During homeostasis, the colonic epithelium is replenished every 3–5 days by rapidly cycling Lgr5+ stem cells. However, various insults can lead to depletion of Lgr5+ stem cells, and colonic epithelium can be regenerated from Lgr5‐negative cells. While studies in the small intestine have addressed the lineage identity of the Lgr5‐negative regenerative cell population, in the colon this question has remained unanswered. Here, we set out to identify which cell(s) contribute to colonic regeneration by performing genetic fate‐mapping studies of progenitor populations in mice. First, using keratin‐19 (Krt19) to mark a heterogeneous population of cells, we found that Lgr5‐negative cells can regenerate colonic crypts and give rise to Lgr5+ stem cells. Notch1+ absorptive progenitor cells did not contribute to epithelial repair after injury, whereas Atoh1+ secretory progenitors did contribute to this process. Additionally, while colonic Atoh1+ cells contributed minimally to other lineages during homeostasis, they displayed plasticity and contributed to epithelial repair during injury, independent of Lgr5+ cells. Our findings suggest that promotion of secretory progenitor plasticity could enable gut healing in colitis.

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Background Colorectal cancer (CRC) is the 2nd leading cause of cancer death in Canada. A major risk factor for this disease is chronic inflammation. Despite the clear link between inflammation and cancer, the exact mechanism by which colitis leads to cancer is unknown. Our group has previously shown that a rare cell type in the gut marked by the expression of doublecortin-like kinase-1 (Dclk1) and known as a tuft cell, is quiescent, long-lived, and resistant to proliferation even upon mutation of the tumor suppressor APC. Interestingly, in the setting of colitis, these APC-mutated tuft cells become powerful cancer-initiating cells, but the mechanism by which this occurs is not known. NF-kB signaling is a major inflammatory pathway active in colitis and that has been linked to colorectal cancer. Inhibition of the NF-kB pathway in intestinal epithelial cells has also been shown to inhibit tumor initiation in a mouse model of colitis-associated cancer (Greten et al., 2004). Aims In the present study, we aim to examine the effect of NF-kB inhibition in tuft cells on colitis-associated cancer. Methods Dclk1CreERT2/APCf/f mice were crossed to IKK-β f/f mice and administered tamoxifen to conditionally knockout the function of both APC and IKK-β in tuft cells. Mice were then exposed to the colitis-inducing agent dextran sodium sulfate (DSS) to induce tumorigenesis. Approximately 16 weeks post-tamoxifen, colonic tumor number and size were analyzed to determine the effect of NF-kB pathway inhibition on tumor initiation and growth, respectively. Extent of inflammation was assessed by myeloperoxidase (MPO) activity and histological damage, and colonic tissue was collected for measurement of inflammatory mediators by qRT-PCR at both acute and long-term time points. Results Interestingly, at baseline we detected increased MPO activity in Dclk1CreERT2/APCf/f/IKK-β f/f mice compared to control mice, suggesting that inhibition of NF-kB in Dclk1+ cells may increase basal colonic inflammation. Consistent with this observation, inhibition of the NF-kB pathway also resulted in an increased number of tuft cell-derived tumors, with no observed change in tumor size. Acutely, we also observed an exacerbation of DSS-colitis in Dclk1CreERT2/APCf/f/IKK-β f/f mice, as detected by elevated MPO activity, increased histological damage, and reduced colon length compared to wildtype (IKK-β +/+) controls. Conclusions These data suggest that Dclk1+ cell-specific NF-kB signaling plays a key protective role against colitis and colitis-associated tumorigenesis. Targeting the NF-kB pathway may reduce the severity of colitis and the incidence of colitis-associated cancer. Funding Agencies CIHR

Background Colorectal cancer is the second leading cause of cancer death in Canada. A major risk factor for the development of colorectal cancer is chronic inflammation leading to colitis-associated cancer (CAC). We previously described a CAC mouse mode in which tumors arise from DCLK1+ tuft cells following loss of the tumor suppressor adenomatous polyposis coli (APC) and induction of colitis. Interestingly, both colitis and CAC display epigenetic changes that modulate gene expression. Specifically, DNA methylation is altered in colitis, but its role in colonic tumorigenesis is not known. We hypothesize that inhibition of DNA methylation in DCLK1+ tuft cells reduces colonic tumorigenesis. Aims In this study, we aim to investigate the role of DNA methylation in CAC by inhibiting DNA methylation by genetic and pharmacologic means. Methods We crossed our Dclk1-CreERT2/Apcf/f mice to DNMT1f/f mice to delete the DNA methyltransferase DNMT1 in DCLK1+ tuft cells. We induced CAC in Dclk1/Apcf/f and Dclk1/Apcf/f/DNMT1f/f mice by administering three doses of tamoxifen followed by 2.5% dextran sodium sulfate (DSS) for five days. Fourteen weeks later we assessed colonic tumor number and size. Lineage tracing of Dclk1+ cells was also examined in colonic tissues from all mice. In a separate cohort of Dclk1/Apcf/f mice, we induced colitis and treated the mice with six doses of the DNA de-methylating drug 5-AZA-2’-deoxycytidine (5-AZA) or vehicle. Ki67 immunostaining was additionally performed to assess cellular proliferation in the colon. Results Deletion of DNMT1 in DCLK1+ cells significantly inhibited the number and size of colonic tumors. Treatment of mice with 5-AZA similarly reduced the overall number of mice with tumors, as well as, the number and size of tumors per mouse. Interestingly, 5-AZA treatment was associated with reduced colonic proliferation as assessed by fewer Ki67+ cells, and quiescent DCLK1+ cells that did not lineage trace. Furthermore, deletion of DNMT1 or treatment with 5-AZA reduced the number of lineage tracing events detected upon exposure to low DSS. Conclusions Our findings demonstrate that loss of Dnmt1 or 5-AZA treatment reduces CAC formation. Furthermore, 5-AZA appears to exert its anti-tumor effects by reducing proliferation and preventing tuft cell stemness. Our data demonstrates that altering DNA methylation plays an important role in CAC. Funding Agencies CIHR

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