Haploinsufficiency of Mir-146a in High-Risk Del(5q) MDS/AML Requires an Intrachromosomal Gene Network Involving p62/TRAF6/NF-κB

Abstract 557

Deletion of chromosome 5q (del(5q)) is one of the most common cytogenetic abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Commonly deleted regions (CDR) have been mapped to 5q31.1 and 5q33.1 in del(5q) AML and MDS, respectively. Although there has been extensive efforts to identify candidate genes within the CDRs and decipher which genes contribute to the high-risk versus low-risk phenotypes, more recent findings indicate that deletions involving the telomeric bands of 5q are associated with more aggressive forms of del(5q) MDS/AML (Jerez et al., JCO, 2012). To determine which genes on the telomeric extremes of chr 5q contribute to the aggressive phenotype associated with these patients, we performed a gene expression and functional analysis of candidate tumor suppressor (TS) genes located between ∼5q34 to 5q35.3. Among the 7 candidate TS genes localized on the telomeric extreme, expression of miR-146a is significantly reduced in del(5q) MDS/AML marrow cells with extended deletions as compared to ones with shorter deletions. In addition, low expression of miR-146a correlates with increased marrow blasts and shorter overall survival of patients with del(5q). To investigate the effects of miR-146a on hematopoietic stem/progenitor cell (HSPC) function, we stably knocked down miR-146a in human CD34+ cord blood and mouse HSPC-enriched cells. HSPC cells with reduced miR-146a exhibited features of malignant transformation, including increased survival, proliferation, and colony replating in methylcellulose. The mechanism underlying reduced miR-146a expression was investigated by performing gene expression profiling on marrow cells from MDS/AML patients with extended and short del(5q). Gene Ontology and Gene Set Enrichment Analysis revealed that patients with low expression of miR-146a exhibited a significant increase in cell cycle, immune response, and NF-kB target genes. These findings are consistent with the model that reduced levels of miR-146a results in derepression of TRAF6, a signaling protein within the innate immune pathway and activator of NF-kB (Starczynowski et al., Nature Medicine 2010). TRAF6 forms a signaling complex with Sequestosome 1 (SQSTM1/p62), a gene located within the telomeric deleted region in del(5q) on band 5q34. Surprisingly, expression of p62 was not reduced, but rather overexpressed in del(5q) MDS/AML patients with the extended deletions and inversely correlated with miR-146a expression. The compensatory expression of p62 is due in part by loss of miR-146a. RNAi-mediated knockdown revealed that p62 and TRAF6 are essential for NF-kB activation and survival of human MDS/AML cell lines with low miR-146a expression. In summary, we identified that miR-146a is a TS-like gene in MDS/AML patients with extended deletions of chr 5q, and that reduced miR-146a expression increases HSPC survival/proliferation and NF-kB signaling via p62 and TRAF6. Therefore, we propose that inhibiting the p62/TRAF6/NFκB intrachromosomal gene network represents a novel targeting strategy in high-risk MDS/AML patients with extended chr 5q deletions.