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Fluorescent Biosensor for Measuring Ras Activity in Living Cells
Ryan Weeks1,2, Xin Zhou1, Tina L Yuan3
1Department of Pharmacology, University of California, San Diego, La Jolla, California 92093, United States.
Abstract:
The small GTPase Ras is a critical regulator of cell growth and proliferation. Its activity is frequently dysregulated in cancers, prompting decades of work to pharmacologically target Ras. Understanding Ras biology and developing effective Ras therapeutics both require probing Ras activity in its native context, yet tools to measure its activities in cellulo are limited. Here, we developed a ratiometric Ras activity reporter (RasAR) that provides quantitative measurement of Ras activity in living cells with high spatiotemporal resolution. We demonstrated that RasAR can probe live-cell activities of all the primary isoforms of Ras. Given that the functional roles of different isoforms of Ras are intimately linked to their subcellular distribution and regulation, we interrogated the spatiotemporal regulation of Ras utilizing subcellularly targeted RasAR and uncovered the role of Src kinase as an upstream regulator to inhibit HRas. Furthermore, we showed that RasAR enables capture of KRasG12C inhibition dynamics in living cells upon treatment with KRasG12C covalent inhibitors, including ARS1620, Sotorasib, and Adagrasib. We found in living cells a residual Ras activity lingers for hours in the presence of these inhibitors. Together, RasAR represents a powerful molecular tool to enable live-cell interrogation of Ras activity and facilitate the development of Ras inhibitors.
Insights
Scientists developed RasAR, a new tool to measure Ras protein activity in living cells. This reporter helps understand Ras regulation in cancer and evaluate new cancer drug effectiveness.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Ras GTPases are key regulators of cell growth, and their dysregulation is common in cancer.
- Targeting Ras is a major goal in cancer therapy, but requires understanding its activity in living cells.
- Current methods for measuring Ras activity in cells are limited.
Purpose of the Study:
- To develop a novel reporter for quantitative, real-time measurement of Ras activity in living cells.
- To investigate the spatiotemporal regulation of Ras isoforms and identify upstream regulators.
- To assess the dynamics of Ras inhibition by covalent KRasG12C inhibitors.
Main Methods:
- Development of a ratiometric Ras activity reporter (RasAR) for live-cell imaging.
- Utilizing subcellularly targeted RasAR to study Ras isoform regulation.
- Monitoring Ras activity in real-time following treatment with KRasG12C inhibitors.
Main Results:
- RasAR enables quantitative, high-resolution measurement of Ras activity across all primary isoforms.
- Src kinase was identified as an upstream inhibitor of HRas.
- RasAR revealed persistent Ras activity for hours even with KRasG12C inhibitors like Sotorasib.
Conclusions:
- RasAR is a versatile tool for interrogating Ras activity in live cells.
- The reporter facilitates understanding of Ras biology and development of Ras-targeted cancer therapies.
- Findings highlight the need for strategies to overcome residual Ras activity during inhibitor treatment.
