From Atoms to Organoids: Understanding the Conformational Landscape and Regulation of Small GTPases

/Abstract In the United States alone, over 250,000 people suffer from Small GTPase (smG) dysfunction linked to cancer, and this number scales to 3.4 million people worldwide. The smG proteins interconvert between the GDP-bound inactive form and the GTP-bound active form, where the active forms bind to effector proteins to trigger downstream signaling. Regulatory proteins such as GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) assist most smG in completing their catalytic cycles. Unlike other smG, RhoA has a unique regulatory process where the Guanine Dissociation Inhibitor (GDI) protein binds the GDP-bound inactive state of RhoA, sequestering it in the cytosol. While there are similarities in the biochemical properties between Ras and Rho proteins, the mutational hotspots in oncogenic RhoA are distinct. Oncogenic gain-of-function mutations of RhoA have been identified in leukemia, lymphoma, and particularly gastric cancer. Although many studies addressed the structure-function relationship of smGs, most structural biology and biophysical approaches employ a reductionist in vitro strategy in which the native environment is ignored. Therefore, there is a gap in understanding how smG performs its activities in its physiological environment and how oncogenic smG behaves in cancer cell environments. In this proposal, I aim to answer three outstanding questions about RhoA function. The hypothesis driving this work is that the mechanism of hydrolysis in oncogenic RhoA is distinct from that in Ras and that the tumor milieu differentially affects conformational states of RhoGTPases. The main objective of this K99/R00 proposal is to develop the tools to study Rho GTPases and exploit them to uncover the activity and regulation of oncogenic RhoA in relevant cancer cell environments and define Rho-GDI interaction as a new therapeutic target. To investigate this hypothesis, during the K99 phase (1) I will elucidate the mechanism of GTP hydrolysis in RhoA and its oncogenic mutants to understand how this is distinct from that in KRas. I will perform time-resolved X-ray crystallography experiments and combine them with NMR- based protein dynamics data to obtain atomic-level details of the transient intermediates formed during GTP hydrolysis; (2) I will answer how cellular environments affect the enzymatic activity of RhoA. Are protein-ligand interactions altered in the cancer cell environment over time? I will implement in-cell NMR and in-organoid NMR methods to measure rates of GTP hydrolysis in gastric tumor cells and protein-ligand binding in gastric-tumor organoids to define these differences in RhoA activity due to changes in the cellular environment. (3 I will interrogate how GDI regulates oncogenic RhoA, identify transient pockets in the RhoA-GDI complex for developing molecular glues that stabilize this interaction, and screen for ligands that bind RhoA in patient-derived organoid models. Upon completion, this work will expand the tools available to study small GTPases in ex-vivo models to target specific oncogenic forms and better understand a key regulatory mechanism in RhoGTPase sequestering. These tools will aid the cancer community, especially the NCI’s Ras Initiative beyond this proposal. My long-term goal is to develop an independent program that connects integrated structural biology with cancer cell metabolism to decipher oncogenic signaling pathways and provide a blueprint for targeting transient pockets in protein-protein interactions. Project Number: 1K99CA300901-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Fatema Bhinderwala | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $141,566 | Activity Code: K99 | Study Section: Special Emphasis Panel[ZRG1 CDPT-N (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11282284