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)

Human thermogenic adipose tissue mitigates metabolic disease, raising much interest in understanding its development and function. Here, we show that human thermogenic adipocytes specifically express a primate-specific long non-coding RNA, LINC00473 which is highly correlated with UCP1 expression and decreased in obesity and type-2 diabetes. LINC00473 is detected in progenitor cells, and increases upon differentiation and in response to cAMP. In contrast to other known adipocyte LincRNAs, LINC00473 shuttles out of the nucleus, colocalizes and can be crosslinked to mitochondrial and lipid droplet proteins. Up- or down- regulation of LINC00473 results in reciprocal alterations in lipolysis, respiration and transcription of genes associated with mitochondrial oxidative metabolism. Depletion of PLIN1 results in impaired cAMP-responsive LINC00473 expression and lipolysis, indicating bidirectional interactions between PLIN1, LINC00473 and mitochondrial oxidative functions. Thus, we suggest that LINC00473 is a key regulator of human thermogenic adipocyte function, and reveals a role for a LincRNA in inter-organelle communication and human energy metabolism.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders. MDS is frequently associated with deletions on chromosome 5q as well as aberrant DNA methylation patterns including hypermethylation of key tumor suppressors. We have previously shown that hypermethylation and silencing of the non-coding RNA VTRNA2-1 are correlated with poor outcomes in acute myeloid leukemia patients. In this study, we find that VTRNA1-2 and VTRNA1-3, both located on chromosome 5q, can be regulated and silenced by promoter DNA methylation, and that the hypomethylating agent 5-aza-2-deoxycytidine causes reactivation these genes. In normal hematopoiesis, we find that vault RNAs (vtRNAs) show differential methylation between various hematopoietic cell populations, indicating that allele-specific methylation events may occur during hematopoiesis. In addition, we show that VTRNA1-3 promoter hypermethylation is frequent in lower risk MDS patients and is associated with a decreased overall survival.

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.

SummarySpecialized adipocytes localized in distinct depots mediate the many physiological functions of adipose tissue. In humans, paucity of thermogenic adipocytes correlates with high metabolic disease risk, raising much interest in the mechanisms by which these cells arise. Here we report molecular signatures associated with adipocyte development in different human depots and identify a long non-coding RNA, LINC00473, as the transcript most closely associated with enrichment of thermogenic adipocytes.LINC00473 expression is low in subjects with obesity or type-2 diabetes and is highly correlated with cAMP signaling and mitochondrial oxidative phosphorylation pathways. LINC00473 is localized in the nucleus and the cytoplasm, and its knockdown impairs induction of UCP1 and mitochondrial respiration.These results reveal that depot-enriched genes that modulate responsiveness to external stimuli, specifically LINC00473, are important determinants of the adipose tissue thermogenic phenotype, and potential targets for metabolic disease therapy.