Abstract 3920: Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma
Authors: Moreno Marta Roman , Kostyrko Kaja , Herrington Kari , Bassik Michael , Jackson Peter , Sweet-Cordero Alejandro 
Published: April 2023

Abstract
While KRAS is among the most frequently mutated oncogenes, our understanding of the mechanisms of KRAS-driven oncogenesis remains limited. A significant remaining gap is a lack of understanding of tissue-specific effectors of Ras activation and the role of specific KRAS mutations in determining downstream vulnerabilities. Previously, we used a combination of proteomics and CRISPR/Cas9 screens in human lung adenocarcinoma (LUAD) cells to identify a KRAS-specific vulnerability induced by the combined loss of RHOA and the long isoform of RAP1GDS1, suggesting a potentially novel approach for targeting KRAS-driven cancer. Here we use biochemical, proteomic, and genetic approaches to dissect the isoform-specific roles of RAP1GDS1 to elucidate its synthetic lethal interaction with RHOA in KRAS-driven LUAD.
We performed AP/MS in KRAS-mutant A549 cells using both the long (RAP1GDS1-607) and the short (RAP1GDS1-558) isoforms of RAP1GDS1 as bait to identify overlapping and isoform-specific RAP1GDS1 interactors. We enriched this analysis with orthologous datasets from DepMap, published protein-protein interaction (PPI) data and RAP1GDS1-specific prenylome. This comprehensive analysis identified a cluster of RAB GTPases, proteins involved in vesicular transport and lysosomal function, as specific interactors of RAP1GDS1-607. We subsequently used the proximity ligation assay (PLA) to confirm the direct interaction between RAP1GDS1-607 and two of the strongest interactors - RAB7A and RAB22A – according to the PPI data. We also depleted either or both RAP1GDS1 isoforms in cells expressing GFP-tagged RAB7A, RAB22A or KRAS and used live cell imaging to determine how loss of RAP1GDS1 changes their subcellular localization. We found that depletion of both RAP1GDS1 isoforms and, to a lesser extent, single loss of RAP1GDS1-607, significantly decreased KRAS localization at the plasma membrane. We also used PLA to confirm that RAP1GDS1-607 has a stronger interaction with KRAS compared to the short isoform. Moreover, we demonstrated that loss of the long RAP1GDS1 isoform significantly decreased the membrane localization levels of GTP-bound KRAS, compared to the control or RAP1GDS1-558 knock-down. Finally, to identify the molecular mechanism of R1G1/RhoA synthetic lethality with KRAS, we performed a Genome Wide-CRISPR screen in R1G1-607 and RhoA knock-out cells. We identified the strongest hits and designed a focused CRISPR library of approximately 400 genes to screen a larger panel of KRAS-mutant LUAD cell lines (H23, A549 and H358). The aim is to identify other genes that are synthetic lethal in combination with RhoA or R1G1-607 loss and genes that could mediate the lethal effect of their combined loss.
In conclusion, these data demonstrate a specific role of the long RAP1GDS1 isoform in the membrane-localization and activation of KRAS, which supports its role as a potential therapeutic target for KRAS mutant tumors.
Citation Format: Marta Roman Moreno, Kaja Kostyrko, Kari Herrington, Michael Bassik, Peter Jackson, Alejandro Sweet-Cordero. Functional analysis of the role of RAP1GDS1 and RhoA in KRAS-driven lung adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3920.