Highlights d TBK1 kinase activity regulates disease progression in an ALS SOD1 mouse model d Loss of TBK1 in motor neurons increases SOD1 aggregation and accelerates disease onset d Loss of TBK1 activity in all cell types accelerates disease onset but extends survival d Loss of TBK1 activity in all cell types reduces the IFN response in microglia

Human achaete scute homolog 2 (HASH2) and its murine ortholog MASH2 are potential targets for colorectal cancer immunotherapy. We assessed immunogenicity and antitumor potential of recombinant MASH2 protein combined with AS15 immunostimulant (recMASH2+AS15) in CB6F1 and Apc+/Min-FCCC mice. CB6F1 mice received 4 injections of recMASH2+AS15 or AS15 alone before challenge with TC1-MASH2 tumor cells (Tumor Challenge). Apc+/Min-FCCC mice received 9 injections of recMASH2+AS15 or vehicle (phosphate buffer saline [PBS] or AS15 alone), before (two independent Prophylactic Studies) or after (Immunotherapy) colon adenomas were detectable by colonoscopy. CB6F1 mice immunized with recMASH2+AS15 had a significantly smaller mean tumor size and improved survival rate compared to controls (104 mm2 vs. 197 mm2 [p = 0.009] and 67% vs. 7% [p = 0.001], respectively). In Prophylactic Study 1, the mean number of colon adenomas was significantly lower in Apc+/Min-FCCC mice receiving recMASH2+AS15 compared to PBS (1.8 [95% confidence interval 1.0–3.3] vs. 5.2 [3.7–7.4], p = 0.003). Fewer microadenomas were observed in recMASH2+AS15 groups compared to PBS in both Prophylactic Studies (Study 1: mean 0.4 [0.2–1.0] vs. 1.5 [0.9–2.4], p = 0.009; Study 2: 0.4 [0.2–0.6] vs. 1.1 [0.8–1.5], p = 0.001). In the Immunotherapy Study, fewer colon adenomas tended to be observed in recMASH2+AS15-treated mice (4.1 [2.9–6.0]) compared to controls (AS15 4.7 [3.3–6.6]; PBS 4.9 [3.5–6.9]; no significant difference). recMASH2+AS15 induced MASH2-specific antibody and CD4+ responses in both mouse models. recMASH2+AS15 partially protected mice against MASH2-expressing tumors and reduced spontaneous colorectal adenomas in Apc+/Min-FCCC mice, indicating that MASH2/HASH2 antigens are targets for colorectal cancer immunotherapy.

Purpose: The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. Methods: Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). Results: A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/β-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. Conclusions: High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.

An urgent need exists to identify efficacious therapeutic preventive interventions for individuals who are at high risk of developing colorectal cancer. To maximize the benefits of preventive intervention, it is vital to identify the time interval during which the initiation of a preventive intervention will lead to an optimal outcome. The goal of the present study was to determine if oncogenic events can be detected in the nonneoplastic colonic mucosa of Apc+/Min-FCCC mice prior to formation of the first adenoma, thus defining an earlier point of intervention along the cancer continuum. Tissues taken at three potential points of intervention were characterized: prior to Apc mutation (wild type Apc+/+-FCCC mice); after initiation but prior to colon adenoma formation (tumor-free Apc+/Min-FCCC mice); and after formation of the first colon adenoma (tumor-bearing Apc+/Min-FCCC mice). Experimentation focused on molecular processes that are dysregulated in early colon lesions: 1) cellular proliferation (proliferative index and size of the proliferative zone); 2) cellular stemness (expression of Ascl2, Grem1, Lgr5 and Muc2); 3) EGFR signaling (expression of Ereg); and 4) inflammation (expression of Mmp9, Ptsg2, and Reg4, as well as secretion of 18 cytokines involved in immune activation and response). Interestingly, the nonneoplastic colonic mucosa of wild type, tumor-free Apc+/Min-FCCC, and tumor-bearing Apc+/Min-FCCC mice did not display significant differences in average epithelial cell proliferation (fold change 0.8–1.3, p≥0.11), mucosal gene expression (fold change 0.8–1.4, p≥0.22), or secretion of specific cytokines from colonic mucosa (fold change 0.2–1.5, p≥0.06). However, the level of cytokine secretion was highly variable, with many (22% of wild type, 31% of tumor-free Apc+/Min-FCCC, and 31% of tumor-bearing Apc+/Min-FCCC) mice categorized as outliers (&gt; 1.5 x interquartile ranges below the first quartile or above the third quartile) due to elevated expression of at least one cytokine. In summary, no differences were observed in proliferation, stemness, and EGFR signaling in the colonic mucosa of wild type vs Apc+/Min-FCCC mice, with low baseline cytokine expression, prior to the formation of the first colon adenoma. The results of this study provide valuable baseline data to inform the design of future cancer prevention studies.

The importance of the host immune system in controlling the growth and development of colorectal cancer (CRC) is highlighted by the prognostic value of immune infiltration, which suggests that cancer immunotherapy could mobilize the immune system for clinical benefit. Human achaete scute homolog 2 (Hash2) and its murine ortholog Mash2 are overexpressed in human and mouse CRC, respectively, and are thus potential targets for immunotherapy. The present studies assessed the ability of a recombinant MASH2 protein combined with the AS15 immunostimulant to induce an immune response and inhibit tumor growth in mouse models. CB6F1 mice received 4 injections of AS15 or MASH2+AS15 and were subsequently injected with TC1/MASH2 tumor cells. In parallel, APC+/Min-FCCC mice that spontaneously develop MASH2-expressing colorectal adenomas were immunized (x9) with phosphate buffered saline (PBS), AS15 alone or MASH2+AS15, either before (prophylaxis) or after (immunotherapy) colon adenomas were detectable by colonoscopy. MASH2-specific antibody and cellular immune responses (CD4+ and CD8+ cells producing cytokines) were measured by ELISA and flow cytometry, respectively. Colorectal adenomas were diagnosed and characterized by histopathology. In both CB6F1 and APC+/Min-FCCC mice, MASH2+AS15 induced strong MASH2-specific antibody and CD4+ responses that were not observed after injection with PBS or AS15. CB6F1 mice immunized with MASH2+AS15 showed slower growth of injected TC1/MASH2 tumors and better survival in comparison to mice injected with AS15 alone. In APC+/Min-FCCC mice, the mean number of colon adenomas was 2.6, 2.4 and 3.8 in the MASH2+AS15, AS15 and PBS groups, respectively (p = 0.059 for MASH2+AS15 vs PBS). In these mice, the mean number of colon microadenomas was ∼3-fold lower in the MASH2+AS15 group as compared to PBS control (MASH2+AS15 [0.4], AS15 [0.6], PBS [1.1]; p = 0.001 for MASH2+AS15 vs PBS). MASH2+AS15 was most effective in inhibiting distal colon microadenomas, with the mean number 3-fold lower than that of the PBS group (0.2 for MASH2+AS15, 0.4 for AS15, 0.6 for PBS, p&lt;0.001 for MASH2+AS15 vs PBS, p = 0.032 for MASH2+AS15 vs AS15). Similarly, in the therapeutic study, colon microadenomas were inhibited to the greatest extent after immunization with MASH2+AS15. In conclusion, MASH2+AS15 immunotherapy leads to partial protection of mice against MASH2-expressing transplantable tumors and strongly reduces the mean number of spontaneous colorectal adenomas in APC+/Min-FCCC mice, by inducing MASH2-specific CD4+ T cells and antibodies. These data are very promising and demonstrate the utility of MASH2 immunotherapy, with responses that may be further improved by combining with immunotherapies that induce antigen-specific CD8+ T cells or with immune checkpoint inhibitors. Funding: GlaxoSmithKline Biologicals SA Citation Format: Clément Rioux, Margie Clapper, Harry Cooper, Jean Michaud, Natalie St Amant, Hossein Koohsari, Laura Workman, Esther Kaunga, Anthony Pilorget, Catherine Gerard. Immunotherapy targeting MASH2 antigen protects mice against MASH2-expressing transplanted tumors and inhibits the development of colorectal adenomas in APC +/Min-FCCC mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2510. doi:10.1158/1538-7445.AM2015-2510