Recurrent RhoGAP gene fusion CLDN18-ARHGAP26 promotes RHOA activation and focal adhesion kinase and YAP-TEAD signalling in diffuse gastric cancer

OBJECTIVE: Genomic studies of gastric cancer have identified highly recurrent genomic alterations impacting RHO signalling, especially in the diffuse gastric cancer (DGC) histological subtype. Among these alterations are interchromosomal translations leading to the fusion of the adhesion protein CLDN18 and RHO regulator ARHGAP26. It remains unclear how these fusion constructs impact the activity of the RHO pathway and what is their broader impact on gastric cancer development. Herein, we developed a model to allow us to study the function of this fusion protein in the pathogenesis of DGC and to identify potential therapeutic targets for DGC tumours with these alterations. DESIGN: We built a transgenic mouse model with LSL-CLDN18-ARHGAP26 fusion engineered into the Col1A1 locus where its expression can be induced by Cre recombinase. Using organoids generated from this model, we evaluated its oncogenic activity and the biochemical effects of the fusion protein on the RHOA pathway and its downstream cell biological effects in the pathogenesis of DGC. RESULTS: We demonstrated that induction of CLDN18-ARHGAP26 expression in gastric organoids induced the formation of signet ring cells, characteristic features of DGC and was able to cooperatively transform gastric cells when combined with the loss of the tumour suppressor geneTrp53. CLDN18-ARHGAP26 promotes the activation of RHOA and downstream effector signalling. Molecularly, the fusion promotes activation of the focal adhesion kinase (FAK) and induction of the YAP pathway. A combination of FAK and YAP/TEAD inhibition can significantly block tumour growth. CONCLUSION: These results indicate that the CLDN18-ARHGAP26 fusion is a gain-of-function DGC oncogene that leads to activation of RHOA and activation of FAK and YAP signalling. These results argue for further evaluation of emerging FAK and YAP-TEAD inhibitors for these deadly cancers

Exome sequencing reveals novel oncogenic mutations in early-onset sporadic rectal cancer

Colorectal cancer (CRC) is an aging related disease with 72% of newly diagnosed cases aged 65 years or older (Cancer Registration Statistics, 2012). Past few decades of research from the West have identified aberrantly activated canonical Wnt/β-catenin signaling and microsatellite instability (MSI) as the major pathways driving CRC tumorigenesis. In India however majority of cases appear to belong to a younger age group with preponderance of rectal cancer. Molecular genetic screening of early-onset sporadic rectal cancer (EOSRC) performed earlier from our laboratory identified a significant proportion of EOSRC to be driven neither by aberrant Wnt signaling nor MSI. We performed whole exome sequencing of 27 tumor/normal pairs obtained from surgically resected rectal adenocarcinomas (microsatellite stable with no aberrant Wnt signaling) using the Illumina HiSeq 2500 platform. We identified recurrently mutated genes in EOSRC including known tumor suppressors (TSG) and oncogenes such as TP53, APC, KRAS, SMAD4 and PIK3CA. More importantly, we discovered mutations in uncharacterized TSGs and oncogenes such as ARID2, FAT3, FAT4, ZEB2 and TRPC7. These included putative oncogenic mutations in the zinc finger domains of ZEB2, a DNA-binding transcriptional repressor that promotes epithelial to mesenchymal transition in tumors. Functional work is underway to characterize the effect of these mutations on CRC tumor progression. APC mutations detected in this study were present in major population of cells, yet were not driving β-catenin to the nucleus. This observation suggests β-catenin degradation independent tumor suppressor function of APC, which is being validated

Identifying regulators of aberrant stem cell and differentiation activity in colorectal cancer using a dual endogenous reporter system

Abstract Aberrant stem cell-like activity and impaired differentiation are central to the development of colorectal cancer (CRC). To identify functional mediators of these key cellular programs, we engineer a dual endogenous reporter system by genome-editing the SOX9 and KRT20 loci of human CRC cell lines to express fluorescent reporters, broadcasting aberrant stem cell-like and differentiation activity, respectively. By applying a CRISPR screen targeting 78 epigenetic regulators with 542 sgRNAs to this platform, we identify factors that contribute to stem cell-like activity and differentiation in CRC. Perturbation single cell RNA sequencing (Perturb-seq) of validated hits nominate SMARCB1 of the BAF complex (also known as SWI/SNF) as a negative regulator of differentiation across an array of neoplastic colon models. SMARCB1 is a dependency and required for in vivo growth of human CRC models. These studies highlight the utility of biologically designed endogenous reporter platforms to uncover regulators with therapeutic potential