Background: Diffuse malignant peritoneal mesothelioma (DMPM) is an aggressive tumor of the lining of the abdomen, characterized by late clinical symptoms and poor prognosis. Systemic chemotherapy together with cytoreductive surgery and intraperitoneal hyperthermic therapy has been introduced as the best treatment option, resulting in an overall 5-year survival rate of approximately 50%. To further increase survival, novel drugs targeting key molecular factors in DMPM are warranted. Analogous to pleural mesothelioma, such a factor may be alternative splicing which can be modulated by inhibiting the SF3B subunit of the spliceosome. This strategy is an emerging therapeutic opportunity for a number of solid tumors and hematological malignancies. In particular, the spliceosome inhibitor Pladienolide B (PB) has low nanomolar IC50 values against a range of cancer cell lines and leukemic cells, but no data are available regarding its antitumor efficacy in DMPM.

Aims: This study investigates (1) the activity of PB (alone or in combination with standard chemotherapeutics) in primary peritoneal mesothelioma cells through in vitro and in vivo assays, and (2) the molecular mechanisms of splicing inhibition through whole-genome RNA-seq and PCR of selected genes involved in apoptosis and invasion.

Methods: The antiproliferative effect of PB was investigated using the SRB assay on two primary mesothelioma cell cultures (MESOII and STO), obtained from resected tumors with well-annotated clinical characteristics. Further in vitro studies were performed to evaluate the pro-apoptotic and anti-invasive activities, while splicing profiles of treated and untreated cells were determined with RNA-seq.

Results: PB impaired DMPM cell growth in a dose-dependent manner, with IC50 values of 1.57 ± 0.30 nM in MESOII and 1.18 ± 0.16 nM in STO (n = 3, mean ± standard deviation).

The specific activity on the spliceosome was demonstrated by PB induced time- and dose-dependent alterations of splicing patterns for several apoptotic genes, such as Mcl-1, Bcl-X, Fas, and for the pro-metastatic tyrosine kinase receptor RON, which was shifted to its un-spliced and non-functional variant. In addition, RNA-seq showed several differentially expressed alternatively spliced genes in PB treated samples. The DMPM cells have also been genetically engineered to express Firefly- and Gaussia- luciferases, enabling monitoring of tumor growth inhibition by PB in in vivo orthotopic models.

Conclusions: These data provide evidence that PB has a strong antitumor activity against relevant models of DPMP, associated with modulation of splicing, induction of apoptosis and inhibition of invasion. RNA-seq represents a powerful tool for the identification of alternatively spliced genes that could serve as useful diagnostic markers as well as potential therapeutic targets for DMPM.

Citation Format: Rocco Sciarrillo, Valentina E. Gomez, Marzia Pennati, Anna Wojtuszkiewicz, Gert-Jan L. Kaspers, Carla Molthoff, Nadia Zaffaroni, Godefridus J. Peters, Gerrit Jansen, Elisa Giovannetti. Spliceosome inhibition as novel strategy against diffuse malignant peritoneal mesothelioma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 332.