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MicroRNA (miRNA)-126 is a known regulator of hematopoietic stem cell quiescence. We engineered murine hematopoiesis to express miRNA-126 across all differentiation stages. Thirty percent of mice developed monoclonal B cell leukemia, which was prevented or regressed when a tetracycline-repressible miRNA-126 cassette was switched off. Regression was accompanied by upregulation of cell-cycle regulators and B cell differentiation genes, and downregulation of oncogenic signaling pathways. Expression of dominant-negative p53 delayed blast clearance upon miRNA-126 switch-off, highlighting the relevance of p53 inhibition in miRNA-126 addiction. Forced miRNA-126 expression in mouse and human progenitors reduced p53 transcriptional activity through regulation of multiple p53-related targets. miRNA-126 is highly expressed in a subset of human B-ALL, and antagonizing miRNA-126 in ALL xenograft models triggered apoptosis and reduced disease burden.

We have previously identified microRNA-126 as a central modulator of B-ALL biology (Nucera et al, Cancer Cell). Ectopic miR-126 expression in murine stem and progenitor cells is sufficient and necessary to induce and maintain a B-ALL with a Philadelphia(Ph)-like gene expression profile. Importantly, miR-126 is expressed in the majority of human Ph+ B-ALL. Interestingly, there is marked intra-tumoral heterogeneity with regards to miR-126 levels, defining a new layer of subclonal architecture different from genetic subclones. By prospectively isolating miR-126(high) and miR-126(low) subpopulations exploiting a lentiviral miRNA reporter vector, we consistently found a more aggressive behavior of the miR-126(high) subset in xenograft models. To detect miR-126-dependent pathways that may explain this more aggressive behavior, we engineered 2 primary human B-ALLs with lentiviral vector cassettes containing (1) a reverse tetracycline transactivator (rTTA) and (2) a Tet-operon driving overexpression of miR-126 (miR-126-OE), a miR-126-3p seed mutant (miR-126-OE-SM) or a miR-126 sponge (miR-126-KD) performing serial passages in mice following enrichment for transduced cells. Vector cassettes were induced in vivo by doxycycline administration to mice that have developed full blown leukemia, and blasts were collected within 72hr. In patient 1, acute miR-126-OE induced apoptosis, while it was well tolerated in the disease from patient 2 that we hence studied in more detail. RNA sequencing highlighted more than 2,000 deregulated genes between miR-126-OE and miR-126-OE-SM or miR-126-KD blasts (qValue 0.2). Genes down-regulated upon miR-126-OE included transcription factors associated with B cell differentiation (PAX5, IKZ1, TCF3, BCL6), cell cycle regulators (CCND2, RBL1, E2F7, EP300) and ribosomal genes. Moreover, pathway analysis revealed significant de-regulation in JAK-STAT signaling, cell proliferation, adhesion/migration, BCR signaling, FOXO signaling and cell metabolism. Strikingly, there was a strong overlap (221 genes) with a miR-126 switch-off signature obtained from the Ph-like B-ALL mouse model. These shared genes were enriched in the categories "cell proliferation", "cancer-related signaling", "FOXO signaling" and "chromatin assembly during cell cycle", pointing to a miR-126-dependent core circuit shared between human Ph+ B-ALL and the mouse model. We started to validate the transcriptomic data at the protein level measuring phosphorylation status and subcellular localization of signaling hubs. Primary B-ALL from patient 2 showed increased phospho-STAT5 shortly after miR-126-OE, which returned to baseline within 6 days, suggesting adaptive responses counteracting a potentially deleterious excess of signaling in this disease. We hypothesize that a surge in signaling strength upon acute induction of miR-126-OE may have contributed to the apoptotic phenotype observed in patient 1. In keeping with this, constitutive miR-126-OE induced apoptosis in an additional 5 Ph+ B-ALL and, consequently, reduced engraftment in NSG mice, similar to knockdown of miR-126. Our results further consolidate an important role of miR-126 in human B-ALL, whereby a tight regulation of this miRNA appears critical for leukemia maintenance. Disclosures No relevant conflicts of interest to declare.

To investigate miRNA function in human acute myeloid leukemia (AML) stem cells (LSC), we generated a prognostic LSC-associated miRNA signature derived from functionally validated subpopulations of AML samples. For one signature miRNA, miR-126, high bioactivity aggregated all in vivo patient sample LSC activity into a single sorted population, tightly coupling miR-126 expression to LSC function. Through functional studies, miR-126 was found to restrain cell cycle progression, prevent differentiation, and increase self-renewal of primary LSC in vivo. Compared with prior results showing miR-126 regulation of normal hematopoietic stem cell (HSC) cycling, these functional stem effects are opposite between LSC and HSC. Combined transcriptome and proteome analysis demonstrates that miR-126 targets the PI3K/AKT/MTOR signaling pathway, preserving LSC quiescence and promoting chemotherapy resistance. Lechman et al. show that miR-126 targets the PI3K/AKT/MTOR signaling pathway to preserve quiescence, increase self-renewal, and promote chemotherapy resistance of acute myeloid leukemia stem cells (LSC). Reducing the miR-126 level impairs LSC maintenance in contrast to expanding normal hematopoietic stem cells.

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.

During organ development and remodeling, macrophages support angiogenesis, not only by secreting proangiogenic growth factors and matrix-remodeling proteases, but also by physically interacting with the sprouting vasculature to assist the formation of complex vascular networks. Recent data further indicate that embryonic and tumor-associated macrophages express similar genetic programs, possibly suggesting convergent functions in organogenesis and tumorigenesis. In this article, we review the role of macrophages in development, tissue injury and regeneration, by focusing on the mechanisms used by subsets of these cells, such as the TIE2-expressing macrophages, to regulate angiogenesis and lymphangiogenesis in both fetal and post-natal life.

We have recently shown that miR-126 expression faithfully identifies the engrafting fraction of bone marrow (BM) and cord blood (Gentner et al, Sci Transl Med 2010), in which it regulates hematopoietic stem cell (HSC) pool size by modulating cell cycle progression (Lechman et al., Cell Stem Cell 2012). Antagonizing miR-126 expands HSC by enhancing PI3K/AKT signaling, without causing their exhaustion or transformation. By reconstituting mice with lentivirally transduced BM cells ectopically expressing miR-126, we noted differential effects in HSC and progenitors: while miR-126 overexpressing (126OE) HSC were more quiescent and outcompeted by HSC with physiologic miR-126 expression, progenitor subsets were increased in number and proliferated more upon 126OE. In particular, B lymphopoiesis was enhanced, with the appearance of 126OE-vector marked, CD45(low)CD19+Ig(-) B cell progenitors in the peripheral blood. This abnormal population was transiently observed in all 126OE (n=45), but in none of the control mice (n=30), and BM B cell precursors and progenitors from 126OE mice showed significantly reduced levels of apoptosis, as well as a differentiation block at the immature B cell stage. At least part of this effect was due to interference of miR-126 with p53 activation. Consequentially, 20-30% of 126OE mice (as compared to 0% of control mice) followed over 1 year developed vector-marked, high-grade B-cell neoplasms which resembled diffuse large B-cell lymphoma, lymphoblastic lymphoma or acute lymphoblastic leukemia (ALL). Strikingly, by using a tetracycline-regulated, conditional miR-126OE vector, we observed full regression of the expanded abnormal B cell population after switching miR-126 expression off by doxycycline administration, demonstrating that miR-126 is an oncogenic driver in hematologic malignancies. To establish the relevance of miR-126 in human disease, we studied primary samples from patients with acute leukemia. We measured miR-126 expression in blasts from 12 adult patients with acute lymphoblastic leukemia (ALL). miR-126 was highly expressed in all studied cases of ALL (Phil+: n=7, Phil-: n=5), often surpassing the levels found in normal HSC. Unlike in HSC, miR-126 expression was independent from expression of its host gene EGFL7, suggesting an ALL-specific regulation of the miR-126 locus. By exploiting a lentiviral miR-126 reporter vector, we quantified miR-126 activity with single cell resolution after xenotransplantation of miR-126 reporter vector transduced ALL blasts into NSG mice. In 7 out of 12 ALL cases, we identified distinct subpopulations exhibiting different levels of miR-126 activity, and miR-126 (hi) ALL cells were more frequently contained in the CD34+ cell fraction. Moreover, high miR-126 expression correlated with faster and higher ALL engraftment in NSG mice. In acute myeloid leukemia (AML), miR-126 is significantly enriched in leukemic stem cells (LSC) where it governs quiescence and chemotherapy resistance (see abstract by Lechman et al), and analogous studies in ALL are ongoing. To further substantiate the role of miR-126 in AML, we studied miR-126 activity in 11 primary AML cases (4 AML with CBF, core binding factor, mutations; 7 AML with normal karyotype) using the reporter vector technology and the NSG xenotransplantation model. Globally, we found highest miR-126 activity in CBF-mutated AML. However, there was substantial heterogeneity in subpopulations of each single disease. Significantly higher miR-126 levels were found in CD34+, and particularly in CD34+CD38- AML cells, a fraction that is enriched for LSC. To see whether there is a correlation between miR-126 expression and disease progression, we measured miR-126 levels in sorted blasts from paired diagnosis/relapse (n=6) and diagnosis/chemotherapy-refractory (n=8) patient samples. Strikingly, we found a significant up-regulation of miR-126 in the disease progression sample, suggesting a mechanistic role for miR-126 in AML persistence after chemotherapy. In summary, these data support a clinically relevant role of miR-126 in the pathogenesis, treatment response and progession of acute leukemias. Authors AG and SN contributed equally, and LN and BG contributed equally. Disclosures: No relevant conflicts of interest to declare.