Alternative splicing (AS) is a critical post-transcriptional event, which affects the number of cellular processes. Aberrant splicing of some genes has been reported in multiple myeloma (MM). However, to date, whole-transcriptome-wide AS study has not been performed. We used deep RNA-sequencing data from 16 normal plasma cells (NPC) and 360 newly-diagnosed MM patients to describe the landscape of the alternative splicing events and the molecular mechanisms driving aberrant AS in MM. Global splicing analysis showed that mutually exclusive exon (MXE) (n=510) and Skipped Exon (SE) (n=417) are the most frequent splicing events in MM compared to NPC. Among these events, ~54% were observed in genes which are not differentially expressed between MM and NPC and 46% of the AS events (SE, MXE, retained intron, alternative 3'/5' sites) were observed in differentially expressed genes targeting 203 unique genes. AS affected RNA transcription regulation genes such as IKZF1, IKZF3, and key regulatory elements in MM including, IRF3, IRF4, or key transcription factors such as MEF2C, XPB1, STAT2, and ILF3. In general, AS targetted DNA replication, cell cycle, and apoptosis pathways. MM subgroups showed a heterogeneity for AS events. Monosomy 14, t(4;14), del1p and del17p had the highest number of unique (not observed in other subgroups) AS events compared to NPC.To understanding the molecular mechanisms driving aberrant alternate splicing we next investigated115 splicing factors (SF) in MM and associated them with AS events. We observed that ~40% of SF were dysregulated (dysregulated expression and/or copy number alteration) in MM compared to NPC, including SRSF, PCBP and RBM families. To understand the key binding regions, we have performed SF binding motif enrichment analysis around AS events and found that SRFS1, SRSF9, and PCB1 motifs to be enriched among the splicing events. Importantly, SRSF1 expression was linked with survival in two independent MM datasets.We therefore explored functional role of SRSF1 in MM with perturbation studies. While upregulation of SRSF1 expression significantly increased the cell growth and survival, conversely downregulation of SRSF1 inhibited the both. To dissect the mechanisms of SRSF1-mediated MM growth induction, we utilized SRSF1 mutants lacking either of the 2 RNA-recognition motifs or the serine/argine-rich C-terminal domain involved in protein-protein interactions, and recruitment of spliceosome components. We also utilized 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. These studies suggested that SRSF1-regulated AS effects MM cell proliferation. We surprisingly also found that even NRS1 mutant failed to promote MM growth, suggesting an important role of cytoplasmic SRSF1 in promoting MM cells proliferation.We next investigated alternative splicing pattern changes induced by SRSF1 knock down.When analyzing cellular functions of SRSF1-regulated splicing events, we found that SRSF1 knock down affect's genes in the RNA processing pathway as well as genes involved in cancer-related functions such as mTOR, E2F and MYC-related pathways. Splicing analysis was corroborated with immunoprecipitation (IP) followed by mass spectrometry (MS) analysis of T7-tagged SRSF1 MM cells.Finally, using genome wide chromatin and transcription landscape mapping techniques, we have found SRSF1 to be under the transcriptional control of oncogenic E2F1 in MM cells. Consistent with these findings, we observed greater in vitro loss of viability in a large panel of MM cell lines compared with PBMCs from healthy volunteers, following exposure to the splicing modulator pladeniolide. In summary, this study for the first time reports a detailed splicing landscape in myeloma and highlights the biological and clinical importance of alternative splicing events. Moreover, these results indicate a functional role and clinical significance of a gene involved in regulation of alternate splicing in MM, highlighting the need to further understand the splicing pattern in myeloma initiation and progression.
Disclosures
Anderson: Takeda: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Oncopep: Other: Scientific Founder; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: Other: Scientific Founder. Avet-Loiseau:takeda: Consultancy, Other: travel fees, lecture fees, Research Funding; celgene: Consultancy, Other: travel fees, lecture fees, Research Funding. Munshi:Adaptive: Consultancy; Abbvie: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Amgen: Consultancy; Celgene: Consultancy.
Molecular Mechanisms of Cereblon-Interacting Small Molecules in Multiple Myeloma Therapy