Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs.

Additional GSEA plots, Seahorse analysis data, and FPKM of hexokinase 1 from RNA-seq analysis.

List of differentially expressed genes from RNA-seq analysis of A673 cells, shEF vs. shLUC.

MetaboAnalyst plot, western blots confirmation of EF, SHMT2, and PHGDH.

Cancer Cell Line Encyclopedia plot for PHGDH and additional SurvExpress plots.

Figure legends for Supplemental Figures S1, S2 and S3.

The fate of pyruvate is a defining feature in many cell types. One major fate is mitochondrial entry via the mitochondrial pyruvate carrier (MPC). We found that diffuse large B cell lymphomas (DLBCLs) consume mitochondrial pyruvate via glutamate-pyruvate transaminase 2 to enable α-ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid cycle in DLBCLs. As a result, MPC inhibition led to decreased glutaminolysis in DLBCLs, opposite to previous observations in other cell types. We also found that MPC inhibition or genetic depletion decreased DLBCL proliferation in an extracellular matrix (ECM)–like environment and xenografts, but not in a suspension environment. Moreover, the metabolic profile of DLBCL cells in ECM is markedly different from cells in a suspension environment. Thus, we conclude that the synergistic consumption and assimilation of glutamine and pyruvate enables DLBCL proliferation in an extracellular environment-dependent manner.

1 Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84112, USA 2 Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA 3 Blais Proteomics Center, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA 4 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA 5 Children’s Medical Center Research Institute, University of Texas (UT) Southwestern Medical Center, Dallas, TX 75390, USA 6 Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390, USA 7 Howard Hughes Medical Institute, University of Utah School of Medicine, Salt Lake City, UT 84112, USA 8 Present address: Department of Medicine, Massachusetts General Hospital, Boston, MA 02114, USA * Correspondence: rutter@biochem.utah.edu (J. Rutter)

The first lineage choice in human embryo development separates trophectoderm from the inner cell mass. Naïve human embryonic stem cells are derived from the inner cell mass and offer possibilities to explore how lineage integrity is maintained. Here, we discover that polycomb repressive complex 2 (PRC2) maintains naïve pluripotency and restricts differentiation to trophectoderm and mesoderm lineages. Through quantitative epigenome profiling, we found that a broad gain of histone H3 lysine 27 trimethylation (H3K27me3) is a distinct feature of naïve pluripotency. We define shared and naïve-specific bivalent promoters featuring PRC2-mediated H3K27me3 concomitant with H3K4me3. Naïve bivalency maintains key trophectoderm and mesoderm transcription factors in a transcriptionally poised state. Inhibition of PRC2 forces naïve human embryonic stem cells into an 'activated' state, characterized by co-expression of pluripotency and lineage-specific transcription factors, followed by differentiation into either trophectoderm or mesoderm lineages. In summary, PRC2-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development.

Epigenetic barriers need to be surmounted in order to increase the efficiency of cardiac reprogramming. A new study now reports that the histone reader PHF7 enhances cardiac reprogramming via recruiting the chromatin remodelling SWI/SNF complex and key transcription factors to the cardiac super enhancers.

We first investigated the expression level of the mitochondrial pyruvate carrier (MPC) in metabolically distinct Diffuse large B-cell lymphoma (DLBCL) subtypes. Although MPC expression and activity was different, we found that both DLBCL subgroups oxidized minimal mitochondrial pyruvate. Mitochondrial pyruvate was instead mainly consumed by glutamate pyruvate transaminase 2 (GPT2) to support α-ketoglutarate (α-KG) production as part of glutamine catabolism. This leads us to discover that glutamine, not glucose, is the major carbon source for the TCA cycle in DLBCLs, and interestingly, this glutaminolysis process is regulated by MPC. Furthermore, we found that MPC inhibition only decreases DLBCL proliferation in a solid environment, but not in a suspension environment. Overall, these data demonstrate that the MPC is required to support adequate glutaminolysis, which is essential for DLBCL proliferation in a 3D environment. This metabolic program unveils a non-canonical connection between the consumption and assimilation of carbohydrates and glutamine.

SUMMARYThe fate of pyruvate, which is modulated by the activity of the mitochondrial pyruvate carrier (MPC), is a defining metabolic feature in many cancers. Diffuse large B-cell lymphomas (DLBCLs) are a genetically and metabolically heterogeneous cancer. Although MPC expression and activity differed between DLBCL subgroups, mitochondrial pyruvate was uniformly consumed by glutamate pyruvate transaminase 2 (GPT2) to support α-ketoglutarate production as part of glutaminolysis. This led us to discover that glutamine exceeds pyruvate as a carbon source for the tricarboxylic acid (TCA) cycle in DLBCLs. Furthermore, we found that MPC inhibition unexpectedly leads to decreased glutaminolysis, which is contrary to previous observations in other cell types. We also discovered that MPC inhibition and depletion only decreased DLBCL proliferation in an extracellular matrix (ECM) environment and in vivo xenografts, but not in the typical DLBCL suspension environment. We also have found that the metabolic profile of DLBCL cells in ECM is markedly different from cells in suspension environment. Thus, we report that besides the canonical glutamate dehydrogenase (GDH)-mediated glutaminolysis, the non-canonical GPT2 mediated consumption and assimilation of glutamine and pyruvate in DLBCLs enables their proliferation in an extracellular environment-dependent manner.HIGHLIGHTSGlutamine, but not glucose, is a major carbon source for the tricarboxylic acid cycle in diffuse large B-cell lymphomas.Mitochondrial pyruvate supports glutaminolysis in diffuse large B-cell lymphomas by supplying pyruvate for glutamate pyruvate transaminase 2-mediated α -ketoglutarate production.Mitochondrial pyruvate carrier inhibition leads to decreased glutaminolysis in diffuse large B-cell lymphomas.α -ketoglutarate production is important for diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment.Mitochondrial pyruvate carrier activity supports diffuse large B-cell lymphoma proliferation in a solid extracellular matrix environment and in mouse xenografts.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear if these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy we generated transgenic mice with inducible cardiomyocyte-specific expression of the glucose transporter (GLUT4). We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in non-diabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset, exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

DNA fluorescence in situ hybridization (DNA FISH) is a powerful method to study chromosomal organization in single cells. At present, there is a lack of free resources of DNA FISH probes and probe design tools which can be readily applied. Here, we describe iFISH, an open-source repository currently comprising 380 DNA FISH probes targeting multiple loci on the human autosomes and chromosome X, as well as a genome-wide database of optimally designed oligonucleotides and a freely accessible web interface ( http://ifish4u.org ) that can be used to design DNA FISH probes. We individually validate 153 probes and take advantage of our probe repository to quantify the extent of intermingling between multiple heterologous chromosome pairs, showing a much higher extent of intermingling in human embryonic stem cells compared to fibroblasts. In conclusion, iFISH is a versatile and expandable resource, which can greatly facilitate the use of DNA FISH in research and diagnostics.

Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. We found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.

Purpose: Serrated polyposis syndrome (SPS) presents with multiple sessile serrated adenoma/ polyps (SSA/Ps) and confers a high risk for development of colon cancer.SSA/Ps have unique histology with dilated basal crypts and typically present with a mucus or stool cap noted on colonoscopy exam.The underlying serrated neoplasia pathway is not entirely understood.Organoid models of colon epithelium can be used to study various genetic changes and therapeutic targets.Patient-derived colon organoid models of SSA/Ps have been reported in only one prior publication.Developing an organoid model of SSA/Ps is challenging, and currently no in vitro model exists to study genetic mechanisms in these polyps.The aim of this study was to develop and begin to examine colon organoid models of SSA/Ps from patients with SPS.Methods: Five SSA/Ps from four patients with SPS were obtained for organoid culture.For comparison, two adenomatous polyps from a patient with Familial Adenomatous Polyposis (FAP) were used to generate organoid cultures.Ascending colon, sigmoid colon and rectal biopsies from these patients' normal-appearing, uninvolved epithelium also produced organoid cultures, along with normal colon tissues from two patients with no hereditary colon cancer predisposition.Organoids were grown and passaged using the standard protocol described by Sato et al for 5 to 15 passages.BRAF V600E mutation, CpG island methylator phenotype (CIMP) and histological analyses were performed on organoids from normal control colon, SSA/Ps and adenomas.Results: Two of five SSA/P organoids were positive for V600E mutation in BRAF and for CIMP.BRAF-mutant SSA/P organoids showed altered morphology, including reduced budding of crypt domains and less cell differentiation.BRAF-mutant SSA/P organoids also showed dilated crypt domains and were positive for Alcian Blue mucin stain inside the dilated crypt lumen (Figure 1).None of the uninvolved colon organoids showed BRAF mutation.FAP adenomas appeared to have similar but likely increased crypt budding than normal colon organoids.BRAF V600E wild type SSA/P and normal colon organoids showed similar morphology.Conclusion: We have developed an organoid model of SSA/Ps that demonstrates genomic aberrations frequently found in SSA/Ps.SSA/P organoids that are BRAF-mutant and CIMP-positive also show unique morphology including mucin-filled dilated crypt domains and reduced differentiation.These results confirm that genomic aberrations and increased mucin production frequently observed in SSA/Ps originate and can be reproduced from colon epithelial stem cells.

The transcription factor Oct1/Pou2f1 promotes poised gene expression states, mitotic stability, glycolytic metabolism and other characteristics of stem cell potency. To determine the effect of Oct1 loss on stem cell maintenance and malignancy, we deleted Oct1 in two different mouse gut stem cell compartments. Oct1 deletion preserved homeostasis in vivo and the ability to establish organoids in vitro, but blocked the ability to recover from treatment with dextran sodium sulfate, and the ability to maintain organoids after passage. In a chemical model of colon cancer, loss of Oct1 in the colon severely restricted tumorigenicity. In contrast, loss of one or both Oct1 alleles progressively increased tumor burden in a colon cancer model driven by loss-of-heterozygosity of the tumor suppressor gene Apc. The different outcomes are consistent with prior findings that Oct1 promotes mitotic stability, and consistent with differentially expressed genes between the two models. Oct1 ChIPseq using HCT116 colon carcinoma cells identifies target genes associated with mitotic stability, metabolism, stress response and malignancy. This set of gene targets overlaps significantly with genes differentially expressed in the two tumor models. These results reveal that Oct1 is selectively required for recovery after colon damage, and that Oct1 has potent effects in colon malignancy, with outcome (pro-oncogenic or tumor suppressive) dictated by tumor etiology.

D-dimer is an indirect marker of fibrinolysis and fibrin turnover; this molecule exhibits unique properties as a biological marker of hemostatic abnormalities as well as an indicator of intravascular thrombosis. D-dimer is a soluble fibrin degradation product that results from the systematic degradation of vascular thrombi through the fibrinolytic mechanism. Because of this, the D-dimer serves as a valuable marker of activation of coagulation and fibrinolysis in a number of clinical scenarios. Most commonly, D-dimer has been extensively investigated for excluding the diagnosis of venous thromboembolism (VTE) and is used routinely for this indication. In addition, D-dimer has been evaluated for determining the optimal duration of anticoagulation in VTE patients, for diagnosing and monitoring disseminated intravascular coagulation, and for monitoring other conditions in which the patient is at high risk of bleeding or thrombosis. Limitations of the assay include D-dimer elevation in a constellation of clinical scenarios (age, pregnancy, and cancer) and lack of clinical standardization.

The transcription factor Oct1/Pou2f1 promotes poised gene expression states, mitotic stability, glycolytic metabolism and other characteristics of stem cell potency. To determine the effect of Oct1 loss on stem cell maintenance and malignancy, we deleted Oct1 in two different mouse gut stem cell compartments. Oct1 deletion preserved homeostasis in vivo and the ability to generate cultured organoids in vitro, but blocked the ability to regenerate after treatment with dextran sodium sulfate, and the ability to maintain organoids after passage. In a chemical model of colon cancer, loss of Oct1 in the colon severely restricted tumorigenicity. In contrast, loss of one or both Oct1 alleles progressively increased tumor burden in a colon cancer model driven by loss of heterozygosity of the tumor suppressor gene Apc. The different outcomes are consistent with prior findings that Oct1 promotes mitotic stability, and consistent with different gene expression signatures associated with the two models. These results reveal that Oct1 is selectively required for gut regeneration, and has potent effects in colon malignancy, with outcome (pro-oncogenic or tumor suppressive) dictated by tumor etiology. Author summary Colorectal cancer is the second leading cause of cancer death in the United States. Approximately 35% of diagnosed patients eventually succumb to disease. The high incidence and mortality due to colon cancer demand a better understanding of factors controlling the physiology and pathophysiology of the gastrointestinal tract. Previously, we and others showed that the widely expressed transcription factor is expressed at higher protein levels in stem cells, including intestinal stem cells. In this study we use a conditional mouse Oct1 ( Pou2f1 ) allele deleted in two different intestinal stem cell compartments. The results indicate that Oct1 loss is dispensable for maintenance of the mouse gut, but required for regeneration. We also tested Oct1 loss in the context of two different mouse colon cancer models. We find that Oct1 loss has opposing effects in the two models, and further that the two models are associated with different gene expression signatures. The differentially expressed genes are enriched for previously identified Oct1 targets, suggesting that differential gene control by Oct1 is one mechanism underlying different outcomes.

CSM-exposed Caco-2 cells and 2D organoids were recultured in CS-free 3D conditions to establish possible irreversible effects of CS-exposure.Cell cluster sizes and polarity were analyzed by microscopy.Expression and subcellular location of occludens-1 and ZO-1 were analyzed by immunofluorescence microscopy.Fluorescein isothiocyanate-dextran of 4 kDa (FD4) was used to analyze tight junction integrity.Effects of CSM on ISC markers ( LGR5), CS markers (CYP1A1) and differentiation markers (VILLIN, MUC-5) are analyzed by quantitative RT-PCR.Results: 24 h CSM exposure (up to 20% CSM) did not affect the polarity or induce leakage of FD4 of fully developed 3D Caco-2 spheroids.In contrast, 3D morphogenesis of Caco-2 spheroids was dose-dependently impaired during 7 day CSM exposure leading to disturbed cell polarity and location of tight junction proteins.These effects penetrated into next cell generations even after short-term CSM exposure of Caco-2 cells.3D morphogenesis of human colonic organoids was not affected by short-or long-term CSM exposure, but it dose-dependently (0-20%) suppresses organoid growth and expression of LGR5.Once exposed to CSM, the organoid growth remained impaired and LGR5 levels reduced, even after reculturing for 10-14 days in CS-free conditions.Conclusions: Short-term exposure of human gut epithelial cells does not acutely affect epithelial barrier integrity.However, it may cause long-lasting effects on ISC function and intestinal epithelial differentiation and polarization, which may predispose for the development of Crohn's disease and colon cancer.