Abstract
Gene expression profile has provided interesting insights into the disease biology, helped develop new risk stratification, and identify novel druggable targets in multiple myeloma (MM). However, there is significant impact of alternative pre-mRNA splicing (AS) as one of the key transcriptome modifier. These spliced variants increases the transcriptomic complexity and its misregulation affect disease behavior impacting therapeutic consideration in various disease processes including cancer.
Our large well annotated deep RNA sequencing data from purified MM cells data from 420 newly-diagnosed patients treated homogeneously have identified 1534 genes with one or more splicing events observed in at least 10% or more patients. Median alternative splicing event per patient was 595 (range 223 - 2735). These observed global alternative splicing events in MM involves aberrant splicing of critical growth and survival genes affects the disease biology as well as overall survival. Moreover, the decrease of cell viability observed in a large panel of MM cell lines after inhibition of splicing at the pre-mRNA complex and stalling at the A complex confirmed that MM cells are exquisitely sensitive to pharmacological inhibition of splicing.
Based on these data, we further focused on understanding the molecular mechanisms driving aberrant alternative splicing in MM. An increasing body of evidence indicates that altered expression of regulatory splicing factors (SF) can have oncogenic properties by impacting AS of cancer-associated genes. We used our large RNA-seq dataset to create a genome wide global alterations map of SF and identified several splicing factors significantly dysregulated in MM compared to normal plasma cells with impact on clinical outcome. The splicing factor Serine and Arginine Rich Splicing Factor 1 (SRSF1), regulating initiation of spliceosome assembly, was selected for further evaluation, as its impact on clinical outcome was confirmed in two additional independent myeloma datasets.
In gain-of (GOF) studies enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells; and conversely, in loss-of function (LOF) studies, downregulation of SRSF1, using stable or doxy-inducible shRNA systems significantly inhibited MM cell proliferation and survival over time. We utilized SRSF1 mutants to dissect the mechanisms involved in the SRSF1-mediated MM growth induction, and observed that the growth promoting effect of SRSF1 in MM cells was mainly due to its splicing activity.
We next investigated the impact of SRSF1 on allelic isoforms of specific gene targets by RNA-seq in LOF and confirmed in GOF studies. Splicing profiles showed widespread changes in AS induced by SRSF1 knock down. The most recurrent splicing events were skipped exon (SE) and alternative first (AF) exon splicing as compared to control cells. SE splice events were primarily upregulated and AF splice events were evenly upregulated and downregulated. Genes in which splicing events in these categories occurred mostly did not show significant difference in overall gene expression level when compared to control, following SRSF1 depletion. When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affects genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells.
We have observed increased levels of SRSF phosphorylation, which regulates it's subcellular localization and activity, in MM cell lines and primary patient MM cells compared to normal donor PBMCs. Moreover, we evaluated the chemical compound TG003, an inhibitor of Cdc2-like kinase (CLK) 1 and 4 that regulate splicing by fine-tuning the phosphorylation of SR proteins. Treatment with TG003 decreased SRSF1 phosphorylation preventing the spliceosome assembly and inducing a dose dependent inhibition of MM cell viability.
In conclusions, here we provide mechanistic insights into myeloma-related splicing dysregulation and establish SRSF1 as a tumor promoting gene with therapeutic potential.
Disclosures
Avet-Loiseau: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Munshi:OncoPep: Other: Board of director.
Cellular stressors may alter islet hormone cell proportions by moderation of alternative splicing patterns
