Abstract 1727: Cytoplasmic EZH2 and the DLC1 tumor suppressor are regulated by the CRM1 nuclear export protein and by specific complex formation between mutant KRAS and RAN-GAP
Authors: Tripathi Brajendra , Hirsh Nicole , Hansen David , Qian Xiaolan , Wang Dunrui , Durkin Marian E. , Lim Ashley , Papageorge Alex G. , Ramalingham Suresh S. , Marcus Adam I. , Lowy Douglas R. 
Published: April 2023

Abstract
The DLC1 tumor suppressor gene encodes a cytoplasmic RHO-GAP protein whose expression is frequently decreased in many cancer types, especially lung cancer. We have identified several cancer-associated post-translational modifications in the DLC1 protein that decrease its tumor suppressor activity and are exploring the upstream mechanisms that regulate the changes as well as the therapeutic potential of reactivating the tumor suppressor activity of DLC1 by inhibiting the post-translational changes. We have made the unexpected observation that lung cancer expresses increased levels of EZH2 in the cytoplasm, where it can methylate DLC1, which can trigger the ubiquitin-proteasomal-dependent degradation of DLC1. The cytoplasmic EZH2 is derived from the nucleus, and inhibition of the CRM1 nuclear export protein or the EZH2 methyl transferase activity can drastically reduce the level of cytoplasmic EZH2 or its activity, which increases the half-life and steady-state level of DLC1 protein several fold. The stabilized DLC1 protein can be reactivated by inhibiting the AKT and SRC kinases, whose phosphorylation of DLC1 can attenuate its tumor suppressor function. A three-drug combination of an EZH2 or a CRM1 inhibitor, together with AKT and SRC kinase inhibitors, can greatly reduce the weight of tumor xenografts from human lung cancer cell lines and induce markers of apoptosis and senescence. Remarkably, isogenic knock-out of DLC1 abolishes most of the anti-tumor activity, implying that DLC1 is the main target of the three-drug treatment, although each inhibitor has many other targets. In addition, we have found that inhibiting endogenous mutant KRAS in the lung cancer lines can phenocopy the CRM1 inhibitor, leading to loss of cytoplasmic EZH2 and increasing the level of DLC1 protein. Conversely, mutant KRAS increases the level of cytoplasmic EZH2 and decreases DLC1. We have investigated the underlying mechanism, and have determined that mutant KRAS regulates nucleocytoplasmic protein shuttling by forming a previously unidentified complex with RAN-GAP (present in primary human tumors and cell lines), which regulates the RAN GTPase, a protein that is required for other proteins to shuttle from the cytoplasm to the nucleus and from the nucleus to the cytoplasm. Formation of the RAS/RAN-GAP complex and its downstream consequences are independent of known RAS signaling from the plasma membrane, as inhibition of MAPK and PI3K does not affect this process. However, efficient complex formation does depend on farnesylation of RAS, as farnesylation-deficient mutants form the complex less efficiently and have reduced consequences for EZH2 and DLC1. Thus, we have identified nucleocytoplasmic protein shuttling as a new function for mutant KRAS and have determined the mechanism depends on complex formation between KRAS and RAN-GAP.
Citation Format: Brajendra Tripathi, Nicole Hirsh, David Hansen, Xiaolan Qian, Dunrui Wang, Marian E. Durkin, Ashley Lim, Alex G. Papageorge, Suresh S. Ramalingham, Adam I. Marcus, Douglas R. Lowy. Cytoplasmic EZH2 and the DLC1 tumor suppressor are regulated by the CRM1 nuclear export protein and by specific complex formation between mutant KRAS and RAN-GAP [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1727.