Distinct MiR-126 Expression Describes a Non-Genetic but Functional Intra-Tumoral Heterogeneity in Primary Human B-ALL and Can be Prospectively Used to Identify Aggressive Subclones in NSG Mice

Intra-tumor heterogeneity is a hallmark of B acute lymphoblastic leukemia (B-ALL) favoring dissociated treatment responses, clonal evolution and disease progression. Heterogeneity at the genetic level has been widely described, but prospective isolation of functionally relevant, rare blast subpopulation within the B-ALL bulk remains challenging. We have found that all human adult B-ALL we tested (n=11) contained up to 4 subpopulations differing in the expression level of a stem cell microRNA, miR-126, which regulates quiescence of normal hematopoietic and AML leukemic stem cells (Lechman et al., Cancer Cell 2016). Transducing primary human B-ALL with a lentiviral miR-126 reporter, we can detect and purify within a single disease distinct B-ALL subsets differing in miR-126 expression. These miR-126_variants do not correspond to distinct genetic subclones, since they harbor, when purified, the same immunoglobulin/TCR rearrangements and show similar variant allele frequencies as the ones found by whole exome sequencing in the disease bulk. Surprisingly, when we xenotransplanted purified miR-126_variants, they stably maintained their associated miR-126 activity throughout tertiary transplantation, suggesting that they are not hierarchically-related but represent distinct, non-genetically-coded subclones. We next transplanted miR-126_variants at limiting dilution to measure their leukemia-initiating cell (LIC) frequency. LIC frequency was similar in miR-126(high) and miR-126(low) subpopulations. However, the miR-126(high) subsets from a given disease (n=2 BCR-ABL1-rearranged B-ALL) had more aggressive growth kinetics than their miR-126(low) counterparts and were enriched during chemotherapy with vincristine and dexamethasone. We then performed RNA sequencing of miR-126_variants comparing the miR-126(high) to the miR-126(low) subpopulation from the same primary B-ALL (n=3 BCR-ABL1-rearranged B-ALL cases). Differentially regulated gene ontologies in miR-126(high) versus (low) subpopulations common to all 3 diseases include PI3K/AKT signaling (a well-known, previously validated miR-126 target pathway), metabolism, cell cycle, TP53 and cell-cell interactions. We started to functionally validate some of these pathways, focusing on metabolic activity. We measured the rate of protein synthesis in miR-126(high) and miR-126(low) subpopulations by OPP incorporation and confirmed a higher rate of protein synthesis in the miR-126(high) subset. Intriguingly, the transcriptional profile of naturally-occurring miR-126_variants in human B-ALL resembled a miR-126 withdrawal signature in an experimental, miR-126-addicted B-ALL mouse model (Nucera et al., Cancer Cell 2016), pointing to a pathogenetic relevance of miR-126 also in the human disease. More in-depth studies on clinical samples, including diagnosis-relapse pairs, are ongoing. In conclusion, miR-126 can be used to prospectively dissect B-ALL heterogeneity, and understanding its molecular targets may open up new treatment approaches directed to specific functional cell states relevant to B-ALL biology.