# De novo designed Ras tools to uncover the mechanisms underlying drug resistance

> **NIH NIH K99** · UNIVERSITY OF WASHINGTON · 2024 · $109,906

## Abstract

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 lo...

## Key facts

- **NIH application ID:** 10947746
- **Project number:** 1K99CA293001-01
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Jason Zhang
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $109,906
- **Award type:** 1
- **Project period:** 2024-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10947746

## Citation

> US National Institutes of Health, RePORTER application 10947746, De novo designed Ras tools to uncover the mechanisms underlying drug resistance (1K99CA293001-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10947746. Licensed CC0.

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