Abstract PR009: RNF43 G659fs is an oncogenic and immune-modulating colorectal cancer mutation and sensitizes tumor cells to PI3K/mTOR inhibition

Introduction: The RNF43 p.G659fs mutation occurs with a frequency of approximately 8% in colorectal cancer (CRC) and is enriched among microsatellite-instability high tumors. However, despite RNF43 being implicated as a Wnt-signaling tumor suppressor, the function of the RNF43 p.G659fs mutation remains undescribed, with no specific therapies directed against it. We hypothesized that large-scale drug screening coupled with functional validation and testing in CRC pre-clinical models would reveal the functional role and selective vulnerabilities of this alteration, leading to new therapeutic approaches in CRC. Methods: We targeted the RNF43 659 site with CRISPR-Cas9 to generate isogenic RNF43659mut clones of CRC cell lines. We leveraged the Broad Institute’s drug repurposing library of over 5,300 compounds to identify selective hits (z5% selective killing in mutant vs WT) in RNF43659mut isogenic cells. These hits were validated in secondary screens and top drugs were tested in patient-derived organoid and xenograft models of the RNF43 p.G659fs mutation. We assessed the functional role of the mutation using cell viability and colony formation assays, quantitative mass spectrometry proteomics, immunoprecipitation and ubiquitination experiments. The immunomodulatory role of RNF43659mut was assessed through bulk and single-cell RNA Seq analysis of mutant cell lines and patient-derived CRC specimens. Results: Using edited cell line models, we demonstrated that RNF43 p.G659fs has a Wnt-independent oncogenic role in CRC. We performed a comprehensive drug screen in isogenic RNF43659mut HT29 CRC cells with a repurposing library of 5,363 compounds and discovered that RNF43659mut is selectively targeted by PI3K/mTOR inhibitors (p = 1.34 x 10-5, chi-square test). Secondary screens in LS513 RNF43659mut-edited and HCT116 RNF43G659fs-overexpressing cells confirmed these results and we further showed that alpelisib and PF-04691502 (a dual PI3K/mTOR inhibitor) selectively killed patient-derived organoids with the RNF43 p.G659Vfs mutation and reduced tumor growth in RNF43659mut CDX mice (p=0.0005, Student’s t-test). RNF43 p.G659fs increased PI3K/AKT signaling in vitro and in vivo through binding, ubiquitinating and degrading p85 and this was reversed by PI3K/mTOR inhibition, RNF43 knockout or RSPO stimulation. Additionally, in pre-clinical models and single-cell studies of human CRCs (>350,000 single cells), we found that RNF43659mut modulates interferon activit