The Distinctive MicroRNA Signature of Acute Myeloid Leukemia with Translocation t(8;16)(p11;p13)/MYST3-CREBBP Is Responsible for RET Overexpression and Is Regulated by Epigenetic Mechanisms

Acute myeloid leukemia (AML) with t(8;16)(p11;p13) and MYST3-CREBBP rearrangement [t(8;16) AML] is an infrequent leukemia subtype with specific clinical and biological characteristics including a specific gene expression profile with overexpression of RET protooncogene. Our group has previously described a specific signature of microRNAs (miRNAs), most of which were downregulated, and found an inverse correlation between RET mRNA expression and the expression of several of these miRNAs. Since MYST3 and CREBBP are chromatin-modifying proteins, we have now investigated the role of epigenetic mechanisms in the downregulation of the miRNAs in our signature and examined RET as a potential target of some of these miRNAs.

To analyze the possible epigenetic regulation of microRNA signature of t(8;16) AML, we first selected those miRNA genes with CpG islands in the promoter regions according to UCSC Genome Browser (http://genome.ucsc.edu). Changes in the expression of selected miRNA levels were studied in cryopreserved cells from a t(8;16) AML patient and in the K562 cell line after treatment for 72 hours with demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC, decitabine) and histone deacetylase inhibitor trichostatin A (TSA). Expression of selected miRNAs after treatment with 5-aza-dC and TSA was analyzed using TaqMan MicroRNA Assays (Applied Biosystems). In addition, the degree of quantitative methylation in the promoter region of these miRNA genes was measured with Human Cancer miRNA Genes, Signature Panel Methyl-Profiler DNA Methylation PCR Array (Qiagen). For the validation of RET as the target of specific miRNAs, we performed a Renilla/Luciferase assay with a modified expression vector psiCHECK-2 containing the 3'UTR region of RET cloned in the 3'UTR region of Renilla gene. Modulation of RET expression by selected miRNAs was analyzed after transfection of the corresponding pre-miRNAs in the K-562 cell line, and luminescence was read at 24 hours.

Since methylation is a common mechanism of the regulation of miRNA expression, we performed a bioinformatic analysis which showed that 40% of the miRNAs in our t(8;16) AML signature harbored CpG islands in their promoter gene region, making them susceptible to silencing by methylation. After treatment with 5-AZA-dc, 60% of these miRNAs were re-expressed more than two fold-change. Interestingly, TSA induced a greater re-expression in 75% of these miRNAs. The degree of methylation status was analyzed in 8 of these miRNAs using the DNA methylation PCR Array. CpG hypermethylation was present to a certain degree in three miRNAs (22% in let-7g, 29% in miR-1, and 56% in miR-126) and to a greater extent (99%) in the miR-23b cluster, which comprises miR-23b, miR-24 and miR-27b.

Due to the strong inverse correlation of several miRNAs of the t(8;16) AML signature and RET mRNA levels, we then focused on miRNA target validation of RET. The analysis of the 3'UTR region of RET with TargetScan v5.1 revealed the presence of target sites for 9 miRNAs of the signature (miR-15b, miR-195, miR-218, miR-15a, miR-34a, miR-424, miR-128, and miR-27a/b), including 2 putative binding sites for miR-218. The Renilla/Luciferase assay, performed with the modified psicheck2 vector containing the 3'UTR of the RET gene, showed that 6 of these 9 miRNAs (miR-218, miR-128, miR-27b, miR-15a, miR-195, and miR-424) produced a significant downregulation of Renilla translation (41–49%), confirming our hypothesis that RET is a target of some of the miRNAs of our t(8;16) AML signature. Interestingly, 4 of the miRNAs targeting RET (miR-218, miR-27b, miR-15a and miR-424) were re-expressed after treatment with 5-AZA-dc and TSA.

Our findings provide greater insight into the genetic and epigenetic abnormalities in t(8;16) AML. Here we have shown that our t(8,16) AML-specific miRNA signature could be a functional consequence of impaired epigenetic mechanisms, including hypermethylation and abnormal histone acetylation. In addition, we provide the first validation of RET as a target of several miRNAs in this t(8;16) AML miRNA signature.

No relevant conflicts of interest to declare.