Abstract 4554: Downstream effects of rigosertib (ON 01910.Na) in cancer cells involves impairment of protein translation via eIF2 and eIF4

Abstract

Abstract Purpose: Rigosertib, a novel synthetic styryl benzyl sulfone, is cytotoxic against a variety of human tumor cell lines in vitro and in vivo. Significantly, rigosertib is currently being tested in a randomized phase III clinical trial in patients with higher risk myelodysplastic syndrome (MDS) who have failed treatment with hypomethylating agents. Previous studies have shown that rigosertib interacts with both the phosphoinositide 3-kinase (PI3K) and polo-like kinase (PLK1) pathways. Because these pathways can affect several cellular functions, we used a combination of unbiased global proteomics and interactomics approaches to assemble a rigosertib target profile. Here, we confirm and elaborate the pathways modulated by rigosertib. Experimental Design: Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics was used to identify the differentially expressed proteins in HeLa cells with and without 8 hours of treatment with 10 μM rigosertib. In addition, rigosertib protein interaction (interactomics) studies were performed to validate the proteomics observations. Results: Using a combination of SILAC and subcellular fractionation we were able to quantify more than 1500 proteins, among which 125 were significantly altered by rigosertib treatment. Quantified proteins were selected and clustered by biological function. We performed pathway and network analysis using Ingenuity® Systems computational pathway analysis. The pathways that were identified at statistically significant levels (p < 0.05) were eukaryotic initiation factor (eIF2) signaling, eIF4 signaling and nuclear erythroid factor 2 (Nrf2)-mediated oxidative stress response. In total, 19 proteins were down-regulated and 2 proteins were up-regulated in the eIF2 signaling pathway, suggesting that rigosertib represses protein translation in tumor cells. These observations have been noted in other experimental systems using different experimental approaches. To validate these findings and to determine extent of interaction of these proteins with rigosertib, we used drug affinity pull-down followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis to confirm that rigosertib interacts with eIF2 and eIF4 via Raf and p70S6K, respectively. Conclusions: Collectively, these results demonstrate that a terminal effect of rigosertib in cancer cells is the inhibition of protein translation mediated by eIF2 and eIF4. Citation Format: Salim Merali, Oscar Perez-Leal, Carlos Barrero. Downstream effects of rigosertib (ON 01910.Na) in cancer cells involves impairment of protein translation via eIF2 and eIF4. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4554. doi:10.1158/1538-7445.AM2014-4554