Allelic Methylation Levels of the Non-Coding RNA Gene VTRNA2-1 Located on Chromosome 5q31.1 Predict Outcome in AML,

Deletions of chromosome 5q are associated with poor outcomes in acute myeloid leukemia (AML), suggesting the presence of tumor suppressor(s) at the locus. Two different critical deleted regions (CDRs) have been identified on 5q. One region is linked to de novo AML and high-risk MDS (CDR1 at 5q31), and a second region to the low-risk MDS 5q- syndrome (CDR2 at 5q32–33). However, definitive identification of putative tumor suppressor genes remains controversial. Since it has recently been shown that several types of hematological malignancies have globally altered expression of non-coding RNAs (ncRNAs), we therefore searched for candidate ncRNA genes in the CDR1 region.

Cell lines were treated with either 5-azacytidine or 5-aza-2`-deoxycytidine. Upregulated microRNAs (miRs) were identified by microarray analysis and confirmed by RT-qPCR analysis. The transcription start site was determined by 5`RACE and promoter methylation status by methylation specific melting curves, pyrosequencing and bisulfite sequencing. ncRNA expression and processing were analysed by RT-qPCR, Northern blotting, and siRNA mediated knock down of Drosha.

We identified the putative miR886, which became induced by azanucleoside treatment in an AML cell line, suggesting that it was regulated by promoter methylation. We found that the processing of miR886 was independent of Drosha, and northern blotting showed that this was a different type of longer ncRNA, annotated vtRNA2-1. These observations were supported by the results from three others groups (Nandy, J Mol Biol 2009; Stadler, Mol Biol Evol 2009; Lee, RNA 2011). The gene that encodes VTRNA2-1 is embedded in a CpG island. The CpG island is fully methylated in 4 different myeloid cell lines (HL60, NB4, U937 and F36P) and these cell lines have no expression of vtRNA2-1. Treatment with 5-azacytidine and 5-aza-2`-deoxycytidine derepressed expression of vtRNA2-1 in the cell lines. vtRNA2-1 is expressed in hematopoietic tissue (including CD34+ cells) from healthy individuals. Surprisingly, 75% of these carry a monoallelicly methylated VTRNA2-1 promoter while 25% carry an unmethylated VTRNA2-1 promoter.

The methylation status of VTRNA2-1 was examined in bone marrow mononuclear cells from 101 AML patients taken at the time of diagnosis. 38 (38%) of these patients carried a hypomethylated promoter, 53 (52%) an intermediate methylated promoter and 10 (10%) a hypermethylated promoter. AML patients with hypomethylation of VTRNA2-1 have a significant better prognosis than patients with intermediate- or hypermethylation of the promotor (p=0.001). VTRNA2-1 methylation was independently associated with a poor survival in Cox proportional-hazards analysis (p=0.043), when testing against age, cytogenetic risk classification and leukocyte count at diagnosis.

It has previously been shown that vtRNA2-1 may be involved in the regulation of the double stranded RNA dependent kinase, PKR. Down regulation of vtRNA2-1 leads to activation of PKR and its downstream targets including NFkB (Lee, RNA 2011). Furthermore, PKR has previously been shown to alter response to chemotherapeutic agents by promoting cell survival (Pataer, Cancer Biol Ther 2009). Since constitutive PKR and NFkB activity are well documented features in AML, we speculate whether this may at least in part be mediated via loss of vtRNA2-1 expression.

Here, we show that VTRNA2-1 may be directly implicated in AML, that expression of vtRNA2-1 is regulated by promoter methylation. Interestingly, we found that the majority of the healthy Danish population (∼75%) carry a monoallelically silenced VTRNA2-1 in normal hematopoietic cells. Our data suggest that the gene dosage of this particular type of ncRNA may play an important role in tumor progression or response to therapy since patients with hypomethylation of both alleles of the VTRNA2-1 promoter have a significantly better prognosis, while those that gain hypermethylation of the second VTRNA2-1 copy have a poorer outcome. Our data, combined with the previous findings, suggest that VTRNA2-1 is a novel tumor suppressor, located on chromosome 5q31.1, which probably acts through PKR.

Jones:Eli Lilly: Consultancy.