Functional analysis of the role of rap1gds1 and RHOA in KRAS-driven lung adenocarcinoma
Marta Román Moreno
School of Medicine; University of California, San Francisco
While KRAS is among the most frequently mutated oncogenes in cancer, 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. Our group has previously used a combination of proteomics and CRISPR/Cas9 screens in human lung adenocarcinoma (LUAD) cells that identified 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. Currently, we are using 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 have 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 the long RAP1GDS1 isoform. We subsequently used proximity ligation assays (PLA) to confirm the direct interaction between RAP1GDS1-607 and two of the strongest interactors - RAB7A and RAB22A – according to the PPI data. We are also performing live cell imaging to determine how loss of RAP1GDS1 changes the subcellular localization of such RABs. We found that depletion of both RAP1GDS1 isoforms and, to a lesser extent, single loss of the long RAP1GDS1 isoform, significantly decreased KRAS localization at the cell membrane. Moreover, PLA confirmed that RAP1GDS1-607 has a stronger interaction with KRAS compared to RAP1GDS1-558. 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 isoform of RAP1GDS1 in the membrane-localization and activation of KRAS, which supports its role as a potential therapeutic target for KRAS mutant tumors.