MLL-Associated Leukemias Drive Expression of MiR-9, Required for Tumorigenesis

Abstract 525

Acute leukemia represents one of the most deadly cancers in the United States. Clinical treatments in leukemia have progressed significantly through the use of therapies targeted specifically to chromosomal translocations. The success of these therapies has provided a model for future treatment in various cancers. However, there are various subtypes of leukemia where five-year survival and relapse rates have poor clinical outcome, indicating that new therapies are needed. A particular leukemia subtype, namely mixed lineage leukemia (MLL)-rearranged leukemia that is a result of chromosomal rearrangements leading to fusions between MLL and partner genes, is associated with a dismal outcome. Therapeutic targeting of MLL rearrangements has proven challenging as there have been dozens of described rearrangements. An emerging hallmark of cancer is the deregulation of non-coding RNAs called microRNAs (miRs). We hypothesized that MLL-associated leukemias have aberrant microRNA expression. We performed microRNA microarray analyses on leukemia patient samples and showed that microRNA-9 (miR-9) is highly upregulated in MLL-associated leukemias. We discovered that expression of miR-9 enhances cell transformation in vitro and tumorigenesis in vivo, and the opposite is true when miR-9 function is inhibited by anti-miR sponges. Interestingly, we observed that mice transplanted with both MLL-AF9 and miR-9 had a significantly higher amount of blast-like cells and immature cells in the bone marrow and fewer in the peripheral blood compared to mice transplanted with MLL-AF9 alone. Furthermore, inhibition of miR-9 function causes a significant defect in stem cell self-renewal and myeloid differentiation. Thus, this suggests that miR-9 has a critical role in stem cell potentiation and myeloid promotion in MLL-associated leukemias. To investigate target genes of miR-9, we correlated microarray expression of miR-9 and those of its putative target genes predicted with multiple prediction algorithms. We identified six potential target genes that exhibit a significantly inverse correlation of expression with miR-9 and are inhibited in MLL-associated leukemia. These identified genes are Transforming growth factor beta-induced protein (TGFBI), E-cadherin (CDH1), Nuclear factor-kappa-B p105 subunit (NFkB1), Ras homolog gene family member H (RHOH), Ring1 and YY1-binding protein (RYBP) and Foxhead Box O3 (FOXO3), We demonstrated that all these targets genes are inhibited upon expression of miR-9, in vitro and in vivo and blocking miR-9 function rescues their expression. As these target genes affect multiple cell processes such as proliferation, stem cell differentiation, chromatin remodeling, and cellular migration, it appears that miR-9 is a potential master regulator during MLL-associated tumorigenesis.