Functional and Clinical Impact of Splicing Factor Dysregulation in Multiple Myeloma

Transcriptome modifiers such as alternative pre-mRNA splicing (AS), long non-coding RNA and microRNA (miRNA) need to be considered in order to provide a more accurate genomic framework for clinical correlation, as well as for high value therapeutic target discovery. Aberrant splicing of numerous genes has been reported in other malignancies, including a small number of genes reported in MM. We have evaluated AS in MM by analyzing clinically annotated high throughput RNA-seq data from 410 newly-diagnosed patients and 18 normal donor plasma cells. We observed a profound and significant AS in MM with over 600 genes showing significant changes in relative isoform abundances (isoform switching) between MM and normal samples. Importantly, unsupervised analysis identified clinically relevant MM subgroups with high and low splicing index respectively and showed significant impact of alternate splicing on overall clinical outcome. Based on these data, we next focused on understanding the molecular mechanisms driving aberrant alternate splicing in myeloma. Several studies provide evidence that an abnormally expressed splicing factor (SF) can have oncogenic properties by impacting alternative splicing of cancer-associated genes.

We detected dysregulated expression of several SFs, including SF3B1, Fox2, SRSF1, NONO, in patients with MM compared to normal plasma cells with impact on outcome, highlighting for the first time the prognostic significance of splicing related factors in myeloma. We further observed that overexpression of some of these SFs increased cell proliferation, enhanced anchorage independent growth in semi-solid medium, and affected tumorigenic potential. We have further investigated role of Serine/Arginine Splicing Factor 1 (SRSF1) in MM by gain of- and loss of- function studies. Enforced expression of SRSF1 in MM cells significantly increased proliferation, especially in the presence of bone marrow stromal cells. Conversely, transient or stable downregulation of SRSF1 with specific siRNA and shRNAs respectively, significantly inhibited MM cell proliferation and cell survival. We have also investigated a small molecular inhibitor of SRSF1 (TG003) and observed inhibition of MM cell growth and survival. The impact of this inhibitor on allelic isoforms of specific gene targets is undergoing.

To dissect the mechanisms involved in the SRSF1-mediated MM growth induction, we used SRSF1 mutants lacking either of the two RNA-recognition motifs (ΔRRM1 or ΔRRM2 mutants) or the serine/argine-rich C-terminal domain (ΔRS mutant) involved in protein-protein interactions, subcellular localization, and recruitment of spliceosome components. We also used a C-terminal fusion of SRSF1 with the nuclear-retention signal of SRSF2 (NRS1 mutant), to force SRSF1 retention in the nucleus and assess the role of its nuclear versus cytoplasmic functions. We surprisingly found that only NRS1 mutant failed to promote MM growth, suggesting an important role of cytoplasmic SRSF1 in promoting MM cells proliferation.

Finally, using genome wide chromatin and transcription landscape mapping techniques, we have found SRSF1 to be under the transcriptional control of E2F1, a transcription factor with significant impact on MM cell growth and survival. A significant reduction in SRSF1 at mRNA and protein levels was observed after E2F1 and/or E2F1 heterodimerization partner Dp1 gene silencing. Moreover, peptide-based strategy to abrogate interaction between Dp1-E2F1 led to decreased SRSF1 expression levels.

These results indicate a functional role and clinical significance of a gene involved in regulation of alternate splicing in MM. The study highlights the need to further understand the splicing pattern in myeloma and also supports the emerging concept that splicing programs, together with transcriptional programs participate in the altered cellular function during tumor initiation and progression.