MiR-193a Is a Negative Regulator of Hematopoietic Stem Cells and Promotes Anti-Leukemic Effects in Acute Myeloid Leukemia
Authors: Krowiorz Kathrin , Bhayadia Raj , Jammal Razan , Pabst Caroline , Pochert Nicole , Rueß Christoph , Miller Christina , Döhner Konstanze , Maetzig Tobias , Döhner Hartmut , Bullinger Lars , Emmrich Stephan , Heckl Dirk , Martins Vera , Rösler Reinhild , Wiese Sebastian , Humphries R. Keith , Rouhi Arefeh , Klusmann Jan-Henning , Kuchenbauer Florian 
Published: October 2019

Abstract
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.