Abstract LB-108: Targeting the Ras-Binding domain of RAS effector proteins by a small molecule inhibitor, Rigosertib
Authors: Divakar SaiKrishna , Caprio Rodrigo Vasqez-Del , Baker Stacey J. , Reddy M.V. Ramana , Ritt Daniel A. , Morrison Deborah K. , Reddy E. Premkumar 
Published: May 2015

Oncogenic activation of RAS genes via point mutations occur in more than 30% of all human cancers, and investigations aimed at understanding the biochemical and biological mechanisms that are critical for the function of these oncogenic proteins is at the forefront of cancer research. Studies have shown that RAS interacts with a large number of effector proteins by a highly conserved mechanism that involves the switch region of RAS and the RAS-binding domains (RBDs) of its effector proteins. Because these interactions play a critical role in oncogenic RAS function, inhibiting this interaction constitutes an attractive and important therapeutic approach. Rigosertib is a novel Styryl Benzyl Sulfone which has completed Phase III clinical trials for Myelodysplastic Syndrome (MDS) and is currently in Phase II trials for Head and Neck cancer. In this abstract, we present evidence that rigosertib interacts with the RBDs of several RAS effector proteins, including RAF, the PI3K family of proteins as well as RalGDS, and blocks their interaction with RAS. We demonstrate that rigosertib binds to the RBDs of various effector proteins using Differential Scanning Fluorimetry (DSF), a thermal-denaturation assay that measures the thermal stability of a target protein and a subsequent increase in protein melting temperature due to the binding of a ligand to the protein A consequence of inhibiting RAS binding to RAF appears to be a block in growth factor-induced activation of RAF kinase activity in vivo. We also show that a result of this block in RAS/RAF interactions is an inability of RAF proteins to form dimers and activate MEK and ERK. This block in the activation of MEK/ERK pathways can be seen in cells that express wild-type RAS and RAF proteins (HeLa), in cells that express a constitutively active form of oncogenic RAS (HeLa-N-RAS-G12D) or in cells that exhibit amplification of EGF receptors (A431). Vemurafenib, an ATP-competitive RAF kinase inhibitor, failed to inhibit the MEK/ERK pathway in all three cell types. Rigosertib also inhibits the phosphorylation of c-RAF serine 338, which has been shown to be essential for the activation of its kinase activity and for its association with and activation of PLK-1. Our results showing that rigosertib-mediated inhibition of PLK-1/RAF interaction could explain the ability of this compound to induce mitotic arrest of human tumor cells.

In addition to the RAF family of proteins, we also demonstrate the binding of rigosertib to the RBDs of the PI3K family of kinases as well as RalGDS, both of which constitute important effectors of RAS. A consequence of the interaction of rigosertib with the RBD domains of PI3Ks appears to be a block in growth factor-induced AKT activation in cells treated with rigosertib. These studies suggest that rigosertib is a first-in-class molecule that targets the multiple RAS-driven signaling pathways by binding to the RBD of Ras effector proteins leading to their inactivation.

Citation Format: SaiKrishna Divakar, Rodrigo Vasqez-Del Caprio, Stacey J. Baker, M.V. Ramana Reddy, Daniel A. Ritt, Deborah K. Morrison, E. Premkumar Reddy. Targeting the Ras-Binding domain of RAS effector proteins by a small molecule inhibitor, Rigosertib. [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 LB-108. doi:10.1158/1538-7445.AM2014-LB-108