Abstract
Introduction: A fundamental role of RNA splicing in human biology and its relevance in cancer is rapidly emerging, evidenced by spliceosome mutations that determine the prognosis of patients with hematologic malignancies. Highly recurrent alterations in genes encoding components of the spliceosome are the most unexpected and dramatic findings in the cancer genome.
Methods and Results: In Multiple myeloma (MM), our ongoing studies (IFM/DFC 2009, >420 patients) demonstrate that splice variant expression can predict MM patient survival outcome better than gene expression alone, implicating a significant role of splicing mechanisms in MM pathophysiology (ASH 2015, 2016). In this cohort, splicing alterations were recurrent in >30% patients, and the predominant (~32%, 495 of 1534) recurrent alterations were intron retentions. To identify the causes of aberrant splicing in MM, patients in the IFM/DFCI cohort were evaluated with respect to their deregulated expression of genes encoding splicing proteins involved at different stages of the splicing process. In this cohort, several core splicing factors and small nuclear ribonucleoproteins (snRNPs) were significantly deregulated (up- or down-regulated), and their expression correlated with patient survival. These findings suggest a role of altered splicing in MM pathophysiology.
Since we observed intron retentions as a predominant recurrent splicing alteration in MM patients, we focused on PTBPs (PTBP1, 2, and 3) proteins that play a critical role in intron excision as lariat structures, before mature transcripts are generated and transported into the cytoplasm. Expression levels of the PTBPs were evaluated in PC from 20 MGUS (monoclonal gammopathy of undetermined significance), 33 SMM (smoldering MM), 41 MM patients, and 5 healthy donor samples (NPC); this cohort is an independent cohort previously published (GDS4968). We observed progressively increased deregulation of PTBPs in clonal PCs from MGUS, SMM, and symptomatic MM, compared to NPC. PTBP1 showed a progressive increase, whereas PTBP3 transcripts exhibited progressive downregulation, with progression from NPC to MM (Fig 1). PTBP1 and PTBP2 expression remained the same in NPC and SMM, with a distinct differential expression of these transcripts with progression to active MM. PTBP2 transcripts were significantly upregulated in symptomatic MM. These findings were validated at the transcript and protein level in 8 MM patients and 10 MM cell lines, and consistent with differential expression of PTBPs in large patient cohorts (n=94 or 420) . A significant upregulation of PTBP2 at the protein level was also detected different levels in MM cell lines (RPMI8226, H929, OPM2 and U266). These data suggest that deregulated expression of PTBPs may play a role in the malignant transformation in MM.
To determine whether overexpression of PTBPs are transforming, we next expressed PTBPs in HEK293 cells. One week after transfection, these cells formed foci in conventional cultures, which were even more distinct in 3D cultures (Fig 2). We also expressed PTBP2 in a RPMI 8226 MM cell line, which expresses only endogenous PTBP2. Like HEK293T cells, RPMI8226 MM cells expressing PTBP2 formed colonies in conventional culture. We also detected increased proliferation of RPMI8226 MM cells overexpressing PTBP2, which was even more evident after co-culturing RPMI8226-PTBP2 with MM patient derived stromal cells. These results suggest that PTBPs may increase cell proliferation, enhance anchorage-independent cell-growth, and have transforming potential, as is observed with PTBP1 overexpression in solid tumors. To evaluate effects of PTBP2 on splicing in the context of MM microenvironment we monitored Gene A splicing in MM cell lines alone and co-cultured with patient derived stroma. We observed that expression of Gene A novel splice variants induced in MM cell lines expressing higher levels of PTBP2, and co-cultured with stromal cells derived from samples taken at diagnosis.
Conclusions: PTBPs represent a target for novel therapeutics directed to normalize the splicing signature in MM cells. Our ongoing studies are not only developing novel therapeutics to modulate splicing in tumor cells, but also identifying splicing alterations in MM microenvironment associated with PTBP deregulation in MM cells. Splice variants of surface proteins on MM stromal cells may represent targets for novel immunotherapies.
Hideshima: Acetylon: Consultancy; C4 Therapeutics: Equity Ownership. Anderson: MedImmune: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees.
