More than half of human colorectal cancers (CRCs) carry either KRAS or BRAF mutations, and are often refractory to approved targeted therapies. We report that cultured CRC cells harboring KRAS or BRAF mutations are selectively killed when exposed to high levels of vitamin C. This effect is due to increased uptake of the oxidized form of vitamin C, dehydroascorbate (DHA), via the GLUT1 glucose transporter. Increased DHA uptake causes oxidative stress as intracellular DHA is reduced to vitamin C depleting glutathione. Thus, ROS accumulates and inactivates glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Inhibiting GAPDH in highly glycolytic KRAS or BRAF mutant cells leads to an energetic crisis and cell death not seen in KRAS and BRAF wild-type cells. In vivo studies indicate that high-dose vitamin C can impair tumor growth in Apc/KrasG12D mutant mouse intestinal cancers. While it is unclear whether human tumors will respond similarly, our results provide a mechanistic rationale for exploring the therapeutic use of vitamin C to treat CRCs with KRAS or BRAF mutations.

Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we find that high fat diet (HFD)-induced obesity augments the numbers and function of Lgr5+ intestinal stem-cells (ISCs) of the mammalian intestine. Mechanistically, HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-d) signature in intestinal stem and (non-ISC) progenitor cells, and pharmacologic activation of PPAR-d recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-d dependent manner. Interestingly, HFD- and agonist-activated PPAR-d signaling endow organoid-initiating capacity to progenitors, and enforced PPAR-d signaling permits these progenitors to form in vivo tumors upon loss of the tumor suppressor Apc. These findings highlight how diet-modulated PPAR-d activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumors.

SummaryThe metastatic process of colorectal cancer (CRC) is not fully understood and effective therapies are lacking. We show that activation of NOTCH1 signaling in the murine intestinal epithelium leads to highly penetrant metastasis (100% metastasis; with >80% liver metastases) in KrasG12D-driven serrated cancer. Transcriptional profiling reveals that epithelial NOTCH1 signaling creates a tumor microenvironment (TME) reminiscent of poorly prognostic human CRC subtypes (CMS4 and CRIS-B), and drives metastasis through transforming growth factor (TGF) β-dependent neutrophil recruitment. Importantly, inhibition of this recruitment with clinically relevant therapeutic agents blocks metastasis. We propose that NOTCH1 signaling is key to CRC progression and should be exploited clinically.

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms HHS Public AccessAuthor manuscript Nat Biotechnol. Author manuscript; available in PMC 2017 November 01. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript (CRC) and metastasis that rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/ Δ;Kras G12D/+ ;Trp53Δ/ Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5+ stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.Recent tumor sequencing studies have identified a large number of candidate genes that are mutated in CRCs and may contribute to carcinogenesis, tumor phenotype, and treatment responses in subsets of patients. 1,2 Traditionally, functional assessment of putative cancerassociated genes in vivo has required the development of genetically engineered mouse models (GEMMs) of CRC through extensive intercrossing or de novo generation of genetargeted mice, which is expensive and time consuming. Most GEMMs of CRC such as the Apc Min mouse 3,4 are also limited by delayed tumor onset (i.e., 2-4 months) and high tumor burden (i.e., 30-100 polyps) in the small intestine, which is a rare location for human intestinal tumors and precludes study of tumor progression beyond early adenomas or longitudinal studies using colonoscopy 5,6 . Tumorigenesis can be localized to the colon with either Apc loss driven by a colon-specific promoter, which is limited by slow tumor growth (i.e., 4-6 months) 7-9 , or somatic deletion of Apc in the distal colon of Apc fl/fl mice with rectal enema of adenoviral Cre, which is requires colonic injury and/or time-consuming surgery 10-12 .In addition to GEMMs, human and mouse cell lines are used to model CRC in vivo. Typical sites of transplantation are the mouse flank or kidney capsule, which do not recapitulate the native stroma of the colon mucosa. 6 Several groups have sought to orthotopically deliver tumor cell lines into the mouse colon, either surgically into the cecal serosa 13 (which is not the relevant tissue layer for CRC development) or into the...

A decline in stem cell function impairs tissue regeneration during aging, but the role of the stem cell supporting niche in aging is not well understood. The small intestine is maintained by actively cycling intestinal stem cells (ISCs) that are regulated by the Paneth cell niche 1,2. Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age due to defects in both stem cells and their niche. The functional decline was caused by decrease in stemness maintaining Wnt signalling due to production of an extracellular Wnt-inhibitor, Notum, in aged Paneth cells. Mechanistically, high mTORC1 activity in old Paneth cells inhibits PPARa activity 3 and lowered PPARa increased Notum expression. Genetic targeting of Notum or Wnt-supplementation restored function of old intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of old stem cells and promoted recovery from chemotherapy induced damage. Our results reveal an unappreciated role for the stem cell niche in aging and demonstrate that targeting of Notum can promote regeneration of old tissues. Tissue turnover and regenerative capacity decrease upon aging in many tissue types 4-6. The intestinal epithelium is one of the fastest renewing tissues in the human body and has been reported to regenerate without loss of self-renewal in long term in vitro organoid culture 7. However,

Excessive consumption of beverages sweetened with high-fructose corn syrup (HFCS) is associated with obesity and with an increased risk of colorectal cancer. Whether HFCS contributes directly to tumorigenesis is unclear. We investigated the effects of daily oral administration of HFCS in adenomatous polyposis coli (APC) mutant mice, which are predisposed to develop intestinal tumors. The HFCS-treated mice showed a substantial increase in tumor size and tumor grade in the absence of obesity and metabolic syndrome. HFCS increased the concentrations of fructose and glucose in the intestinal lumen and serum, respectively, and the tumors transported both sugars. Within the tumors, fructose was converted to fructose-1-phosphate, leading to activation of glycolysis and increased synthesis of fatty acids that support tumor growth. These mouse studies support the hypothesis that the combination of dietary glucose and fructose, even at a moderate dose, can enhance tumorigenesis.

consults and holds stock in Ideaya, and cofounded and holds stock in Cedilla Therapeutics. G.G. receives research funding from IBM and Pharmacyclics and is an inventor on multiple patent applications related to bioinformatic tools, including applications related to MuTect, ABSOLUTE, MSMuTect, MSMutSig and MSIClass. Y.E.M. is an inventor on patent applications related to the bioinformatic tools, MSMuTect, MSMutSig and MSIClass. The Broad Institute filed a US patent application related to the target described in this manuscript.

Colorectal cancers (CRCs) 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 combines targeting of 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 mutant (MT) and BRAF MT but not wild-type (WT) CRC cells. This specific susceptibility results from TORC1/2 inhibition leading to suppression of MCL-1 expression in mutant, but not WT CRCs, leading to abrogation of BIM/MCL-1 complexes. This combination strategy leads to tumor regressions in both KRAS MT colorectal cancer xenograft and genetically-engineered mouse models of CRC, but not in the corresponding KRAS WT CRC models. These data suggest that the combination of BCL-2/XL inhibitors with TORC1/2 inhibitors constitutes a promising targeted therapy strategy to treat these recalcitrant cancers.