Abstract 5875: Analysis of clonal expansion in epithelium affected by ulcerative colitis reveals frequent mutations affecting IL-17 signaling pathway and novel cancer vulnerability

Chronic inflammation is a major cause of morbidity and mortality in the human population and is associated with a substantial cancer risk. It has recently been demonstrated that extensive tissue remodeling can take place even in apparently normal tissues in an age-dependent manner and is mediated by expansion of numerous clones carrying common cancer-related mutations. Given the inflammation-associated cancer risks, clonal expansion may also play a role in inflammation-associated tissue remodeling. However, it is largely unknown whether this actually happens, with what mutations it occurs if ever, and how it modifies cancer development. To address these issues, we studied ulcerative colitis (UC) which is a chronic inflammatory bowel disease. Long-standing UC is associated with an increased risk (15-20%) of colitis-associated colorectal cancer (CAC). We performed whole exome sequencing (WES) of single crypts (n=441) and bulk crypts (n=569) from patients with UC and control individuals, as well as CAC samples (n=99). WES of single crypts from control individuals revealed UC crypts showed a more than 3 times higher mutation rate than non-UC crypts. We analyzed history of crypt expansion in clusters of single crypts. From mutations detected in each crypt, we reconstructed phylogenetic trees of clusters of crypts. Non-UC trees showed many early branches before 20 years of age, whereas, UC trees were characterized by a prominently long trunk with many short branches, suggesting crypts rapidly expanded from a common ancestral crypt during the past few years. Bulk-crypt analysis from surgically resected UC rectums showed many expanded clones, among which the maximum clone was as large as 19 cm2 in size. While no driver gene was detected in control individuals, the dN/dS analysis on 399 non-dysplasia UC samples revealed 14 driver genes, within which NFKBIZ were most frequently mutated, followed by PIGR and ZC3H12A. These drivers displayed a surprising overlap to signaling pathways converging on NFKBIZ. Among these pathways most prominent was IL-17 signaling, in which IL-17 receptors (IL17RA/IL17RC) and its adaptor, TRAF3IP2 were significantly mutated in UC epithelium. A driver role of NFKBIZ mutations was confirmed by growth advantage of Nfkbiz-KO cells compared with control cells under IL-17A stimulation in vitro or in a DSS-induced colitis model. Despite a substantial overlap of driver genes between CAC and UC epithelium, their frequencies significantly differed between both tissues. NFKBIZ mutations, which is most prevalent in UC non-dysplasia, were never found in 99 CAC samples. We confirmed a negative role of NFKBIZ mutations by compromised cell-competition of NFKBIZ-disrupted colorectal cancer cells and significantly attenuated colitis-induced tumor formation in Nfkbiz-deficient mice. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which unexpectedly reveal cancer vulnerability potentially utilized for therapeutics of colorectal cancer. Citation Format: Nobuyuki Kakiuchi, Kenichi Yoshida, Motoi Uchino, Takako Kihara, Akaki Kotaro, Yoshikage Inoue, Kenji Kawada, Satoshi Nagayama, Akira Yokoyama, Tomonori Hirano, Yasuhide Takeuchi, Hiroyuki Miyoshi, Yoshiharu Sakai, Hironori Haga, Seiichi Hirota, Hiroki Ikeuchi, Osamu Takeuchi, Satoru Miyano, Hiroshi Seno, Seishi Ogawa. Analysis of clonal expansion in epithelium affected by ulcerative colitis reveals frequent mutations affecting IL-17 signaling pathway and novel cancer vulnerability [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5875.