CETSA profiling unveils novel targets engaged by anti-tumor drug rigosertib to inhibit RAS-MAPK signaling and trigger NLRP3 inflammasome activation

Abstract Background: The importance of styryl benzyl sulfones that induce G2/M cell cycle arrest and apoptosis in tumor cells has raised interest in cancer therapeutics. Rigosertib (ON 01910.Na) has been characterized as a disruptor of multiple signaling pathways including RAS-MAPK signaling through multiple mechanisms. The aim of our study was to further elucidate the mechanism of action and to identify potential novel targets engaged by rigosertib. Methods: Thermal shift, NanoBiT, in vitro tubulin polymerization and a variety of biochemical, molecular and cell biological assays were performed to characterize the binding of rigosertib to previously proposed targets and its effect on RAS-MAPK signaling and tumor cell survival. A mass spectrometry-based Cellular Thermal Shift Assay (CETSA-MS) that serves as a key tool to identify the targets in the entire proteome engaged by chemical ligands or small molecules of interest at physiological levels in cells, was conducted in multiple cell types to identify potential new protein targets of rigosertib. Results: Our data suggest that rigosertib exerts its inhibitory effect on RAS-MAPK signaling through ROS induced activation of JNK signaling. While we confirm that rigosertib might affect microtubules at higher concentrations, the main cellular target(s) responsible for the induction of stress and JNK-mediated inhibition of RAS-RAF-MEK signaling needs to be further characterized. By performing comparative CETSA profiling of rigosertib and colchicine, a classical anti mitotic drug that binds to soluble tubulin, we are able to identify unique targets of rigosertib in multiple tumor cell types. In particular, we identify two ROS-related proteins, ERO1A and NQO2 that potentially contribute to the induction of ROS-dependent JNK activation. NEK7, a relevant protein for microtubule organization and essential for NLRP3 inflammasome activation, is another potential target for rigosertib. Indeed, rigosertib activates NLRP3-dependent inflammatory responses via NEK7 and Caspase-1 to trigger IL-1β secretion. Conclusion: These results suggest that rigosertib may represent an effective compound for inhibition of RAS-MAPK signaling through ROS-mediated JNK activation. Our findings reveal a rigosertib-dependent mechanism for NLRP3 activation that might be the cause of rigosertib synergistic effect with immune checkpoint blockers to induce cell death in advanced KRAS-mutated non-small cell lung cancer patients. This MOA suggests rigosertib can modulate the mutated RAS pathway no matter the mutation present, now confirmed with clinical data. Further clinical trials in a number of RAS mutated cancers are continuing. Citation Format: Petros Kechagioglou, Camille Dupont, Hajime Yurugi, Alexey Chernobrovkin, Kristina Riegel, Stephen Cosenza, Steven M. Fruchtman, Krishnaraj Rajalingam. CETSA profiling unveils novel targets engaged by anti-tumor drug rigosertib to inhibit RAS-MAPK signaling and trigger NLRP3 inflammasome activation [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr A020.