P318: loss of scarna12 results in aberrant splicing of target genes and downregulation of p53 in b-cell precursor acute lymphoblastic leukemia (bcp-all)

Background: B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is characterized by the rapid proliferation of B-cell precursors in the bone marrow (BM). Despite successful treatment of pediatric BCP-ALL (treatment success rate >90%), adult BCP-ALL still has a dismal prognosis, with limited treatment options and cure rates of less than 40%. The genetic landscape of BCP-ALL is heterogeneous and about 80% of patients is typified by the presence of various recurrent chromosomal abnormalities, many of which are associated with age and prognosis. Up to 20% of BCP-ALL patients have a normal karyotype. Aberrant splicing is a common event in BCP-ALL. However, mutations in splicing factors in BCP-ALL are rare and the mechanisms behind the splicing defects are largely unknown. Small nucleolar RNAs (snoRNAs) are implicated in human cancer, including different types of leukemia. SnoRNAs are responsible for 2’O-methylation and/or pseudouridylation of ribosomal RNAs, transfer RNAs, and small nuclear RNAs (snRNAs). Aberrant functions of selected snoRNAs have been studied in various hematological malignancies, however, aberrant roles of snoRNAs in BCP-ALL are unknown. Aims: We hypothesized that deregulation of snoRNAs and their downstream targets drives leukemogenesis of BCP-ALL. Methods: We performed small RNA next generation sequencing (NGS) to identify aberrantly expressed snoRNAs in a panel of 64 BCP-ALL BM samples at diagnosis, ten BCP-ALL cell lines and four FACS-sorted B-cell precursor subsets from normal BM. We subsequently evaluated the functional consequences of the most prominent deregulated snoRNA. Results: We found that the expression of specific snoRNA subsets are up- and downregulated during normal precursor B-cell differentiation. In addition, we identified aberrantly expressed snoRNAs in BCP-ALL, of which 14 snoRNAs were downregulated and 12 were upregulated. Small cajal body-specific RNA (scaRNA)-12 was the most downregulated snoRNA with the lowest adjusted p-value (7,3-Fold, Padj=3,5e-10) in BCP-ALL. ScaRNAs are a subclass of snoRNAs that localize to the Cajal body, a nucleic organelle involved in the biogenesis of small nuclear ribonucleoproteins and post-transcriptional modification of small nuclear RNAs (snRNA), which are part of the spliceosome. ScaRNA12 mediates the pseudouridylation of uridine 46 of the U5 snRNA (U5:46). As expected, pseudouridylation of the U5:U46 in BCP-ALL cell lines, with low levels of scaRNA12, was strongly reduced compared to normal B-cell precursors. Retroviral scaRNA12 overexpression in a BCP-ALL cell line enhanced pseudouridylation of U5:U46, indicating that U5:U46 is a genuine target of scaRNA12. Since snRNAs are involved in splicing, we performed RNA-seq of our BCP-ALL patient cohort, normal B-cell precursors, and scaRNA12-overexpressing and control BCP-ALL cell lines. Our data showed thousands of aberrant splice variations in BCP-ALL samples. ScaRNA12 overexpression reverted the splicing defects of a small mRNA subset in BCP-ALL cell lines and resulted in upregulation of p53 and modulation of MDM2 protein expression, two genes that are commonly mutated or deregulated in human cancer. Summary/Conclusion: We demonstrated that a subset of snoRNAs is specifically deregulated in BCP-ALL. ScaRNA12 is commonly depleted in BCP-ALL and may be a novel tumor suppressor by regulating p53 expression. Our results highlight the potential role for snoRNA in BCP-ALL leukemogenesis and may pave the way for new therapeutic targets in the disease. Keywords: B cell subsets, Alternative splicing, p53, Acute lymphoblastic leukemia