Baseline serum and stool microbiome biomarkers predict clinical efficacy and tissue molecular response after ritlecitinib induction therapy in ulcerative colitis.

BACKGROUND AND AIMS: Ritlecitinib, an oral JAK3/TEC family kinase inhibitor, was well- tolerated and efficacious in the phase 2b VIBRATO study in participants with moderate-to-severe ulcerative colitis (UC). The aim of this study was to identify baseline serum and microbiome markers that predict subsequent clinical efficacy and to develop noninvasive serum signatures as potential real-time noninvasive surrogates of clinical efficacy after ritlecitinib. METHODS: Tissue and peripheral blood proteomics, transcriptomics, and fecal metagenomics were performed on samples before and after 8-week oral ritlecitinib induction therapy (20 mg, 70 mg, 200 mg, or placebo once daily, N=39, 41, 33, and 18, respectively). Linear mixed models were used to identify baseline and longitudinal protein markers associated with efficacy. The combined predictivity of these proteins was evaluated using a logistic model with permuted efficacy data. Differential expression of fecal metagenomic was used to differentiate responders and nonresponders. RESULTS: Peripheral blood serum proteomics identified 4 baseline serum markers (LTA, CCL21, HLA-E, MEGF10) predictive of modified clinical remission (MR), endoscopic improvement (EI), histologic remission (HR), and integrative score of tissue molecular improvement. In responders, 37 serum proteins significantly changed at Week 8 compared with baseline (FDR<0.05); of these, changes in 4 (IL4R, TNFRSF4, SPINK4, and LAIR-1) predicted concurrent EI and HR responses. Fecal metagenomics analysis revealed baseline and treatment response signatures that correlated with EI, MR, and tissue molecular improvement. CONCLUSIONS: Blood and microbiome biomarkers stratify endoscopic, histologic, and tissue molecular response to ritlecitinib, which may help guide future precision medicine approaches to UC treatment.

Oral Ritlecitinib and Brepocitinib for Moderate-to-Severe Ulcerative Colitis: Results From a Randomized, Phase 2b Study.

BACKGROUND & AIMS: The efficacy and safety of ritlecitinib (oral JAK3/TEC family kinase inhibitor) and brepocitinib (oral TYK2/JAK1 inhibitor) as induction therapy were assessed in patients with active, moderate-to-severe ulcerative colitis. METHODS: This phase 2b, parallel-arm, double-blind umbrella study randomized patients with moderate-to-severe ulcerative colitis to receive 8-week induction therapy with ritlecitinib (20, 70, 200 mg), brepocitinib (10, 30, 60 mg), or placebo once daily. The primary endpoint was total Mayo Score (TMS) at week 8. RESULTS: Of 319 randomized patients, 317 received ritlecitinib (n = 150), brepocitinib (n = 142), or placebo (n = 25). The placebo-adjusted mean TMSs (90% confidence interval) at week 8 were -2.0 (-3.2 to -0.9), -3.9 (-5.0 to -2.7), and -4.6 (-5.8 to -3.5) for ritlecitinib 20, 70, and 200 mg, respectively (P = .003, P < .001, P < .001), and -1.8 (-2.9 to -0.7), -2.3 (-3.4 to -1.1), and -3.2 (-4.3 to -2.1) for brepocitinib 10, 30, and 60 mg, respectively (P = .009, P = .001, P < .001). Estimates (90% confidence interval) for placebo-adjusted proportions of patients with modified clinical remission at week 8 were 13.7% (0.5%-24.2%), 32.7% (20.2%-45.3%), and 36.0% (23.6%-48.6%) for ritlecitinib 20, 70, and 200 mg, respectively, and 14.6% (1.9%-25.7%), 25.5% (11.0%-38.1%), and 25.5% (11.0%-38.1%) for brepocitinib 10, 30, and 60 mg, respectively. Adverse events were mostly mild, and there were no serious cases of herpes zoster infection. Infections were observed with brepocitinib (16.9% [12.5%-23.7%]), ritlecitinib (8.7% [5.2%-13.4%]), and placebo (4.0% [0.2%-17.6%]). One death due to myocardial infarction (ritlecitinib) and 1 thromboembolic event (brepocitinib) occurred; both were considered unrelated to study drug. CONCLUSIONS: Ritlecitinib and brepocitinib induction therapies were more effective than placebo for the treatment of moderate-to-severe active ulcerative colitis, with an acceptable short-term safety profile. CLINICALTRIALS: gov number: NCT02958865.

Antitumor Necrosis Factor-like Ligand 1A Therapy Targets Tissue Inflammation and Fibrosis Pathways and Reduces Gut Pathobionts in Ulcerative Colitis.

BACKGROUND: The first-in-class treatment PF-06480605 targets the tumor necrosis factor-like ligand 1A (TL1A) molecule in humans. Results from the phase 2a TUSCANY trial highlighted the safety and efficacy of PF-06480605 in ulcerative colitis. Preclinical and in vitro models have identified a role for TL1A in both innate and adaptive immune responses, but the mechanisms underlying the efficacy of anti-TL1A treatment in inflammatory bowel disease (IBD) are not known. METHODS: Here, we provide analysis of tissue transcriptomic, peripheral blood proteomic, and fecal metagenomic data from the recently completed phase 2a TUSCANY trial and demonstrate endoscopic improvement post-treatment with PF-06480605 in participants with ulcerative colitis. RESULTS: Our results revealed robust TL1A target engagement in colonic tissue and a distinct colonic transcriptional response reflecting a reduction in inflammatory T helper 17 cell, macrophage, and fibrosis pathways in patients with endoscopic improvement. Proteomic analysis of peripheral blood revealed a corresponding decrease in inflammatory T-cell cytokines. Finally, microbiome analysis showed significant changes in IBD-associated pathobionts, Streptococcus salivarius, S. parasanguinis, and Haemophilus parainfluenzae post-therapy. CONCLUSIONS: The ability of PF-06480605 to engage and inhibit colonic TL1A, targeting inflammatory T cell and fibrosis pathways, provides the first-in-human mechanistic data to guide anti-TL1A therapy for the treatment of IBD.

Anti-TL1A Antibody PF-06480605 Safety and Efficacy for Ulcerative Colitis: A Phase 2a Single-Arm Study.

BACKGROUND & AIMS: An immune component of inflammatory bowel disease is up-regulated tumor necrosis factor-like ligand 1A (TL1A). Anti-TL1A antibodies such as PF-06480605, a fully human immunoglobulin G1 monoclonal antibody, may have therapeutic potential. METHODS: This Phase 2a, multicenter, single-arm, open-label study (TUSCANY) evaluated safety, tolerability, efficacy, pharmacokinetics, and immunogenicity in PF-06480605-treated participants with moderate to severe ulcerative colitis (UC). Participants received 500 mg intravenous PF-06480605 every 2 weeks, 7 doses total, with a 3-month follow-up period. Primary safety and efficacy endpoints were the incidence of adverse events (AEs) and week 14 endoscopic improvement (EI) (Mayo endoscopic subscore = 0 or 1), respectively. Secondary endpoints included total soluble TL1A (free/drug-bound) (sTL1A), incidence of anti-drug and neutralizing antibodies, PF-06480605 concentrations, and changes in fecal calprotectin and high-sensitivity C-reactive protein. Histology was assessed at week 14. RESULTS: The study enrolled 50 participants; 42 completed. Of 109 treatment-emergent AEs, 18 were treatment-related. The most common AEs were UC disease exacerbation and arthralgia (6 participants each). Four serious AEs, no deaths, and no malignancies were reported. Week 14 EI was observed in a statistically significant proportion of participants (38.2% [uniformly minimum-variance unbiased estimator, per protocol population]). Minimal histologic disease was observed after treatment (Robarts Histopathology Index ≤5: 33.3%; Geboes Index ≤3.2: 47.6%). sTL1A increase over time from baseline indicated sustained target engagement. Forty-one participants (82%) tested positive for anti-drug antibodies and 5 (10%) for neutralizing antibodies. CONCLUSIONS: PF-06480605 demonstrated an acceptable safety profile and statistically significant EI in participants with moderate to severe UC, warranting further study in a larger participant cohort. Tissue histopathology analyses support this conclusion. TRIAL REGISTRATION NUMBER: https://clinicaltrials.gov/NCT02840721.

First-in-human, randomized dose-escalation study of the safety, tolerability, pharmacokinetics, pharmacodynamics and immunogenicity of PF-06480605 in healthy subjects.

AIMS: Human genetic, tissue expression, proteomics, transcriptomics and nonclinical studies implicate tumour necrosis factor α-like ligand 1A (TL1A) as a novel target in inflammatory bowel disease (IBD). PF-06480605, a fully human immunoglobulin G1 monoclonal antibody, targets TL1A. This first-in-human, Phase 1, dose-escalation study assessed safety, tolerability, pharmacokinetics, pharmacodynamics and immunogenicity of intravenous (IV) and subcutaneous (SC) PF-06480605 in healthy subjects (NCT01989143). METHODS: Ninety-two subjects were randomized to single ascending doses (SAD), PF-06480605 1 mg, 3 mg, 10 mg, 30 mg, 100 mg, 300 mg, 600 mg or 800 mg IV, or multiple ascending doses (MAD), PF-06480605 3 × 500 mg IV, or 3 × 30 mg, 3 × 100 mg, or 3 × 300 mg SC every 2 weeks for three doses, or placebo. Safety, tolerability, pharmacokinetics, immunogenicity profiles and total TL1A, anti-drug antibody (ADA) and neutralizing antibody (NAb) levels were assessed at pre-determined times. RESULTS: PF-06480605 SAD up to 800 mg IV and MAD up to 300 mg ×3 SC and 500 mg ×3 IV were well tolerated. Overall, there were 45 and 44 treatment-emergent adverse events in SAD and MAD cohorts, respectively, and no deaths or serious adverse events. PF-06480605 exposure generally increased dose-dependently. ADA and NAb levels did not impact safety, pharmacokinetics, or pharmacodynamics at higher doses. Target engagement was demonstrated through dose-dependent differences in serum total soluble TL1A concentrations for PF-06480605 vs placebo cohorts. CONCLUSIONS: PF-06480605 was generally well tolerated, and binding of soluble TL1A was maintained throughout the dose interval, supporting further study of PF-06480605 in patients with IBD and other inflammatory conditions.

PKM2 is not required for colon cancer initiated by APC loss.

BACKGROUND: Cancer cells express the M2 isoform of the glycolytic enzyme pyruvate kinase (PKM2). PKM2 expression is not required for some cancers, and PKM2 loss can promote cancer progression; however, PKM2 has been reported to be essential in other tumor contexts, including a proposed non-metabolic role in ß-catenin nuclear translocation. PKM2 is expressed in colon cancers where loss of the Apc tumor suppressor results in ß-catenin nuclear translocation and aberrant activation of the canonical Wnt signaling pathway. Whether PKM2 is required in this colon cancer context has not been investigated. RESULTS: Colon tumorigenesis was induced in mice harboring conditional Apc and Pkm2 alleles, and tumor progression was monitored by serial colonoscopy. PKM2 deletion had no effect on overall survival, the number of mice that developed tumors, or the number of tumors that developed per animal. Immunohistochemical analysis demonstrated PKM2 expression in wild-type tumors and the expected loss of PKM2 expression in tumors from Pkm2 conditional mice. Loss of PKM2 resulted in pyruvate kinase M1 expression but had no effect on nuclear ß-catenin staining. These findings are consistent with tumor growth and activated Wnt signaling despite PKM2 loss in this model. We also found a large fraction of human colon cancers had very low or undetectable levels of PKM2 expression. CONCLUSIONS: PKM2 is not required for Apc-deficient colon cancer or for nuclear translocation of ß-catenin in Apc-null tumor cells. These findings suggest that PKM2 expression is not required for colon tumor formation or progression.

Resistance to dual blockade of the kinases PI3K and mTOR in KRAS-mutant colorectal cancer models results in combined sensitivity to inhibition of the receptor tyrosine kinase EGFR.

Targeted blockade of aberrantly activated signaling pathways is an attractive therapeutic strategy for solid tumors, but drug resistance is common. KRAS is a frequently mutated gene in human cancer but remains a challenging clinical target. Inhibitors against KRAS signaling mediators, namely, PI3K (phosphatidylinositol 3-kinase) and mTOR (mechanistic target of rapamycin), have limited clinical efficacy as single agents in KRAS-mutant colorectal cancer (CRC). We investigated potential bypass mechanisms to PI3K/mTOR inhibition in KRAS-mutant CRC. Using genetically engineered mouse model cells that had acquired resistance to the dual PI3K/mTOR small-molecule inhibitor PF-04691502, we determined with chemical library screens that inhibitors of the ERBB [epidermal growth factor receptor (EGFR)] family restored the sensitivity to PF-04691502. Although EGFR inhibitors alone have limited efficacy in reducing KRAS-mutant tumors, we found that PF-04691502 induced the abundance, phosphorylation, and activity of EGFR, ERBB2, and ERBB3 through activation of FOXO3a (forkhead box O 3a), a transcription factor inhibited by the PI3K to AKT pathway. PF-04691502 also induced a stem cell-like gene expression signature. KRAS-mutant patient-derived xenografts from mice treated with PF-04691502 had a similar gene expression signature and exhibited increased EGFR activation, suggesting that this drug-induced resistance mechanism may occur in patients. Combination therapy with dacomitinib (a pan-ERBB inhibitor) restored sensitivity to PF-04691502 in drug-resistant cells in culture and induced tumor regression in drug-resistant allografts in mice. Our findings suggest that combining PI3K/mTOR and EGFR inhibitors may improve therapeutic outcome in patients with KRAS-mutant CRC.

Overview of genetically engineered mouse models of colorectal carcinoma to enable translational biology and drug development.

Preclinical models for colorectal cancer (CRC) are critical for translational biology and drug development studies to characterize and treat this condition. Mouse models of human cancer are particularly popular because of their relatively low cost, short life span, and ease of use. Genetically engineered mouse models (GEMMs) of CRC are engineered from germline or somatic modification of critical tumor suppressor genes and/or oncogenes that drive mutations in human disease. Detailed in this overview are the salient features of several useful colorectal cancer GEMMs and their value as tools for translational biology and preclinical drug development.

Oncogenic transformation of diverse gastrointestinal tissues in primary organoid culture.

The application of primary organoid cultures containing epithelial and mesenchymal elements to cancer modeling holds promise for combining the accurate multilineage differentiation and physiology of in vivo systems with the facile in vitro manipulation of transformed cell lines. Here we used a single air-liquid interface culture method without modification to engineer oncogenic mutations into primary epithelial and mesenchymal organoids from mouse colon, stomach and pancreas. Pancreatic and gastric organoids exhibited dysplasia as a result of expression of Kras carrying the G12D mutation (Kras(G12D)), p53 loss or both and readily generated adenocarcinoma after in vivo transplantation. In contrast, primary colon organoids required combinatorial Apc, p53, Kras(G12D) and Smad4 mutations for progressive transformation to invasive adenocarcinoma-like histology in vitro and tumorigenicity in vivo, recapitulating multi-hit models of colorectal cancer (CRC), as compared to the more promiscuous transformation of small intestinal organoids. Colon organoid culture functionally validated the microRNA miR-483 as a dominant driver oncogene at the IGF2 (insulin-like growth factor-2) 11p15.5 CRC amplicon, inducing dysplasia in vitro and tumorigenicity in vivo. These studies demonstrate the general utility of a highly tractable primary organoid system for cancer modeling and driver oncogene validation in diverse gastrointestinal tissues.

Cross-species analysis of genetically engineered mouse models of MAPK-driven colorectal cancer identifies hallmarks of the human disease.

Effective treatment options for advanced colorectal cancer (CRC) are limited, survival rates are poor and this disease continues to be a leading cause of cancer-related deaths worldwide. Despite being a highly heterogeneous disease, a large subset of individuals with sporadic CRC typically harbor relatively few established 'driver' lesions. Here, we describe a collection of genetically engineered mouse models (GEMMs) of sporadic CRC that combine lesions frequently altered in human patients, including well-characterized tumor suppressors and activators of MAPK signaling. Primary tumors from these models were profiled, and individual GEMM tumors segregated into groups based on their genotypes. Unique allelic and genotypic expression signatures were generated from these GEMMs and applied to clinically annotated human CRC patient samples. We provide evidence that a Kras signature derived from these GEMMs is capable of distinguishing human tumors harboring KRAS mutation, and tracks with poor prognosis in two independent human patient cohorts. Furthermore, the analysis of a panel of human CRC cell lines suggests that high expression of the GEMM Kras signature correlates with sensitivity to targeted pathway inhibitors. Together, these findings implicate GEMMs as powerful preclinical tools with the capacity to recapitulate relevant human disease biology, and support the use of genetic signatures generated in these models to facilitate future drug discovery and validation efforts.

Colon-specific tumorigenesis in mice driven by Cre-mediated inactivation of Apc and activation of mutant Kras.

Several genetically engineered mouse (GEM) models of colorectal cancer have been developed and are a mainstay in our efforts to identify means of preventing and treating this disease. Many of these models involve a germline disruption of the adenomatous polyposis coli (Apc) tumor suppressor gene and share the limitation that the great preponderance of tumors appear in the small rather than large intestine. In recent years efforts have been made to increase the similarity of these models to human sporadic colorectal cancer by disrupting Apc in a tissue-specific fashion using the Cre-Lox system so that the genetic aberrations are confined to the colonic epithelium. These models have shown great promise but reproducible and high penetrance colon-specific tumorigenesis has not yet been achieved without invasive techniques to introduce the Cre enzyme. We therefore sought to create a new model with high penetrance colon-specific tumorigenesis but without the need for exogenous Cre administration. We utilized existing mice possessing a conditional knock out for the Apc gene and a latent activated Kras allele and crossed them with mice expressing Cre recombinase solely in the large intestine. Using this approach we generated mice that developed 1-9 colonic adenomas per mouse (average 4.3) but without any tumors in the small intestine or cecum. No invasive tumors were observed. Despite the apparent lack of invasion, the geographical correctness, complete penetrance and intermediate tumor burden make this model a promising addition to our toolkit for the study of colorectal cancer treatment and prevention.

Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer.

PI3K inhibition in combination with other agents has not been studied in the context of PIK3CA wild-type, KRAS mutant cancer. In a screen of phospho-kinases, PI3K inhibition of KRAS mutant colorectal cancer cells activated the MAPK pathway. Combination PI3K/MEK inhibition with NVP-BKM120 and PD-0325901 induced tumor regression in a mouse model of PIK3CA wild-type, KRAS mutant colorectal cancer, which was mediated by inhibition of mTORC1, inhibition of MCL-1, and activation of BIM. These findings implicate mitochondrial-dependent apoptotic mechanisms as determinants for the efficacy of PI3K/MEK inhibition in the treatment of PIK3CA wild-type, KRAS mutant cancer.

mTOR inhibition specifically sensitizes colorectal cancers with KRAS or BRAF mutations to BCL-2/BCL-XL inhibition by suppressing MCL-1.

Colorectal cancers harboring KRAS or BRAF mutations are refractory to current targeted therapies. Using data from a high-throughput drug screen, we have developed a novel therapeutic strategy that targets the apoptotic machinery using the BCL-2 family inhibitor ABT-263 (navitoclax) in combination with a TORC1/2 inhibitor, AZD8055. This combination leads to efficient apoptosis specifically in KRAS- and BRAF-mutant but not wild-type (WT) colorectal cancer cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT, colorectal cancers, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS-mutant colorectal cancer xenograft and genetically engineered mouse models of colorectal cancer, but not in the corresponding KRAS-WT colorectal cancer models. These data suggest that the combination of BCL-2/BCL-XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.

Concomitant BRAF and PI3K/mTOR blockade is required for effective treatment of BRAF(V600E) colorectal cancer.

PURPOSE: BRAF(V600E) mutations are associated with poor clinical prognosis in colorectal cancer (CRC). Although selective BRAF inhibitors are effective for treatment of melanoma, comparable efforts in CRC have been disappointing. Here, we investigated potential mechanisms underlying this resistance to BRAF inhibitors in BRAF(V600E) CRC. EXPERIMENTAL DESIGN: We examined phosphoinositide 3-kinase (PI3K)/mTOR signaling in BRAF(V600E) CRC cell lines after BRAF inhibition and cell viability and apoptosis after combined BRAF and PI3K/mTOR inhibition. We assessed the efficacy of in vivo combination treatment using a novel genetically engineered mouse model (GEMM) for BRAF(V600E) CRC. RESULTS: Western blot analysis revealed sustained PI3K/mTOR signaling upon BRAF inhibition. Our BRAF(V600E) GEMM presented with sessile serrated adenomas/polyps, as seen in humans. Combination treatment in vivo resulted in induction of apoptosis and tumor regression. CONCLUSIONS: We have established a novel GEMM to interrogate BRAF(V600E) CRC biology and identify more efficacious treatment strategies. Combination BRAF and PI3K/mTOR inhibitor treatment should be explored in clinical trials.

Development of a colon cancer GEMM-derived orthotopic transplant model for drug discovery and validation.

PURPOSE: Effective therapies for KRAS-mutant colorectal cancer (CRC) are a critical unmet clinical need. Previously, we described genetically engineered mouse models (GEMM) for sporadic Kras-mutant and non-mutant CRC suitable for preclinical evaluation of experimental therapeutics. To accelerate drug discovery and validation, we sought to derive low-passage cell lines from GEMM Kras-mutant and wild-type tumors for in vitro screening and transplantation into the native colonic environment of immunocompetent mice for in vivo validation. EXPERIMENTAL DESIGN: Cell lines were derived from Kras-mutant and non-mutant GEMM tumors under defined media conditions. Growth kinetics, phosphoproteomes, transcriptomes, drug sensitivity, and metabolism were examined. Cell lines were implanted in mice and monitored for in vivo tumor analysis. RESULTS: Kras-mutant cell lines displayed increased proliferation, mitogen-activated protein kinase signaling, and phosphoinositide-3 kinase signaling. Microarray analysis identified significant overlap with human CRC-related gene signatures, including KRAS-mutant and metastatic CRC. Further analyses revealed enrichment for numerous disease-relevant biologic pathways, including glucose metabolism. Functional assessment in vitro and in vivo validated this finding and highlighted the dependence of Kras-mutant CRC on oncogenic signaling and on aerobic glycolysis. CONCLUSIONS: We have successfully characterized a novel GEMM-derived orthotopic transplant model of human KRAS-mutant CRC. This approach combines in vitro screening capability using low-passage cell lines that recapitulate human CRC and potential for rapid in vivo validation using cell line-derived tumors that develop in the colonic microenvironment of immunocompetent animals. Taken together, this platform is a clear advancement in preclinical CRC models for comprehensive drug discovery and validation efforts.

Synthetic lethal interaction of combined BCL-XL and MEK inhibition promotes tumor regressions in KRAS mutant cancer models.

KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers.

Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth.

Approximately 2% of colorectal cancer is linked to pre-existing inflammation known as colitis-associated cancer, but most develops in patients without underlying inflammatory bowel disease. Colorectal cancer often follows a genetic pathway whereby loss of the adenomatous polyposis coli (APC) tumour suppressor and activation of ß-catenin are followed by mutations in K-Ras, PIK3CA and TP53, as the tumour emerges and progresses. Curiously, however, 'inflammatory signature' genes characteristic of colitis-associated cancer are also upregulated in colorectal cancer. Further, like most solid tumours, colorectal cancer exhibits immune/inflammatory infiltrates, referred to as 'tumour-elicited inflammation'. Although infiltrating CD4(+) T(H)1 cells and CD8(+) cytotoxic T cells constitute a positive prognostic sign in colorectal cancer, myeloid cells and T-helper interleukin (IL)-17-producing (T(H)17) cells promote tumorigenesis, and a 'T(H)17 expression signature' in stage I/II colorectal cancer is associated with a drastic decrease in disease-free survival. Despite its pathogenic importance, the mechanisms responsible for the appearance of tumour-elicited inflammation are poorly understood. Many epithelial cancers develop proximally to microbial communities, which are physically separated from immune cells by an epithelial barrier. We investigated mechanisms responsible for tumour-elicited inflammation in a mouse model of colorectal tumorigenesis, which, like human colorectal cancer, exhibits upregulation of IL-23 and IL-17. Here we show that IL-23 signalling promotes tumour growth and progression, and development of a tumoural IL-17 response. IL-23 is mainly produced by tumour-associated myeloid cells that are likely to be activated by microbial products, which penetrate the tumours but not adjacent tissue. Both early and late colorectal neoplasms exhibit defective expression of several barrier proteins. We propose that barrier deterioration induced by colorectal-cancer-initiating genetic lesions results in adenoma invasion by microbial products that trigger tumour-elicited inflammation, which in turn drives tumour growth.

In vivo optical molecular imaging of matrix metalloproteinase activity following celecoxib therapy for colorectal cancer.

We present an optical molecular imaging approach to measure the efficacy of the cyclooxygenase-2 (COX-2) inhibitor celecoxib on tumor growth rate through its effect on matrix metalloproteinase (MMP) activity. A xenograft model of colorectal cancer was generated in nude mice, which were then randomized to receive celecoxib versus vehicle. MMP activity was measured by an enzyme-activatable optical molecular probe. A novel genetically engineered mouse (GEM) model of colorectal cancer was also used to assess celecoxib's effect on MMP activity, which was measured by quantitative fluorescence colonoscopy. Subcutaneously implanted xenograft tumors were 84% (SD 20.2%) smaller in volume in the treatment group versus the control group. Moreover, treated animals exhibited only a 7.6% (SEM 9%) increase in MMP activity versus 106% (SEM 8%) for untreated animals. There was an apparent linear relationship (r  =  .91) between measured MMP activity and tumor growth rate. Finally, in the GEM model experiment, treated murine tumors remained relatively unchanged in volume and MMP activity; however, untreated tumors grew significantly and showed an increase in MMP activity. This method may provide for the improved identification of patients for whom COX-2 inhibition therapy is indicated by allowing one to balance the patient's cardiovascular risk with the cancer's responsiveness to celecoxib.

Priceless GEMMs: genetically engineered mouse models for colorectal cancer drug development.

To establish effective drug development for colorectal cancer (CRC), preclinical models that are robust surrogates for human disease are crucial. Mouse models are an attractive platform because of their relatively low cost, short life span, and ease of use. There are two main categories of mouse CRC models: xenografts derived from implantation of CRC cells or tumors in immunodeficient mice; and genetically engineered mouse models (GEMMs) derived from modification of human cancer predisposition genes, resulting in spontaneous tumor formation. Here, we review xenografts and GEMMs and focus on their potential application in translational research. Furthermore, we describe newer GEMMs for sporadic CRC that are particularly suitable for drug testing. Finally, we discuss recent advances in small-animal imaging, such as optical colonoscopy, which allow in vivo assessment of tumors. With the increasing sophistication of GEMMs, our preclinical armamentarium provides new hope for the ongoing war against CRC.

EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib.

UNLABELLED: BRAF mutations occur in 10-15% of colorectal cancers (CRCs) and confer adverse outcome. While RAF inhibitors such as vemurafenib (PLX4032) have proven effective in BRAF mutant melanoma, they are surprisingly ineffective in BRAF mutant CRCs, and the reason for this disparity remains unclear. Compared to BRAF mutant melanoma cells, BRAF mutant CRC cells were less sensitive to vemurafenib, and P-ERK suppression was not sustained in response to treatment. Although transient inhibition of phospho-ERK by vemurafenib was observed in CRC, rapid ERK re-activation occurred through EGFR-mediated activation of RAS and CRAF. BRAF mutant CRCs expressed higher levels of phospho-EGFR than BRAF mutant melanomas, suggesting that CRCs are specifically poised for EGFR-mediated resistance. Combined RAF and EGFR inhibition blocked reactivation of MAPK signaling in BRAF mutant CRC cells and markedly improved efficacy in vitro and in vivo. These findings support evaluation of combined RAF and EGFR inhibition in BRAF mutant CRC patients. SIGNIFICANCE: BRAF valine 600 (V600) mutations occur in 10% to 15% of colorectal cancers, yet these tumors show a surprisingly low clinical response rate (~5%) to selective RAF inhibitors such as vemurafenib, which have produced dramatic response rates (60%­80%) in melanomas harboring the identical BRAF V600 mutation. We found that EGFR-mediated MAPK pathway reactivation leads to resistance to vemurafenib in BRAF-mutant colorectal cancers and that combined RAF and EGFR inhibition can lead to sustained MAPK pathway suppression and improved efficacy in vitro and in tumor xenografts.