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Inhibition of the PARP superfamily tankyrase enzymes suppresses Wnt/β-catenin signalling in tumour cells. Here, we describe here a novel, drug-like small molecule inhibitor of tankyrase MSC2504877 that inhibits the growth of APC mutant colorectal tumour cells. Parallel siRNA and drug sensitivity screens showed that the clinical CDK4/6 inhibitor palbociclib, causes enhanced sensitivity to MSC2504877. This tankyrase inhibitor-CDK4/6 inhibitor combinatorial effect is not limited to palbociclib and MSC2504877 and is elicited with other CDK4/6 inhibitors and toolbox tankyrase inhibitors. The addition of MSC2504877 to palbociclib enhances G 1 cell cycle arrest and cellular senescence in tumour cells. MSC2504877 exposure suppresses the upregulation of Cyclin D2 and Cyclin E2 caused by palbociclib and enhances the suppression of phospho-Rb, providing a mechanistic explanation for these effects. The combination of MSC2504877 and palbociclib was also effective in suppressing the cellular hyperproliferative phenotype seen in Apc defective intestinal stem cells in vivo . However, the presence of an oncogenic Kras p.G12D mutation in mice reversed the effects of the MSC2504877/palbociclib combination, suggesting one molecular route that could lead to drug resistance.

Background Dysregulated Notch signalling contributes to breast cancer development and progression, but validated tools to measure the level of Notch signalling in breast cancer subtypes and in response to systemic therapy are largely lacking. A transcriptomic signature of Notch signalling would be warranted, for example to monitor the effects of future Notch-targeting therapies and to learn whether altered Notch signalling is an off-target effect of current breast cancer therapies. In this report, we have established such a classifier. Methods To generate the signature, we first identified Notch-regulated genes from six basal-like breast cancer cell lines subjected to elevated or reduced Notch signalling by culturing on immobilized Notch ligand Jagged1 or blockade of Notch by γ -secretase inhibitors, respectively. From this cadre of Notch-regulated genes, we developed candidate transcriptomic signatures that were trained on a breast cancer patient dataset (the TCGA-BRCA cohort) and a broader breast cancer cell line cohort and sought to validate in independent datasets. Results An optimal 20-gene transcriptomic signature was selected. We validated the signature on two independent patient datasets (METABRIC and Oslo2), and it showed an improved coherence score and tumour specificity compared with previously published signatures. Furthermore, the signature score was particularly high for basal-like breast cancer, indicating an enhanced level of Notch signalling in this subtype. The signature score was increased after neoadjuvant treatment in the PROMIX and BEAUTY patient cohorts, and a lower signature score generally correlated with better clinical outcome. Conclusions The 20-gene transcriptional signature will be a valuable tool to evaluate the response of future Notch-targeting therapies for breast cancer, to learn about potential effects on Notch signalling from conventional breast cancer therapies and to better stratify patients for therapy considerations.

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I–IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab. The heterogeneity of colorectal cancer has important clinical and therapeutic implications. Here the authors analysed the responses of a large biobank of organoids and xenografts derived from colorectal patients to a panel of clinically relevant therapeutic agents to identify genes signatures associated with drug response.

Chemoproteomic studies have revealed that a Wnt-pathway inhibitor, CCT251545, is a potent and selective small-molecule chemical probe that inhibits the Mediator complex–associated protein kinases CDK8 and CDK19 through a type 1 binding mode and modulates the growth of Wnt-dependent tumors. There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex–associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway–regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1 SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo . Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.

Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors. Healthy cells in the human body can become cancerous if they gain genetic mutations that allow them to rapidly grow and divide. Some types of cancer respond better to drug treatments than others and tumors often develop resistance to a particular drug treatment after a while. Because of this, researchers are always searching for new molecules to develop into anticancer drugs. Recently, a team of researchers identified some small molecules that could inactivate two closely related proteins called CDK8 and CDK19. CDK8 is essential for the WNT signaling pathway – which enables cells to communicate with one another – and has been extensively studied in various cancers. Previous studies indicate that this protein can either promote or inhibit the growth of tumors, depending on the type and stage of the cancer. Furthermore, CDK8 regulates a type of molecular switch called a “super-enhancer”, which controls the activity of many genes. In contrast, the role of CDK19 in cells was not as well understood. Here Clarke, Ortiz-Ruiz et al. investigated whether two different classes of small molecules that target CDK8 and CDK19 (referred to as “prototype CDK8/19 drugs”) could inhibit the growth of cancers, and whether they have any harmful side effects on healthy cells. For the experiments, human cancer cells were implanted into mice. Treating these mice with prototype CDK8/19 drugs inhibited the activity of CDK8 and CDK19 in the cancer cells and slowed the growth of colorectal tumors. A type of blood cancer called acute myeloid leukaemia was particularly sensitive to the drugs. However, Clarke, Ortiz-Ruiz et al. also observed that the prototype drugs altered the activity of many genes with roles in healthy tissues such as immune, bone and stem cells. Further experiments in mice and cells grown in the laboratory confirmed that these prototype drugs have adverse effects on healthy intestinal and bone marrow stem cells and trigger changes to immune cells. These concerning side effects were also evident when the prototype drugs were tested in rats and dogs. Furthermore, the experiments indicate that there is not a suitable range of doses of these drugs in which the therapeutic benefits outweigh the toxic side effects. Clarke, Ortiz-Ruiz et al. conclude that the clinical development of CDK8/19 drugs will be extremely challenging and that their prototype drugs would not currently be suitable for use as cancer treatments. However, the small molecules they describe will be important probes in research to study exactly how CDK8/19 regulate gene activity in both healthy cells and cancers.

The four members of the mannose receptor family (the mannose receptor, the M‐type phospholipase A 2 receptor, DEC‐205 and Endo180) share a common extracellular arrangement of an amino‐terminal cysteine‐rich domain followed by a fibronectin type II (FNII) domain and multiple C‐type lectin‐like domains (CTLDs). In addition, all have a short cytoplasmic domain, which mediates their constitutive recycling between the plasma membrane and the endosomal apparatus, suggesting that these receptors function to internalize ligands for intracellular delivery. We have generated mice with a targeted deletion of Endo180 exons 2–6 and show that this mutation results in the efficient expression of a truncated Endo180 protein that lacks the cysteine‐rich domain, the FNII domain and CTLD1. Analysis of embryonic fibroblasts reveals that this mutation does not disrupt the C‐type lectin activity that is mediated by CTLD2, but results in cells that have a defect in collagen binding and internalization and an impaired migratory phenotype.

The Notch signalling pathway is highly evolutionarily conserved and regulates differentiation and homeostasis in most organs. Given the critical role of Notch signalling for normal development, dysregulated Notch signalling is frequently linked to pathogenesis of disease and cancer. Hence, developing Notch-targeting therapeutics is warranted but has been challenging and Notch inhibitors have not yet reached broad clinical use. In this report, we identify potential Notch inhibitors, using a novel cell-based Notch reporter system for unbiased screening of compounds reducing Notch signalling. A library of 37.966 small organic compounds was screened for inhibitor candidates, followed by a counter screen to eliminate γ-secretase inhibitor-like compounds and an orthogonal screen based on the role of Notch signalling in myogenic differentiation. This triage led to the identification of five Notch inhibitor candidate hits with different chemical backbones and unrelated to previous Notch antagonists. One candidate hit showed structural similarities to dihydroorotate dehydrogenase (DHODH) inhibitors, and we provide evidence that inhibition of DHODH activity reduces Notch signalling. In conclusion, our data support the notion that DHODH inhibition may be an interesting avenue to explore for the development of novel Notch inhibitors.

Aberrant activation of the Wnt signalling pathway is required for tumour initiation and survival in the majority of colorectal cancers. The development of inhibitors of Wnt signalling has been the focus of multiple drug discovery programs targeting colorectal cancer and other malignancies associated with aberrant pathway activation. However, progression of new clinical entities targeting the Wnt pathway has been slow. One challenge lies with the limited predictive power of 2D cancer cell lines because they fail to fully recapitulate intratumoural phenotypic heterogeneity. In particular, the relationship between 2D cancer cell biology and cancer stem cell function is poorly understood. By contrast, 3D tumour organoids provide a platform in which complex cell-cell interactions can be studied. However, complex 3D models provide a challenging platform for the quantitative analysis of drug responses of therapies that have differential effects on tumour cell subpopulations. Here, we generated tumour organoids from colorectal cancer patients and tested their responses to inhibitors of Tankyrase (TNKSi) which are known to modulate Wnt signalling. Using compounds with 3 orders of magnitude difference in cellular mechanistic potency together with image-based assays, we demonstrate that morphometric analyses can capture subtle alterations in organoid responses to Wnt inhibitors that are consistent with activity against a cancer stem cell subpopulation. Overall our study highlights the value of phenotypic readouts as a quantitative method to asses drug-induced effects in a relevant preclinical model.