miRNAs Function As Biomarker in Pediatric AML and Regulate Gene-Silencing in AML-Relevant Pathways

Abstract 570

microRNAs (miRNAs) are small (21-24 nt), non-coding and highly conserved molecules, which are involved in several important regulatory processes like cell growth, proliferation, differentiation, immune response and apoptosis. Thus, their involvement in the pathogenesis of several diseases, including acute myeloid leukemia (AML) is not surprising. Several studies address the miRNA expression changes in adulthood AML, however, comprehensive studies in AML of children and adolescents are missing so far. We investigated the miRNA expression profiles of different AML subtypes from pediatric patients, in order to identify differentially expressed miRNAs. Subsequently, appropriate cell line models were used for global biochemical identification of miRNA targeting structures.

miRNA expression profiles of 102 pediatric AML patient samples were identified using microarray technology, and analyzed by unsupervised hierarchical cluster analysis and statistical testing. AML subtypes with translocations t(8;21) and t(15;17) can be separated from each other, solely based on their miRNA expression profile, while other translocations involving mixed-lineage leukemia (MLL) rearrangements are interspersed and lack a characteristic miRNA signature. Only six and seven miRNAs are differentially expressed between AML samples with translocations t(8;21) and t(15;17), respectively, and all other AML subtypes. This is surprising, since patients of different AML subtypes, investigated in this study, differ greatly in their clinical presentation. Differentially expressed miRNAs contain lineage specific miRNAs (miR-223), oncogenic miRNAs (miR-21) and more ubiquitously expressed miRNAs (let-7b/c, miR-100, −125b and −181a/b) with no designated characteristics. Furthermore, these differentially expressed miRNAs were not described as abundant in adult AML patients.

To gain further insights into the function of differentially expressed miRNAs, we established a modified PAR-CLIP method termed PAR-CLIP-Array (Photo-activatable-Ribonucleoside-Enhanced Crosslinking-Immunoprecipitation and Microarray Hybridization) for global identification of Ago-associated miRNAs and their mRNA-targets. On average 25% of mRNAs in AML cell lines bearing the AML1/ETO or PML/RARα translocation were identified in Argonaute complexes and carry at least one miRNA binding site and thus are under miRNA control. 60% and 27% of miRNAs and mRNAs, respectively, overlap between the four analyzed Argonaute proteins, while 50% and 52% (46 miRNAs and 241 mRNAs) were associated with one Argonaute protein specifically. However, pathway classification of Ago-associated target-mRNAs indicate more than 90% overlap between the Argonaute proteins and thus are indicative of a concerted action of these four proteins in 150 pathways identified. Moreover, miR-181a/b, up-regulated in t(15;17)-positive AML patients, were detected in association with the four human Argonaute proteins in NB4 cells and show binding sites for the protein kinase PDPK1 potentially leading to inhibition of AKT, whereas eight other Ago-associated miRNA sequence families (seqgrp-miR-98, −130a, −19a, −25, −27a, −301a, −361 and −320) in association with Ago3 are able to repress the upstream tumor suppressor TSC1 leading to activation of the mTOR pathway and increased cell growth. In addition, the repression of the MAP kinase phosphatase DUSP6 by four Ago-associated miRNA sequence families (seqgrp-miR-29a, −17, −125a and −98) leads to activation of proliferative genes in the MAPK pathway of both, t(8;21)- and t(15;17)-positive AML.

In summary, miRNAs represent suitable biomarkers for differentiation of AML subtypes of pediatric AML patients. Furthermore, our studies show that the four human Argonaute proteins cooperate in the regulation of AML-relevant signaling pathways providing new insights into AML biology and may present the starting point for novel therapeutic interventions.