stem cells.