Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs—such as LINC00173 in granulocytes—and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy.

MicroRNAs (miRNAs) play a pivotal role in the regulation of hematopoiesis and development of leukemia. Great interest emerged in modulating miRNA expression for therapeutic purposes. In order to identify miRNAs, which specifically suppress leukemic growth of acute myeloid leukemia (AML) with t(8;21), inv(16) or mixed lineage leukemia (MLL) rearrangement by inducing differentiation, we conducted a miRNA expression profiling in a cohort of 90 cytogenetically characterized, de novo pediatric AML cases. Four miRNAs, specifically downregulated in MLL-rearranged, t(8;21) or inv(16) AMLs, were characterized by their tumor-suppressive properties in cell lines representing those respective cytogenetic groups. Among those, forced expression of miR-9 reduced leukemic growth and induced monocytic differentiation of t(8;21) AML cell lines in vitro and in vivo. The tumor-suppressive functions of miR-9 were specifically restricted to AML cell lines and primary leukemic blasts with t(8;21). On the other hand, these functions were not evident in AML blasts from patients with MLL rearrangements. We showed that miR-9 exerts its effects through the cooperation with let-7 to repress the oncogenic LIN28B/HMGA2 axis. Thus, miR-9 is a tumor suppressor-miR which acts in a stringent cell context-dependent manner.

Purpose Dysregulated microRNAs are implicated in the pathogenesis and aggressiveness of acute myeloid leukemia (AML). We describe the effect of the hematopoietic stem-cell self-renewal regulating miR-193b on progression and prognosis of AML. Methods We profiled miR-193b-5p/3p expression in cytogenetically and clinically characterized de novo pediatric AML (n = 161) via quantitative real-time polymerase chain reaction and validated our findings in an independent cohort of 187 adult patients. We investigated the tumor suppressive function of miR-193b in human AML blasts, patient-derived xenografts, and miR-193b knockout mice in vitro and in vivo. Results miR-193b exerted important, endogenous, tumor-suppressive functions on the hematopoietic system. miR-193b-3p was downregulated in several cytogenetically defined subgroups of pediatric and adult AML, and low expression served as an independent indicator for poor prognosis in pediatric AML (risk ratio ± standard error, -0.56 ± 0.23; P = .016). miR-193b-3p expression improved the prognostic value of the European LeukemiaNet risk-group stratification or a 17-gene leukemic stemness score. In knockout mice, loss of miR-193b cooperated with Hoxa9/Meis1 during leukemogenesis, whereas restoring miR-193b expression impaired leukemic engraftment. Similarly, expression of miR-193b in AML blasts from patients diminished leukemic growth in vitro and in mouse xenografts. Mechanistically, miR-193b induced apoptosis and a G1/S-phase block in various human AML subgroups by targeting multiple factors of the KIT-RAS-RAF-MEK-ERK (MAPK) signaling cascade and the downstream cell cycle regulator CCND1. Conclusion The tumor-suppressive function is independent of patient age or genetics; therefore, restoring miR-193b would assure high antileukemic efficacy by blocking the entire MAPK signaling cascade while preventing the emergence of resistance mechanisms.

The microRNA (miRNA) family miR-193 consists of two members, miR-193a and miR-193b, which share identical seed regions and are therefore thought to be functionally redundant. However, the target spectrum of a miRNA is defined by the mRNA spectrum of the tissue, where it is expressed as well as dynamic strand preferences (miRNA-5p vs miRNA-3p) (Kuchenbauer et al., Blood, 2011). We and others previously showed that miR-193b is a tumor suppressor in acute myeloid leukemia (AML) (Bhayadia et al., JCO, 2018) and a potent regulator of hematopoietic stem cell expansion (Haetscher et al., Nat Com, 2015). However, the role of miR-193a in normal and malignant hematopoiesis is still unclear. First, we profiled the expression of miR-193a-5p and miR-193a-3p in human hematopoietic subpopulations from healthy donors via quantitative real-time PCR (qRT-PCR). Unlike miR-193b, which is enriched in hematopoietic stem cells (HSC) (Haetscher et al., Nat Com, 2015), miR-193a expression was restricted to differentiated cells of the myeloid lineage (granulocytes, monocytes, and erythrocytes). To investigate the function of miR-193a during normal hematopoiesis, we analyzed its effect on a murine myeloid progenitor cell line (32D cells) upon engineered overexpression of miR-193a in combination with granulocyte-stimulating growth factor (G-CSF) treatment (n=3-7). MiR-193a strongly promoted granulocytic differentiation of 32D cells already after two days compared to the control arm (p=0.006) as assessed by flow cytometry and morphological analysis. To analyze the effect of miR-193a on HSC function, we ectopically expressed miR-193a in highly purified E-SLAM (CD45+EPCR+CD48-CD150+) cells and transplanted them into lethally irradiated recipient mice (n=5 mice/arm). HSCs overexpressing miR-193a failed to reconstitute hematopoiesis (p=0.038). The negative effect on stem cell properties could be translated to human CD34+ cord blood (CB) cells, where miR-193a overexpression significantly reduced colony counts. Taken together, these results suggest, that miR-193a has pro-differentiation and anti-stemness functions. To assess the role of miR-193a in AML, we profiled miR-193a-5p and miR-193a-3p in two cohorts of de novo pediatric (n=187) and de novo adult AML patients (n=40) by qRT-PCR. We found that both miRNA strands (miR-193a-5p and miR-193a-3p) are present at similar levels, which is in contrast to the almost undetectable levels of miR-193b-5p strand, hinting at additional functional roles for miR-193a through its 5p arm. MiR-193a was significantly downregulated in adult and pediatric AML compared to healthy donor samples, suggesting a possible tumor suppressor function. To investigate the role of miR-193a in AML, we engineered transplantable, primary murine AML cell lines based on retroviral overexpression of Hoxa9/Meis1 (aggressive, short latency in vivo) with low endogenous miR-193a levels. Restoration of miR-193a by lentiviral overexpression delayed Hoxa9/Meis1 mediated leukemogenesis in vivo (p=0.01, n=8-10). Furthermore, miR-193a overexpression reduced leukemic growth of human AML cell lines (n=6) and decreased colony-forming capacity of primary AML patient samples (n=4; p=0.029) in vitro. To identify novel targets of miR-193a, we performed a proteomics screen in human NOMO1 AML cells overexpressing miR-193a compared to an empty control vector (n=5). We identified and verified stathmin (STMN1), a tubulin-associated, intracellular phosphoprotein previously linked to proliferation of AML cells, as a novel putative miR-193a target, further explaining its tumor suppressor effect. Taken together, we characterized miR-193a as a positive regulator of myeloid differentiation and negative modulator of HSCs. Based on the balanced presence of both miRNA arms, we hypothesize that each arm is functionally active and has different functions such as pro-differentiation and anti-stemness. Furthermore, this work is the first characterization of a miRNA family that exerts cooperative effects at both early and late hematopoietic differentiation stages, highlighting a novel mechanism of balancing anti-stemness and pro-differentiation. Disclosures Döhner: Bristol Myers Squibb: Research Funding; AROG Pharmaceuticals: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Pfizer: Research Funding; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Bullinger:Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Bayer Oncology: Research Funding; Sanofi: Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Speakers Bureau.