The Ras family of proteins contain four major isoforms, and altogether these proteins are constitutively activated in a third of cancers. In the past decade, inhibitors to mutant Ras (RasG12C) have been developed, but most patients administered RasG12C inhibitors (RasG12Ci) relapse. Interestingly, these Ras inhibitor resistant tumors have Ras signaling reactivated and the signaling mechanisms underlying this drug resistance are unknown. To uncover these drug resistance mechanisms, I developed Ras activity sensors and Ras activity dependent proximity labelers, applied them to RasG12C-addicted cancer cells treated with RasG12Ci, and observed that RasG12Ci blocked mutant Ras signaling at the plasma membrane while wildtype (WT) Ras is activated at endomembranes to fuel oncogenic signaling and cell growth. While these results are preliminary as these studies were done in 2D cell culture and do not delineate which particular Ras isoforms enable RasG12Ci resistance, these exciting findings beg the question of whether cancer cells can evade other recently developed Ras inhibitors targeting RasG12C, G12D, G12R, or G12S by also reactivating Ras signaling. Therefore, the objective of this K99/R00 proposal is to expand the molecular toolkit for Ras and utilize these tools to profile and uncover the molecular mechanisms driving Ras inhibitor resistance. The central hypothesis driving this work is that WT Ras compensation for mutant Ras inhibition is a general feature cancer cells employ to evade Ras inhibitors. Profiling the subcellular Ras activities during Ras inhibitor treatment and uncovering the molecular components driving this reorganization of Ras signaling will allow better understanding of Ras inhibitor resistance and illumination of new therapeutic targets. To investigate this hypothesis, the following specific aims will be addressed: (1) Developing and applying Ras sensors in complex cancer cell models (K99); (2) De novo design of Ras isoform selective tools (K99/R00); and (3) Profiling and dissecting the mechanisms underpinning Ras inhibitor resistance (R00). In the proposed research, I will protein engineer current and new Ras tools (sensors, proximity labelers, perturbators) along with microscopy and proteomic techniques to determine how Ras inhibitors impact compartmentalized Ras signaling. The expected outcomes are (1) an expansion of tools that can be applied to in vivo models and probe specific Ras isoforms and (2) a better understanding of how Ras inhibitors operate and how drug resistance can occur. Of note, I believe these new Ras tools will be of great interest to the cancer community (e.g. NCI’s Ras initiative) and can be useful for other applications beyond the scope of this proposal such as diagnostics and therapeutics. Towards completion of the proposed work, I will be trained in protein design methods and complex cancer models and guided by an advisory committee composed of experts in cancer, Ras signaling, and cell culture. The long-term goal of this project is to develop an independent research program that bridges protein design with cancer cell biology to understand how oncogenic signaling pathways rewire themselves during oncogenesis and drug resistance.

The proposed research is relevant to public health as it will lead to better understanding of the etiology and drug resistance of Ras-driven cancers (a third of all cancers). By developing tools to profile, track, and perturb specific Ras isoforms in physiologically relevant cancer models, I propose to use these tools to understand how cancer cells re-organize Ras signaling during Ras inhibitor treatment to reactive oncogenic cellular programs. This work will provide critically needed tools and address how compartmentalized oncogenic signaling can dynamically adapt during drug treatment, thus is well-suited to meet the priorities outlined by NCI.