Publications by authors named "Ömer H Yilmaz"

86 Publications

Reprogramming of H3K9bhb at regulatory elements is a key feature of fasting in the small intestine.

Cell Rep 2021 11;37(8):110044

Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address:

β-hydroxybutyrate (β-OHB) is an essential metabolic energy source during fasting and functions as a chromatin regulator by lysine β-hydroxybutyrylation (Kbhb) modification of the core histones H3 and H4. We report that Kbhb on histone H3 (H3K9bhb) is enriched at proximal promoters of critical gene subsets associated with lipolytic and ketogenic metabolic pathways in small intestine (SI) crypts during fasting. Similar Kbhb enrichment is observed in Lgr5 stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb is associated with active chromatin states and that fasting enriches for an H3K9bhb-H3K27ac signature at active metabolic gene promoters and distal enhancer elements. Intestinal knockout of Hmgcs2 results in marked loss of H3K9bhb-associated loci, suggesting that local production of β-OHB is responsible for chromatin reprogramming within the SI crypt. We conclude that modulation of H3K9bhb in SI crypts is a key gene regulatory event in response to fasting.
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http://dx.doi.org/10.1016/j.celrep.2021.110044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8668154PMC
November 2021

Agrin Loss in Barrett's Esophagus-Related Neoplasia and Its Utility as a Diagnostic and Predictive Biomarker.

Clin Cancer Res 2021 Nov 16. Epub 2021 Nov 16.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts.

Purpose: There is an unmet need for identifying novel biomarkers in Barrett's esophagus that could stratify patients with regards to neoplastic progression. We investigate the expression patterns of extracellular matrix (ECM) molecules in Barrett's esophagus and Barrett's esophagus-related neoplasia, and assess their value as biomarkers for the diagnosis of Barrett's esophagus-related neoplasia and to predict neoplastic progression.

Experimental Design: Gene-expression analyses of ECM matrisome gene sets were performed using publicly available data on human Barrett's esophagus, Barrett's esophagus-related dysplasia, esophageal adenocarcinoma (ADCA) and normal esophagus. Immunohistochemical expression of basement membrane (BM) marker agrin (AGRN) and p53 was analyzed in biopsies of Barrett's esophagus-related neoplasia from 321 patients in three independent cohorts.

Results: Differential gene-expression analysis revealed significant enrichment of ECM matrisome gene sets in dysplastic Barrett's esophagus and ADCA compared with controls. Loss of BM AGRN expression was observed in both Barrett's esophagus-related dysplasia and ADCA. The mean AGRN loss in Barrett's esophagus glands was significantly higher in Barrett's esophagus-related dysplasia and ADCA compared with non-dysplastic Barrett's esophagus (NDBE; < 0.001; specificity = 82.2% and sensitivity = 96.4%). Loss of AGRN was significantly higher in NDBE samples from progressors compared with non-progressors ( < 0.001) and identified patients who progressed to advanced neoplasia with a specificity of 80.2% and sensitivity of 54.8%. Moreover, the combination of AGRN loss and abnormal p53 staining identified progression to Barrett's esophagus-related advanced neoplasia with a specificity and sensitivity of 86.5% and 58.7%.

Conclusions: We highlight ECM changes during Barrett's esophagus progression to neoplasia. BM AGRN loss is a novel diagnostic biomarker that can identify patients with NDBE at increased risk of developing advanced neoplasia.
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http://dx.doi.org/10.1158/1078-0432.CCR-21-2822DOI Listing
November 2021

Low neoantigen expression and poor T-cell priming underlie early immune escape in colorectal cancer.

Nat Cancer 2021 Oct 30;2(10):1071-1085. Epub 2021 Sep 30.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Immune evasion is a hallmark of cancer, and therapies that restore immune surveillance have proven highly effective in cancers with high tumor mutation burden (TMB) (e.g., those with microsatellite instability (MSI)). Whether low TMB cancers, which are largely refractory to immunotherapy, harbor potentially immunogenic neoantigens remains unclear. Here, we show that tumors from all patients with microsatellite stable (MSS) colorectal cancer (CRC) express clonal predicted neoantigens despite low TMB. Unexpectedly, these neoantigens are broadly expressed at lower levels compared to those in MSI CRC. Using a versatile platform for modulating neoantigen expression in CRC organoids and transplantation into the distal colon of mice, we show that low expression precludes productive cross priming and drives immediate T cell dysfunction. Strikingly, experimental or therapeutic rescue of priming rendered T cells capable of controlling tumors with low neoantigen expression. These findings underscore a critical role of neoantigen expression level in immune evasion and therapy response.
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http://dx.doi.org/10.1038/s43018-021-00247-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8562866PMC
October 2021

Combined Supplementation with Vitamin B-6 and Curcumin is Superior to Either Agent Alone in Suppressing Obesity-Promoted Colorectal Tumorigenesis in Mice.

J Nutr 2021 Dec;151(12):3678-3688

Vitamins & Carcinogenesis Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA.

Background: Obesity increases the colorectal cancer risk, in part by elevating colonic proinflammatory cytokines. Curcumin (CUR) and supplemental vitamin B-6 each suppress colonic inflammation.

Objectives: We examined whether the combination of CUR and vitamin B-6 amplifies each supplement's effects and thereby suppress obesity-promoted tumorigenesis.

Methods: Male Friend Virus B (FVB) mice (4-week-old; n = 110) received 6 weekly injections of azoxymethane beginning 1 week after arrival. Thereafter, they were randomized to receive a low-fat diet (10% energy from fat), a high-fat diet (HFD; 60% energy from fat), a HFD containing 0.2% CUR, a HFD containing supplemental vitamin B-6 (24 mg pyridoxine HCl/kg), or a HFD containing both CUR and supplemental vitamin B-6 (C + B) for 15 weeks. Colonic inflammation, assessed by fecal calprotectin, and tumor metrics were the primary endpoints. The anti-inflammatory efficacy of the combination was also determined in human colonic organoids.

Results: HFD-induced obesity produced a 2.6-fold increase in plasma IL-6 (P < 0.02), a 1.9-fold increase in fecal calprotectin (P < 0.05), and a 2.2-fold increase in tumor multiplicity (P < 0.05). Compared to the HFD group, the C + B combination, but not the individual agents, decreased fecal calprotectin (66%; P < 0.01) and reduced tumor multiplicity and the total tumor burden by 60%-80% (P < 0.03) in an additive fashion. The combination of C + B also significantly downregulated colonic phosphatidylinositol-4,5-bisphosphate 3-kinase, Wnt, and NF-κB signaling by 31%-47% (P < 0.05), effects largely absent with the single agents. Observations that may explain how the 2 agents work additively include a 2.8-fold increased colonic concentration of 3-hydroxyanthranillic acid (P < 0.05) and a 1.3-fold higher colonic concentration of the active coenzymatic form of vitamin B-6 (P < 0.05). In human colonic organoids, micromolar concentrations of CUR, vitamin B-6, and their combination suppressed secreted proinflammatory cytokines by 41%-93% (P < 0.03), demonstrating relevance to humans.

Conclusions: In this mouse model, C + B is superior to either agent alone in preventing obesity-promoted colorectal carcinogenesis. Augmented suppression of procancerous signaling pathways may be the means by which this augmentation occurs.
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http://dx.doi.org/10.1093/jn/nxab320DOI Listing
December 2021

Dietary suppression of MHC class II expression in intestinal epithelial cells enhances intestinal tumorigenesis.

Cell Stem Cell 2021 11 15;28(11):1922-1935.e5. Epub 2021 Sep 15.

The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA; Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02140, USA.

Little is known about how interactions of diet, intestinal stem cells (ISCs), and immune cells affect early-stage intestinal tumorigenesis. We show that a high-fat diet (HFD) reduces the expression of the major histocompatibility complex class II (MHC class II) genes in intestinal epithelial cells, including ISCs. This decline in epithelial MHC class II expression in a HFD correlates with reduced intestinal microbiome diversity. Microbial community transfer experiments suggest that epithelial MHC class II expression is regulated by intestinal flora. Mechanistically, pattern recognition receptor (PRR) and interferon-gamma (IFNγ) signaling regulates epithelial MHC class II expression. MHC class II-negative (MHC-II-) ISCs exhibit greater tumor-initiating capacity than their MHC class II-positive (MHC-II+) counterparts upon loss of the tumor suppressor Apc coupled with a HFD, suggesting a role for epithelial MHC class II-mediated immune surveillance in suppressing tumorigenesis. ISC-specific genetic ablation of MHC class II increases tumor burden cell autonomously. Thus, HFD perturbs a microbiome-stem cell-immune cell interaction that contributes to tumor initiation in the intestine.
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http://dx.doi.org/10.1016/j.stem.2021.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8650761PMC
November 2021

Live cell tagging tracking and isolation for spatial transcriptomics using photoactivatable cell dyes.

Nat Commun 2021 08 17;12(1):4995. Epub 2021 Aug 17.

Institute for Medical Engineering & Science, MIT, Cambridge, MA, USA.

A cell's phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT-Spatially PhotoActivatable Color Encoded Cell Address Tags-to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function.
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http://dx.doi.org/10.1038/s41467-021-25279-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8371137PMC
August 2021

Transcriptome-wide Effects of Aspirin on Patient-derived Normal Colon Organoids.

Cancer Prev Res (Phila) 2021 Dec 13;14(12):1089-1100. Epub 2021 Aug 13.

Center for Public Health Genomics, Department of Public Health Sciences, University of Virginia, Charlottesville, Virginia.

Mechanisms underlying aspirin chemoprevention of colorectal cancer remain unclear. Prior studies have been limited because of the inability of preclinical models to recapitulate human normal colon epithelium or cellular heterogeneity present in mucosal biopsies. To overcome some of these obstacles, we performed aspirin treatment of colon organoids derived from normal mucosal biopsies to reveal transcriptional networks relevant to aspirin chemoprevention. Colon organoids derived from 38 healthy individuals undergoing endoscopy were treated with 50 μmol/L aspirin or vehicle control for 72 hours and subjected to bulk RNA sequencing. Paired regression analysis using DESeq2 identified differentially expressed genes (DEG) associated with aspirin treatment. Cellular composition was determined using CIBERSORTx. Aspirin treatment was associated with 1,154 significant ( < 0.10) DEGs prior to deconvolution. We provide replication of these findings in an independent population-based RNA-sequencing dataset of mucosal biopsies (BarcUVa-Seq), where a significant enrichment for overlap of DEGs was observed ( < 2.2E). Single-cell deconvolution revealed changes in cell composition, including a decrease in transit-amplifying cells following aspirin treatment ( = 0.01). Following deconvolution, DEGs included novel putative targets for aspirin such as ( = 0.055), a negative regulator of Wnt signaling. Weighted gene co-expression network analysis identified 12 significant modules, including two that contained hubs for and , the latter being previously implicated in aspirin chemoprevention. In summary, aspirin treatment of patient-derived colon organoids using physiologically relevant doses resulted in transcriptome-wide changes that reveal altered cell composition and improved understanding of transcriptional pathways, providing novel insight into its chemopreventive properties. PREVENTION RELEVANCE: Numerous studies have highlighted a role for aspirin in colorectal cancer chemoprevention, though the mechanisms driving this association remain unclear. We addressed this by showing that aspirin treatment of normal colon organoids diminished the transit-amplifying cell population, inhibited prostaglandin synthesis, and dysregulated expression of novel genes implicated in colon tumorigenesis.
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http://dx.doi.org/10.1158/1940-6207.CAPR-21-0041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8639779PMC
December 2021

The CD155/TIGIT axis promotes and maintains immune evasion in neoantigen-expressing pancreatic cancer.

Cancer Cell 2021 10 5;39(10):1342-1360.e14. Epub 2021 Aug 5.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address:

The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8 T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.
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http://dx.doi.org/10.1016/j.ccell.2021.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8511341PMC
October 2021

High-fat diet-activated fatty acid oxidation mediates intestinal stemness and tumorigenicity.

Cell Rep 2021 06;35(10):109212

Department of Biology, The David H. Koch Institute for Integrative Cancer Research at MIT, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA. Electronic address:

Obesity is an established risk factor for cancer in many tissues. In the mammalian intestine, a pro-obesity high-fat diet (HFD) promotes regeneration and tumorigenesis by enhancing intestinal stem cell (ISC) numbers, proliferation, and function. Although PPAR (peroxisome proliferator-activated receptor) nuclear receptor activity has been proposed to facilitate these effects, their exact role is unclear. Here we find that, in loss-of-function in vivo models, PPARα and PPARδ contribute to the HFD response in ISCs. Mechanistically, both PPARs do so by robustly inducing a downstream fatty acid oxidation (FAO) metabolic program. Pharmacologic and genetic disruption of CPT1A (the rate-controlling enzyme of mitochondrial FAO) blunts the HFD phenotype in ISCs. Furthermore, inhibition of CPT1A dampens the pro-tumorigenic consequences of a HFD on early tumor incidence and progression. These findings demonstrate that inhibition of a HFD-activated FAO program creates a therapeutic opportunity to counter the effects of a HFD on ISCs and intestinal tumorigenesis.
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http://dx.doi.org/10.1016/j.celrep.2021.109212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8258630PMC
June 2021

High-fat diet activates a PPAR-δ program to enhance intestinal stem cell function.

Cell Stem Cell 2021 04;28(4):598-599

The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA. Electronic address:

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http://dx.doi.org/10.1016/j.stem.2021.03.001DOI Listing
April 2021

100 Years of Exploiting Diet and Nutrition for Tissue Regeneration.

Cell Stem Cell 2021 03;28(3):370-373

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Pathology, Massachusetts General Hospital Boston and Harvard Medical School, Boston, MA 02114, USA. Electronic address:

In this forum piece, we review progress in exploiting diet and nutrition for enhancing tissue regeneration with a particular emphasis on how dietary composition and diet-induced physiology influence adult stem cell biology.
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http://dx.doi.org/10.1016/j.stem.2021.02.014DOI Listing
March 2021

LGR5 in Barrett's Esophagus and its Utility in Predicting Patients at Increased Risk of Advanced Neoplasia.

Clin Transl Gastroenterol 2020 12 22;12(1):e00272. Epub 2020 Dec 22.

Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA.

Introduction: The expression of LGR5, a known stem cell marker, is poorly understood in Barrett's esophagus (BE) and related neoplasia. The aim of this study was to evaluate LGR5 in BE and related neoplasia and to evaluate its utility as a potential biomarker of progression to advanced neoplasia.

Methods: We evaluated total 137 patients, including 119 with BE and 18 with normal gastroesophageal mucosa for expression of LGR5 using RNA in situ hybridization; this also included 28 progressors and 30 nonprogressors. The LGR5 stain was evaluated using 1 qualitative and 2 quantitative parameters, using manual and automated platforms.

Results: Surface LGR5 expression was mainly seen in high-grade dysplasia (12/18) compared with low-grade dysplasia (1/8) and nondysplastic BE (0/17) (P < 0.0001). In contrast to nondysplastic BE, low- and high-grade dysplasia showed a higher percentage of mean number of LGR5-positive crypts per patient (P < 0.0001) and an increase in the mean number of LGR5 transcripts per cell (P < 0.0001). The mean percentage of LGR5-positive crypts per patient and the mean number of LGR5 transcripts per cell were also significantly higher in nondysplastic BE from progressor compared with nonprogressor (P < 0.0001, P = 0.014). The sensitivity and specificity of LGR5 for distinguishing progressor from nonprogressor were 50% and 87%, respectively.

Discussion: BE-related advanced neoplasia shows an expansion of the LGR5-positive cellular compartment, supporting its role as a stem cell marker in this disease. Quantitative LGR5 expression and surface epithelial reactivity are novel biomarkers of increased risk of progression to advanced neoplasia in BE.
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http://dx.doi.org/10.14309/ctg.0000000000000272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8345923PMC
December 2020

Nutritional Control of Intestinal Stem Cells in Homeostasis and Tumorigenesis.

Trends Endocrinol Metab 2021 01 1;32(1):20-35. Epub 2020 Dec 1.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA; Department of Biology, MIT, Cambridge, MA, USA; Broad Institute of Harvard and MIT, Cambridge, MA, USA; Departments of Pathology, Gastroenterology, and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, USA. Electronic address:

Food and nutrition have a profound impact on organismal health and diseases, and tissue-specific adult stem cells play a crucial role in coordinating tissue maintenance by responding to dietary cues. Emerging evidence indicates that adult intestinal stem cells (ISCs) actively adjust their fate decisions in response to diets and nutritional states to drive intestinal adaptation. Here, we review the signaling mechanisms mediating the dietary responses imposed by caloric intake and nutritional composition (i.e., macronutrients and micronutrients), fasting-feeding patterns, diet-induced growth factors, and microbiota on ISCs and their relevance to the beginnings of intestinal tumors.
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http://dx.doi.org/10.1016/j.tem.2020.11.003DOI Listing
January 2021

Enhancing chemotherapy response through augmented synthetic lethality by co-targeting nucleotide excision repair and cell-cycle checkpoints.

Nat Commun 2020 08 17;11(1):4124. Epub 2020 Aug 17.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.
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http://dx.doi.org/10.1038/s41467-020-17958-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431578PMC
August 2020

Regenerative Reprogramming of the Intestinal Stem Cell State via Hippo Signaling Suppresses Metastatic Colorectal Cancer.

Cell Stem Cell 2020 10 29;27(4):590-604.e9. Epub 2020 Jul 29.

Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA. Electronic address:

Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies.
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http://dx.doi.org/10.1016/j.stem.2020.07.003DOI Listing
October 2020

Strategies for Measuring Induction of Fatty Acid Oxidation in Intestinal Stem and Progenitor Cells.

Methods Mol Biol 2020 ;2171:53-64

Department of Biological Chemistry and Pharmacology, The Ohio State University, Columbus, OH, USA.

This protocol describes a multipronged approach that we have created to determine the transcriptional induction of fatty acid oxidation (FAO) genes in Lgr5 intestinal stem cells and a subsequent metabolomics-based approach for assessing fatty acid utilization in the mammalian intestinal crypt. More specifically, we describe methods for crypt isolation followed by a FACS-based purification of stem and progenitor populations and RNA-sequencing analysis. Using this workflow, we can determine both basal gene expression profiles of key metabolic genes as well as corresponding changes in response to altered metabolic states, such as fasting. Subsequently, we describe a complementary metabolomics-based approach that we have developed to assess fatty acid uptake and utilization in the crypt using C stable isotope tracing. Combining these approaches, one can gain a better understanding of substrate utilization and the preceding transcriptional changes that accommodate these reactions in physiologic states of low carbohydrate utilization or during overabundance of dietary lipids.
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http://dx.doi.org/10.1007/978-1-0716-0747-3_4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8065345PMC
March 2021

A Platinum(IV) Prodrug-Perfluoroaryl Macrocyclic Peptide Conjugate Enhances Platinum Uptake in the Brain.

J Med Chem 2020 07 26;63(13):6741-6747. Epub 2020 Jun 26.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

Effective delivery to the brain limits the development of novel glioblastoma therapies. Here, we introduce conjugation between platinum(IV) prodrugs of cisplatin and perfluoroaryl peptide macrocycles to increase brain uptake. We demonstrate that one such conjugate shows efficacy against glioma stem-like cells. We investigate the pharmacokinetics of this conjugate in mice and show that the amount of platinum in the brain after treatment with the conjugate is 15-fold greater than with cisplatin after 5 h.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7992430PMC
July 2020

Region-Specific Proteome Changes of the Intestinal Epithelium during Aging and Dietary Restriction.

Cell Rep 2020 04;31(4):107565

Leibniz Institute on Aging - Fritz Lipmann Institute (FLI), Jena, Germany. Electronic address:

The small intestine is responsible for nutrient absorption and one of the most important interfaces between the environment and the body. During aging, changes of the epithelium lead to food malabsorption and reduced barrier function, thus increasing disease risk. The drivers of these alterations remain poorly understood. Here, we compare the proteomes of intestinal crypts from mice across different anatomical regions and ages. We find that aging alters epithelial immunity, metabolism, and cell proliferation and is accompanied by region-dependent skewing in the cellular composition of the epithelium. Of note, short-term dietary restriction followed by refeeding partially restores the epithelium by promoting stem cell differentiation toward the secretory lineage. We identify Hmgcs2 (3-hydroxy-3-methylglutaryl-coenzyme A [CoA] synthetase 2), the rate-limiting enzyme for ketogenesis, as a modulator of stem cell differentiation that responds to dietary changes, and we provide an atlas of region- and age-dependent proteome changes of the small intestine.
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http://dx.doi.org/10.1016/j.celrep.2020.107565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446723PMC
April 2020

A novel histological index for evaluation of environmental enteric dysfunction identifies geographic-specific features of enteropathy among children with suboptimal growth.

PLoS Negl Trop Dis 2020 01 13;14(1):e0007975. Epub 2020 Jan 13.

Department of Pathology, University of Virginia, Charlottesville, VA, United States of America.

Background: A major limitation to understanding the etiopathogenesis of environmental enteric dysfunction (EED) is the lack of a comprehensive, reproducible histologic framework for characterizing the small bowel lesions. We hypothesized that the development of such a system will identify unique histology features for EED, and that some features might correlate with clinical severity.

Methods: Duodenal endoscopic biopsies from two cohorts where EED is prevalent (Pakistan, Zambia) and North American children with and without gluten sensitive enteropathy (GSE) were processed for routine hematoxylin & eosin (H&E) staining, and scanned to produce whole slide images (WSIs) which we shared among study pathologists via a secure web browser-based platform. A semi-quantitative scoring index composed of 11 parameters encompassing tissue injury and response patterns commonly observed in routine clinical practice was constructed by three gastrointestinal pathologists, with input from EED experts. The pathologists then read the WSIs using the EED histology index, and inter-observer reliability was assessed. The histology index was further used to identify within- and between-child variations as well as features common across and unique to each cohort, and those that correlated with host phenotype.

Results: Eight of the 11 histologic scoring parameters showed useful degrees of variation. The overall concordance across all parameters was 96% weighted agreement, kappa 0.70, and Gwet's AC 0.93. Zambian and Pakistani tissues shared some histologic features with GSE, but most features were distinct, particularly abundance of intraepithelial lymphocytes in the Pakistani cohort, and marked villous destruction and loss of secretory cell lineages in the Zambian cohort.

Conclusions: We propose the first EED histology index for interpreting duodenal biopsies. This index should be useful in future clinical and translational studies of this widespread, poorly understood, and highly consequential disorder, which might be caused by multiple contributing processes, in different regions of the world.
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http://dx.doi.org/10.1371/journal.pntd.0007975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980693PMC
January 2020

Agrin in the Muscularis Mucosa Serves as a Biomarker Distinguishing Hyperplastic Polyps from Sessile Serrated Lesions.

Clin Cancer Res 2020 03 18;26(6):1277-1287. Epub 2019 Dec 18.

David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts.

Purpose: Sessile serrated lesions (SSL) are precursors to colon carcinoma, and their distinction from other polyps, in particular hyperplastic polyps (HP), presents significant diagnostic challenges. We evaluated expression patterns in colonic polyps of previously identified colon carcinoma-associated extracellular matrix (ECM) proteins to identify markers distinguishing SSLs from other polyps.

Experimental Design: Gene-expression analyses of ECM proteins were performed using publicly available data on preneoplastic colonic polyps. In parallel, we evaluated by IHC the expression of agrin (AGRN) in over 400 colonic polyps, including HP, SSL with and without dysplasia, traditional serrated adenomas (TSA), and tubular adenomas (TA), and compared the consistency of standard histologic diagnosis of SSLs by experienced gastrointestinal pathologists with that of AGRN IHC.

Results: Differential gene expression analysis and IHC identified AGRN, serine peptidase inhibitor (SERPINE2), and TIMP metallopeptidase inhibitor 1 (TIMP1) elevated in SSLs and HPs but decreased in TAs and absent in normal colon. AGRN-positive basal laminae were noted in all TA, TSA, HP, and SSL in distinguishable patterns, whereas other polyps and normal mucosa were negative. SSL with or without dysplasia consistently showed IHC staining for AGRN in the muscularis mucosae, which was absent in HP, TSA, TA, and other polyps. In contrast, histologic evaluation showed only weak interobserver agreement (kappa value = 0.493) in distinguishing SSLs.

Conclusions: Muscularis mucosae-based AGRN immunostaining is a novel biomarker to distinguish SSL from HP, TSA, and TA, with a specificity of 97.1% and sensitivity of 98.9% and can assist in diagnosis of morphologically challenging colonic polyps.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073301PMC
March 2020

Epithelial NOTCH Signaling Rewires the Tumor Microenvironment of Colorectal Cancer to Drive Poor-Prognosis Subtypes and Metastasis.

Cancer Cell 2019 09;36(3):319-336.e7

Cancer Research UK Beatson Institute, Glasgow, UK; Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK. Electronic address:

The 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 Kras-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.
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http://dx.doi.org/10.1016/j.ccell.2019.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853173PMC
September 2019

High-fat diet in a mouse insulin-resistant model induces widespread rewiring of the phosphotyrosine signaling network.

Mol Syst Biol 2019 08;15(8):e8849

The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

Obesity-associated type 2 diabetes and accompanying diseases have developed into a leading human health risk across industrialized and developing countries. The complex molecular underpinnings of how lipid overload and lipid metabolites lead to the deregulation of metabolic processes are incompletely understood. We assessed hepatic post-translational alterations in response to treatment of cells with saturated and unsaturated free fatty acids and the consumption of a high-fat diet by mice. These data revealed widespread tyrosine phosphorylation changes affecting a large number of enzymes involved in metabolic processes as well as canonical receptor-mediated signal transduction networks. Targeting two of the most prominently affected molecular features in our data, SRC-family kinase activity and elevated reactive oxygen species, significantly abrogated the effects of saturated fat exposure in vitro and high-fat diet in vivo. In summary, we present a comprehensive view of diet-induced alterations of tyrosine signaling networks, including proteins involved in fundamental metabolic pathways.
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http://dx.doi.org/10.15252/msb.20198849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6674232PMC
August 2019

Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet.

Cell 2019 08;178(5):1115-1131.e15

Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA 02139, USA; Department of Biology, MIT, Cambridge, MA 02139, USA; Department of Pathology, Massachusetts General Hospital Boston and Harvard Medical School, Boston, MA 02114, USA; Klarman Cell Observatory, Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address:

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (βOHB), distinguishes self-renewing Lgr5 stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes βOHB levels in Lgr5 ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous βOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, βOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through βOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of βOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.
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http://dx.doi.org/10.1016/j.cell.2019.07.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6732196PMC
August 2019

Cholangiolar pattern and albumin in situ hybridisation enable a diagnosis of intrahepatic cholangiocarcinoma.

J Clin Pathol 2020 Jan 17;73(1):23-29. Epub 2019 Aug 17.

Depatment of Pathology, Massachusetts General Hospital, Boston, Massachusetts, USA

Aims: The histological distinction of intrahepatic cholangiocarcinoma (ICC) from metastatic adenocarcinoma remains a challenge. The primary goal was to evaluate the diagnostic value of morphology and albumin expression in the diagnosis of ICC.

Methods: We evaluated morphological patterns in 120 ICCs and 677 non-hepatic adenocarcinomas and performed in situ hybridisation (ISH) stain for albumin in the former cohort (retrospective cohort). We also identified 119 samples from primary and metastatic lesions, the validation cohort, in which albumin ISH was performed as part of the diagnostic workup. Targeted sequencing was performed on selected cases. We also mined existing expression profiling data including cases from The Cancer Genome Atlas (TCGA) (41 760 unique samples).

Results: In the retrospective cohort, 45% of ICCs and <1% of non-hepatic adenocarcinomas showed a cholangiolar pattern; albumin ISH was positive in 93% of ICCs with significant intratumorous heterogeneity. In the validation cohort, 29% of ICCs showed a cholangiolar pattern and 88% expressed albumin, while all metastatic non-hepatic neoplasms were negative (n=37) (sensitivity 88% and specificity 100%). Targetable genetic alterations ( mutations and fusions) were identified in 31% of ICCs (10 of 32). An analysis of the TCGA data validated the specificity of the albumin assay.

Conclusions: The cholangiolar pattern and albumin RNA ISH distinguishes ICC from metastatic adenocarcinoma with high specificity. Given the high prevalence of targetable mutations in ICC, albumin RNA ISH is an essential component in the workup of tumours of uncertain origin. A specific diagnosis of ICC could trigger molecular testing and uncover targetable genetic alterations.
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http://dx.doi.org/10.1136/jclinpath-2019-206055DOI Listing
January 2020

MYC promotes tryptophan uptake and metabolism by the kynurenine pathway in colon cancer.

Genes Dev 2019 09 15;33(17-18):1236-1251. Epub 2019 Aug 15.

Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.
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http://dx.doi.org/10.1101/gad.327056.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719621PMC
September 2019

Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium.

Nature 2019 07 10;571(7765):398-402. Epub 2019 Jul 10.

Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland.

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche. Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor α (PPAR-α), and lowered PPAR-α activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.
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http://dx.doi.org/10.1038/s41586-019-1383-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151802PMC
July 2019

Gut organoids: mini-tissues in culture to study intestinal physiology and disease.

Am J Physiol Cell Physiol 2019 09 19;317(3):C405-C419. Epub 2019 Jun 19.

The David H. Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology, Cambridge, Massachusetts.

In vitro, cell cultures are essential tools in the study of intestinal function and disease. For the past few decades, monolayer cellular cultures, such as cancer cell lines or immortalized cell lines, have been widely applied in gastrointestinal research. Recently, the development of three-dimensional cultures known as organoids has permitted the growth of normal crypt-villus units that recapitulate many aspects of intestinal physiology. Organoid culturing has also been applied to study gastrointestinal diseases, intestinal-microbe interactions, and colorectal cancer. These models are amenable to CRISPR gene editing and drug treatments, including high-throughput small-molecule testing. Three-dimensional intestinal cultures have been transplanted into mice to develop versatile in vivo models of intestinal disease, particularly cancer. Limitations of currently available organoid models include cost and challenges in modeling nonepithelial intestinal cells, such as immune cells and the microbiota. Here, we describe the development of organoid models of intestinal biology and the applications of organoids for study of the pathophysiology of intestinal diseases and cancer.
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http://dx.doi.org/10.1152/ajpcell.00300.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766612PMC
September 2019

Breakthrough Moments: Genome Editing and Organoids.

Cell Stem Cell 2019 06;24(6):841-842

The David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, USA. Electronic address:

Six years ago, Schwank et al. (2013) adapted CRISPR-Cas9 and organoid technology to repair genetic diseases in patient-derived tissues. We shine a spotlight on how this work has inspired the development of tools to study and correct genetic diseases in experimental systems, with the ultimate goal of treating human disease.
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http://dx.doi.org/10.1016/j.stem.2019.05.008DOI Listing
June 2019
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