Inhibition of microRNA 10a, an Autophagy Modulator, Increases Chemosensitivity to Ara-C in Acute Myeloid Leukemia

Aims: Leukemic relapse due to chemoresistance is a major obstacle in curing acute myeloid leukemia (AML), and the activation of pro-survival autophagy pathways has been implicated in this process. The microRNA-10a (miR-10a), a post-transcriptional regulator of several cell cycle and autophagy-related genes, is highly expressed in normal karyotype (NK) AML, especially those with nucleophosmin1 mutations ( NPM1^c+ ). This study aimed to investigate whether miR-10a modulation could affect autophagy and chemosensitivity in these cells.

Results: Lentiviral transduction of a miR-10a inhibitor into the miR-10a-high NPM1^c+ OCI-AML3 cell line significantly inhibited autophagy as signified by a marked decrease in autophagy marker LC3-II accumulation, compared to cells transduced with a scrambled control inhibitor (SCR). This was accompanied by significantly increased sensitivity to Ara-C (IC₅₀ = 8.2µM vs 20.0µM; p <0.0001) characterised by a 2-fold increase in apoptosis ( p = 0.035; quantified by Annexin V binding). Conversely, overexpression of miR-10a in the miR-10a-low, NPM1-wildtype MV4-11 AML cell line resulted in increased autophagy as demonstrated by a 3-fold increase in LC3-II ( p = 0.001) and conferred pro-survival effects, including reduced apoptosis ( p = 0.04) and markedly decreased Ara-C sensitivity (IC₅₀ = 8.7 µM vs 2.7µM; p = 0.03).

We previously demonstrated that miR-10a directly targets TFAP2C, a key inhibitor of CDKN1A-encoding cell cycle inhibitor p21. p21 is an important promoter of cell cycle arrest that precedes autophagy in response to stress signalling from the p53 signalling network. miR-10a inhibition was associated with a marked reduction in p21 expression, particularly in those cells in which p53 was activated either by treatment with MDM2 antagonist Nutlin-3a ( p = 0.02) or by serum deprivation ( p =0.005), and was associated with aberrant cell cycling in response to Ara-C treatment. Inhibition of CDKN1A specifically by siRNA showed similar effects to miR-10a inhibition, with increased cell death in response to Ara-C treatment ( p <0.05).

The observed increase in chemosensitivity in vitro was replicated in vivo in NSG mice engrafted with miR-10a inhibitor-transduced OCI-AML3 cells and treated with a course of Ara-C (50mg/kg for 3 weeks; n = 6 per treatment group). Treated mice had significantly improved survival (83% of miR-10a inhibited mice surviving at 30 days post-engraftment vs 50% of controls; hazard ratio = 6.6; p = 0.02) and had significantly fewer leukemic cells detectable in the bone marrow 24h after the end of treatment (26.7% of cells compared to 54.5% in animals engrafted with control inhibitor-transduced OCI-AML3 cells; p = 0.04).

Conclusion: To our knowledge, this study provides for the first time evidence that miR-10a confers chemoresistance to Ara-C in NK-AML, likely via its effects on the p21/p53-mediated activation of autophagy and may in part explain treatment failure observed in the clinic.