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(a) Barplot of GISTIC2.0 chromosome 20q gene level copy number calls in TCGA COADREAD data and (b) chromosome 20q genes covered by MSK-IMPACT. (c) Interquartile range (IGR) of gene level RNASeq expression z-score by copy number group from 2a. (d) MSK-IMPACT and TCGA tumor purity estimates.

(a) Plot of SCNA in a CRC with 20q amplification as detected by MSK-IMPACT and (b) high resolution SNP analysis on Oncoscan confirmed the results without showing focal amplifications

Here, comprehensive analysis was performed on the molecular and clinical features of colorectal carcinoma harboring chromosome 20q amplification. Tumor and normal DNA from patients with advanced colorectal carcinoma underwent next-generation sequencing via MSK-IMPACT, and a subset of case samples was subjected to high-resolution microarray (Oncoscan). Relationships between genomic copy number and transcript expression were assessed with The Cancer Genome Atlas (TCGA) colorectal carcinoma data. Of the colorectal carcinoma patients sequenced (n = 401) with MSK-IMPACT, 148 (37%) had 20q gain, and 30 (7%) had 20q amplification. In both the MSK-IMPACT and TCGA datasets, BCL2L1 was the most frequently amplified 20q oncogene. However, SRC was the only recognized 20q oncogene with a significant inverse relationship between mRNA upregulation and RAS/RAF mutation (OR, −0.4 ± 0.2, P = 0.02). In comparison with 20q diploid colorectal carcinoma, 20q gain/amplification was associated with wild-type KRAS (P < 0.001) and BRAF (P = 0.01), microsatellite stability (P < 0.001), distal primary tumors (P < 0.001), and mutant TP53 (P < 0.001), but not stage. On multivariate analysis, longer overall survival from the date of metastasis was observed with chromosome 20q gain (P = 0.02) or amplification (P = 0.04) compared with diploid 20q.Implications: 20q amplification defines a subset of colorectal cancer patients with better overall survival from the date of metastasis, and further studies are warranted to assess whether the inhibition of 20q oncogenes, such as SRC, may benefit this subset of patients. Mol Cancer Res; 15(6); 708–13. ©2017 AACR.

Anticancer drug development campaigns often fail due to an incomplete understanding of the therapeutic index differentiating the efficacy of the agent against the cancer and its on-target toxicities to the host. To address this issue, we established a versatile preclinical platform in which genetically defined cancers are produced using somatic tissue engineering in transgenic mice harboring a doxycycline-inducible short hairpin RNA against the target of interest. In this system, target inhibition is achieved by the addition of doxycycline, enabling simultaneous assessment of efficacy and toxicity in the same animal. As proof of concept, we focused on CDK9—a cancer target whose clinical development has been hampered by compounds with poorly understood target specificity and unacceptable toxicities. We systematically compared phenotypes produced by genetic Cdk9 inhibition to those achieved using a recently developed highly specific small molecule CDK9 inhibitor and found that both perturbations led to robust antitumor responses. Remarkably, nontoxic levels of CDK9 inhibition could achieve significant treatment efficacy, and dose-dependent toxicities produced by prolonged CDK9 suppression were largely reversible upon Cdk9 restoration or drug withdrawal. Overall, these results establish a versatile in vivo target validation platform that can be employed for rapid triaging of therapeutic targets and lend support to efforts aimed at advancing CDK9 inhibitors for cancer therapy.

Metastasis-initiating cells with stem-like properties drive cancer lethality, yet their origins and relationship to primary-tumor-initiating stem cells are not known. We show that L1CAM+ cells in human colorectal cancer (CRC) have metastasis-initiating capacity, and we define their relationship to tissue regeneration. L1CAM is not expressed in the homeostatic intestinal epithelium, but is induced and required for epithelial regeneration following colitis and in CRC organoid growth. By using human tissues and mouse models, we show that L1CAM is dispensable for adenoma initiation but required for orthotopic carcinoma propagation, liver metastatic colonization and chemoresistance. L1CAMhigh cells partially overlap with LGR5high stem-like cells in human CRC organoids. Disruption of intercellular epithelial contacts causes E-cadherin–REST transcriptional derepression of L1CAM, switching chemoresistant CRC progenitors from an L1CAMlow to an L1CAMhigh state. Thus, L1CAM dependency emerges in regenerative intestinal cells when epithelial integrity is lost, a phenotype of wound healing deployed in metastasis-initiating cells. The authors identify a population of L1CAM-positive cells that, following loss of epithelial integrity, promote intestinal tissue regeneration and mediate metastasis initiation and chemoresistance in colorectal cancer.

Somatic mutations in the KRAS oncogene are associated with poor outcomes in locally advanced rectal cancer but the underlying biologic mechanisms are not fully understood. We profiled mRNA in 76 locally advanced rectal adenocarcinomas from patients that were enrolled in a prospective clinical trial and investigated differences in gene expression between KRAS mutant (KRAS‐mt) and KRAS‐wild‐type (KRAS‐wt) patients. We found that KRAS‐mt tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix. We validated our findings using samples from The Cancer Genome Atlas (TCGA) and also by performing immunohistochemistry (IHC) and immunofluorescence (IF) in orthogonal cohorts. Using in vitro and in vivo models, we show that oncogenic KRAS signaling within the epithelial cancer cells modulates the activity of the surrounding fibroblasts in the tumor microenvironment., Oncogenic KRAS is associated with poor outcomes in locally advanced rectal cancer (LARC) but the underlying biologic mechanisms are not fully understood. We investigated differences in gene expression profiles between KRAS mutant and KRAS wild‐type LARC tumors. We found that KRAS mutant tumors display lower expression of genes related to the tumor stroma and remodeling of the extracellular matrix.

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation, and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to patients. We established a biorepository of 65 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic, or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and their ex vivo responses to clinically relevant chemotherapy and radiation treatment correlated with the clinical responses noted in individual patients’ tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive RC followed by metastasis to lung and liver. Importantly, engrafted tumors displayed the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based, ex vivo platform coupled with in vivo endoluminal propagation in animals.

Rectal cancer (RC) is a challenging disease to treat that requires chemotherapy, radiation, and surgery to optimize outcomes for individual patients. No accurate model of RC exists to answer fundamental research questions relevant to individual patients. We established a biorepository of 32 patient-derived RC organoid cultures (tumoroids) from patients with primary, metastatic, or recurrent disease. RC tumoroids retained molecular features of the tumors from which they were derived, and theirex vivoresponses to clinically relevant chemotherapy and radiation treatment correlate well with responses noted in individual patients’ tumors. Upon engraftment into murine rectal mucosa, human RC tumoroids gave rise to invasive rectal cancer followed by metastasis to lung and liver. Importantly, engrafted tumors closely reflected the heterogenous sensitivity to chemotherapy observed clinically. Thus, the biology and drug sensitivity of RC clinical isolates can be efficiently interrogated using an organoid-based,in vitroplatform coupled with endoluminal propagation in animals.

Metastatic cancers relapse due to the emergence of stemlike clones capable of reversible quiescence, tumour reinitiation, and therapy resistance termed metastasis-initiating cells (MICs). The origins of MICs and their relationship to primary tumor-initiating cells are not known, largely due to a lack of representative models of MICS. To directly scrutinize MICs in patient metastases, we established organoid cultures from therapy-resistant, residual colorectal cancer (CRC) liver metastases of patients undergoing cancer surgery. We identify the neuronal cell adhesion molecule L1 (L1CAM) as a critical mediator and marker of colorectal cancer (CRC) MICs. L1CAM+ cells are quiescent in well-structured neoplastic glands in vivo, but when dissociated from their epithelial niche, drive organoid regeneration and xenograft tumor reinitiation. FACS-sorted L1CAM+ cells preferentially regenerate heterogeneous organoids containing both L1CAM+ and L1CAM- progeny. Single-cell mRNA sequencing of 10,000 cells from four patient-derived organoids reveals that L1CAMhigh MICs in human CRC organoids partially overlap with a subset of LGR5high cancer stem cells (CSCs). The number of LGR5-expressing cells decreases, while the number of L1CAM-expressing cells increases in metastases in comparison with primary tumors in patients. We show that L1CAM is required for organoid formation, intestinal epithelial repair following colitis in vivo, regeneration of orthotopic rectal tumors, and metastatic colonization of the liver, but is dispensable for intestinal epithelial homeostasis or tumor initiation. Mechanistically, disruption of intercellular epithelial contacts inhibits an E-cadherin-REST signaling axis and enables transcriptional derepression of L1CAM. Our work underscores the distinct requirements of the CSCs that initiate tumor growth and MICs that drive lethal metastatic relapse. We identify the loss of epithelial integrity, an obligatory step of metastasis, as a crucial molecular driver of the transcriptional plasticity required for the emergence of prometastatic traits. Further, we define L1CAM as a crucial vulnerability of MICs that could be exploited therapeutically to treat patients with metastatic cancer. Citation Format: Karuna Ganesh, Harihar Basnet, Yasemin Kaygusuz, Ashley Laughney, Lan He, Roshan Sharma, Kevin O'Rourke, Vincent Reuter, Yun-Han Huang, Ignas Masilionis, Mesruh Turkekul, Ekrem Er, Katja Manova-Todorova, Leonard Saltz, Martin Weiser, Julio Garcia-Aguilar, Richard Koche, Scott Lowe, Dana Peer, Jinru Shia, Joan Massague. L1CAM defines the regenerative origin of metastasis initiating cells in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A12.

Tetraploid cells, which are most commonly generated by errors in cell division, are genomically unstable and have been shown to promote tumorigenesis. Recent genomic studies have estimated that ∼40% of all solid tumors have undergone a genome-doubling event during their evolution, suggesting a significant role for tetraploidy in driving the development of human cancers. To safeguard against the deleterious effects of tetraploidy, nontransformed cells that fail mitosis and become tetraploid activate both the Hippo and p53 tumor suppressor pathways to restrain further proliferation. Tetraploid cells must therefore overcome these antiproliferative barriers to ultimately drive tumor development. However, the genetic routes through which spontaneously arising tetraploid cells adapt to regain proliferative capacity remain poorly characterized. Here, we conducted a comprehensive gain-of-function genome-wide screen to identify microRNAs (miRNAs) that are sufficient to promote the proliferation of tetraploid cells. Our screen identified 23 miRNAs whose overexpression significantly promotes tetraploid proliferation. The vast majority of these miRNAs facilitate tetraploid growth by enhancing mitogenic signaling pathways (e.g., miR-191-3p); however, we also identified several miRNAs that impair the p53/p21 pathway (e.g., miR-523-3p), and a single miRNA (miR-24-3p) that potently inactivates the Hippo pathway via down-regulation of the tumor suppressor gene NF2. Collectively, our data reveal several avenues through which tetraploid cells may regain the proliferative capacity necessary to drive tumorigenesis.

Tetraploid cells, which are most commonly generated by errors in cell division, are genomically unstable and have been shown to promote tumorigenesis. Recent genomic studies have estimated that ∼40% of all solid tumors have undergone a genome-doubling event during their evolution, suggesting a significant role for tetraploidy in driving the development of human cancers. To safeguard against the deleterious effects of tetraploidy, non-transformed cells that fail mitosis and become tetraploid activate both the Hippo and p53 tumor suppressor pathways to restrain further proliferation. Tetraploid cells must therefore overcome these anti-proliferative barriers to ultimately drive tumor development. However, the genetic routes through which spontaneously arising tetraploid cells adapt to regain proliferative capacity remain poorly characterized. Here, we conducted a comprehensive, gain-of-function genome-wide screen to identify miRNAs that are sufficient to promote the proliferation of tetraploid cells. Our screen identified 23 miRNAs whose overexpression significantly promotes tetraploid proliferation. The vast majority of these miRNAs facilitate tetraploid growth by enhancing mitogenic signaling pathways (e.g. miR-191-3p); however, we also identified several miRNAs that impair the p53/p21 pathway (e.g. miR-523-3p), and a single miRNA (miR-24-3p) that potently inactivates the Hippo pathway via downregulation of the tumor suppressor gene NF2. Collectively, our data reveal several avenues through which tetraploid cells may regain the proliferative capacity necessary to drive tumorigenesis.

SummaryThe adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC.

CRISPR-Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR-Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9(D10A) (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively. Further, we show that this inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and, thus, provides a flexible and fast platform to study loss-of-function phenotypes in vivo.

576 Background: KRAS mutation ( KRASmut) is associated with aggressive biological behavior and resistance to chemoradiotherapy in colorectal cancer (CRC). The tumor microenvironment is a critical component framing the biological behavior of cancers. We have recently shown that a KRASmut modulates the tumor microenvironment by reducing the expression of extracellular matrix (ECM) genes in CRC. The effect of a KRASmut on integrins, the epithelial cell receptors for ECM proteins, remains largely unknown. Here, we investigated the impact of KRASmut on integrin expression in CRC and the effect of integrin beta 4 (ITGB4) expression on CRC phenotype. Methods: The genomic profile of 79 locally advanced rectal cancers (LARC) was characterized by the MSK-IMPACT DNA assay and RNA sequencing by Hi-Seq platform. The transcriptomic changes associated with KRAS in the LARC cohort was validated in the TCGA colon cancer dataset. Expression of ITGB4 was investigated by immunofluorescence (IF) in 39 colon cancer specimens by counting ITGB4-positive cells in an E-cadherin positive epithelial population. The relationship between KRAS and ITGB4 was also explored by manipulating KRAS expression in human cell lines and genetically engineered mouse models (GEMMs). ITGB4 knockout in HCT116 CRC cell lines and organoids from GEMMs were generated with CRISPR/Cas9. ITGB4 expression was confirmed using qRT-PCR and western blotting. Cell proliferation was assessed with the MTT assay. Cell invasion and migration were assessed in a trans-well system. Results: ITGB4 gene expression was increased in KRASmut compared to KRASwildtype in LARC and TCGA. Increased ITGB4 expression in KRASmut colon cancers was also validated by IF (p = 0.0029). Introduction of KRASmut in HCT116 CRCs and GEMMs increased ITGB4 expression by 1.5 to 2 fold. Knockout of ITGB4 reduced the migration and invasion of HCT116 CRC cells but did not alter proliferation. Conclusions: KRASmutincreases the expression of ITGB4 in CRC. Increased ITGB4 expression is associated with CRC cell invasion and migration. These results inform the biology of tumor progression in KRASmut CRC tumors.

Here, comprehensive analysis was performed on the molecular and clinical features of colorectal carcinoma harboring chromosome 20q amplification. Tumor and normal DNA from patients with advanced colorectal carcinoma underwent next-generation sequencing via MSK-IMPACT, and a subset of case samples was subjected to high-resolution microarray (Oncoscan). Relationships between genomic copy number and transcript expression were assessed with The Cancer Genome Atlas (TCGA) colorectal carcinoma data. Of the colorectal carcinoma patients sequenced (n = 401) with MSK-IMPACT, 148 (37%) had 20q gain, and 30 (7%) had 20q amplification. In both the MSK-IMPACT and TCGA datasets, BCL2L1 was the most frequently amplified 20q oncogene. However, SRC was the only recognized 20q oncogene with a significant inverse relationship between mRNA upregulation and RAS/RAF mutation (OR, −0.4 ± 0.2, P = 0.02). In comparison with 20q diploid colorectal carcinoma, 20q gain/amplification was associated with wild-type KRAS (P < 0.001) and BRAF (P = 0.01), microsatellite stability (P < 0.001), distal primary tumors (P < 0.001), and mutant TP53 (P < 0.001), but not stage. On multivariate analysis, longer overall survival from the date of metastasis was observed with chromosome 20q gain (P = 0.02) or amplification (P = 0.04) compared with diploid 20q. Implications: 20q amplification defines a subset of colorectal cancer patients with better overall survival from the date of metastasis, and further studies are warranted to assess whether the inhibition of 20q oncogenes, such as SRC, may benefit this subset of patients. Mol Cancer Res; 15(6); 708–13. ©2017 AACR.

Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.

Metastatic cancers invariably relapse due to the emergence of resistant tumor clones capable of self-renewal, entry into and exit from quiescence, tumor re-initiation and therapy resistance. The origins of such metastasis propagating cells (MPCs), which ultimately cause cancer death, are not well-understood. To directly scrutinize MPCs in patient metastases, we established ex vivo organoid cultures from surgically resected, chemoresistant residual colorectal cancer (CRC) liver metastases. We show that the neuronal cell-adhesion molecule L1CAM, which is ectopically expressed in many cancer types and strongly associated with poor prognosis, is a marker of MPCs. L1CAM+ cells are largely quiescent in structured neoplastic glands in tumors, but when dissociated from their epithelial niche, proliferate to regenerate heterogeneous organoids or xenografts containing both L1CAM+ and L1CAM- progeny. To define the relationship between L1CAM+ MPCs and Lgr5+ intestinal stem cells, we performed single cell mRNA sequencing on ~15,000 CRC organoid-derived cells from four patients. We identified only partial overlap between Lgr5+ and L1CAM+ cells. Lgr5high cells consistent with homeostatic stem cells, have low L1CAM levels, while Lgr5low transit amplifying progenitor-like cells have high L1CAM levels. In addition, we identify a separate population of L1CAMhighLgr5- cells. The data suggest that human CRC metastases are derived from an L1CAM+ population of transit-amplifying, partially differentiated cells. L1CAM is not expressed in intact human or mouse intestinal crypts during homeostasis. However, when the intestinal epithelium is disrupted by dextran sodium sulfate-mediated colitis, L1CAM is strongly induced in cells in the middle of regenerating crypts. Intestinal epithelium specific deletion of L1CAM causes profound weight loss, poor tissue healing and reduces survival in DSS-treated mice. In turn, L1CAM knockdown/knockout in mouse or human CRC cells inhibits regeneration of organoids in vitro, subcutaneous tumors and orthotopic liver metastases in vivo. Mechanistically, L1CAM RNA expression is normally silenced in non-neuronal cells by the transcriptional repressor REST. We show that disruption of epithelial integrity by organoid dissociation or E-cadherin knockdown reduces REST binding to an L1CAM intronic enhancer, thus inducing L1CAM expression. Our results suggest that L1CAM is dispensable for epithelial homeostasis, but is required for normal and neoplastic epithelial regeneration when tissue integrity is disrupted. During cancer progression, disseminated tumor cells at the invasion front of primary tumors, in the circulation, or in isolated residual disease following therapy, induce and depend on L1CAM for survival and eventual regrowth. Thus, L1CAM represents a crucial vulnerability of disseminated and residual MPCs that could be exploited therapeutically to treat patients with metastatic cancer. Citation Format: Karuna Ganesh, Harihar Basnet, Kevin P. O'Rourke, Ashley M. Laughney, Lan He, Eduard Batlle, Scott W. Lowe, Dana Pe'er, Jinru Shia, Joan Massague. Regenerative origin of colorectal metastasis stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4990.

In this protocol we describe our modifications to a method to isolate, culture and maintain mouse intestinal stem cells as crypt-villus forming organoids. These cells, isolated either from the small or large intestine, maintain self-renewal and multilineage differentiation potential over time. This provides investigators a tool to culture wild type or transformed intestinal epithelium, and a robust assay for stem cell tissue homeostasis in vitro.

Epithelial regeneration is critical for barrier maintenance and organ function after intestinal injury. The intestinal stem cell (ISC) niche provides Wnt, Notch, and epidermal growth factor (EGF) signals supporting Lgr5+ crypt base columnar ISCs for normal epithelial maintenance1,2. However, little is known about the regulation of the ISC compartment after tissue damage. Utilizing ex vivo organoid cultures, we provide evidence that innate lymphoid cells (ILCs), potent producers of Interleukin-22 (IL-22) after intestinal injury3,4, increased the growth of murine small intestine (SI) organoids in an IL-22-dependent fashion. Recombinant IL-22 directly targeted ISCs, augmenting the growth of both murine and human intestinal organoids, increasing proliferation, and promoting ISC expansion. IL-22 induced Stat3 phosphorylation in Lgr5+ ISCs, and Stat3 was critical for both organoid formation and IL-22-mediated regeneration. Treatment with IL-22 in vivo after murine allogeneic bone marrow transplantation (BMT) enhanced recovery of ISCs, increased epithelial regeneration, and reduced intestinal pathology and mortality from graft vs. host disease (GVHD). Atoh1-deficient organoid culture demonstrated that IL-22 induced epithelial regeneration independent of the Paneth cell niche. Our findings reveal a fundamental mechanism by which the immune system is able to support intestinal epithelium, activating ISCs to promote regeneration.

International audience; Genetically unstable tetraploid cells can promote tumorigenesis. Recent estimates suggest that ∼37% of human tumors have undergone a genome-doubling event during their development. This potentially oncogenic effect of tetraploidy is countered by a p53-dependent barrier to proliferation. However, the cellular defects and corresponding signaling pathways that trigger growth suppression in tetraploid cells are not known. Here, we combine RNAi screening and in vitro evolution approaches to demonstrate that cytokinesis failure activates the Hippo tumor suppressor pathway in cultured cells, as well as in naturally occurring tetraploid cells in vivo. Induction of the Hippo pathway is triggered in part by extra centrosomes, which alter small G protein signaling and activate LATS2 kinase. LATS2 in turn stabilizes p53 and inhibits the transcriptional regulators YAP and TAZ. These findings define an important tumor suppression mechanism and uncover adaptive mechanisms potentially available to nascent tumor cells that bypass this inhibitory regulation.

Background: Treatment for locally advanced rectal cancer (LARC) consists of neoadjuvant chemoradiotherapy (NCRT) followed by radical excision. Patients with tumors carrying a mutant KRAS are less likely to respond to NCRT compared to KRAS wild type tumors. We hypothesized that an RNA-based signature differentiating KRAS mutant and wild type patients could serve as an indicator of the biological process associated with response to NCRT. We found that the RNA-based signature is enriched for stromal and immune genes. Furthermore, the stromal component of the signature is a predictor of response to NCRT. Methods: Tumors from 120 LARC patients enrolled in a multicenter phase 2 trial studying response to NCRT were tested for KRAS status by Sanger Sequencing or Memorial Sloan Kettering (MSK)-IMPACT assay and gene expression was quantified by sequencing. Colorectal cancer (CRC) patients from MSK (n = 95) and TCGA (n = 261), previously annotated for KRAS mutation status and gene expression, were used for validation. A KRAS-inducible mouse model and CRC patient-derived xenografts (PDXs) were utilized to determine the cell of origin for the gene expression signature. Stromal enrichment was assessed with the ESTIMATE stromal gene signature. Immunohistochemistry (IHC) was completed for Periostin (POSTN), a stromal marker from the RNA-signature. Variant Allele Frequency (VAF) was used to measure the abundance of KRAS, TP53 and Adenomatous Polyposis Coli (APC) mutant alleles in tumors, and was quantified by targeted exome sequencing with the MSK-IMPACT assay. Results: Analysis of the KRAS-associated gene signature showed significant stromal inactivation in KRAS mutant patients. The signature was validated in the MSK and TCGA cohorts. The stromal signature was recapitulated in a KRAS inducible mouse model. Human CRC PDXs in mouse indicated that the signature arose from murine stroma and not human epithelium. Consistent with the stromal signature, IHC for POSTN, a stromal marker, was significantly lower in the KRAS mutant tumors compared with the KRAS wild type tumors (p Conclusions: This study shows that a KRAS mutation in CRC is associated with a lower expression of a stromal signature and that this signature is derived from the tumor microenvironment. This study indicates that CRC KRAS mutant tumors and a stromal subtype are closely related. Understanding this relationship may play a key role in elucidating the mechanism by which a KRAS mutant tumor is resistant to standard therapy. Citation Format: Raphael Pelossof, Moshe Elkatebts, Oliver Chow, Lauren Fairchild, Kevin O’Rourke, Jesse J. Smith, Chin-Tung Chen, Samuel Brook, Maurizio Scaltriti, Jinru Shia, Philip Paty, Christina Leslie, Scott Lowe, Jose Baselga, Julio Garcia-Aguilar. KRAS mutation status is associated with stromal inactivation in colorectal cancer and predicts poor response to neoadjuvant chemoradiotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4078.

Colorectal cancer (CRC) accounts for almost 10% of all cancer mortality in the United States. The Adenomatous Polyposis Coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. Using a novel mouse model in which Apc can be conditionally suppressed using a doxycycline-regulated shRNA, we previously showed that Apc suppression in adenomas and carcinomas drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. However, this study was unable to explore Apc action in metastasis, which is the most common cause of death in CRC patients, since models do not live long enough for metastatic dissemination to occur. To produce a system that would enable further disease progression, we generated a comprehensive panel of colon organoid cultures that reflect iterative combinations of the 3 most common genetic lesions in human CRC: Apc-mut, Apc-mut;KrasG12D, Apc-mut;p53-mut and Apc-mut;KrasG12D;p53-mut. These cultures can be transplanted orthotopically into the colon of recipient mice or seeded in the circulation to assess primary tumor behavior and metastatic dissemination. We show that Apc silencing in organoid culture results in the dysregulated proliferation of stem and progenitor cells but this is reversible upon Apc restoration or pharmacologic inhibition of the Wnt pathway. When engrafted into the colon of recipient mice, these cells create ∼1-3 focal colon tumors that can be tracked using colonoscopy, ultrasound and bioluminescence imaging, and are largely indistinguishable from tumors that develop in transgenic models. Most importantly, triple mutant (Apc/Kras/p53) organoids form invasive adenocarcinoma and colonize the lung following tail vein injection, providing a setting to dissect the importance of Wnt pathway activity in various stages of disease progression. Ultimately, the ability to study cellular and molecular phenotypes in vitro, and assess tumor development in vivo, provides a flexible system to explore the genetic factors underlying CRC progression and metastasis, and overcomes many of the previous technical challenges of modelling advanced CRC. Citation Format: Kevin P. O’Rourke, Lukas E. Dow, Scott W. Lowe. Apc restoration promotes cellular differentiation and reestablishes crypt homeostasis in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 666.

Reviews the relevance of the Minuteman III Intercontinental Ballistic Missile with regard to Post Cold War deterrence with regard to military and political utility.

Streptococcus mutans is the primary causative agent of human dental caries, a ubiquitous infectious disease for which effective treatment strategies remain elusive. We investigated a 25-kDa SloR metalloregulatory protein in this oral pathogen, along with its target genes that contribute to cariogenesis. Previous studies have demonstrated manganese- and SloR-dependent repression of the sloABCR metal ion transport operon in S. mutans . In the present study, we demonstrate that S. mutans coordinates this repression with that of certain virulence attributes. Specifically, we noted virulence gene repression in a manganese-containing medium when SloR binds to promoter-proximal sequence palindromes on the S. mutans chromosome. We applied a genome-wide approach to elucidate the sequences to which SloR binds and to reveal additional “class I” genes that are subject to SloR- and manganese-dependent repression. These analyses identified 204 S. mutans genes that are preceded by one or more conserved palindromic SloR recognition elements (SREs). We cross-referenced these genes with those that we had identified previously as SloR and/or manganese modulated in microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) experiments. From this analysis, we identified a number of S. mutans virulence genes that are subject to transcriptional upregulation by SloR and noted that such “class II”-type regulation is dependent on direct SloR binding to promoter-distal SREs. These observations are consistent with a bifunctional role for the SloR metalloregulator and implicate it as a target for the development of therapies aimed at alleviating S. mutans -induced caries formation.

628 Background: KRAS mutation in colorectal cancer (CRC) is characterized by an altered transcriptional profile when compared to wild-type KRAS tumors. The list of differentially expressed genes overlaps significantly with a stromal fibroblast activation (SFA) signature present across multiple carcinomas. We have reported low expression of SFA genes in KRAS mutant CRC compared to KRAS wild type tumors. Here we sought to confirm the variation of the SFA signature with KRAS mutation and infer its origin in the stromal component of the tumor using experimental models. Methods: The SFA signature was assessed in an inducible-KRAS murine CRC model using RNA-sequencing, and in a CRC cell line with and without a transduced KRAS mutant vector by microarray analysis. Finally, RNA-sequencing of CRC patient-derived xenografts (PDXs) was used to determine whether the SFA signature was being expressed in the tumor epithelium or the surrounding stroma by leveraging the ability to align sequenced reads to the mouse and human genomes separately. Results: The SFA signature was identified in the inducible-KRAS mouse model, matching human cohort observations of decreased SFA gene expression in KRAS mutant CRC. On the other hand, KRAS transduction did not recapitulate the SFA signature in a CRC cell line, suggesting that the presence of stroma may be required for the expression of the SFA signature. Finally, RNA-seq reads for SFA signature genes in CRC PDXs immediately after implantation aligned primarily to the human genome but in later passages of the same PDXs aligned only to the mouse genome. These data suggest that the SFA transcriptional program is associated with the stroma rather than the epithelial tumor cells. Conclusions: KRAS mutation in CRC is associated with a gene expression signature derived from the tumor stroma. These findings suggest that KRAS mutation in the epithelial tumor cells may impact the tumor microenvironment in CRC.

Tetraploid cells, which are a common byproduct of cell division failures, are genetically unstable and have the capacity to facilitate tumorigenesis. Consequently, p53-dependent tumor suppression mechanisms exist to limit the continued proliferation of these cells by promoting G1 cell cycle arrest. However, unlike other common cellular insults that activate p53 and promote G1 arrest, such as the DNA damage response, the mechanisms governing G1 arrest in response to tetraploidy remain largely unexplored. To gain insight into these mechanisms, we have developed a novel genome-wide RNAi screening assay to comprehensively identify proteins that are necessary to activate or maintain G1 cell cycle arrest after the induction of tetraploidy. Results from this screen, in combination with complementary bioinformatic approaches, have begun to illuminate the nature of the stresses associated with tetraploidy. More importantly, this work is shedding light on the adaptive mechanisms that tetraploid and near-tetraploid tumors may have evolved in order to overcome these stresses. These findings have the potential to lead to the identification of new pathways that can be targeted by chemotherapeutics to specifically reinforce G1 arrest in grossly aneuploid cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2941. doi:10.1158/1538-7445.AM2011-2941

Colorectal cancer (CRC) is a leading cause of death in the developed world, yet facile preclinical models that mimic the natural stages of CRC progression are lacking. Through the orthotopic engraftment of colon organoids we describe a broadly usable immunocompetent CRC model that recapitulates the entire adenoma-adenocarcinoma-metastasis axis in vivo. The engraftment procedure takes less than 5 minutes, shows efficient tumor engraftment in two-thirds of mice, and can be achieved using organoids derived from genetically engineered mouse models (GEMMs), wild-type organoids engineered ex vivo, or from patient-derived human CRC organoids. In this model, we describe the genotype and time-dependent progression of CRCs from adenocarcinoma (6 weeks), to local disseminated disease (11-12 weeks), and spontaneous metastasis (>20 weeks). Further, we use the system to show that loss of dysregulated Wnt signaling is critical for the progression of disseminated CRCs. Thus, our approach provides a fast and flexible means to produce tailored CRC mouse models for genetic studies and pre-clinical investigation.