microRNA-126 Orchestrates a Stem Cell-like Program in AML and B-ALL and Can be Exploited to Dissect Disease Heterogeneity

MicroRNA-126 reinforces hematopoietic stem cell (HSC) quiescence by dampening PI3K-AKT signaling. We recently reported key functions of miR-126 in acute leukemia: in human AML, it was required to maintain leukemic stem cell (LSC) quiescence (Lechman et al, Cancer Cell 2016), while its ectopic expression in mouse HSC induced leukemia (75% B-ALL, 25% AML) that fully regressed when switching a tetracycline-repressible miR-126 cassette off (Nucera et al, Cancer Cell 2016). RNA sequencing showed that miR-126 targeted cell cycle, apoptosis and p53 response genes, prevented differentiation and sustained oncogenic/pro-survival pathways typically associated with stem and progenitor cells (Kit, Wnt, Thy1, Jak/Stat, Bcl2). We quantified miR-126 expression levels in a cohort of 45 newly diagnosed AML patients (n=38 de novo, of which 37% favorable, 34% int-1, 16% int-2, 13% adverse according to the ELN classification) presenting at the San Raffaele Hospital between 2010 and 2015 using a robust digital droplet PCR assay. In addition to core-binding factor (CBF) mutated AML, we found that the group of AML with chromosomal aneuploidy showed significantly elevated miR-126 levels, suggesting that this subgroup is characterized by high LSC frequencies and/or a specific need to suppress p53 responses, a hypothesis supported by the data obtained in our mouse model. To measure miR-126 at single cell resolution, we stably transduced primary AML blasts (n=5 diseases) with a lentiviral miR-126 reporter vector, xenotransplanted them into NSG mice and quantified miR-126 activity by FACS in the engrafted cells recovered from the mice's bone marrow. Across different genetic subgroups including CBF leukemia, cells with the highest miR-126 activity were enriched in the CD34+ or CD34+CD38- fractions, consistent with LSC. To facilitate prospective LSC isolation based on miRNA activity, we screened a series of combination miRNA reporters incorporating response elements for LSC-enriched and LSC-depleted miRNAs and have now identified an optimized construct that highlights easily sortable, distinct subpopulations that are currently undergoing functional validation. Applying this tool to AML with complex and monosomal karyotype, we are addressing whether high miR-126 expression refers to elevated LSC frequency or is a specific feature of this clinically relevant AML entity. We next applied the miR-126 reporter to n=15 primary, human B-ALL measuring miR-126 activity in the xenograft. Surprisingly, we identified well-separated blast subpopulations that differed in miR-126 activity within single diseases. Heterogeneity for miR-126 appeared to be a general feature of B-ALL as we detected 2-3 subpopulations in most of the diseases studied. We purified miR-126(high) and miR-126(low) B-ALL subpopulations from the primografts and verified up to 1log differences in miR-126 expression, while we detected equal levels of the BCR-ABL fusion transcript in all subpopulations from Philadelphia+ B-ALL confirming their neoplastic nature. When transplanting miR-126(high) and miR-126(low) subpopulations into secondary or tertiary recipients, post-sorting miR-126 levels were tightly maintained indicating that miR-126 levels were static rather than dynamically regulated in distinct B-ALL subpopulations, as expected from a subclonal architecture. RNAseq performed on miR-126(high) and miR-126(low) human B-ALL fractions evidenced a miR-126 signature reminiscent of the one obtained in the B-ALL mouse model and cord blood CD34+ cells, uncovering physiological miR-126 activity in human primary B-ALL. Taken together, these data support a broad, pathogenetically important role for miR-126 in human AML and B-ALL that goes beyond LSC and open up opportunities to better understand leukemia disease biology, dissect intratumoral heterogeneity and therapeutically target resistant and refractory disease, considering that miR-126 knockdown expands normal HSC while depleting the leukemia. Disclosures Ciceri: MolMed SpA: Consultancy.