Spatiotemporally resolved colorectal oncogenesis in mini-colons ex vivo.

Three-dimensional organoid culture technologies have revolutionized cancer research by allowing for more realistic and scalable reproductions of both tumour and microenvironmental structures1-3. This has enabled better modelling of low-complexity cancer cell behaviours that occur over relatively short periods of time4. However, available organoid systems do not capture the intricate evolutionary process of cancer development in terms of tissue architecture, cell diversity, homeostasis and lifespan. As a consequence, oncogenesis and tumour formation studies are not possible in vitro and instead require the extensive use of animal models, which provide limited spatiotemporal resolution of cellular dynamics and come at a considerable cost in terms of resources and animal lives. Here we developed topobiologically complex mini-colons that are able to undergo tumorigenesis ex vivo by integrating microfabrication, optogenetic and tissue engineering approaches. With this system, tumorigenic transformation can be spatiotemporally controlled by directing oncogenic activation through blue-light exposure, and emergent colon tumours can be tracked in real-time at the single-cell resolution for several weeks without breaking the culture. These induced mini-colons display rich intratumoural and intertumoural diversity and recapitulate key pathophysiological hallmarks displayed by colorectal tumours in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, mini-colons can be used to identify tumorigenic determinants and pharmacological opportunities. As a whole, our study paves the way for cancer initiation research outside living organisms.

Bcl-xL targeting eliminates ageing tumor-promoting neutrophils and inhibits lung tumor growth.

Elevated peripheral blood and tumor-infiltrating neutrophils are often associated with a poor patient prognosis. However, therapeutic strategies to target these cells are difficult to implement due to the life-threatening risk of neutropenia. In a genetically engineered mouse model of lung adenocarcinoma, tumor-associated neutrophils (TAN) demonstrate tumor-supportive capacities and have a prolonged lifespan compared to circulating neutrophils. Here, we show that tumor cell-derived GM-CSF triggers the expression of the anti-apoptotic Bcl-xL protein and enhances neutrophil survival through JAK/STAT signaling. Targeting Bcl-xL activity with a specific BH3 mimetic, A-1331852, blocked the induced neutrophil survival without impacting their normal lifespan. Specifically, oral administration with A-1331852 decreased TAN survival and abundance, and reduced tumor growth without causing neutropenia. We also show that G-CSF, a drug used to combat neutropenia in patients receiving chemotherapy, increased the proportion of young TANs and augmented the anti-tumor effect resulting from Bcl-xL blockade. Finally, our human tumor data indicate the same role for Bcl-xL on pro-tumoral neutrophil survival. These results altogether provide preclinical evidence for safe neutrophil targeting based on their aberrant intra-tumor longevity.

Notch signaling drives intestinal graft-versus-host disease in mice and nonhuman primates.

Notch signaling promotes T cell pathogenicity and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) in mice, with a dominant role for the Delta-like Notch ligand DLL4. To assess whether Notch's effects are evolutionarily conserved and to identify the mechanisms of Notch signaling inhibition, we studied antibody-mediated DLL4 blockade in a nonhuman primate (NHP) model similar to human allo-HCT. Short-term DLL4 blockade improved posttransplant survival with durable protection from gastrointestinal GVHD in particular. Unlike prior immunosuppressive strategies tested in the NHP GVHD model, anti-DLL4 interfered with a T cell transcriptional program associated with intestinal infiltration. In cross-species investigations, Notch inhibition decreased surface abundance of the gut-homing integrin α4ß7 in conventional T cells while preserving α4ß7 in regulatory T cells, with findings suggesting increased ß1 competition for α4 binding in conventional T cells. Secondary lymphoid organ fibroblastic reticular cells emerged as the critical cellular source of Delta-like Notch ligands for Notch-mediated up-regulation of α4ß7 integrin in T cells after allo-HCT. Together, DLL4-Notch blockade decreased effector T cell infiltration into the gut, with increased regulatory to conventional T cell ratios early after allo-HCT. Our results identify a conserved, biologically unique, and targetable role of DLL4-Notch signaling in intestinal GVHD.

Resistance mechanism to Notch inhibition and combination therapy in human T-cell acute lymphoblastic leukemia.

Gain-of-function mutations in NOTCH1 are among the most frequent genetic alterations in T-cell acute lymphoblastic leukemia (T-ALL), highlighting the Notch signaling pathway as a promising therapeutic target for personalized medicine. Yet, a major limitation for long-term success of targeted therapy is relapse due to tumor heterogeneity or acquired resistance. Thus, we performed a genome-wide CRISPR-Cas9 screen to identify prospective resistance mechanisms to pharmacological NOTCH inhibitors and novel targeted combination therapies to efficiently combat T-ALL. Mutational loss of phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) causes resistance to Notch inhibition. PIK3R1 deficiency leads to increased PI3K/AKT signaling, which regulates cell cycle and the spliceosome machinery, both at the transcriptional and posttranslational level. Moreover, several therapeutic combinations have been identified, in which simultaneous targeting of the cyclin-dependent kinases 4 and 6 (CDK4/6) and NOTCH proved to be the most efficacious in T-ALL xenotransplantation models.

Notch1 signaling is limited in healthy mature kidneys in vivo.

OBJECTIVE: A Delta-Notch signaling component, Notch1, is involved in the normal development and multiple disorders of the kidney. Although the increase in Notch1 signaling is crucial to these pathogeneses, the basal signaling level in 'healthy' mature kidneys is still unclear. To address this question, we used an artificial Notch1 receptor fused with Gal4/UAS components in addition to the Cre/loxP system and fluorescent proteins in mice. This transgenic reporter mouse system enabled labeling of past and ongoing Notch1 signaling with tdsRed or Cre recombinase, respectively. RESULTS: We confirmed that our transgenic reporter mouse system mimicked the previously reported Notch1 signaling pattern. Using this successful system, we infrequently observed cells with ongoing Notch1 signaling only in Bowman's capsule and tubules. We consider that Notch1 activation in several lines of disease model mice was pathologically significant itself.

Notch Signaling Pathway in Tooth Shape Variations throughout Evolution.

Evolutionary changes in vertebrates are linked to genetic alterations that often affect tooth crown shape, which is a criterion of speciation events. The Notch pathway is highly conserved between species and controls morphogenetic processes in most developing organs, including teeth. Epithelial loss of the Notch-ligand Jagged1 in developing mouse molars affects the location, size and interconnections of their cusps that lead to minor tooth crown shape modifications convergent to those observed along Muridae evolution. RNA sequencing analysis revealed that these alterations are due to the modulation of more than 2000 genes and that Notch signaling is a hub for significant morphogenetic networks, such as Wnts and Fibroblast Growth Factors. The modeling of these tooth crown changes in mutant mice, via a three-dimensional metamorphosis approach, allowed prediction of how Jagged1-associated mutations in humans could affect the morphology of their teeth. These results shed new light on Notch/Jagged1-mediated signaling as one of the crucial components for dental variations in evolution.

Disruption of stem cell niche-confined R-spondin 3 expression leads to impaired hematopoiesis.

Self-renewal and differentiation of stem and progenitor cells are tightly regulated to ensure tissue homeostasis. This regulation is enabled both remotely by systemic circulating cues, such as cytokines and hormones, and locally by various niche-confined factors. R-spondin 3 (RSPO3) is one of the most potent enhancers of Wnt signaling, and its expression is usually restricted to the stem cell niche where it provides localized enhancement of Wnt signaling to regulate stem cell expansion and differentiation. Disruption of this niche-confined expression can disturb proper tissue organization and lead to cancers. Here, we investigate the consequences of disrupting the niche-restricted expression of RSPO3 in various tissues, including the hematopoietic system. We show that normal Rspo3 expression is confined to the perivascular niche in the bone marrow. Induction of increased systemic levels of circulating RSPO3 outside of the niche results in prominent loss of early B-cell progenitors and anemia but surprisingly has no effect on hematopoietic stem cells. Using molecular, pharmacologic, and genetic approaches, we show that these RSPO3-induced hematopoietic phenotypes are Wnt and RSPO3 dependent and mediated through noncanonical Wnt signaling. Our study highlights a distinct role for a Wnt/RSPO3 signaling axis in the regulation of hematopoiesis, as well as possible challenges related to therapeutic use of RSPOs for regenerative medicine.

Stromal Notch ligands foster lymphopenia-driven functional plasticity and homeostatic proliferation of naive B cells.

In lymphopenic environments, secondary lymphoid organs regulate the size of B and T cell compartments by supporting the homeostatic proliferation of mature lymphocytes. The molecular mechanisms underlying these responses and their functional consequences remain incompletely understood. To evaluate homeostasis of the mature B cell pool during lymphopenia, we turned to an adoptive transfer model of purified follicular B cells into Rag2-/- mouse recipients. Highly purified follicular B cells transdifferentiated into marginal zone-like B cells when transferred into Rag2-/- lymphopenic hosts but not into wild-type hosts. In lymphopenic spleens, transferred B cells gradually lost their follicular phenotype and acquired characteristics of marginal zone B cells, as judged by cell surface phenotype, expression of integrins and chemokine receptors, positioning close to the marginal sinus, and an ability to rapidly generate functional plasma cells. Initiation of follicular to marginal zone B cell transdifferentiation preceded proliferation. Furthermore, the transdifferentiation process was dependent on Notch2 receptors in B cells and expression of Delta-like 1 Notch ligands by splenic Ccl19-Cre+ fibroblastic stromal cells. Gene expression analysis showed rapid induction of Notch-regulated transcripts followed by upregulated Myc expression and acquisition of broad transcriptional features of marginal zone B cells. Thus, naive mature B cells are endowed with plastic transdifferentiation potential in response to increased stromal Notch ligand availability during lymphopenia.

A leukemia-protective germline variant mediates chromatin module formation via transcription factor nucleation.

Non-coding variants coordinate transcription factor (TF) binding and chromatin mark enrichment changes over regions spanning >100 kb. These molecularly coordinated regions are named "variable chromatin modules" (VCMs), providing a conceptual framework of how regulatory variation might shape complex traits. To better understand the molecular mechanisms underlying VCM formation, here, we mechanistically dissect a VCM-modulating noncoding variant that is associated with reduced chronic lymphocytic leukemia (CLL) predisposition and disease progression. This common, germline variant constitutes a 5-bp indel that controls the activity of an AXIN2 gene-linked VCM by creating a MEF2 binding site, which, upon binding, activates a super-enhancer-like regulatory element. This triggers a large change in TF binding activity and chromatin state at an enhancer cluster spanning >150 kb, coinciding with subtle, long-range chromatin compaction and robust AXIN2 up-regulation. Our results support a model in which the indel acts as an AXIN2 VCM-activating TF nucleation event, which modulates CLL pathology.

Generation of a Gal4-dependent gene recombination and illuminating mouse.

Cell labeling technologies, including the Cre/loxP system, are powerful tools in developmental biology. Although the conventional Cre/loxP system has been extensively used to label the expression of specific genes, it is less frequently used for labeling protein-protein interactions owing to technical difficulties. In the present study, we generated a new Gal4-dependent transgenic reporter mouse line that expressed Cre recombinase and a near-infrared fluorescent protein, miRFP670. To examine whether this newly generated transgenic mouse line is applicable in labeling of protein-protein interaction, we used a previously reported transgenic mouse lines that express Notch1 receptor with its intracellular domain replaced with a yeast transcription factor, Gal4. Upon the binding of this artificial Notch1 receptor and endogenous Notch1 ligands, Gal4 would be cleaved from the cell membrane to induce expression of Cre recombinase and miRFP670. Indeed, we observed miRFP670 signal in the mouse embryos (embryonic day 14.5). In addition, we examined whether our Cre recombinase was functional by using another transgenic mouse line that express dsRed after Cre-mediated recombination. We observed dsRed signal in small intestine epithelial cells where Notch1 signal was suggested to be involved in the crypt stem cell maintenance, suggesting that our Cre recombinase was functional. As our newly generated mouse line required only the functioning of Gal4, it could be useful for labeling several types of molecular activities in vivo.

Tcf1 is essential for initiation of oncogenic Notch1-driven chromatin topology in T-ALL.

NOTCH1 is a well-established lineage specifier for T cells and among the most frequently mutated genes throughout all subclasses of T cell acute lymphoblastic leukemia (T-ALL). How oncogenic NOTCH1 signaling launches a leukemia-prone chromatin landscape during T-ALL initiation is unknown. Here we demonstrate an essential role for the high-mobility-group transcription factor Tcf1 in orchestrating chromatin accessibility and topology, allowing aberrant Notch1 signaling to convey its oncogenic function. Although essential, Tcf1 is not sufficient to initiate leukemia. The formation of a leukemia-prone epigenetic landscape at the distal Notch1-regulated Myc enhancer, which is fundamental to this disease, is Tcf1-dependent and occurs within the earliest progenitor stage even before cells adopt a T lymphocyte or leukemic fate. Moreover, we discovered a unique evolutionarily conserved Tcf1-regulated enhancer element in the distal Myc-enhancer, which is important for the transition of preleukemic cells to full-blown disease.

Notch1 Deficiency Induces Tumor Cell Accumulation Inside the Bronchiolar Lumen and Increases TAZ Expression in an Autochthonous Kras LSL-G12V Driven Lung Cancer Mouse Model.

Purpose: Abrogation of Notch signaling, which is pivotal for lung development and pulmonary epithelial cell fate decisions was shown to be involved in the aggressiveness and the differentiation of lung carcinomas. Additionally, the transcription factors YAP and TAZ which are involved in the Hippo pathway, were recently shown to be tightly linked with Notch signaling and to regulate the cell fate in epidermal stem cells. Thus, we aim to elucidate the effects of conditional Notch1 deficiency on carcinogenesis and TAZ expression in lung cancer. Methods: We investigated the effect of conditional Cre-recombinase mediated Notch1 knock-out on lung cancer cells in vivo using an autochthonous mouse model of lung adenocarcinomas driven by Kras LSL-G12V and comprehensive immunohistochemical analysis. In addition, we analyzed clinical samples and human lung cancer cell lines for TAZ expression and supported our findings by publicly available data from The Cancer Genome Atlas (TCGA). Results: In mice, we found induction of papillary adenocarcinomas and protrusions of tumor cells from the bronchiolar lining upon Notch1 deficiency. Moreover, the mutated Kras driven lung tumors with deleted Notch1 showed increased TAZ expression and focal nuclear translocation which was frequently observed in human pulmonary adenocarcinomas and squamous cell carcinomas of the lung, but not in small cell lung carcinomas. In addition, we used data from TCGA to show that putative inactivating NOTCH1 mutations co-occur with KRAS mutations and genomic amplifications in lung adenocarcinomas. Conclusion: Our in vivo study provides evidence that Notch1 deficiency in mutated Kras driven lung carcinomas contributes to lung carcinogenesis in a subgroup of patients by increasing TAZ expression who might benefit from TAZ signaling blockade.

The E protein-TCF1 axis controls γδ T cell development and effector fate.

TCF1 plays a critical role in T lineage commitment and the development of αß lineage T cells, but its role in γδ T cell development remains poorly understood. Here, we reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element in the Tcf7 locus, and this axis regulates both γδ T lineage commitment and effector fate. Indeed, the level of TCF1 expression plays an important role in setting the threshold for γδ T lineage commitment and modulates the ability of TCR signaling to influence effector fate adoption by γδ T lineage progenitors. This finding provides mechanistic insight into how TCR-mediated repression of E proteins promotes the development of γδ T cells and their adoption of the interleukin (IL)-17-producing effector fate. IL-17-producing γδ T cells have been implicated in cancer progression and in the pathogenesis of psoriasis and multiple sclerosis.

Notch signaling promotes disease initiation and progression in murine chronic lymphocytic leukemia.

NOTCH1 gain-of-function mutations are recurrent in B-cell chronic lymphocytic leukemia (B-CLL), where they are associated with accelerated disease progression and refractoriness to chemotherapy. The specific role of NOTCH1 in the development and progression of this malignancy is unclear. Here, we assess the impact of loss of Notch signaling and pathway hyperactivation in an in vivo mouse model of CLL (IgH.TEµ) that faithfully replicates many features of the human pathology. Ablation of canonical Notch signaling using conditional gene inactivation of RBP-J in immature hematopoietic or B-cell progenitors delayed CLL induction and reduced incidence of mice developing disease. In contrast, forced expression of a dominant active form of Notch resulted in more animals developing CLL with early disease onset. Comparative analysis of gene expression and epigenetic features of Notch gain-of-function and control CLL cells revealed direct and indirect regulation of cell cycle-associated genes, which led to increased proliferation of Notch gain-of-function CLL cells in vivo. These results demonstrate that Notch signaling facilitates disease initiation and promotes CLL cell proliferation and disease progression.

A third Notch in colorectal cancer progression and metastasis.

In this issue of JEM, Varga et al. (https://doi.org/10.1084/jem.20191515) describe a mouse model of invasive and metastatic colorectal cancer (CRC) closely resembling the human consensus molecular subtype (CMS) 4 associated with the poorest overall survival of the four CMSs. Transcriptomic and bioinformatic analysis combined with pharmacological and genetic studies identified Notch3 as a promoter of tumor progression and metastasis. NOTCH3 expression was up-regulated in CMS4 CRC patients and associated with tumor staging, lymph node and distant metastasis. These findings feature NOTCH3 as putative therapeutic target for advanced CMS4 CRC patients.

Pharmacological disruption of the Notch transcription factor complex.

Notch pathway signaling is implicated in several human cancers. Aberrant activation and mutations of Notch signaling components are linked to tumor initiation, maintenance, and resistance to cancer therapy. Several strategies, such as monoclonal antibodies against Notch ligands and receptors, as well as small-molecule γ-secretase inhibitors (GSIs), have been developed to interfere with Notch receptor activation at proximal points in the pathway. However, the use of drug-like small molecules to target the downstream mediators of Notch signaling, the Notch transcription activation complex, remains largely unexplored. Here, we report the discovery of an orally active small-molecule inhibitor (termed CB-103) of the Notch transcription activation complex. We show that CB-103 inhibits Notch signaling in primary human T cell acute lymphoblastic leukemia and other Notch-dependent human tumor cell lines, and concomitantly induces cell cycle arrest and apoptosis, thereby impairing proliferation, including in GSI-resistant human tumor cell lines with chromosomal translocations and rearrangements in Notch genes. CB-103 produces Notch loss-of-function phenotypes in flies and mice and inhibits the growth of human breast cancer and leukemia xenografts, notably without causing the dose-limiting intestinal toxicity associated with other Notch inhibitors. Thus, we describe a pharmacological strategy that interferes with Notch signaling by disrupting the Notch transcription complex and shows therapeutic potential for treating Notch-driven cancers.

Canonical Notch signaling controls the early thymic epithelial progenitor cell state and emergence of the medullary epithelial lineage in fetal thymus development.

Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). However, the mechanisms controlling cTEC and mTEC production from the common TEPC are not understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPCs at the expense of cTEC differentiation. Finally, we uncover a cross-regulatory relationship between NOTCH and FOXN1, a master regulator of TEC differentiation. These data establish NOTCH as a potent regulator of TEPC and mTEC fate during fetal thymus development, and are thus of high relevance to strategies aimed at generating/regenerating functional thymic tissue in vitro and in vivo.

GCNT1-Mediated O-Glycosylation of the Sialomucin CD43 Is a Sensitive Indicator of Notch Signaling in Activated T Cells.

Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.

Neutrophils suppress tumor-infiltrating T cells in colon cancer via matrix metalloproteinase-mediated activation of TGFß.

High T-cell infiltration in colorectal cancer (CRC) correlates with a favorable disease outcome and immunotherapy response. This, however, is only observed in a small subset of CRC patients. A better understanding of the factors influencing tumor T-cell responses in CRC could inspire novel therapeutic approaches to achieve broader immunotherapy responsiveness. Here, we investigated T cell-suppressive properties of different myeloid cell types in an inducible colon tumor mouse model. The most potent inhibitors of T-cell activity were tumor-infiltrating neutrophils. Gene expression analysis and combined in vitro and in vivo tests indicated that T-cell suppression is mediated by neutrophil-secreted metalloproteinase activation of latent TGFß. CRC patient neutrophils similarly suppressed T cells via TGFß in vitro, and public gene expression datasets suggested that T-cell activity is lowest in CRCs with combined neutrophil infiltration and TGFß activation. Thus, the interaction of neutrophils with a TGFß-rich tumor microenvironment may represent a conserved immunosuppressive mechanism in CRC.