Introduction: RNF43 is a transmembrane E3 ubiquitin ligase and WNT signaling suppressor that is commonly mutated in colorectal cancer (CRC). A C-terminal RNF43 hotspot mutation, RNF43_G659fs, occurs in approximately 36% (55/151) of microsatellite-instability (MSI)-high CRCs, but its underlying mechanism and function remain poorly understood. This study investigated the functional role of RNF43_G659fs in order to evaluate potential novel therapeutic approaches for tumors harboring this mutation.

Methods: Isogenic RNF43117mut and RNF43659mut cell line models were generated using the CRISPR/Cas9 system to evaluate CRC tumorigenesis and WNT dependency. RNF43659mut was screened with a novel high-throughput drug repurposing library that employed a set of 5363 small molecules to identify compounds capable of selectively inhibiting RNF43659mut cell growth. Small molecules that selectively killed the RNF43659mut cells were validated in organoid models. Proteomic analysis, RNA-Seq and gene set enrichment analysis (GSEA) were performed to characterize mechanistic interactions and related signaling pathways of RNF43659mut in CRC.

Results: Unlike N-terminal RNF43 frameshift mutations, we observed that RNF43659mut conferred a growth advantage over RNF43WT cells independent of WNT signaling. Furthermore, RNF43659mut and RNF43WT exhibited differential drug responses in the high-throughput drug repurposing screen which revealed that RNF43659mut cells were vulnerable to PI3K/AKT/mTOR inhibitors, including BYL-719 (Alpelisib). Enhanced AKT and mTOR activation was observed in RNF43659mut cell and attenuated by BYL-719 treatment in a dose-dependent manner. These results were subsequently validated in patient-derived organoid models. Furthermore, immunoprecipitation and proteomic analysis revealed interactions between RNF43_G659fs and p85, a negative regulator of PI3K. We also demonstrated that the RNF43_G659fs mutant activated PI3K/AKT/mTOR signaling through binding and degradation of p85. Consistent with the role of PI3K in immunomodulation, our RNA-Seq results showed that the RNF43_G659fs mutation was positively related to NF-kB activation (Normalized Enrichment Score=1.842, p<0.01) and inversely related to Interferon-alpha/Interferon-gamma response pathways (Normalized Enrichment Score= -1.992, p<0.01; Normalized Enrichment Score= -1.577, p<0.01, respectively), indicating its role in tumor microenvironment remodeling.

Conclusion: This study confirms that RNF43659mut is an essential driver mutation in CRC and provides evidence that patients harboring RNF43_G659fs-mutant tumors may respond favorably to PI3K inhibition.

Citation Format: Lishan Fang, Dane Ford-Roshon, Max Russo, Casey O'Brien, Carino Gurjao, Maximilien Grandclaudon, Steven M. Corsello, Srivatsan Raghavan, Namrata Udeshi, James Berstler, Ewa Sicinska, Kimmie Ng, Marios Giannakis. RNF43 G659fs is an oncogenic mutation in colorectal cancer and sensitizes tumor cells to PI3K/mTOR inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 960.