Abstract 2089: A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications

Abstract The majority of RAS oncogenic activity is thought to be mediated by its canonical signaling, mainly via RAS-GTP interactions with its effectors RAF and PI3K at the plasma membrane, which respectively activate MEK/ERK and AKT/mTOR pathways. However, in this study, we have identified a new, fundamental, pro-oncogenic, non-canonical activity of RAS-GTP that increases the Exportin 1 (XPO1)-dependent export of nuclear proteins into the cytoplasm and is independent of RAF/MEK and PI3K/AKT signaling. The nuclear protein export machinery is known to depend on a trimeric protein complex composed of RAN-GTP:XPO1:nuclear protein cargo. Unexpectedly, we have found that RAS-GTP forms a stable complex with RAN GTPase-Activating Protein 1 (RAN-GAP) at the nuclear envelope, which facilitates the release of nuclear protein cargo by increasing the hydrolysis of RAN-GTP to RAN-GDP. We have determined that RAS-GTP and RAN-GAP form this complex in primary human lung tumors, patient derived xenografts, and human lung cancer cell lines. Interestingly, the RAS-GTP:RAN-GAP complex is found at the nuclear envelope and in the cytoplasm, but not at the plasma membrane. The RAS-dependent step mediated by the RAS-GTP:RAN-GAP complex acts downstream of XPO1 by increasing RAN-GTP hydrolysis, which promotes protein cargo release. Our fundamental findings have important clinical implications. For example, in human lung cancer models that harbor a KRAS-G12C mutation, the combined inhibition of MEK and PI3K (MEKi + PI3Ki) does not inhibit tumor growth to the same degree as a KRAS-G12C inhibitor. Remarkably, adding an XPO1 inhibitor to MEKi + PI3Ki inhibits tumor growth to the same degree as the KRAS-G12C inhibitor. One critical pro-oncogenic target of the RAS-GTP:RAN-GAP complex is nuclear export of the EZH2 methyltransferase, whose increase in the cytoplasm leads to a direct methylation of the cytoplasmic DLC1 tumor suppressor protein, leading to ubiquitin-dependent proteasomal degradation of DLC1 protein. Analysis of a KRAS mutant cell line from which the DLC1 gene has been disrupted by CRISPR-Cas9 technology indicates that DLC1 makes an important contribution to the growth inhibition by inhibitors that reduce nuclear protein export. These findings establish nuclear protein export as a critical, pro-oncogenic, non-canonical RAS function that is mediated by the RAS-GTP:RAN-GAP complex and provide a mechanistic explanation for poor clinical responses to combined inhibition of MEK and PI3K, which do not regulate the nuclear protein activity identified in this study. Citation Format: Brajendra K. Tripathi, Nicole H. Hirsh, David Hansen, Xiaolan Qian, Marian E. Durkin, Dunrui Wang, Alex G. Papageorge, Ross Lake, Yvonne A. Evrard, Adam Marcus, Suresh S. Ramalingam, Mary Dasso, Karen Vousden, James H. Doroshow, Kylie J. Walters, Douglas R. Lowy. A pro-oncogenic RAS-GTP:RAN-GAP complex facilitates nuclear protein export and has clinical implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2089.