P3.03-001 Targeting Cullin Ubiquitin Ligase Leads to Growths Arrest in Malignant Pleural Mesothelioma Cells: Topic: Mesothelioma Transitional

Abstract

Mutation of the tumor suppressor gene NF2 was detected in 30-40% of malignant pleural mesothelioma (MPM) patients. NF2 suppresses tumorigenesis in part by inhibiting Cullin4 ubiquitin ligase. Cullin4A (CUL4A) gene amplification and its’ overexpression has been detected in MPM cell lines and tumors. We hypothesized that cullin4 is a potential treatment target for MPM. Cullins’ activity can be blocked by the inhibition of neddylation, a post-translational modification for cullins. In this study we assessed the efficacy of pevonedistat, an inhibitor of protein neddylation. Thirteen MPM cell lines and 3 MPM primary cells grown in monolayer (2D) were employed to assess the efficacy of pevonedistat in vitro compared to normal mesothelial cells, using MTT assay. The expression of cullins was assessed by quantitative real time PCR and western blot. Cell cycle was analyzed by flow cytometry. Four cell lines were cultured in multicellular spheroid (3D) format and measured for viability by acid phosphatase assay. Five MPM cell lines overexpressing CUL4A are highly sensitive to pevonedistat (figure1). The treatment induced G2 cell cycle arrest and accumulation of cells containing >4N DNA content, representing cells undergoing DNA re-replication. DNA re-replication is known to be mediated by the accumulation of a DNA replication licensing factor, CDT1. Indeed, higher CDT1 accumulation was detected in the sensitive compared to the resistant cell lines. All primary cells showed no CUL4A overexpression compared to normal mesothelial cells, nonetheless 2 of them were sensitive to pevonedistat. Interestingly, these cells exhibited higher levels of neddylated (activated) CUL4A and higher CTD1 accumulation following the treatment. Cells lines overexpressing CUL4A remained sensitive to pevonedistat when grown in 3D spheroids. Inhibition of cullins by pevonedistat induced growth arrest preferentially in MPM cells overexpressing CUL4A in 2D and 3D cultures. The major mechanism seems to be mediated by DNA re-replication induced by CDT1 accumulation.