Members of the microRNA-106a-363 Cluster Associate with Unfavorable Outcome in Adult Acute Myeloid Leukemia Patients and Promote Leukemogenesis invivo through Increased Metabolic Activity
Authors: Sperb Nadine , Krowiorz Kathrin , Grasedieck Sarah , Rueß Christoph , Pochert Nicole , Schneider Edith , Johansson Pegah , Malmberg Erik Delsing , Arabanian Laleh , Maetzig Tobias , Fogelstrand Linda , Döhner Hartmut , Palmqvist Lars , Rösler Reinhild , Wiese Sebastian , Calzia Enrico , Rouhi Arefeh , Kuchenbauer Florian 
Published: October 2019

Abstract
The miR-106a-363 cluster, encoding six miRNAs (miR-106a, miR-18b, miR-20b, miR-19b, miR-92a and miR-363), is a paralogue of the oncogenic miR-17-92a polycistron and its role in leukemia is at present largely unknown. We aimed to investigate the putative oncogenic role of the miR-106a-363 cluster in adult acute myeloid leukemia (AML) and to dissect the contributions of its individual members to disease formation and progression.
First, we analyzed the expression of each miRNA in AML patient samples as well as their clinical relevance. To determine the association of the miR-106a-363 cluster in AML with active disease, we quantified all six miRNAs individually in AML patient samples at initial diagnosis (n=33) and in AML patients in complete remission after chemotherapy (n=6). Hereof, miR-106a-5p, miR-19b-3p and miR-92a-3p levels were significantly lower in remission samples (p=0.0015, p=0.0013 and p=0.0004, respectively), confirming that these miRNAs are upregulated in AML. Stratifying AML patients within the LAML miRNA-Seq dataset of The Cancer Genome Atlas (TCGA) Research Network (n=187) (Ley et al., NEJM, 2013) according to their cytogenetic risk group demonstrated that all members of the cluster, except for miR-18b-5p, significantly associated with adverse cytogenetics. In addition, with the exception of miR-18b-5p, all members associated with an inferior overall survival (OS) in AML patients within the TCGA-LAML dataset, further supporting a pro-leukemogenic role for the cluster. Of note, miR-106a-5p was the most abundantly expressed unique miRNA of the polycistron, both in the TCGA patient cohort and in 11 myeloid leukemia cell lines quantified by quantitative real-time PCR (qRT-PCR).
Since the miR-106a-363 cluster is associated with high risk AML, we hypothesized that increased levels of the entire cluster as well as individual members would significantly shorten the survival time in a murine transplantation model mimicking aggressive AML. Therefore, we engineered transplantable, primary murine AML cell lines based on retroviral overexpression of Hoxa9 and Meis1 exhibiting a median disease latency of 39 days (n=14) after syngeneic transplantation in mice. Enforced lentiviral expression of miR-106a-363 (n=13, p<0.0001), miR-106a (n=15, p=0.0003), miR-18b (n=8, p<0.0001), miR-20b (n=13, p<0.0001) and miR-363 (n=13, p<0.0001) in Hoxa9/Meis1 cells significantly accelerated leukemogenesis compared to the control arm. The most pronounced anemia (p=0.03) and the most immature phenotype, based on a significantly higher proportion of c-kit+ (p=0.0147) and a concurrent lower percentage of Mac-1+ and Gr-1+ (p=0.0051) cells, were observed in mice transplanted with Hoxa9/Meis1/miR-106a cells.
Based on these results, we focused on the mechanism by which miR-106a contributed to the pathogenesis of AML and performed a proteomics screen comparing Hoxa9/Meis1/miR-106a and Hoxa9/Meis1/control cells. In particular, mitochondrial respiration processes, such as oxidative phosphorylation and electron transport chain components were induced by miR-106a as shown by Gene Set Enrichment Analysis. Preliminary results using high-resolution respirometry further indicated an increased number of mitochondria in Hoxa9/Meis1/miR-106a cells, supporting these findings.
In conclusion, we highlight the previously unrecognized oncogenic contribution of the miR-106a-363 polycistron in adult AML. Functional dissection of this cluster, in particular miR-106a, revealed a new therapeutic angle for high risk AML.

Disclosures
Döhner: Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Celator: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Celator: Consultancy, Honoraria; Pfizer: Research Funding; Sunesis: Consultancy, Honoraria, Research Funding; AROG Pharmaceuticals: Research Funding; Agios: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Seattle Genetics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.