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Alternative splicing in multiple myeloma
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Figure 6. Patients with high levels of novel splice loci are associated with poor outcome. (A and B) Kaplan-Meier curves in high novel splicing (top 20%) (n=117) versus low/medium novel splicing (bottom 60%) (n=464) patient samples: (A) progression-free survival; (B) overall survival. (C and D) Outcome of patients with a t(4;14) with low/medium (n=70), and high (n=5) novel splice groups: (C) progression-free survival; (D) overall survival.
tions in SF3B1 affect a large set of genes that are specific to MM.
We found that many of the genes undergoing alterna- tive splicing in MM are genes related to mRNA processing and splicing such as PPIE, SUGP1 and the small nuclear ribonucleoproteins, SNRPN and SNRPC. We also detect a large number of genes related to protein ubiquitination and RNA decay. Implying that disruption to mRNA splic- ing, due to the hotspot mutations in SF3B1, activates these genes and pathways. Similar enrichment to protein ubiq- uitination and RNA decay pathways have been observed in myelodysplasia with mutations in splicing factors.35,36 We hypothesize that in an effort to remove aberrant pro- teins and transcripts they themselves are aberrantly spliced perpetuating the cellular dysregulation.
We detected significant disruption to the splicing and expression of MZB1, resulting in differential expression at the transcript level as well as differential splicing and the introduction of novel transcripts due to novel splice sites. MZB1 in B cells is an endoplasmic reticulum-localized protein and plays a role in protein folding and antibody secretion.37,38 MZB1 has been shown to have a prognostic value in many mature B-cell diseases such as CLL, follicu- lar lymphoma, and diffuse large B-cell lymphoma, where higher expression is associated with poorer survival.39 Additional experiments will be needed to see if changes to
MZB1 isoform ratios, as well as to the altered splicing of its transcripts, have similar prognostic value in MM.
The results of both GSEA identified common deregulat- ed pathways in both the mutated SF3B1 and high splice groups, namely UPR and protein secretion pathways. The UPR is a key pathway in many cancers that is important in many secretory tissues such as plasma cells due to its protective role in avoiding endoplasmic reticulum (ER) stress induced apoptosis.40,41 The SF3B1 mutant group had increased splicing aberrations, but we identified signatures of decreased UPR activity. We would expect that there would be an enrichment in these pathways because these splicing aberrations have the potential to increase non- viable transcripts.
Based on the importance of splicing associated with SF3B1 mutations, we characterized abnormal splicing and potential association with clinically relevant features of MM. We observed a significant difference in novel splicing sites in the pooled normal samples compared to all other groups, suggesting that there is a general increase in aber- rant splicing in MM. In addition, there were clear differ- ences in aberrant or alternative splicing in the different translocation groups. In our ongoing quest to refine the classification of MM subgroups, the identification of an association of t(4;14) samples with less aberrant splicing and the t(11;14) subgroup being associated with more
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