Colon Organoids Research Articles

Interleukin-22 regulates neutrophil recruitment in ulcerative colitis and is associated with resistance to ustekinumab therapy

The function of interleukin-22 (IL-22) in intestinal barrier homeostasis remains controversial. Here, we map the transcriptional landscape regulated by IL-22 in human colonic epithelial organoids and evaluate the biological, functional and clinical significance of the IL-22 mediated pathways in ulcerative colitis (UC). We show that IL-22 regulated pro-inflammatory pathways are involved in microbial recognition, cancer and immune cell chemotaxis; most prominently those involving CXCR2+ neutrophils. IL-22-mediated transcriptional regulation of CXC-family neutrophil-active chemokine expression is highly conserved across species, is dependent on STAT3 signaling, and is functionally and pathologically important in the recruitment of CXCR2+ neutrophils into colonic tissue. In UC patients, the magnitude of enrichment of the IL-22 regulated transcripts in colonic biopsies correlates with colonic neutrophil infiltration and is enriched in non-responders to ustekinumab therapy. Our data provide further insights into the biology of IL-22 in human disease and highlight its function in the regulation of pathogenic immune pathways, including neutrophil chemotaxis. The transcriptional networks regulated by IL-22 are functionally and clinically important in UC, impacting patient trajectories and responsiveness to biological intervention.

324 (PB104) - Quantitative analysis of EGFR expression and distribution upon drug treatment in colorectal organoid models

Background: Human organoids are patient-derived tissues that can be expanded in vitro by stimulating the (cancer) stem cells with WNT pathway activators, treatment with differentiation inhibitors and by providing growth factors in an extracellular matrix environment. These organoids form three-dimensional (3D) multicellular structures that polarize and self-organize and express relevant disease-related markers, which makes an organoid biobank ideal for testing the efficacy and mode of action of compounds and antibodies across a diverse patient population. Here, our colorectal organoid biobank and high content imaging platform were used to characterize EGFR dynamics upon drug treatment. Methods: An established biobank of normal colon and colorectal cancer organoid models was characterized for its sensitivity to the growth factor EGF, EGFR-inhibitors (Cetuximab), and standard-of-care compounds. To study the distribution of the EGF receptor, antibodies followed by immunofluorescence staining were used to detect the localization and distribution upon growth factor stimulation. (Inducible) knockdown organoid models with shRNA constructs targeting EGFR were generated to validate the findings. Results: The 3D organoid screening and our proprietary image analysis platform allows the in-depth detection and distinction of growth factor responses that not necessarily only relate to proliferation. By making use of this system, the sensitivity of organoid models to EGF stimulation that are independent of EGF for their outgrowth could be determined. By combining the detection of the target, EGFR, with the detection of targeting EGFR-inhibitory antibodies by immunofluorescence followed by image analysis, the redistribution of the receptor upon EGF-stimulation and in response to antibody exposure could be visualized and quantified. This revealed that EGFR is internalized in the first hours upon EGF stimulation. Along with making use of (inducible) knockdown organoids, it also revealed that different EGFR-targeting antibodies have different effects on the redistribution, internalization, and degradation of EGFR. Conclusions: The high content imaging platform in combination with high throughput screening in 3D-cultured organoid models allows compound and antibody testing in physiologically relevant patient-derived models to not only study the sensitivity to novel compounds, but also for patient stratification and to study the mode of action or understand the underlying relevant biology. The outcome of this work demonstrated that organoids are versatile, multicellular structures that are highly suitable for studying the expression and redistribution of disease-relevant targets. No conflict of interest.

Gene expression responses reflecting 5-FU-induced toxicity: Comparison between patient colon tissue and 3D human colon organoids

Capecitabine is a chemotherapeutic drug that is widely used as a monotherapy option in advanced cancer patients. After administration, it is converted into its active metabolite 5-fluorouracil (5-FU), a cytotoxic compound that may also induce adverse side effects in the gastrointestinal (GI) tract. Although these side effects can interfere with the continuation of the chemotherapy, diagnostic tools to detect early onset and prevention strategies are not available. In this explorative case study, we aim to identify differentially expressed genes (DEGs) that provide insight into the molecular mechanisms of toxicity induced by 5-FU in healthy colon tissue of breast cancer patients receiving capecitabine. Gene expression responses observed in patients were compared with those established in an in vitro model of healthy colon organoids. Colon biopsies from two patients with advanced breast cancer were collected before and after the treatment with capecitabine and used for RNA sequencing to determine transcriptomic responses. Differential expression analysis resulted in 31 affected genes, showing that the most affected pathways were transport of small molecules, cellular responses to stress, folate metabolism, NF-kB signalling pathway and immune system responses. The most biologically relevant genes were haemoglobin subunits encoding genes, involved in several processes; ATP12A, SLC26A3 and AQP8, involved in the transport of ions and water; TRIM31, a regulator of NF-kB signalling pathway; MST1P2 and MST1L, stimulators of macrophages. Comparison of human in vitro and in vivo responses showed that the gene expression of TRIM31 was similarly altered in the colon organoids exposed to 5-FU. Therefore, this gene constitutes a potential biomarker of colon toxicity that might be used in future in vitro drug safety design and screening.

Novel Histone Deacetylase 6 Inhibitor Confers Anti-inflammatory Effects and Enhances Gut Barrier Function.

The purpose of the current study was to examine the anti-inflammatory effects of CKD-506, a novel histone deacetylase 6 inhibitor, on human peripheral blood mononuclear cells (PBMCs) and CD4+ T cells and to explore the relationship between CKD-506 and gut epithelial barrier function. Lipopolysaccharide-stimulated human PBMCs from inflammatory bowel disease (IBD) patients were treated with CKD-506, and tumor necrosis factor (TNF)-α expression was measured using an enzyme-linked immunosorbent assay. The proliferation of CD4+ T cells from IBD patients was evaluated using flow cytometric analysis. The effects of CKD-506 on gut barrier function in a cell line and colon organoids, based on examinations of mRNA production, goblet cell differentiation, and E-cadherin recovery, were investigated using quantitative reverse transcription polymerase chain reaction, immunofluorescence, and a fluorescein isothiocyanate-dextran permeability assay. Secretion of TNF-α, a pivotal pro-inflammatory mediator in IBD, by lipopolysaccharide-triggered PBMCs was markedly decreased by CKD-506 treatment in a dose-dependent manner and to a greater extent than by tofacitinib or tubastatin A treatment. E-cadherin mRNA expression and goblet cell differentiation increased significantly and dose-dependently in HT-29 cells in response to CKD-506, and inhibition of E-cadherin loss after TNF-α stimulation was significantly reduced both in HT-29 cells and gut organoids. Caco-2 cells treated with CKD-506 showed a significant reduction in barrier permeability in a dose-dependent manner. The present study demonstrated that CKD-506 has anti-inflammatory effects on PBMCs and CD4 T cells and improves gut barrier function, suggesting its potential as a small-molecule therapeutic option for IBD.

DNA sensor-associated type I interferon signaling is increased in ulcerative colitis and induces JAK-dependent inflammatory cell death in colonic organoids.

DNA sensor pathways can initiate inflammasome, cell death, and type I interferon (IFN) signaling in immune-mediated inflammatory diseases (IMIDs), including type I interferonopathies. We investigated the involvement of these pathways in the pathogenesis of ulcerative colitis (UC) by analyzing the expression of DNA sensor, inflammasome, and type I IFN biomarker genes in colonic mucosal biopsy tissue from control (n = 31), inactive UC (n = 31), active UC (n = 33), and a UC single-cell RNA-Seq dataset. The effects of type I IFN (IFN-β), IFN-γ, and TNF-α on gene expression, cytokine production, and cell death were investigated in human colonic organoids. In organoids treated with cytokines alone, or in combination with NLR family pyrin domain-containing 3 (NLRP3), caspase, or JAK inhibitors, cell death was measured, and supernatants were assayed for IL-1β/IL-18/CXCL10. The expression of DNA sensor pathway genes-PYHIN family members [absent in melanoma 2 (AIM2), IFI16, myeloid cell nuclear differentiation antigen (MNDA), and pyrin and HIN domain family member 1 (PYHIN1)- as well as Z-DNA-binding protein 1 (ZBP1), cyclic GMP-AMP synthase (cGAS), and DDX41 was increased in active UC and expressed in a cell type-restricted pattern. Inflammasome genes (CASP1, IL1B, and IL18), type I IFN inducers [stimulator of interferon response cGAMP interactor 1 (STING), TBK1, and IRF3), IFNB1, and type I IFN biomarker genes (OAS2, IFIT2, and MX2) were also increased in active UC. Cotreatment of organoids with IFN-β or IFN-γ in combination with TNFα increased expression of IFI16, ZBP1, CASP1, cGAS, and STING induced cell death and IL-1β/IL-18 secretion. This inflammatory cell death was blocked by the JAK inhibitor tofacitinib but not by inflammasome or caspase inhibitors. Increased type I IFN activity may drive elevated expression of DNA sensor genes and JAK-dependent but inflammasome-independent inflammatory cell death of colonic epithelial cells in UC.NEW & NOTEWORTHY This study found that patients with active UC have significantly increased colonic gene expression of cytosolic DNA sensor, inflammasome, STING, and type I IFN signaling pathways. The type I IFN, IFN-β, in combination with TNF-α induced JAK-dependent but NLRP3 and inflammasome-independent inflammatory cell death of colonic organoids. This novel inflammatory cell death phenotype is relevant to UC immunopathology and may partially explain the efficacy of the JAKinibs tofacitinib and upadacitinib in patients with UC.

Inhibition of GABAA receptors in intestinal stem cells prevents chemoradiotherapy-induced intestinal toxicity.

Lethal intestinal tissue toxicity is a common side effect and a dose-limiting factor in chemoradiotherapy. Chemoradiotherapy can trigger DNA damage and induce P53-dependent apoptosis in LGR5+ intestinal stem cells (ISCs). Gamma-aminobutyric acid (GABA) and its A receptors (GABAAR) are present in the gastrointestinal tract. However, the functioning of the GABAergic system in ISCs is poorly defined. We found that GABAAR α1 (GABRA1) levels increased in the murine intestine after chemoradiotherapy. GABRA1 depletion in LGR5+ ISCs protected the intestine from chemoradiotherapy-induced P53-dependent apoptosis and prolonged animal survival. The administration of bicuculline, a GABAAR antagonist, prevented chemoradiotherapy-induced ISC loss and intestinal damage without reducing the chemoradiosensitivity of tumors. Mechanistically, it was associated with the reduction of reactive oxygen species-induced DNA damage via the L-type voltage-dependent Ca2+ channels. Notably, flumazenil, a GABAAR antagonist approved by the U.S. Food and Drug Administration, rescued human colonic organoids from chemoradiotherapy-induced toxicity. Therefore, flumazenil may be a promising drug for reducing the gastrointestinal side effects of chemoradiotherapy.

Establishment of organoid models based on a nested array chip for fast and reproducible drug testing in colorectal cancer therapy

The conventional microwell-based platform for construction of organoid models exhibits limitations in precision oncology applications because of low-speed growth and high variability. Here, we established organoid models on a nested array chip for fast and reproducible drug testing using 50% matrigel. First, we constructed mouse small intestinal and colonic organoid models. Compared with the conventional microwell-based platform, the mouse organoids on the chip showed accelerated growth and improved reproducibility due to the nested design of the chip. The design of the chip provides miniaturized and uniform shaping of the matrigel that allows the organoid to grow in a concentrated and controlled manner. Next, a patient-derived organoid (PDO) model from colorectal cancer tissues was successfully generated and characterized on the chip. Finally, the PDO models on the chip, from three patients, were implemented for high-throughput drug screening using nine treatment regimens. The drug sensitivity testing on the PDO models showed good quality control with a coefficient of variation under 10% and a Z' factor of more than 0.7. More importantly, the drug responses on the chip recapitulate the heterogeneous response of individual patients, as well as showing a potential correlation with clinical outcomes. Therefore, the organoid model coupled with the nested array chip platform provides a fast and reproducible means for predicting drug responses to accelerate precise oncology.Graphic abstract

Cytokine responsive networks in human colonic epithelial organoids unveil a molecular classification of inflammatory bowel disease.

Interactions between the epithelium and the immune system are critical in the pathogenesis of inflammatory bowel disease (IBD). In this study, we mapped the transcriptional landscape of human colonic epithelial organoids in response to different cytokines responsible for mediating canonical mucosal immune responses. By profiling the transcriptome of human colonic organoids treated with the canonical cytokines interferon gamma, interleukin-13, -17A, and tumor necrosis factor alpha with next-generation sequencing, we unveil shared and distinct regulation patterns of epithelial function by different cytokines. An integrative analysis of cytokine responses in diseased tissue from patients with IBD (n= 1,009) reveals a molecular classification of mucosal inflammation defined by gradients of cytokine-responsive transcriptional signatures. Our systems biology approach detected signaling bottlenecks in cytokine-responsive networks and highlighted their translational potential as theragnostic targets in intestinal inflammation.

Bacillus subtilis programs the differentiation of intestinal secretory lineages to inhibit Salmonella infection.

The role of intestinal microbiota on fate determination of intestinal epithelial cells has not been extensively examined. In this study, we explore the effect of Bacillus subtilis on programmed intestinal epithelial differentiation. We find that B.subtilis stimulates the differentiation of intestinal secretory cells. Moreover, B.subtilis inhibits the Notch pathway to reduce the expression of hairy and enhancer of split 1, thereby shifting intestinal stem cell differentiation toward a secretory cell fate. Moreover, we demonstrate that the programming effect of B.subtilis on intestinal differentiation is Toll-like receptor 2 pathway dependent. B.subtilis is associated with increased numbers of Paneth and goblet cells in the intestine. This results in the production of antimicrobial peptides to protect the intestinal mucosal barrier against Salmonella typhimurium. This study demonstrates that B.subtilis contributes to the differentiation of secretory cells by affecting Notch pathway signaling to maintain the intestinal barrier.

SOC-VI-02 Establishment and validation of co-culture system of human colon organoids and THP-1 derived macrophages

SOC-VI-02 Establishment and validation of co-culture system of human colon organoids and THP-1 derived macrophages

The phenotype of the gut region is more stably retained than developmental stage in piglet intestinal organoids.

Intestinal organoids are innovative in vitro tools to study the digestive epithelium. The objective of this study was to generate jejunum and colon organoids from suckling and weaned piglets in order to determine the extent to which organoids retain a location-specific and a developmental stage-specific phenotype. Organoids were studied at three time points by gene expression profiling for comparison with the transcriptomic patterns observed in crypts in vivo. In addition, the gut microbiota and the metabolome were analyzed to characterize the luminal environment of epithelial cells at the origin of organoids. The location-specific expression of 60 genes differentially expressed between jejunum and colon crypts from suckling piglets was partially retained (48%) in the derived organoids at all time point. The regional expression of these genes was independent of luminal signals since the major differences in microbiota and metabolome observed in vivo between the jejunum and the colon were not reproduced in vitro. In contrast, the regional expression of other genes was erased in organoids. Moreover, the developmental stage-specific expression of 30 genes differentially expressed between the jejunum crypts of suckling and weaned piglets was not stably retained in the derived organoids. Differentiation of organoids was necessary to observe the regional expression of certain genes while it was not sufficient to reproduce developmental stage-specific expression patterns. In conclusion, piglet intestinal organoids retained a location-specific phenotype while the characteristics of developmental stage were erased in vitro. Reproducing more closely the luminal environment might help to increase the physiological relevance of intestinal organoids.

Zranb1-mutant mice display abnormal colonic mucus production and exacerbation of DSS-induced colitis

Expression of mucin MUC2, a component of the colonic mucus layer, plays a crucial role in intestinal homeostasis. Here, we describe a new regulator of MUC2 expression, the deubiquitinase ZRANB1 (Trabid). A ZRANB1 mutation changing cysteine to serine in amino acid position 443, affects ubiquitination. To analyze ZRANB1 function in the intestine, we generated Zranb1 C443S mutant knock-in (Zranb1C443S/C443S) mice using the CRISPR/Cas9 system. Zranb1C443S/C443S mice exhibited decreased mRNA expression and MUC2 production. Colonic organoids from Zranb1C443S/C443S mice displayed decreased Muc2 mRNA expression following differentiation into goblet cells. Finally, we analyzed dextran sulfate sodium-induced colitis to understand ZRANB1's role in intestinal inflammation. Zranb1C443S/C443S mice with colitis exhibited significant weight loss, reduced colon length, and worsening clinical and pathological scores, indicating that ZRANB1 contributes to intestinal homeostasis. Together, these results suggest that ZRANB1 regulates MUC2 expression and intestinal inflammation, which may help elucidating the pathogenesis of inflammatory bowel disease and developing new therapeutics targeting ZRANB1.

Insights into Early Onset Colorectal Cancer through Analysis of Normal Colon Organoids of Familial Adenomatous Polyposis Patients.

Simple SummaryThe increase in early onset colorectal cancer (EOCRC) rates places a new, heavy burden on a younger population. Current studies in EOCRC are often affected by factors such as increased cellular heterogeneity, which mask biological signal. We overcome this using the colon organoid model, which is composed of cells from which CRC is believed to originate. We perform gene expression analysis on colon organoids derived from healthy and familial adenomatous polyposis patients, who are genetically predisposed to develop CRC at a young age, to identify differences that may be used for early monitoring of CRC development. We contextualize our findings in the framework of existing CRC and EOCRC data. We also show that ethanol, a CRC risk factor, may play an important role in further driving aberrant gene expression at EOCRC relevant genes. Functional analysis of these genes may shed new insight into EOCRC.Early onset colorectal cancer (EOCRC) rates have increased in recent decades. While lowering the recommended age for routine colonoscopies to 45 may reduce this burden, such measures do not address those who develop CRC before that age. Additional measures are needed to identify individuals at-risk for CRC. To better define transcriptomic events that precede the development of CRC, we performed RNA-sequencing analysis in colon organoids derived from seven healthy and six familial adenomatous polyposis (FAP) patients. This led to the identification of 2635 significant differentially expressed genes (FDR < 0.05). Through secondary analysis of publicly available datasets, we found that these genes were enriched for significant genes also present in FAP CRC and non-hereditary CRC datasets, including a subset that were unique to EOCRC. By exposing FAP colon organoids to a three-day ethanol treatment, we found that two EOCRC-relevant genes were also targets of CRC related lifestyle factors. Our data provides unique insight into the potential, early mechanisms of CRC development in colon epithelial cells, which may provide biomarkers for patient monitoring. We also show how modifiable lifestyle factors may further alter genes relevant to EOCRC, adding weight to the hypothesis that such factors represent an important contributor to increased EOCRC incidence.

Regulation of the p38-MAPK pathway by hyperosmolarity and by WNK kinases

p38-MAPK is a stress-response kinase activated by hyperosmolarity. Here we interrogated the pathways involved. We show that p38-MAPK signaling is activated by hyperosmotic stimulation in various solutions, cell types and colonic organoids. Hyperosmolarity sensing is detected at the level of the upstream activators of p38-MAPK: TRAF2/ASK1 (but not Rac1) and MKK3/6/4. While WNK kinases are known osmo-sensors, we found, unexpectedly, that short (2 h) inhibition of WNKs (with WNK463) led to elevated p38-MAPK activity under hyperosmolarity, which was mediated by WNK463-dependent stimulation of TAK1 or TRAF2/ASK1, the upstream activators of MKK3/6/4. However, this effect was temporary and was reversed by long-term (2 days) incubation with WNK463. Accordingly, 2 days (but not 2 h) inhibition of p38-MAPK or its upstream activators ASK1 or TAK1, or WNKs, diminished regulatory volume increase (RVI) following cell shrinkage under hyperosmolarity. We also show that RVI mediated by the ion transporter NKCC1 is dependent on p38-MAPK. Since WNKs are known activators of NKCC1, we propose a WNK- > NKCC1- > p38-MAPK pathway that controls RVI. This pathway is augmented by NHE1. Additionally, hyperosmolarity inhibited mTORC1 activation and cell proliferation. Thus, activation of p38-MAPK and WNKs is important for RVI and for cell proliferation.

DNA methylation analysis of normal colon organoids from familial adenomatous polyposis patients reveals novel insight into colon cancer development

BackgroundFamilial adenomatous polyposis (FAP) is an inherited colorectal cancer (CRC) syndrome resulting from germ line mutations in the adenomatous polyposis coli (APC) gene. While FAP accounts for less than 1% of all CRC cases, loss of APC expression is seen in > 80% of non-hereditary CRCs. To better understand molecular mechanisms underlying APC-driven CRC, we performed an epigenome-wide analysis of colon organoids derived from normal-appearing colons of FAP patients versus healthy subjects to identify differentially methylated regions (DMRs) that may precede the onset of CRC.ResultsWe identified 358 DMRs when comparing colon organoids of FAP patients to those of healthy subjects (FDR < 0.05, |mean beta difference| = 5%). Of these, nearly 50% of DMRs were also differentially methylated in at least one of three CRC tumor and normal adjacent tissue (NAT) cohorts (TCGA-COAD, GSE193535 and ColoCare). Moreover, 27 of the DMRs mapped to CRC genome-wide association study (GWAS) loci. We provide evidence suggesting that some of these DMRs led to significant differences in gene expression of adjacent genes using quantitative PCR. For example, we identified significantly greater expression of five genes: Kazal-type serine peptidase inhibitor domain 1 (KAZALD1, P = 0.032), F-Box and leucine-rich repeat protein 8 (FBXL8, P = 0.036), TRIM31 antisense RNA 1 (TRIM31-AS1, P = 0.036), Fas apoptotic inhibitory molecule 2 (FAIM2, P = 0.049) and (Collagen beta (1–0)galactosyltransferase 2 (COLGALT2, P = 0.049). Importantly, both FBXL8 and TRIM31-AS1 were also significantly differentially expressed in TCGA-COAD tumor versus matched NAT, supporting a role for these genes in CRC tumor development.ConclusionsWe performed the first DNA methylome-wide analysis of normal colon organoids derived from FAP patients compared to those of healthy subjects. Our results reveal that normal colon organoids from FAP patients exhibit extensive epigenetic differences compared to those of healthy subjects that appear similar to those exhibited in CRC tumor. Our analyses therefore identify DMRs and candidate target genes that are potentially important in CRC tumor development in FAP, with potential implications for non-hereditary CRC.

Identification of Quiescent LGR5+ Stem Cells in the Human Colon

In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells invivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability invivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-β signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating. Our results highlight the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.

N6-adenomethylation of GsdmC is essential for Lgr5+ stem cell survival to maintain normal colonic epithelial morphogenesis.

Gut epithelial morphogenesis is maintained by intestinal stem cells. Here, we report that depletion of N6-adenosine methyltransferase subunit Mettl14 from gut epithelial cells in mice impaired colon mucosal morphogenesis, leading to increased mucosal permeability, severe inflammation, growth retardation, and premature death. Mettl14 ablation triggered apoptosis that depleted Lgr5+ stem cells and disrupted colonic organoid growth and differentiation, whereas the inhibition of apoptosis rescued Mettl14-deleted mice and organoids. Mettl14 depletion disrupted N6-adenomethylation on GsdmC transcripts and abolished GsdmC expression. Reconstitution of Mettl14-deleted organoids or mice with GSDMC rescued Lgr5 expression and prevented apoptosis and mouse premature death, whereas GSDMC silence eliminated LGR5 and triggered apoptosis in human colonic organoids and epithelial cells. Mechanistically, Mettl14 depletion eliminated mitochondrial GsdmC, disrupted mitochondrial membrane potential, and triggered cytochrome c release that activates the pro-apoptotic pathway. In conclusion, GsdmC N6-adenomethylation protects mitochondrial homeostasis and is essential for Lgr5+ cell survival to maintain normal colonic epithelial regeneration.

Aggregation of cryopreserved mid-hindgut endoderm for more reliable and reproducible hPSC-derived small intestinal organoid generation.

SummaryA major technical limitation hindering the widespread adoption of human pluripotent stem cell (hPSC)-derived gastrointestinal (GI) organoid technologies is the need for de novo hPSC differentiation and dependence on spontaneous morphogenesis to produce detached spheroids. Here, we report a method for simple, reproducible, and scalable production of small intestinal organoids (HIOs) based on the aggregation of cryopreservable hPSC-derived mid-hindgut endoderm (MHE) monolayers. MHE aggregation eliminates variability in spontaneous spheroid production and generates HIOs that are comparable to those arising spontaneously. With a minor modification to the protocol, MHE can be cryopreserved, thawed, and aggregated, facilitating HIO production without de novo hPSC differentiation. Finally, aggregation can also be used to generate antral stomach organoids and colonic organoids. This improved method removes significant barriers to the implementation and successful use of hPSC-derived GI organoid technologies and provides a framework for improved dissemination and increased scalability of GI organoid production.

Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1.

Aldehyde dehydrogenases (ALDHs) are promising cancer drug targets, as certain isoforms are required for the survival of stem-like tumor cells. We have discovered selective inhibitors of ALDH1B1, a mitochondrial enzyme that promotes colorectal and pancreatic cancer. We describe bicyclic imidazoliums and guanidines that target the ALDH1B1 active site with comparable molecular interactions and potencies. Both pharmacophores abrogate ALDH1B1 function in cells; however, the guanidines circumvent an off-target mitochondrial toxicity exhibited by the imidazoliums. Our lead isoform-selective guanidinyl antagonists of ALDHs exhibit proteome-wide target specificity, and they selectively block the growth of colon cancer spheroids and organoids. Finally, we have used genetic and chemical perturbations to elucidate the ALDH1B1-dependent transcriptome, which includes genes that regulate mitochondrial metabolism and ribosomal function. Our findings support an essential role for ALDH1B1 in colorectal cancer, provide molecular probes for studying ALDH1B1 functions and yield leads for developing ALDH1B1-targeting therapies.

Abstract 1416: Aurora kinase is synthetic lethal with APC loss of function in engineered human colon organoid models

Abstract Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide. The disease-initiating event in most CRC cases is the loss of function (LOF) of the adenomatous polyposis coli (APC) gene, accounting for 70-80% of CRC. Strategies to target cells that harbor the truncation mutation in APC, which leads to its LOF, may provide the opportunity to intercept tumorigenesis in CRC patients. High-risk CRC populations include patients with familial adenomatous polyposis (FAP), the inherited disorder caused by germline APC mutations. Aurora Kinase (AK), a family of serine/threonine kinases, is essential for proper cell division, and overexpression of AK has been associated with multiple heme and solid tumor malignancies, including CRC. Aurora kinases have been well-studied, both preclinically and clinically, as a target for therapeutic intervention across multiple cancers; however, the role of AK in CRC disease progression has not been fully explored. Here we report a synthetic lethal dependency between APC and AK which was identified in a human isogenic colon organoid model containing a genetically engineered APC LOF (APCLOF). Aurora A-specific, Aurora B-specific, and Pan-AK inhibitors were evaluated in isogenic APCLOF or APCWT colon organoids derived from multiple donors. All AK inhibitors demonstrated selective growth inhibition of the APCLOF organoid. The strongest synthetic lethal effect was observed with a pan-AK inhibitor showing greater than 1000-fold selectivity in the APCLOF colon organoids compared to APCWT. Interestingly, this activity diminished as oncogenic driver mutations in KRAS and/or p53 genes were introduced into the APCLOF organoids. These findings suggest that therapeutic benefit of AK inhibitors would be greater in early APCLOF driven adenomas as opposed to advanced CRC. Despite these encouraging results indicating therapeutic opportunity in early adenoma disease, on-target genotoxicity was observed in vitro through increases in micronuclei at pharmacologic doses. Therefore, although there is a high unmet need in FAP for therapeutic options, potential genotoxicity associated with AK inhibitor treatments may limit utility as an intervention strategy. Citation Format: Dana S. Gaffney, Eileen Vesely, David Pocalyko, Kurtis Bachman, Karla Wiehagen, Glenn Cowley, Victoria Wong, Prashanth Gokare, Peggy Guzzie-Peck, Sandy Weiner, Ann De Smedt, Jacqueline Kinyamu-Akunda, Tammy Bush, Zhuming Zhang, Robert Schulingkamp, Sylvia F. Boj, Rene Overmeer, Carla Verissimo, Roshni Nair. Aurora kinase is synthetic lethal with APC loss of function in engineered human colon organoid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1416.