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Supplementary Figure 1

Primers used for targeted gene profiling on selected RAS/MAPK pathway genes either on Fluidigm or by qRT-PCR on Roche Lightcycler 480

Supplementary Figure 3

Supplementary Figure 2

KRAS mutation status of cell lines included in in vitro combination screen

The RAS-regulated RAF–MEK1/2–ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

Supplementary Figure 6

Supplementary materials and methods

Supplementary Figure 5

Cell lines defined as sensitive to AZD0364 from 747 cell panel screen

Response of 747 cell lines to treatment with AZD0364 for 72 hours.

Supplementary Figure Legends

The RAS-regulated RAF–MEK1/2–ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

Background/Aims Ankylosing spondylitis (AS) is a chronic inflammatory arthritis leading to long term disability. It is frequently diagnosed late when irreversible damage has already occurred. Unlike most autoimmune rheumatological diseases, there are no known autoantibodies associated with AS; this can confound the diagnostic challenge as diagnosis depends not only on symptoms but on finding evidence of active inflammation at the time of assessment or damage from previously active disease. Our aim was to determine the extent to which autoantibodies may be pathognomonic of AS. Methods A systematic literature review was conducted to identify all articles reporting on autoantibodies in AS. The protocol was preregistered on PROSPERO. Articles were screened and reviewed independently by two reviewers. Disagreements were solved by consensus. Data were extracted and the MINORS (Methodological Index for Non-Randomised Studies) tool was used to assess and compare the quality of the studies. Data were pooled in a narrative synthesis. Results 743 papers were identified after removal of duplicates. Following review of abstracts, 31 full text articles were assessed for eligibility and 18 were excluded, leaving 13 studies to be included in the final systematic review. 12 articles looked at 14 biomarkers; one article looked at 2 high density nucleic acid protein arrays expressing 3,498 proteins. Samples sizes were small and the papers were found to be of modest quality. AS patients showed a wide-ranging autoantibody response across studies. One study found that 60% of autoantibodies detected were found only in the AS cohort versus the rheumatoid arthritis cohort and heathy controls. Antibodies against HLA-B27, pANCA, CD74, OmpC, collagens, PPM1A, noggin, sclerostin, Klebsiella pneumoniae and Breg cells were found more commonly in AS patients than in controls (both healthy controls and those with other autoimmune diseases). Antibodies to human tTG and anti DFS70 were not found to be associated with AS and there was conflicting evidence regarding the association of antibodies to the cell-wall mannan of Saccharomyces cerevisiae (ASCA) in AS from different papers. Conclusion Although many autoantibodies have been found among persons with AS, there is currently no evidence that any are specifically and independently associated with the disease. We therefore conclude that as yet no autoantibodies can be considered to be pathognomonic for AS. However, some autoantibodies may be clinically significant and combined with other biomarkers, such as HLA-B27, may prove fruitful in reducing the delay to diagnosis. Further work should seek to explore other avenues, in particular autoantibodies in closely related diseases, such as inflammatory bowel disease, to try to determine autoantibodies that may aid in the earlier diagnosis of this chronic disease. Disclosure E.J. Davies: None. G.T. Jones: None. R. Sengupta: Honoraria; Received grants, honoraria, and expenses for attendance at advisory board meetings or conferences and for giving lectures from AbbVie, Biogen, Celgene, Lilly, MSD, Novartis, Roche and UCB. Grants/research support; Received grants, honoraria, and expenses for attendance at advisory board meetings or conferences and for giving lectures from AbbVie, Biogen, Celgene, Lilly, MSD, Novartis, Roche and UCB.

Objectives It has been over 10 years since the first report of autoantibodies directed against phenylalanyl tRNA synthetase (anti-Zo) in a patient with features of the anti-synthetase syndrome. In that time no further cases have been published. Here we aim to characterize more fully the clinical phenotype of anti-Zo–associated myositis by describing the clinical features of nine patients. Methods Anti-Zo was identified by protein-immunoprecipitation in patients referred for extended spectrum myositis autoantibody testing at our laboratory. Results were confirmed by immunodepletion using a reference serum. Medical records were retrospectively reviewed to provide detailed information of the associated clinical phenotype for all identified patients. Where possible, HLA genotype was imputed using Illumina protocols. Results Nine patients with anti-Zo were identified. The median age at disease onset was 51 years, and six patients were female. Seven patients had evidence of inflammatory muscle disease, seven of interstitial lung disease and six of arthritis. The reported pattern of interstitial lung disease varied with usual interstitial pneumonia, non-specific interstitial pneumonia and organizing pneumonia all described. Other features of the anti-synthetase syndrome such as RP and mechanics hands were common. HLA data was available for three patients, all of whom had at least one copy of the HLA 8.1 ancestral haplotype. Conclusion Patients with anti-Zo presenting with features of the anti-synthetase syndrome and interstitial lung disease is a common finding. Like other myositis autoantibodies, there is likely to be a genetic association with the HLA 8.1 ancestral haplotype.

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. We have generated a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene. Targeted ObLiGaRe resulted in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse can be utilized for robust and tunable genome editing allowing for flexibility, speed and uniformity at reduced cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

Test compounds used on in vitro model systems are conventionally delivered to cell culture wells as fixed concentration bolus doses; however, this poorly replicates the pharmacokinetic (PK) concentration changes seen in vivo and reduces the predictive value of the data. Herein, proof-of-concept experiments were performed using a novel microfluidic device, the Microformulator, which allows in vivo like PK profiles to be applied to cells cultured in microtiter plates and facilitates the investigation of the impact of PK on biological responses. We demonstrate the utility of the device in its ability to reproduce in vivo PK profiles of different oncology compounds over multiweek experiments, both as monotherapy and drug combinations, comparing the effects on tumour cell efficacy in vitro with efficacy seen in in vivo xenograft models. In the first example, an ERK1/2 inhibitor was tested using fixed bolus dosing and Microformulator-replicated PK profiles, in 2 cell lines with different in vivo sensitivities. The Microformulator-replicated PK profiles were able to discriminate between cell line sensitivities, unlike the conventional fixed bolus dosing. In a second study, murine in vivo PK profiles of multiple Poly(ADP-Ribose) Polymerase 1/2 (PARP) and DNA-dependent protein kinase (DNA-PK) inhibitor combinations were replicated in a FaDu cell line resulting in a reduction in cell growth in vitro with similar rank ordering to the in vivo xenograft model. Additional PK/efficacy insight into theoretical changes to drug exposure profiles was gained by using the Microformulator to expose FaDu cells to the DNA-PK inhibitor for different target coverage levels and periods of time. We demonstrate that the Microformulator enables incorporating PK exposures into cellular assays to improve in vitro–in vivo translation understanding for early therapeutic insight.

The prognosis of metastatic uveal melanoma (UM) is among the worst of all human cancers. The identification of near-ubiquitous GNAQ/GNA11 mutations and the activation of MAPK signaling in UM have raised hopes of more effective, targeted therapies, based on MEK inhibition, for example. We evaluated the potential of drug combinations to increase the efficacy of the MEK inhibitor selumetinib (AZD6244, ARRY-142886), in UM cell lines and Patient-Derived Xenografts. We first evaluated the combination of selumetinib and DTIC. We found that DTIC did not improve the in vitro or in vivo antitumor efficacy of selumetinib, consistent with the outcome of the SUMIT clinical trial assessing the efficacy of this combination in UM. We then tested additional selumetinib combinations with the chemotherapy agent docetaxel, the ERK inhibitor AZ6197, and the mTORC1/2 inhibitor, vistusertib (AZD2014). Combinations of selumetinib with ERK and mTORC1/2 inhibitors appeared to be the most effective in UM PDX models.

There are a number of small-molecule inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway that have either been approved or are in clinical development for oncology across a range of disease indications. The inhibition of ERK1/2 is of significant current interest, as cell lines with acquired resistance to BRAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition in preclinical models. This article reports on our recent work to identify novel, potent, and selective reversible ERK1/2 inhibitors from a low-molecular-weight, modestly active, and highly promiscuous chemical start point, compound 4. To guide and inform the evolution of this series, inhibitor binding mode information from X-ray crystal structures was critical in the rapid exploration of this template to compound 35, which was active when tested in in vivo antitumor efficacy experiments.

The RAS-regulated RAF-MEK1/2-ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in

The RAS/MAPK pathway is a major driver of oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in the BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the clinical responses observed through the use of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors, where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. We describe our structure-based design approach leading to the discovery of AZD0364, a potent and selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency (IC50 = 6 nM) as well as excellent physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties and has demonstrated encouraging antitumor activity in preclinical models.

BRAF and MEK1/2 inhibitors are effective in melanoma but resistance inevitably develops. Despite increasing the abundance of pro-apoptotic BIM and BMF, ERK1/2 pathway inhibition is predominantly cytostatic, reflecting residual pro-survival BCL2 family activity. Here, we show that uniquely low BCL-XL expression in melanoma biases the pro-survival pool towards MCL1. Consequently, BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, driving profound tumour cell death that requires BAK/BAX, BIM and BMF, and inhibiting tumour growth in vivo. Combination of ERK1/2 pathway inhibitors with BCL2/BCL-w/BCL-XL inhibitors is stronger in CRC, correlating with a low MCL1:BCL-XL ratio; indeed the MCL1:BCL-XL ratio is predictive of ERK1/2 pathway inhibitor synergy with MCL1 or BCL2/BCL-w/BCL-XL inhibitors. Finally, AZD5991 delays acquired BRAFi/MEKi resistance and enhances the efficacy of an ERK1/2 inhibitor in a model of acquired BRAFi + MEKi resistance. Thus combining ERK1/2 pathway inhibitors with MCL1 antagonists in melanoma could improve therapeutic index and patient outcomes., BRAF or MEK1/2 inhibitors are cytostatic in melanoma and the surviving cells develop drug resistance. This study shows that the pro-survival pool is biased towards MCL1 in melanoma so that BRAF or MEK1/2 inhibitors are synthetic lethal with the MCL1 inhibitor AZD5991, improving tumour growth inhibition.

Assessing the signalling pathway dependencies of tumours that arise autochthonously in genetically engineered mouse models (GEMMs) of breast cancer is particularly challenging due to the high degree of intra- and inter-tumour heterogeneity, as well as the long latency of tumour development in such models. Use of transplantable tumour lines derived from autochthonous tumours (‘Mouse Derived Xenografts’ or MDXs) is one possible solution and has been used successfully in models of BRCA1-associated triple negative breast cancer. However, their potential in ER+ breast cancer models has not been addressed. Here, we assess the utility of orthotopic transplantable tumour lines derived from an autochthonous ER+Blg-cre Ptenfl/flp53fl/flbreast cancer model. We show that initial tumour implantation and early passage results in the development of lines of progeny with heterogeneous histopathological phenotypes which is coincident with an accumulation of, or selection for,de novomutations. Importantly, these lines also display different dependencies on the key pathways that drive tumourigenesis, which can lead to inherent resistance to treatment with pharmacological agents targeting these pathways and makes them important models to test strategies to overcome such resistance.

For personalized medicine it is crucial that a preclinical model captures the complex tumor biology in vitro in order to individually predict in vivo therapy of tumors. Precision-cut tumor slices maintain tissue heterogeneity with regard to different cell types and preserved native microenvironment. To enable the use of tumor slices as preclinical model that fulfills these criteria we developed a perfusion air culture (PAC) system with continuous and precisely controlled oxygen, medium and drug supply. In the PAC system, precision-cut tumor slices are kept in-between two organotypic supports fixed in a special chamber and placed inside of a 50 mL tube with air exchange capacity housed in a standard CO2-incubator. To evaluate the PAC system, cultured tumor slices from mouse xenografts (MCF7, H1437) and primary human ovarian tumors (phOVT) were compared to in vivo source tissues using immunohistochemistry for morphology, proliferation, DNA damage, apoptosis, and transcriptional biomarkers for cellular stress response. Results show that viability and morphology of the tumor slices are preserved for more than 7 days in the PAC system. We also compared the PAC system with the commonly used static Millipore filter (MF) system which cultures slices on a filter support at an air-liquid interface and gives rise to intra-slice gradients. Both, mouse xenografts and 9 of 15 phOVT tissue slices showed a gradient of cell proliferation and biomarker expression in the MF system while no gradient was detected in slices cultured in the PAC system. Analysis of the culture media revealed lower glucose consumption and lactate production in the PAC system as compared to the MF system indicating more efficient oxygen supply. To analyze therapy response, Cisplatin was applied to phOVT tumor slices for 3 days. Cisplatin treatment was accompanied by minor increase of γ-H2AX in both MF and PAC systems while only in the PAC system strongly enhanced cleavage of caspase-3 was observed, indicating that the PAC system is suitable to assess functional response to drug treatment. To test whether the PAC system is also suitable for the detection of immune response in tumor slices, we analyzed the immune cells of tissue slices before and after cultivation. The patient specific immune cells and their composition is preserved throughout the culture period in the PAC system. In conclusion, cultivation of tumor tissue slices in the PAC system provides an ex vivo model that preserves tumor heterogeneity and native microenvironment. Because the PAC system facilitates homogenous and precisely controlled supply of oxygen, nutrients and drugs, it allows long-term culture of tumor tissue and analysis of therapy response - including immune therapy. We conclude that the newly developed PAC system is suitable to perform patient specific ex vivo tests and thus allows personalized therapy adaption. Citation Format: Meng Dong, Kathrin Böpple, Bernd Winkler, Markus Kleih, Julia Schüler, Emma Davies, Tauno Metsalu, Heike Walles, Hans-Georg Kopp, Frank Essmann, Walter E. Aulitzky. A preclinical model using perfusion air culture of tumor tissue slices for personalized medicine [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5085.

Background: The tumour stroma -or tumour microenvironment- is an important constituent of solid cancers and it is thought to be one of the main obstacles to quantitative translation of drug activity between the preclinical and clinical phases of drug development. The tumour-stroma relationship has been described as being both pro- and antitumour in multiple studies. However, the causality of this complex biological relationship between the tumour and stroma has not yet been explored in a quantitative manner in complex tumour morphologies.Methods: To understand how these stromal and microenvironmental factors contribute to tumour physiology and how oxygen distributes within them, we have developed a lattice-based multiscalar cellular automaton model. This model uses principles of cytokine and oxygen diffusion as well as cell motility and plasticity to describe tumour-stroma landscapes. Furthermore, to calibrate the model, we propose an innovative modelling platform to extract model parameters from multiple in-vitro assays. This platform provides a novel way to extract meta-data that can be used to complement in-vivo studies and can be further applied in other contexts.Results: Here we show the necessity of the tumour-stroma opposing relationship for the model simulations to successfully describe the in-vivo stromal patterns of the human lung cancer cell lines Calu3 and Calu6, as models of clinical and preclinical tumour-stromal topologies. This is especially relevant to drugs that target the tumour microenvironment, such as antiangiogenics, compounds targeting the hedgehog pathway or immune checkpoint inhibitors, and is potentially a key platform to understand the mechanistic drivers for these drugs.Conclusion: The tumour-stroma automaton model presented here enables the interpretation of complex in-vitro data and uses it to parametrise a model for in-vivo tumour-stromal relationships.

Cowden syndrome (CS) is a rare autosomal dominant cancer-prone disorder caused by germ-line mutation of the phosphatase and tensin homolog mutated on chromosome 10 ( PTEN ) tumor-suppressor gene. Affected patients commonly develop juvenile polyps, and show an elevated risk of developing colorectal cancers. The etiology of these peculiar polyps remains unclear, although previous work has suggested somatic PTEN alterations in the stroma of juvenile polyps. After a long latency period, we find epithelial-specific PTEN deletion to cause formation of juvenile polyps in the colorectum without stromal PTEN loss. More important, we find that these lesions closely recapitulate all of the characteristic histopathological features of juvenile polyps seen in patients with CS, including stromal alterations and dysplastic transformation to colorectal carcinoma. The stromal alterations we identify after epithelial-specific PTEN loss suggest that PTEN may be involved in altered epithelial-mesenchymal cross talk, which, in turn, predisposes to colorectal neoplasia and polyposis. Our transgenic model is the first to recapitulate colorectal juvenile polyposis in patients with CS. We conclude that stromal PTEN loss is not a prerequisite for the formation of juvenile polyps, and that colorectal juvenile polyps in CS are bona fide neoplastic precursor lesions.

Carcinomas of the biliary tract are aggressive malignancies in humans. Loss of the tumour suppressor PTEN has previously been associated with cholangiocarcinoma development in a murine model. Activation of KRAS is reported in up to one-third of human cholangiocarcinomas and 50% of gall bladder carcinomas. In this study we aimed to test the potential interaction between PTEN and KRAS mutation in biliary tract malignancy. We used an inducible Cre–LoxP-based approach to coordinately delete PTEN and activate KRAS within the adult mouse biliary epithelium. We found that activation of KRAS alone has little effect upon biliary epithelium. Loss of PTEN alone results in the development of low-grade neoplastic lesions, following long latency and at low incidence. Combination of both mutations causes rapid development of biliary epithelial proliferative lesions, which progress through dysplasia to invasive carcinoma. We conclude that activation of the PI3′K pathway following loss of PTEN is sufficient to drive slow development of low-grade biliary lesions in mice. In contrast, mutational activation of KRAS does not result in a similar phenotype, despite a prediction that this should activate both the RAF–MEK–ERK and PI3′-kinase pathways. However, mutation of both genes results in rapid tumourigenesis, arguing that PTEN normally functions as a ‘brake’ on the PI3′-kinase pathway, limiting the influence of KRAS activation. Mutation of both genes creates a ‘permissive’ environment, allowing the full effects of both mutations to be manifested. These data reveal an in vivo synergy between these mutations and provides a new mouse model of biliary tract malignancy

Two-dimensional (2D) culture of cancer cells in vitro does not recapitulate the three-dimensional (3D) architecture, heterogeneity and complexity of human tumors. More representative models are required that better reflect key aspects of tumor biology. These are essential studies of cancer biology and immunology as well as for target validation and drug discovery. The Innovative Medicines Initiative (IMI) consortium PREDECT (www.predect.eu) characterized in vitro models of three solid tumor types with the goal to capture elements of tumor complexity and heterogeneity. 2D culture and 3D mono- and stromal co-cultures of increasing complexity, and precision-cut tumor slice models were established. Robust protocols for the generation of these platforms are described. Tissue microarrays were prepared from all the models, permitting immunohistochemical analysis of individual cells, capturing heterogeneity. 3D cultures were also characterized using image analysis. Detailed step-by-step protocols, exemplary datasets from the 2D, 3D, and slice models, and refined analytical methods were established and are presented.

Colorectal cancer is one of the most common cancers in the western world and its incidence is steadily increasing. Understanding the basic biology of both the normal intestine and of intestinal tumorigenesis is vital for developing appropriate and effective cancer therapies. However, relatively little is known about the normal intestinal stem cell or the hypothetical intestinal cancer stem cell, and there is much debate surrounding these areas. This review briefly describes our current understanding of the properties of both the intestinal stem cell and the intestinal cancer stem cell. We also discuss recent theories regarding the origin of the intestinal cancer stem cell, and the signals required for its maintenance and proliferation. Finally, we place the relevance of cancer stem cell research into context by discussing potential clinical applications of targeting the intestinal cancer stem cell.

ERK1/2 is a key protein in the MAPK pathway, regulating phenotypes such as proliferation and migration. Upstream mutations (e.g., KRAS mutations in non-small-cell lung (NSCLC)) can cause the pathway to become constitutively activated, driving tumor growth. AZD0364 is a potent, selective inhibitor of ERK's kinase activity against its cytosolic substrate p90RSK. It is currently in preclinical development, where it has shown dose-dependent, anti-tumor activity in xenograft models of KRAS-mutant NSCLC, including Calu-6 (where it shows regression) and A549. Treatment with AZD0364 demonstrates rapid and near-complete inhibition of phospho-p90RSK. In addition, prolonged inhibition with AZD0364 causes a gradual downregulation of p90RSK protein over time, without any corresponding change in p90RSK mRNA. Here we present a pharmacokinetic/pharmacodynamic (PK/PD) model that links AZD0364 concentration to inhibition of ERK activity through both a direct inhibition of phospho-p90RSK and an indirect down-regulation of total-p90RSK protein. Anti-proliferative and pro-apoptotic effects on efficacy are linked to changes in p90RSK. The model leads to two key implications (i) repeated dosing will cause apparent potency to improve over time, since the pool of available substrate (i.e., p90RSK) is itself being reduced and (ii) recovery of signaling to baseline will depend not on washout of the inhibitor but on protein synthesis rates. Protein half-lives appear quite different between tumor models of KRAS-mutant NSCLC, with A549 (~20h) significantly slower than Calu-6 (~4h). The model provides a conceptual framework on which to link the timescale of PD changes with those seen in efficacy. Taken together, this means that while a new PD steady-state is achieved in Calu-6 in a few days, it also recovers quickly, necessitating constant cover (daily dosing) to drive regression. On the other hand, while A549 is more robust to inhibition, and slower to reach steady-state inhibition (~2 weeks), it is also slower to recover, so that intermittent schedules can achieve efficacy similar to those achievable with daily dosing. Citation Format: Francis D. Gibbons, Linda Sandin, Lyndsey Hanson, Rebecca Whiteley, Paul Farrington, Nicola Lindsay, Emma Davies, J Elizabeth Pease, Vikki Flemington. A PK/PD model quantitatively describes inhibition and down-regulation of p90RSK by ERK inhibitor AZD0364 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4913.

Purpose: The prognosis of metastatic uveal melanoma (UM) patients remains one of the worst observed in human cancers. The identification of frequently mutated GNAQ/GNA11 and the activation of MAPK signaling in UM raise the possibility of using efficient targeted therapies such as MEK inhibition. The aim of our study was to evaluate drug combinations that might enhance the efficacy of the MEK inhibitor, selumetinib (AZD6244, ARRY-142886), using patient-derived xenografts. Materials: Five UM PDXs were used in this study, i.e., MP34, MP46, MP55, MP77, and MM26, all mutated for GNAQ or GNA11. Selumetinib was administered alone or combined with chemotherapies (dacarbazine and docetaxel) or targeted therapies: the ERK inhibitor, AZ6197 and the mTORC1/2 inhibitor, vistusertib (AZD2014). All targeted therapies were administered orally five days/week at the following doses: selumetinib at 25 mg/kg/day BID, AZ6197 at 50 mg/kg QD, AZD2014 at 15mg/kg QD. Dacarbazine was administered at 40 mg/kg for 5 consecutive days every 28 days and docetaxel at 15 mg/kg weekly. Both were administered intraperitoneally. Results: The combination of selumetinib and dacarbazine did not show improvement of antitumor efficacy in MP34, MM26 and MP55.The results showed a slight increase of overall response rate when selumetinib was combined with docetaxel, in comparison to the treatment with selumetinib or docetaxel alone. Importantly, we observed a significant antitumor effect when selumetinib was used in combination with AZ6197 or AZD2014 with respect to the monotherapies. Preliminary pharmacodynamics studies showed variation of the expression of p-ERK and also p-AKT and/or p-S6 when UM PDX were treated with AZ6197 or AZD2014 alone or in combination with selumetinib. Conclusion: Our data suggest that ERK and mTORC1/2 targeting could represent promising therapeutic approaches for the treatment of metastatic UM patients. Citation Format: Didier Decaudin, Rania EL Botty, Béré Diallo, Gerald Massonnet, Justine Fleury, Adnan Naguez, Marine Dubois, Emma J. Davies, Aaron Smith, Joanne Wilson, Colin Howes, Paul Smith, Nathalie Cassoux, Pascale Mariani, Sophie Piperno-Neumann, Sergio Roman Roman, Fariba Nemati. Selumetinib-based therapy in uveal melanoma patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5782.

The RAS/MAPK pathway is a major driver in oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as key central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the initial clinical responses observed to BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma, however resistance frequently develops by reactivation of the pathway. Direct targeting of ERK1/2, may provide another therapeutic option in tumours with mutations in BRAF or RAS genes. Importantly, ERK1/2 inhibition may have clinical utility in overcoming acquired resistance to RAF and MEK inhibitors where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. Starting from our published work,1 we will describe for the first time, a scaffold hopping approach leading to the identification of AZD0364, a pre-clinical ERK1/2 inhibitor candidate drug. Driven by conformational modelling and structure-based design, and by utilising novel sulfamidate ring opening chemistry, a high lipophilicity efficiency core was identified. Structure based, multi-parameter based optimisation of this improved core ultimately led to AZD0364. AZD0364 exhibits high cellular potency against a direct downstream substrate on the MAPK pathway (e.g. inhibition of phospho-p90RSK1 in BRAFV600E mutant A375 cells, IC50 = 6 nM). The molecule is a highly selective kinase inhibitor (10/329 kinases tested are inhibited at >50% at a 1 µM) and has long residence time on the protein (as determined by SPR on human unphosphorylated-ERK2: pKd = 10; t1/2 = 277 mins). The good in vitro potency and selectivity is complemented by excellent physico-chemical properties (maximum absorbable dose estimated to be >4 g) and good oral pharmacokinetics across species, leading to a low predicted dose to man. In xenograft models, AZD0364 inhibits phospho-p90RSK1 in tumors in a dose-dependent manner. AZD0364 induces regressions in the KRAS mutant NSCLC Calu 6 xenograft model. AZD0364 can also be combined safely and effectively with the MEK1/2 inhibitor selumetinib in KRAS mutant NSCLC xenograft models. 1Richard A. Ward et. al. Structure-Guided Discovery of Potent and Selective Inhibitors of ERK1/2 from a Modestly Active and Promiscuous Chemical Start Point, J. Med. Chem. 2017, 60, 3438−3450. Citation Format: Iain Simpson, Mark J. Anderton, David M. Andrews, Jason Breed, Emma Davies, Judit E. Debreczeni, Vikki Flemington, Francis D. Gibbons, Mark A. Graham, Philip Hopcroft, Tina Howard, Julian Hudson, Clifford D. Jones, Christopher Jones, Nicola Lindsay, J Elizabeth Pease, Philip Rawlins, Karen Roberts, Steve Swallow, Steve St-Gallay, Michael E. Tonge, Richard A. Ward. Discovery of AZD0364, a potent and selective oral inhibitor of ERK1/2 that is efficacious in both monotherapy and combination therapy in models of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1647.

The RAS/MAPK pathway is a major driver in oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in BRAF or RAS genes. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, single-agent pathway inhibitors have demonstrated limited clinical benefit. Therefore, combined inhibition of multiple nodes within the RAS/MAPK pathway may be necessary to effectively suppress pathway signaling and achieve meaningful clinical benefit, specifically in patients with KRAS mutant tumors. AZD0364 is a potent and highly selective inhibitor of ERK1 and ERK2. AZD0364 exhibits high cellular potency against a direct substrate (e.g., inhibition of phosphorylation of p90RSK in BRAF mutant A375 cells, IC50 = 6 nM) and is highly (10/329 kinases tested are inhibited at >50% at 1 µM). In an unbiased cell proliferation screen of 750 tumor cell lines, >50% of cell lines that are sensitive to AZD0364 have RAS/MAPK pathway genetic alterations such as BRAF, NRAS or KRAS mutations. In a subset of KRAS mutant NSCLC cell lines, combined treatment of AZD0364 and selumetinib (AZD6244, ARRY-142886) is highly synergistic. This combination results in deeper and more durable suppression of the RAS/MAPK pathway that is not achievable with single-agent treatment, as assessed by phospho-p90RSK, change in transcriptional signatures and induction of apoptotic biomarkers. The AZD0364 and selumetinib combination also significantly suppresses RAS/MAPK pathway output and tumor growth in vivo to a greater extent than achievable with either agent given as a monotherapy. This combination is well tolerated in vivo and delivers 65% tumor regression in the NCI H358 KRAS mutant NSCLC xenograft model. This combination also results in significant tumor regressions in both A549 and HCT116 KRAS mutant xenografts. These data demonstrate that combined AZD0364 and selumetinib is well tolerated, effectively suppresses RAS/MAPK pathway signalling and delivers durable regressions in preclinical models. The combination of ERK and MEK inhibition represents a viable clinical approach to target KRAS mutant tumors. Citation Format: Vikki Flemington, Iain Simpson, Jason Breed, Emma Davies, Francis Gibbons, Phillip Hopcroft, Nicola Lyndsay, Christopher Jones, Clifford Jones, David Robinson, Claire Rooney, Karen Roberts, Linda Sandin, Richard Ward, Pei Zhang, Elizabeth Pease. Combination of the novel ERK inhibitor AZD0364 with the MEK inhibitor selumetinib significantly enhances antitumor activity in KRAS mutant tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1856.

Precision-cut slices of in vivo tumours permit interrogation in vitro of heterogeneous cells from solid tumours together with their native microenvironment. They offer a low throughput but high content in vitro experimental platform. Using mouse models as surrogates for three common human solid tumours, we describe a standardised workflow for systematic comparison of tumour slice cultivation methods and a tissue microarray-based method to archive them. Cultivated slices were compared to their in vivo source tissue using immunohistochemical and transcriptional biomarkers, particularly of cellular stress. Mechanical slicing induced minimal stress. Cultivation of tumour slices required organotypic support materials and atmospheric oxygen for maintenance of integrity and was associated with significant temporal and loco-regional changes in protein expression, for example HIF-1α. We recommend adherence to the robust workflow described, with recognition of temporal-spatial changes in protein expression before interrogation of tumour slices by pharmacological or other means.

The RAS/MAPK pathway is a major driver in oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as key central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the initial positive clinical responses observed to BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops by reactivation of the RAS/MAPK pathway. Direct targeting of ERK1/2 may provide another therapeutic option in tumors with mutations in BRAF or RAS genes and, importantly, may overcome acquired resistance to RAF and MEK inhibitors where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. Here we describe the discovery and characterization of compound 35 (Ward et al., J Med Chem 2017 27;60), also known as AZ6197. AZ6197 is a highly potent and selective inhibitor of ERK1 and ERK2, with IC50 of Citation Format: Vikki Flemington, Iain Simpson, Emma Davies, David Robinson, Nicola Lindsay, Lyndsey Hanson, Philip Hopcroft, Michael Tonge, Karen Roberts, Richard Ward. Discovery and characterization of AZ6197, a potent and selective ERK1/2 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B156.

A type VI acromioclavicular joint injury with a supracoracoid location of the distal end of the clavicle (VIa) may be associated with low energy injuries and, in association with a clavicle fracture, can successfully be treated with reduction of the dislocation, fixation of the clavicle, and may not require reconstruction of the acromioclavicular ligaments. An infracoracoid location (VIb) is highly suggestive of a higher energy injury.

Mutation or loss of the genes PTEN and KRAS have been implicated in human colorectal cancer (CRC), and have been shown to co-occur despite both playing a role in the PI3' kinase (PI3'K) pathway. We investigated the role of these genes in intestinal tumour progression in vivo, using genetically engineered mouse models, with the aim of generating more representative models of human CRC. Intestinal-specific deletion of Pten and activation of an oncogenic allele of Kras was induced in wild-type (WT) mice and mice with a predisposition to adenoma development (Apc(fl/+) ). The animals were euthanized when they became symptomatic of a high tumour burden. Histopathological examination of the tissues was carried out, and immunohistochemistry used to characterize signalling pathway activation. Mutation of Pten and Kras resulted in a significant life-span reduction of mice predisposed to adenomas. Invasive adenocarcinoma was observed in these animals, with evidence of activation of the PI3'K pathway but no metastasis. However, mutation of Pten and Kras in WT animals not predisposed to adenomas led to perturbed homeostasis of the intestinal epithelium and the development of hyperplastic polyps, dysplastic sessile serrated adenomas and metastasizing adenocarcinomas with serrated features. These studies demonstrate synergism between Pten and Kras mutations in intestinal tumour progression, in an autochthonous and immunocompetent murine model, with potential application to preclinical drug testing. In particular, they show that Pten and Kras mutations alone predispose mice to the spectrum of serrated lesions that reflect the serrated pathway of CRC progression in humans.

Uveal melanomas (UM) constitute the most common primary intraocular tumors in adults and are characterized by a constitutive activation of the MAPK pathway due to mutations of the GTPase genes GNAQ or GNA11 in almost 80% of cases. The most commonly used treatments for UM are alkylating agents such as dacarbazine (DTIC) and temozolomide (TMZ). The MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) has shown clinical activity compared to DTIC/TMZ in a recent Phase II clinical trial and has recently completed a Phase III clinical trial in combination with DTIC (NCT01974752). In parallel with this trial we sought to evaluate the efficacy of DTIC + selumetinib in UM patient-derived xenografts (PDXs). Three models were included in the study (MP34, MP55, and MM26), all bearing a GNAQ or GNA11 mutation. Selumetinib was administered orally at 25 mg/kg/day, 5 days a week, and DTIC at a dose of 40 mg/kg/day on Days 1 to 5 every 4 weeks. A significant tumor growth inhibition (TGI) of 54% was observed in the MP34 model but not in the two remaining PDXs. In one model, MM26, DTIC induced a strong TGI of about 99% with 6/9 complete remissions (CRs). The combination of selumetinib + DTIC did not significantly increase efficacy compared to monotherapy in any of the models; in the MM26 PDX, the combination induced a similar TGI (99%) and CR rate (5/9) as DTIC alone. In this experiment, after two courses of DTIC + selumetinib, selumetinib was continued alone, showing a significant increased growth delay (p In conclusion, we have observed that response of UM PDX models to DTIC was not increased when combined with selumetinib; these results are similar to those seen in the Phase III study of this drug combination. The observation that MEK inhibition was effective in delaying progression in the DTIC-sensitive PDX and published clinical studies demonstrating MEK inhibitor monotherapy activity indicate that MEK inhibition may have value as a treatment for UM; perhaps in the adjuvant setting in two specific clinical situations, i.e. patients with irradiated or enucleated high-risk primary intraocular or surgically resected metastatic UM. Citation Format: Béré Diallo, Gerald Massonnet, Rania El-Botty, Chloé Raymondie, Guillaume Carita, Sergio Roman-Roman, Paul Smith, Emma Davies, Didier Decaudin, Fariba Némati. The MEK1/2 inhibitor selumetinib (AZD6244; ARRY-142886) appears as an efficient targeted therapy when used in an adjuvant setting in patient-derived xenografts of uveal melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2087.