P490: SNORNA-DERIVED RNAS (SDRNAS) ARE IMPORTANT DRIVERS OF LEUKEMOGENESIS AND LEUKEMIC MAINTENANCE IN AML

Abstract

Background: Small non-coding RNAs play important roles in leukemogenesis. The canonical function of small nucleolar RNAs (snoRNAs) is to facilitate 2’-O-methylation of rRNA via a target site-specific sequence and thus promote ribosomal biogenesis and function. Recently, we have shown that a subset of small non-coding RNAs, i.e. C/D box snoRNAs are overexpressed in acute myeloid leukemia (AML) and required for AML1/ETO-driven leukemia initiation and maintenance. Depletion of snoRNP protein components or even single snoRNAs can potently inhibit AML cell growth or decrease clonogenicity, and the levels of many snoRNAs positively correlate with leukemic stem cell frequency in AML patients. Over the past years, it has been shown that snoRNAs can be further processed into shorter snoRNA-derived RNA fragments (sdRNAs), but the expression patterns of sdRNAs in AML, as well as mechanistic and functional implications, remain widely elusive. Aims: In this study, we investigated sdRNA expression patterns, functions and mechanisms in AML and healthy hematopoiesis. Methods: We prospectively characterized snoRNA and sdRNA expression in 96 AML patient samples, as well as in healthy hematopoietic stem and progenitor cells and differentiated white blood cells by small RNA sequencing. mRNA sequencing was performed to investigate connections between snoRNAs, sdRNAs and the transcriptome. Further, we depleted AML cells of different snoRNA loci by CRISPR/Cas9-based knock-out and performed lentiviral rescue overexpression of the respective sdRNA or full-length snoRNA. Results: We discovered that more than 120 snoRNAs are further processed into sdRNA fragments in AML. Single snoRNAs often evolve into multiple sdRNA isoforms. Interestingly, sdRNA expression varied significantly between different AML patients. Correlation of sdRNA and full-length snoRNA expression with patient characteristics, clinical outcome and mutational status revealed numerous implications. Those include e.g. a distinct pattern of enriched sdRNAs in NPM1-mutated patients. Expression of several sdRNAs was associated with poor overall, event-free and relapse-free survival in AML. We identified sdRNAs that were overexpressed in AML as compared to HSCs and found characteristic expression patterns among differentiated white blood cells. Notably, ratios of sdRNAs and their host snoRNA were not stable across different samples, suggesting an active mechanism regulating sdRNA processing and thus their downstream pathways. Gene set enrichment analyses based on mRNA and sdRNA correlations suggested distinct cell type specific properties. Forced expression of several sdRNAs rescued the reduced clonogenic potential observed upon depletion of the respective host snoRNA locus. This effect was often identical or even more pronounced than the rescue with the respective snoRNA precursor. Of note, overexpression of distinct sdRNAs in AML cell lines enhanced clonogenic potential. Summary/Conclusion: SnoRNA-derived sdRNAs are a common feature of AML with characteristic expression patterns determined by mutation status and clinical features. Targeting certain sdRNAs or factors involved in sdRNA processing or function might constitute promising novel therapeutic leverage points in AML.