MiR-34b Promoter Methylation and Regulation of CREB Expression In Myeloid Transformation

Abstract 538

The cAMP response element binding protein (CREB) is a nuclear transcription factor downstream of various stimuli and is critical for the pathogenesis of leukemia. CREB overexpression promotes abnormal proliferation, cell cycle progression, and clonogenic potential in vitro and in vivo. We found that CREB deregulation in Acute Myeloid Leukemia (AML) is due to both genomic amplification and aberrant miRNA expression. CREB has been shown to be a direct target of the microRNA, miR-34b. The inverse correlation between CREB and miR-34b expression has been described in myeloid leukemic cell lines. Mir-34b restoration reduced CREB levels and leukemia proliferation in vitro. One reason for the lower expression of miR-34b in myeloid leukemia cell lines is the hypermethylation of its promoter. Our goal was to characterize the role of miR-34b in AML progression using primary cells and mouse models. We also studied the regulation of miR-34b expression in cells from patients with AML and myelodysplastic syndromes (MDS).

Primary AML cells transiently overexpressing miR-34b had decreased clonogenicity, as well as increase in apoptosis (9.9 vs. 25.5%, p<0.001). Primary leukemia cells from AML patients (n=3) treated with the demethylating agent 5-aza-2′-deoxycytidine showed a rise in miR-34b expression after 16 hours (RQ=7±2.6). We also observed a concomitant decrease in CREB protein expression and its target genes. In vivo, miR-34b overexpression resulted in decreased CREB expression and suppression of leukemia growth in flank tumor models with HL-60 and K562 cells injected into NOD-SCID IL-2receptor gamma null (NSG) mice, measured by bioluminescence and tumor volume (n=10 per group). These results demonstrated that miR-34b is an important tumor-suppressor through downregulation of CREB. We next investigated miR-34b expression in a large series of AML patients (n=118), a group of MDS patients (n= 49), and healthy bone marrows (HL-BM) (n=17) by quantitative PCR. Our results demonstrated lower miR-34b expression in blast cells from AML patients at diagnosis compared to HL-BM. The lower miR-34b expression in AML patients correlated with elevated CREB levels, similar to myeloid leukemia cell lines. The expression levels of miR-34b in bone marrow from MDS patients were intermediate between AML patients and HL-BM. These results suggest that miR-34b regulates CREB and is involved in the evolution of MDS to AML. In an effort to understand the mechanism of miR-34b downregulation in primary AML and MDS BM cells, miR-34b promoter methylation was determined using MS-PCR in both patient cohorts. The miR-34b promoter was found to be methylated in 65% (78/118) of AML patients at diagnosis, while it was unmethylated in all MDS samples (49/49). In particular, 3 MDS patients that evolved to AML had miR-34b promoter hypermethylation exclusively at the onset of AML. We further tested this hypothesis by downregulating miR-34b in primary HL-BM and fetal liver cells by using both oligonucleotides and a lentiviral transduction. An increase in CREB mRNA and several CREB target genes (for example cyclin B1, cyclin E2, p21) was observed. Moreover, the cell cycle profile demonstrated increased numbers of cells in S phase compared to negative controls. Methylcellulose colony formation was also increased in HL-BM and fetal liver cells transduced with a miR-34b inhibitor compared to controls (197 vs. 101, p<0.001). Therefore, we conclude that miR-34b promoter methylation is critical for the pathogenesis of AML through regulation of CREB-dependent pathways.