Splicing Factor SRSF1 Promotes Tumorigenesis Via Oncogenic Splice-Switching and Predicts Poor Prognosis in Multiple Myeloma

Abstract

ObjectiveDysregulation of alternative splicing (AS) triggers many tumors, yet its contribution to multiple myeloma (MM) has not been elucidated. Our study aims to reveal the role of AS in MM progression, and to explore the molecular mechanisms of the candidate splicing factor (SF), thus valuates its feasibility as a treatment target and prognostic factor.Design and results 1.Gene set enrichment analysis (GSEA) revealed a significant difference in the expression pattern of the alternative splicing pathway genes, notably enriched in MM patients (Figure 1A). And, the splicing factor SRFS1 expression showed concordant differential expression in MM versus normal donors in two datasets (GSE6477, GSE13591). Kaplan-Meier analysis showed that high SRSF1 expression in CD138 + MM cells was associated with decreased PFS and poor OS (GSE9782) (Figure 1B). Moreover, the SRSF1 levels appeared a progressive increase in the progression of plasma cell dyscrasias (MGUS, SMM, MM and PCL) (Figure 1C). A cohort of 57 newly diagnosed MM patients was uniformly treated and followed. Univariate and multivariate analyses indicated that high SRSF1 expression was correlated with decreased PFS and OS. The c-indices of the Cox model fit with the combination of RISS and SRSF1 were higher than those of models fit without SRSF1, suggesting that SRSF1 improved the prognostic stratification of patients with MM.2.In vitro, SRSF1 downregulation in MM cells resulted in decreased proliferation, increased apoptosis and cell cycle arrest in G1 phase. In orthotopic MM xenograft models, bioluminescent imaging monitoring revealed a decreased burden of disease and a prolongation in overall survival in mice injected with SRSF1-shRNA cells versus the control (Figure 1D-F).3.Pearson correlation analysis suggested that the expression of TF YY1 was positively correlated with SRSF1 in five sets of GEO (Figure 1G). CHIP-seq BigWig files were downloaded from GSE31477 and Integrative Genomics Viewer (IGV) tracks displayed the binding site of YY1 at SRSF1 promoter locus from chr17: 56084421 to 56084751 (Figure 1H). CHIP-qPCR assay confirmed that endogenous YY1 directly interacted with the core element of the SRSF1 promoter in myeloma cells. Silencing of YY1 expression decreased the luciferase activity of the reporter construct carrying wild-type SRSF1 promoter region but not mutant in the YY1 binding site. Consistent with the effects of SRSF1, downregulation YY1 displayed anticancer activity in myeloma cells. Kaplan-Meier analysis showed that high YY1 expression in CD138 + myeloma cells was associated with decreased PFS and poor OS.4.RNA-seq analysis revealed a total of 7678 SRSF1-regulated AS events in LP-1 cell with an FDR cut-off of <0.05. Among them, the index of percent spliced in percentage-spliced-in (PSI) was upregulated in 4098 AS events and downregulated in 3580 AS via the downregulation of SRSF1. Changes in alternative splicing events were analyzed by replicate multivariate analysis of transcript splicing (rMATS) to identify downstream key genes regulated by SRSF1. Various types of AS events, including skipped exons (SEs), alternative 5‘ ss exons (A5SSs), alternative 3‘ ss exons (A3SSs), retained introns (RIs), and mutually exclusive exons (MXEs), can be regulated by SRSF1, and of these, SE is the most common type of SRSF1-regulated AS events (Figure 1I-O). The KEGG Pathway and GO analysis revealed that SRSF1-regulated AS events were involved in spliceosome, cell cycle and cellular process. Among the most significant AS events, we noted RBBP6, which isoforms have been reported to exhibit opposite effect on oncogenic activity.5.RNA-seq and subsequent validation indicated that SRSF1 knockdown caused a significantly decreased iso1 and increased iso3 of RBBP6. An opposite effect on myeloma proliferation between iso1 and iso3 was observed upon overexpression of individual proteins. Moreover, the level of RBBP6 iso1 is associated with poor prognosis, whereas iso3 expression is associated with favorable prognosis in MM patientsConclusionOur study uncovered that SRSF1-regulated aberrant AS facilitated myeloma progression through creating reducing normal isoforms and increasing oncogenic isoforms (Figure 1P). Moreover, SRSF1 and its critical splicing target could act as an independent prognostic factor for MM patients. Thus, SRSF1 might be considered as a valuable therapeutic target and a potential prognostic biomarker for MM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.