PS978 DISCOVERY OF NOVEL C/D BOX SNORNAS THAT ARE REQUIRED FOR LEUKEMIA CELL PROLIFERATION

Abstract

Background:The vast majority of the human genome is transcribed, but the function of many transcripts remain obscure. It is likely, that cancer specific transcripts exist that are overlooked by conventional pipeline analysis.Aims:We tried to identify novel non coding RNA transcripts with C/D box snoRNA properties based on sequence homology. We specifically analysed AML patient samplesMethods:To understand the role of snoRNAs in leukemogenesis, we analyzed the snoRNA expression pattern in an initial cohort of 63 AML patients (Zhou, 2017) and an additional cohort of 90 AML patients. Abundant regions with coverage were annotated using snoRNABase (LBME), the recently published updated snoRNAome (Jorjani, 2016) and tracks from ENSEMBL, UCSC, RNAcentral and DASHR databases. Regions without annotation were analyzed for C/D box motifs to discover putative C/D box snoRNAs. For further analysis only regions with at least one reads per million (RPM) in at least one patient sample were included.Results:Next generation sequencing data revealed that 248 of the 269 known C/D box snoRNAs (92%) were expressed in one or more patient samples. For the H/ACA box snoRNAs we could identify 103 of 112 (92%). In addition to this established functionally well annotated core‐set of C/D box snoRNA we confirmed another set of 76 C/D box snoRNA to be expressed in AML patient samples. These 76 snoRNA recently have been described as novel C/D‐box snoRNA based on the analysis of ENCODE small RNA‐seq data. Importantly, our in silico analysis identified 45 novel putative C/D box snoRNAs which had not been described so far. All of these putative C/D box snoRNAs contain the box C/D motifs and were mostly located in introns of known genes. In further analyses, we observed increased expression of putative snoRNAs was found in patients with intermediate molecular risk (7 snoRNAs, p≤0.05) and in patients with a poor response to chemotherapy (9 snoRNAs, p≤0.05). The novel snoRNAs were more prominently expressed in in NPM1 wildtype AML compared to NPM1 mutant AML. Overall, 13 putative C/D box snoRNA were downregulated during myeloid differentiation of HL60 cells. To study the function of the snoRNA we performed a CRISPR/Cas9 knockout of five regions in Kasumi1 and U937 cell lines. The knockout of four out of five novel snoRNAs impaired proliferation and colony growth.Summary/Conclusion:Multiple novel C/D box snoRNAs were identified from primary AML specimens. These show a specific expression pattern in myeloid differentiation in distinct AML subtypes. The functional relevance of these snoRNAs suggests that so far overlooked non coding RNA sequences are of high importance in AML.