Deletions of chromosome 5q are associated with poor outcomes in acute myeloid leukemia (AML) suggesting the presence of tumor suppressor(s) at the locus. However, definitive identification of putative tumor suppressor genes remains controversial. Here we show that a 106-nucleotide noncoding RNA vault RNA2-1 (vtRNA2-1), previously misannotated as miR886, could potentially play a role in the biology and prognosis of AML. vtRNA2-1 is transcribed by polymerase III and is monoallelically methylated in 75% of healthy individuals whereas the remaining 25% of the population have biallelic hypomethylation. AML patients without methylation of VTRNA2-1 have a considerably better outcome than those with monoallelic or biallelic methylation (n ‫؍‬ 101, P ‫؍‬ .001). We show that methylation is inversely correlated with vtRNA2-1 expression, and that 5-azanucleosides induce vtRNA2-1 and down-regulate the phosphorylated RNA-dependent protein kinase (pPKR), whose activity has been shown to be modulated by vtRNA2-1. Because pPKR promotes cell survival in AML, the data are consistent with vtRNA2-1 being a tumor suppressor in AML. This is the first study to show that vtRNA2-1 might play a significant role in AML, that it is either mono-or biallelically expressed in the blood cells of healthy individuals, and that its methylation state predicts outcome in AML. (Blood. 2012; 119(1):206-216)

BackgroundThe hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2′deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT). While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA), which may potentially affect the activities of the drugs in patients.Methodology/Principal FindingsA hematopoietic (HL-60; acute myeloid leukemia) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2) and lasting (day 8) effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells.Conclusions/SignificanceOverall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.

A thorough understanding of the idiopathic hypereosinophilic syndrome (IHES) and further optimization of diagnostic work-up procedures are warranted. We analyzed purified eosinophils from patients with IHES by next-generation whole-exome sequencing and compared DNA methylation profiles from reactive eosinophilic conditions to known clonal and suspected clonal eosinophilia. Somatic missense mutations in cancer-related genes were detected in three IHES patients. These included the spliceosome gene PUF60 and the cadherin gene CDH17. Furthermore, reactive eosinophilia samples could be differentiated from known- and suspected clonal eosinophilia samples based on 285 differentially methylated CpG sites corresponding to 128 differentially methylated genes. Using Ingenuity pathway analysis, we found that differentially methylated genes were highly enriched in functional pathways such as cancer, cell death and survival, and hematological disease. Our data show that a subset of IHES may be of clonal origin not related to the classical molecular aberrations of FGFR, PDGFRA/B, or T-cells, and that the initiating hits could be point mutations in a variety of genes, including spliceosome mutations or hypermethylated tumor suppressor genes. In addition, we identified a DNA methylation signature that is relevant for distinguishing clonal and suspected clonal eosinophilia from reactive eosinophilia per se, which may be useful in daily clinical work.

3450 Introduction: 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. Methods: 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. Results: 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. Discussion: 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. Disclosures: Jones: Eli Lilly: Consultancy.