Dysregulation of alternative splicing contributes to multiple myeloma pathogenesis

Dysregulation of alternative splicing (AS) triggers many tumours, understanding the roles of splicing events during tumorigenesis would open new avenues for therapies and prognosis in multiple myeloma (MM). Molecular, genetic, bioinformatic and statistic approaches are used to determine the mechanism of the candidate splicing factor (SF) in myeloma cell lines, myeloma xenograft models and MM patient samples. GSEA reveals a significant difference in the expression pattern of the alternative splicing pathway genes, notably enriched in MM patients. Upregulation of the splicing factor SRSF1 is observed in the progression of plasma cell dyscrasias and predicts MM patients’ poor prognosis. The c-indices of the Cox model indicated that SRSF1 improved the prognostic stratification of MM patients. Moreover, SRSF1 knockdown exerts a broad anti-myeloma activity in vitro and in vivo. The upregulation of SRSF1 is caused by the transcription factor YY1, which also functions as an oncogene in myeloma cells. Through RNA-Seq, we systematically verify that SRSF1 promotes the tumorigenesis of myeloma cells by switching AS events. Our results emphasise the importance of AS for promoting tumorigenesis of MM. The candidate SF might be considered as a valuable therapeutic target and a potential prognostic biomarker for MM.


INTRODUCTION
Multiple myeloma (MM) is a cancer of neoplastic plasma cells that is typically accompanied by clonal proliferation of malignant plasma cells in the bone marrow microenvironment, monoclonal protein in the blood or urine, and associated organ dysfunction [1]. The introduction of proteasome inhibitors, immunomodulatory drugs, antibodies targeting cell surface molecules, high-dose therapy and autologous stem-cell transplantation (ASCT) has markedly improved outcomes in patients with multiple myeloma [2]. Despite multiple therapeutic advances, almost all people with multiple myeloma eventually become resistant to treatment and die of the disease [3]. It is preceded by monoclonal gammopathy of undetermined significance (MGUS), an asymptomatic premalignant accumulation of clonal plasma cells that shares genetic features with MM [4]. Multiple myeloma is difficult to treat because physicians have conventionally delayed therapy until end-organ damage, which in myeloma consists of bone destruction, anaemia and renal failure, is detected [2,5]. It is time to improve understanding of molecular abnormalities related to disease initiation and progression, and of how genes were altered in tumour cells, enabled the development of new drugs, including interventions based on biomarkers for personalised therapeutic treatment of specific molecular subtypes.
Alternative splicing (AS) is a key control point in the generation of distinct mRNA and protein isoforms from a single gene [6].
Cancer cells have general as well as cancer type-specific and subtype-specific alterations in the splicing process that can have prognostic value and contribute to every hallmark of cancer progression [7]. Splicing perturbations are common in cancer and are associated with altered expression of the components of the splicing machinery [8]. The spliceosome, composed of five small nuclear ribonucleoproteins (snRNPs) and numerous additional proteins. RNA-binding motif (RBM) proteins, arginine-serine-rich (SR) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs) are the major class of alternative splicing decisions [6].
SR family characterised by RNA-recognition motifs (RRM) and an RS domain is composed of 12 members, which can bind directly to a pre-mRNA and elicit changes in its AS [9]. Transcriptome-wide analyses have uncovered numerous splicing-related RBPs that are differentially expressed in tumours when compared with normal tissues [10]. However, the role of alternative splicing in myeloma is still not completely understood, so it is worthy of exploration. Therefore, we sought to elucidate the expression, clinical relevance, biological function, and underlying mechanism of alternative splicing in MM. In this study, three main questions are addressed: (a) does alternative splicing events facilitate tumorigenesis and progression of MM, (b) What is the candidate mechanism underlying the aberrant AS in MM, (c) can we considered the crucial splicing factor SRSF1 as a promising therapeutic target and a prognostic factor for MM?

MATERIALS AND METHODS Cell cultures
MM.1S and LP-1 cells were cultured at 37°C in an atmosphere containing 5% CO 2 and in RPMI-1640 medium (Hyclone, USA) supplemented with 10% foetal bovine serum (FBS). 293T cells maintained in DMEM high glucose medium plus 10% FBS. Cell line authentication was performed by cell line characterisation core using short tandem repeat profiling (Genetic Testing Biotechnology Corporation, China).

Small-interfering RNA (siRNA) transfection
Small-interfering RNAs and scrambled control non-targeting siRNA were synthesised by GenePharma (Shanghai, China). To downregulate SRSF1 and YY1, small-interfering RNAs were transfected into cells, respectively. The transfections were performed per instructions provided by the manufacturer (Neon™). The transfected cells were incubated at 37°C with 5% CO 2 for 48 h. The sequences of siRNAs are shown in Supplementary Table 1.

RNA sequencing
Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer's protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with RNA Integrity Number (RIN) ≥ 7 were subjected to the subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Then these libraries were sequenced on the Illumina sequencing platform (HiSeqTM 2500 or Illumina HiSeq X Ten) and 125 bp/150 bp paired-end reads were generated.

Statistical analysis
Survival analysis was performed to investigate the associations between the censored outcomes and the clinical variables and the expression measures of SRSF1 and YY1 using the R package survival. PFS and OS were the primary outcomes of interest. Pearson correlation analysis was employed to determine the correlation between the expression of YY1 and SRSF1. Student's t test was performed to analyse the difference of mean values between the two groups for other continuous outcomes. All statistical analyses were performed in R (version 3.2.3). A P value lower than 0.05 was considered as significant.

Aberrant alternative splicing in MM
To investigate whether dysregulated alternative splicing events were implicated in MM initiation and progression, gene set enrichment analysis (GSEA) between healthy donors (HD) and MM patients were performed in Gene Expression Omnibus (GEO) database (GSE6691). This revealed a significant difference in the expression pattern of the alternative splicing pathway genes, notably enriched in MM patients (Fig. 1a). AS is generally regulated by splicing factors and the main cause of splicing dysregulation is ascribed to the aberrant expression or activity of splicing factors [11]. And 17 splicing factors have been recognised to function as proto-oncogenes or tumour suppressors [12]. To identify crucial splicing factors in MM initiation and progression, we performed two rounds of screening consecutively (Fig. 1b). The expression level of the 17 splicing factors mentioned above were analysed and five of them showed concordant differential expression in MM versus normal donors in two datasets (Fig. 1b  and Supplementary Table 3). Then, five selected genes were subjected to survival analysis to determine their prognostic significance in MM. Kaplan-Meier analysis showed that SRSF1 expression in CD138 + MM cells was associated with PFS and OS (Fig. 1c), but not other four genes ( Supplementary Fig. 1). We furtherly used Cox survival regression to jointly fit all the clinical variables captured by revised international staging system (R-ISS) and SRSF1 expression. When adjusting for the clinical factors, it was no longer significant for OS. Nevertheless, SRSF1 retained its significance as a prognostic factor for PFS in the univariate setting (Table 1). To further explore the role of SRSF1 in plasma cell tumour transformation, we analysed the SRSF1 expression in patients with plasma cell disease at different stages (MGUS, SMM, MM and PCL) and PCs of normal donor (NC). Among these splicing factors, only the expression level of SRSF1 appeared a progressive increase in the progression of plasma cell dyscrasias (Fig. 1d). The above results provided the critical clue about aberrant alternative splicing might facilitate myeloma development and progression, and SRSF1 may be the crucial splicing factor for AS in MM.

SRSF1 increases the tumorigenic potentials of MM
Increasing evidence demonstrates that SRSF1 is a prototypical splicing factor overexpressed in many tumours and exerts oncogenic roles via control of AS of cancer-related genes [7,[13][14][15][16]. The emerging roles of SRSF1 in AS regulation in cancer are opening up a new therapeutic avenue. Prompted by the above findings, we examined whether SRSF1 exerted oncogenic functions in MM. To unveil the potential function of SRSF1 in MM, we transiently silenced endogenous SRSF1 expression with two independent siRNAs in MM.1S and LP-1 cells. Knockdown efficiency of SRSF1 in MM.1S and LP-1 cells were confirmed by western blotting and qPCR ( Fig. 2a and Supplementary Fig. 2A).
Compared with the control siRNA, SRSF1 siRNAs significantly inhibited the growth of myeloma cells ( Fig. 2b and Supplementary  Fig. 2B). Notably, knockdown of SRSF1 caused G1-phase arrest and promoted apoptosis in myeloma cells (Fig. 2c, d and Supplementary Fig. 2c-f). To evaluate the tumorigenic effect of SRSF1, LP-1 cells transfected with SRSF1-shRNA or control plasmid-constructed lentivirus were injected via the tail vein into the NSG (NOD-SCID IL-2receptor gamma null) mice. Consistent with the in vitro results, bioluminescent imaging monitoring revealed a decreased burden of disease and a prolongation in overall survival in mice injected with SRSF1-shRNA cells compared to the controls ( Fig. 2e-g). Collectively, these findings prove that SRSF1 functioned as a potential proto-oncogene to facilitate tumorigenesis and its inhibition could impede myeloma progression.

SRSF1 is positively regulated by the transcription factor YY1
Large-scale genomic studies have uncovered that cancer cells are capable of hijacking the expression of SR proteins, leading to dysfunctional gene splicing and tumour-specific dependencies [8,17]. In order to elucidate the mechanisms mediating the upregulation of SRSF1 in myeloma, we investigated the transcription factors which could bind to the promoter region of SRSF1 using the UCSC (University of California Santa Cruz) genome browser. We first screened out 99 transcription factors that may bind to the promoter region of SRSF1. From these transcription factors, we selected 25 transcription factors with high predicted scores (Cluster Score > = 500) for subsequent analysis. Correlation analysis was conducted to explore the relationship between 25 transcription factors and SRSF1. We found that five transcription factors which were correlated with SRSF1 overlapped in the five sets of data (Fig. 3a). Furthermore, the results of Pearson   correlation analysis indicated that among the five transcription factors, only the expression of YY1 (YY1 transcription factor) in multiple sets of data had the highest correlation coefficient (r > = 0.25, P < 0.05) with SRSF1 (Fig. 3b), suggesting that YY1 probably has a regulatory role in SRSF1. We next sought to determine the regulation role of YY1 on the transcription of SRSF1. YY1 silencing led to a reduced level of SRSF1 (Fig. 3c, d). In order to further define the specific site of YY1 regulating SRSF1, CHIP-seq BigWig files were downloaded from GSE31477 and loaded in Integrative Genomics Viewer (IGV) to generate representative image tracks of the YY1 binding site at SRSF1 locus, we noticed that IGV tracks displayed the binding site of YY1 at SRSF1 promoter locus from chr17: 56084421 to 56084751 (Fig. 3e). To further validate this finding, we characterised the binding regions of SRSF1 promoter by CHIP-qPCR and observed that the expression of SRSF1 promoter regions pulled down with anti-YY1 were significantly reduced upon YY1 knockdown (Fig. 3f). We further performed dual-luciferase reporter assay to determine the interactions. Consistently, YY1 silencing led to a reduced luciferase activity of the reporter constructs carrying wild-type SRSF1 promoter region, supporting a direct interaction between YY1 and SRSF1 promoter regions (Fig. 3g). Taken all together, these data indicated that YY1 plays a pivotal role in the regulation of SRSF1.
Dysregulated YY1 promotes myeloma progression and is associated with a poor prognosis Since YY1 acted as a crucial regulator of the transcription of SRSF1, we next attempted to gain further insights into the effects of the YY1 in MM. We assessed the expression profile of YY1 and observed a significant increase of YY1 in the progression of plasma cell dyscrasias (Fig. 4a). Likewise, Kaplan-Meier analysis indicated that high YY1 expression was a predictor of poor prognosis for both OS and PFS in MM patients, indicating that YY1 probably tend to participate in tumour growth (Fig. 4b). We also used Cox survival regression to jointly fit all the clinical variables captured by revised international staging system (R-ISS) and YY1 expression (Supplementary Table 4). To verify this hypothesis, loss- b Pearson correlation analysis was performed to detect the correlation between SRSF1 and YY1 in MM patients (GSE16122, GSE2113, GSE2658, GSE6477, GSE13591). r coefficient of determination. c Control siRNA or YY1 siRNAs were transfected into LP-1 and MM.1S cells by electro-transfection. The cells were collected after 48 h transfection and the YY1 expression level was detected by qPCR and western blot. d qPCR and western blot assay were performed to determine the expression of SRSF1 upon YY1 knockdown. e IGV tracks displaying the reads coverage of YY1 and SRSF1. f CHIP assay was performed to verify whether YY1 can bind to the promoter region of SRSF1 to regulate the transcription of SRSF1. g The dual-luciferase assay was performed to detect the combination between YY1 and the promoter region of SRSF1. The pGL3 luciferase reporter vector carrying YY1 and SRSF1 promoter region binding site.
of-function studies were performed to identify the roles of YY1 in MM cell proliferation, apoptosis and cell cycle progression. The CCK-8 assays showed that YY1 knockdown led to significantly decreased cell proliferation (Fig. 4c). In parallel, knockdown of YY1 resulted in a significantly induced apoptosis ( Fig. 4d and Supplementary Fig. 3A). Correspondingly, the flow cytometry analysis showed that the percentage of G1 increased and the percentage of S phase decreased in YY1 knockdown cells, further pointing to the critical role of YY1 in MM ( Fig. 4e and Supplementary Fig. 3B). These above data suggest that YY1 effectively enhances tumour progression in MM cells by promoting the SRSF1 expression.

Global landscape alternative splicing events regulated by SRSF1
Previous studies demonstrate that the splicing factor SRSF1 is a crucial family member of SR (serine/arginine-rich) proteins [11]. SRSF1 plays a dual role in alternative splicing, as represented by promoting splicing by binding to exonic splicing enhancers and inhibiting splicing by binding to intronic elements [18]. In order to investigate SRSF1-regulated AS (alternative splicing) events involved in myeloma, we conducted high-throughput sequencing of RNA (RNA-Seq) on LP-1 cells after SRSF1 depletion. A total of 2349 genes were identified with significant expression change in SRSF1-shRNA cells (Fig. 5a, b). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis showed that these genes were associated with cell cycle, mRNA regulation and development processes (Fig. 5c, d and Supplementary Table 5 and 6). GSEA revealed that differentially expressed genes were involved in cell killing and cell growth pathways ( Fig. 5e and Supplementary Table 7). These data support that SRSF1 is involved in multiple steps of RNA regulation and cell growth in MM. Furthermore, RNA-seq analysis revealed a total of 7678 SRSF1regulated AS events in LP-1 cell with an FDR cut-off of <0.05 ( Fig. 5f, g). 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. Especially, the majority of these AS events belonged to the skipped exon (SE) category, implying a preference of SRSF1 regulating AS events of SE (Fig. 5f, g). Among AS events, the index of percent spliced in (PSI) was upregulated in 4098 AS events and downregulated in 3580 AS events via the knockdown of SRSF1 (Fig. 5h and Supplementary Table 8). ClueGO and CluePedia further revealed that the SRSF1-regulated AS participated in multiple molecular pathways, and most of these were involved in RNA metabolic processes ( Supplementary Fig. 4 and Supplementary Table 9). Overall, SRSF1 tend to promote exon inclusion and is critical to maintain the AS of genes related to RNA processes and cell growth in MM.

DISCUSSION
Our current understanding of regulatory roles of splicing factors in cancers is mainly based in the limited information about the changes in gene copy number and/or expression [6,8,19], however, the detailed mechanisms and biological consequences are still elusive. In particular, it is unknown whether aberrant AS plays vital roles in the development of myeloma remains unknown. In this study, we provided convincing evidence that upregulation of the splicing factor SRSF1 could act as an oncoprotein in MM, and its expression was mediated by the oncogenic transcription factor YY1. In addition, our present work represented a comprehensive study of the splicing factor SRSF1 and its downstream AS landscape in myeloma. We concluded that SRSF1 promoted myelosis by controlling the AS of tumourassociated genes, further highlighting the importance of AS as a crucial contributor to tumorigenesis. Alternative splicing is frequently regulated by trans-acting splicing factors, which bind to sequence motifs that are associated with the promotion (enhancers) or repression (silencers) of splicing [19]. Splicing enhancers and silencers are bound by diverse RNA-binding proteins, exemplified by the serine/argininerich proteins (SR proteins) and heterogeneous nuclear ribonucleoproteins (hnRNPs) [12]. Some SR proteins can act as oncoproteins when overexpressed in the correct cellular context. For instance, insightful reports from other scholars have demonstrated that SR splicing factor 1 (SRSF1; also known as ASF/SF2) is overexpressed in extracranial tumours and plays oncogenic roles via control of the AS of several tumour-related genes [7,14,15,20]. Notably, titration of SRSF1 activity using decoy oligonucleotides containing SRSF1-binding sites results in the inhibition of cancer cell growth and apoptosis in vitro and in mouse xenograft models in glioblastoma [21]. Consistent with previous studies, we have presented a number of findings demonstrating the significant evidence that SRSF1 was appeared a progressive increase during the progression of plasma cell     dyscrasias and its overexpression was associated with a poorer survival. The subsequent functional study verified that SRSF1 knockdown caused the proliferation inhibition, G1-phase arrest and the increase of apoptosis of MM cells in vitro and suppressed the tumour growth of myeloma xenografts in vivo. These findings indicated that SRSF1 played a critical role in the pathogenesis of myeloma and tumour cell proliferation.
Several lines of evidences have demonstrated deleterious outcomes when transcription factors become dysfunctionally activated or inactivated, leading to cellular malfunction, instability, and in some cases, tumorigenesis [22]. Previous studies have proposed a pro-oncogenic action of SRSF1 on the basis of its upregulation in different types of human cancer and its ability to drive the oncogenic transformation of fibroblasts and epithelial cells via enhanced proliferation and compromised apoptosis when overexpressed [23][24][25]. Moreover, treatment with SRPK1 inhibitor inhibited phosphorylation of SRSF1, resulting in potent inhibition of blood vessel growth in models of choroidal angiogenesis in vivo [26,27]. In MM, identifying additional SRSF1 splicing targets involved in transformation, and gaining a deeper understanding of how SRSF1 levels are regulated, should be important priorities towards the development of therapeutic strategies that specifically target relevant AS isoforms.
Based on the dysregulated expression of SRSF1, we further explored the transcription factors which could bind to the promoter region of SRSF1 and alter the transcriptional regulation on SRSF1. The previous study identify SRSF1 is a direct target of the transcription-factor oncoprotein MYC in lung tumours [28]. However, this study did not include other transcription factors except MYC. It is highly possible that the other more important transcription factors may regulate SRSF1 transcription. To overcome this possible drawback, we analysed all the transcription factors that could bind to the promoter region of SRSF1 in order to more accurately reveal the transcription factors that regulate the SRSF1 transcript. According to the results of bioinformatics analysis, we focused on YY1 (Yin Yang 1). YY1 is a 65-kDa member of the GLI-kruppel family of zinc finger transcription factors, which regulate various developmental and differentiation processes [29]. Most processes mediated by YY1 are cancer-related, strongly implicating its importance in cancer development and progression [30]. Indeed, overexpression of YY1 has been observed in various types of cancers [31,32]. Hence, it is important to divulge how transcription factors such as YY1 function in SRSF1 transcription processes and ultimately shape the growth and viability of MM cells. Subsequent experiments confirmed that YY1 could positively regulate SRSF1 expression by directly binding to its promoter region. Thus, YY1 is a novel transcriptional activator of SRSF1 expression. Moreover, consistent with other studies [30,33,34], we were able to show that YY1 as oncoproteins enhanced tumour progression in MM cells.
Alternative splicing can be categorised into five major events, which include skipped exons, alternative 3′ or 5′ splice site selection, mutually exclusive exons, and intron retention [8]. Analysis of 8705 patients from The Cancer Genome Atlas (TCGA) identified a significant amount of aberrant transcripts in tumour cells, including exon skipping and alternative 3′ splice sites when compared with normal human tissues [35]. SRSF1 regulates AS of target genes involved in apoptosis, cell motility, proliferation, and other cellular functions [23]. In parallel, SRSF1 overexpression promotes expression of isoforms that stimulate translation and cell proliferation, by increasing phosphorylation of translation activators, such as S6 or eIF4E, or by inhibiting translational repressors, such as 4EBP1 [15,36]. SRSF1 can also interact with and activate WNT signalling pathway to promote tumorigenesis [13]. However, whether SRSF1 participate in splicing events in MM is still unknown. In an attempt to determine the alternative splice events regulated by SRSF1 in MM, we performed RNA-seq upon SRSF1 depletion. Our study provided convincing evidence that SRSF1 played a significant role in regulating pre-mRNA splicing events, especially in the skipping exon category, in MM. Meanwhile, we also found that SRSF1 regulated some AS events of genes associated with cell killing, cell growth and RNA metabolic processes. In support, we found that SRSF1 regulated RNA splicing leading to tumour cell proliferation.
The evidence gathered from the present studies has placed SRSF1 as the key regulator in pre-mRNA splicing of MM, therefore, reveals exciting and important mechanistic insights into how transcription factor YY1 directly regulates SRSF1 transcription, further pointing to the critical role SRSF1 in the splicing regulation of genes participated in cell cycle regulation and apoptosis pathway.

DATA AVAILABILITY
The data generated in this study are publicly available in Gene Expression Omnibus (GEO) at GSE160724. Other detailed assays are available in the supplemental methods. The data supporting the findings of this study can be found in the article, or available from the corresponding author upon reasonable request.