The Landscape of Small Nucleolar RNA Expression in Multiple Myeloma

Background Small nucleolar RNAs (snoRNAs) are non-coding RNAs involved in the maturation of ribosome RNAs and various other biological processes. Emerging evidence demonstrates that snoRNAs play an essential role in tumor initiation and progression. However, their role in multiple myeloma (MM) pathogenesis remains poorly characterized. To date, the profiling of small RNAs in MM has mainly focused on microRNAs and approaches are confined to PCR- or array-based techniques. Here, we used next generation sequencing (NGS) to explore the snoRNA expression patterns of MM patients and to identify the association between snoRNAs and mRNAs, molecular subgroups of MM and clinical parameters.

Methods Purified CD138+ cells from 71 newly diagnosed MMs and 4 refractory/relapsed MMs were included. Three CD19+ B cell samples from healthy donors were used as control. Total RNA was isolated with the AllPrep DNA/RNA kit (Qiagen, Germany). The mRNA and snoRNA NGS libraries were prepared using the Illumina TruSeq stranded mRNA and the NEBNext multiplex small RNA library kit, respectively. Sequencing of mRNA and snoRNA was performed on an Illumina NextSeq and NovaSeq 6000 PE 100 S1 platform, respectively. Paired-end RNA-seq reads were mapped to the hg19 reference genome using STAR. Gene expressions were quantified using featureCounts. Differentially expressed mRNAs and snoRNAs were identified using DESeq2. The Kaplan-Meier method was used for survival analyses. Progression-free survival (PFS) time was measured from enrollment to relapse or death from any cause, whichever occurred first.

Results Using NGS, we observed expression of 625 full-length snoRNAs. Compared to normal B cells high levels of snoRNA expression were detected in MM plasma cells. Looking for associations between snoRNA expression and molecular subgroups of MM, we found an upregulation of 50 snoRNAs in patients with a hyperdiploid karyotype. The pattern of upregulation supported a regulatory mechanism in this molecular subgroup that goes beyond a pure copy-number effect. Even in patients with multiple trisomies the overwhelming majority (82%) of upregulated snoRNAs were located on chromosome 15, including the SNORD115 family on 15q11. In patients with gain of 1q21, all snoRNAs located on 1q25 were upregulated and the highest expression level of those snoRNAs appeared in patients with amp1q, indicating a copy-number effect. SNORD78, which was recently shown to impact outcome in prostate cancer and lung cancer, was significantly overexpressed in gain1q patients and defined a subgroup with inferior PFS (p =0.019) and overall survival (p = 0.011). SCARNA22, which was upregulated in patients with a t(4;14), was another snoRNA with a negative impact on PFS (31.06 months vs unreached, p = 0.007). To further characterize MM with upregulation of prognostically relevant snoRNAs, we analyzed bulk RNAseq of CD138-enriched MM cells and performed a pathway enrichment analysis. Comparing SNORD78 high expression group with the low group, we found 537 upregulated genes, e.g.SH2D2A, and CDK1, and 229 downregulated genes. Differentially expressed genes were enriched for cell cycle and chromosome segregation. For SCARNA22, we found 963 up- and 219 down-regulated genes. These showed enrichment for cell adhesion (upregulated in SCARNA22^high) and protein targeting to the membrane or the endoplasmic reticulum (downregulated in SCARNA22^high).

Conclusion In this study we provide first insights into the expression patterns of snoRNAs in MM by deep sequencing. We identified snoRNAs dysregulated in MM subgroups, with two of them being associated with poor outcome. This study also shows that expression of particular snoRNAs is associated with specific chromosome gains, which is a common element in MM.

Disclosures No relevant conflicts of interest to declare.

Raab:Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Heidelberg Pharma: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Mueller-Tidow:BiolineRX: Research Funding; Pfizer: Research Funding.