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A) Structure of JNJ-42541707, a structurally related compound to JNJ-42756493. B) 72h growth inhibition (IC50) of JNJ-42541707 against 236 cancer cell lines from multiple origins color coded based on FGFR1,2,4 mRNA overexpression and FGFR WT.

In vitro kinase inhibition by DiscoverX KinomeScan assay. For details see (19)

Inhibitory activity of Brivanib and JNJ-42756943 in kinase (top) and BaF3 kinase dependent proliferation (bottom) assays and ratio of FGFRs/VEGFR2 activities.

JNJ-42756493 anti-proliferative activity against cancer cells lines from multiple origins.. Detailed data supporting Figure 2.

Multitargeted kinase inhibitors have shown clinical efficacy in a range of cancer types. However, two major problems associated with these drugs are the low fraction of patients for which these treatments provide initial clinical benefit and the occurrence of resistance during prolonged therapy. Several types of predictive biomarkers have been suggested, such as expression level and phosphorylation status of the major targeted kinase(s), mutational status of the kinases involved and of key components of the downstream signaling cascades, and gene expression signatures. In this work, we describe the development of a response prediction platform that does not require prior knowledge of the relevant kinases targeted by the inhibitor; instead, a phosphotyrosine peptide profile using peptide arrays with a kinetic readout is derived in lysates in the presence and absence of a kinase inhibitor. We show in a range of cell lines and in xenograft tumors that this approach allows for the stratification of responders and nonresponders to a multitargeted kinase inhibitor. [Mol Cancer Ther 2009;8(7):1846–55]

Purpose: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a second-generation class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy.Experimental Design: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors.Results:In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC50, 0.16 nmol/L).Conclusions: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising second-generation HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies. (Clin Cancer Res 2009;15(22):684151)

Supplementary Figure 1 from Response prediction to a multitargeted kinase inhibitor in cancer cell lines and xenograft tumors using high-content tyrosine peptide arrays with a kinetic readout

Supplementary Data from JNJ-26481585, a Novel Second-Generation Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity

Supplementary Figure 2 from Response prediction to a multitargeted kinase inhibitor in cancer cell lines and xenograft tumors using high-content tyrosine peptide arrays with a kinetic readout

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010–20. ©2017 AACR.

Background Traditional Map based Cloning approaches, used for the identification of desirable alleles, are extremely labour intensive and years can elapse between the mutagenesis and the detection of the polymorphism. High throughput sequencing based Mapping-by-sequencing approach requires an ordered genome assembly and cannot be used with fragmented, un-scaffolded draft genomes, limiting its application to model species and precluding many important organisms. Results We addressed this gap in resource and presented a computational method and software implementations called CHERIPIC (Computing Homozygosity Enriched Regions In genomes to Prioritise Identification of Candidate variants). We have successfully validated implementation of CHERIPIC using three different types of bulk segregant sequence data from Arabidopsis, maize and barley, respectively. Conclusions CHERIPIC allows users to rapidly analyse bulk segregant sequence data and we have made it available as a pre-packaged binary with all dependencies for Linux and MacOS and as Galaxy tool. Electronic supplementary material The online version of this article (10.1186/s12859-018-2515-5) contains supplementary material, which is available to authorized users.

To cope with the ever-increasing amount of sequence data generated in the field of genomics, the demand for efficient and fast database searches that drive functional and structural annotation in both large- and small-scale genome projects is on the rise. The tools of the BLAST+ suite are the most widely employed bioinformatic method for these database searches. Recent trends in bioinformatics application development show an increasing number of JavaScript apps that are based on modern frameworks such as Node.js. Until now, there is no way of using database searches with the BLAST+ suite from a Node.js codebase. We developed blastjs, a Node.js library that wraps the search tools of the BLAST+ suite and thus allows to easily add significant functionality to any Node.js-based application. blastjs is a library that allows the incorporation of BLAST+ functionality into bioinformatics applications based on JavaScript and Node.js. The library was designed to be as user-friendly as possible and therefore requires only a minimal amount of code in the client application. The library is freely available under the MIT license at https://github.com/teammaclean/blastjs .

The Japanese Paeonia japonica (Makino) Miyabe & Takeda (Paeoniaceae) is illustrated. Its taxonomy, distribution, and cultivation requirements are discussed and a full description of the species is provided.

The Turkish peony, Paeonia turcica Davis & Cullen (Paeoniaceae) is illustrated; its botanical history, medicinal and ethnobotanical uses, relationships, distribution and cultural requirements are discussed and a full description of the species is provided.

Sarcoidosis is a rare genetic autoimmune disease that can attack any organ and tissue type in the body. Although it has been recognized for over 100 years the aetiology remains unknown. The group most at risk is black females under the age of 40, but no group is immune to it. The disease is commonly asymptomatic at diagnosis and often resolves without medication; however, where it does manifest it can become chronic and can be fatal. Corticosteroids are the preferred treatment although there are alternative approaches. The disease can develop in different systems simultaneously, and sarcoidosis of the lungs, skin, eyes, heart, the central nervous and musculoskeletal systems are explored in this article. The practicalities of nursing are discussed, and the work of the nurse Imogene King (1981) is shown as a useful strategy to address chronic sarcoidosis.

The results of biomechanical testing of the WorldSID prototype dummy are presented in this paper. The WorldSID dummy is a new, advanced Worldwide Side Impact Dummy that has the anthropometry of a mid-sized adult male. The first prototype of this dummy has been evaluated by the WorldSID Task Group against previously established corridors for its critical body regions. The response corridors are defined in the International Organization of Standardization (ISO) Technical Report 9790. The prototype is the first version of the WorldSID dummy to be built and tested. This dummy has been subjected to a rigorous program of testing to evaluate, first and foremost its biofidelity, but also its repeatability. Following this initial evaluation, any required modifications will be incorporated into a pre-production version of the WorldSID dummy so that it rates "good" to "excellent" on the ISO dummy biofidelity scale - a rating exceeding that of all current side impact dummies. Also, the overall WorldSID repeatability must not exceed a coefficient of variation of 7% at injury assessment level and this has to be verified for the different body regions. The dummy's head, neck, thorax, abdomen and pelvis were evaluated against the ISO technical report requirements. Testing included drop tests, pendulum impacts, and sled tests. The biofidelity rating of the WorldSID prototype was calculated using the weighted biomechanical test response procedure developed by ISO. The paper presents the results of the testing, which give a very positive indication of the dummy's potential. Based on this evaluation of the dummy biofidelity, the WorldSID prototype dummy exhibits a biofidelity rating of 6.15 that corresponds to an ISO biofidelity classification of "fair". In addition, the dummy shows good repeatability with a global coefficient of variation of 3.30% for the pendulum and rigid sled tests.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients undergo primary, adjuvant or neoadjuvant radiotherapy treatment with image-guided radiotherapy (IGRT) being widely used to provide more accurate treatment plans and reduced side effects. Patients who have activating mutations in the epidermal growth factor receptor (EGFR) gene are treated with EGFR tyrosine kinase inhibitors (EGFRi). However, resistance to EGFRi occurs in the majority of patients with the main clinical drivers being secondary gatekeeper mutations (T790M) or amplification of genes such as c-MET and Her2. The small animal radiation research platform (SARRP), which allows the treatment of rodent models of cancer more accurately with planned protocols similar to those utilised in the clinic, was used in combination with EGFRi, to assess the efficacy of combination treatment strategies of irradiation (IR) in EGFRi resistant NSCLC xenografts. METHODS: Resistance to EGFRi in the NSCLC cell line HCC827, which harbours an activating EGFR mutation, was generated through repeated exposure to Gefitinib or Erlotinib. Resistant models were characterised for further mutations in the EGFR gene by direct sequencing and for c-MET, Axl and Her2 over-expression/genomic amplification by quantitative PCR. EGFRi resistant or parental HCC827 cells were implanted subcutaneously into nude mice, xenograft growth was monitored by calliper measurements and treatment initiated when the mean tumour volume reached ∼200mm3. An image guided IR dose of 3Gy/mouse/day (30Gy total) was targeted directly to the xenografts whilst sparing the surrounding normal tissue. Mice were treated with IR either alone or in combination with Erlotinib or Crizotinib; response to treatment was evaluated by tumour volume measurement. RESULTS: EGFR-TKI resistant HCC827 cell lines were generated following successive cycles of treatment. Amplification of c-MET was identified in a number of clones, but no Her2 amplification or gatekeeper mutations; therefore Crizotinib, a c-MET inhibitor, was selected for evaluation in these models. When assessed in vivo, treatment of parental HCC827 xenografts with targeted IR or Erlotinib treatment resulted in tumour regression. However, in the c-MET driven resistant model, both the IR and Erlotinib response was significantly attenuated, resulting in tumour re-growth. Combination testing with Crizotinib restored the efficacy of both IR and Erlotinib to naïve treatment levels confirming the role of c-MET in resistance. CONCLUSIONS: Resistance to EGFRi and IR in a HCC827 model is driven by c-MET amplification. Treatment with Crizotinib restored sensitivity to both EGFRi and IR demonstrating that pre-clinical models of resistance are invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent/overcome resistance to EGFRi. Citation Format: Andrew McKenzie, Nektaria Papadopoulou, Lucy Page, Simon Jiang, Jane Wrigley, Yinfei Yin, Qian Shi, Martin Page, Ian Wilson, Rajendra Kumari. Overcoming acquired EGFRi resistance in NSCLC with targeted beam irradiation in combination with targeted agents. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 765. doi:10.1158/1538-7445.AM2015-765

Introduction: PIM kinases play an important role in a number of aspects of the regulation of cell cycle and proliferation. Over expression of PIM kinases is associated with oncogenicity in a number of haematological and solid tumours. Recent evidencesuggests that PIM expression is upregulated by inhibition of signalling on the AKT/PI3K/mTOR pathway. We set out to examine the effects of inhibition of PIM kinases in a number of solid tumor cell lines and to explore the effects of concomitant inhibition of the AKT/PI3K/mTOR pathway with novel agents having dual PIM kinase and PI3k inhibition. Representative cell lines for colorectal, lung, oesophageal and prostate tumors (HCT116, A549 and H460, OE21, PC3 respectively) were chosen for testing in a cytotoxicity assay. Compounds tested were GDC-0941, a prototypical PI3K inhibitor, AZD 1208, a pan-PIM inhibitor (i.e. equi-active on all 3 PIM isoforms), IBL- 201 and IBL-202 (dual-acting PI3K/PIM inhibitors) and IBL-301 and IBL-302 (triple-acting PI3K/PIM/mTOR inhibitors). Rapamycin the prototypical mTOR inhibitor and cisplatin were also tested as reference compounds. Results: The pan-PIM compound AZD1208 showed efficacy in all cell lines in the micromolar range. Rapamycin was also active across similar concentrations. GDC-0941, the PI3K inhibitor exhibited moderate efficacy across the cell line panel . The dual and triple inhibitors showed cytotoxic effects at sub micromolar concentrations, with the OE21 and PC3 oesophageal and prostate lines respectively showing the most sensitive responses. Conclusion: These results suggest that in selected solid tumor cell lines combined inhibition of PIM and PI3 kinases can lead to an increased cytotoxic effect. Further studies are under way to determine if this observation is corroborated in vivo. Combined inhibition of PIM and PI3 kinases with a single molecule may offer significant opportunities in the clinic for efficacy in key tumour subsets and to circumvent acquired resistance via feedback mechanisms. Citation Format: Michael O'Neil, C. Blanco Aparicio, S. Jiang, S. Martinez, A. McKenzie, Martin Page, J. Pastor. Combined inhibition of PIM and PI3 kinases shows an enhanced efficacy in a number of solid tumour cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4524. doi:10.1158/1538-7445.AM2014-4524

Fibroblast growth factors (FGFs) and their receptors (FGFR1 through 4) regulate a variety of key cellular processes, including proliferation, migration, survival, and differentiationa. Aberrant activation of FGF/FGFR is strongly implicated in oncogenic signalling in many tumor types. This has stimulated the development of a number of FGFR inhibitors, with diverse kinase inhibition and pharmacological profiles that are currently being evaluated in clinical studies. We conducted a fragment screening campaign and this resulted in identification of a 6-aminoquinoxalinyl fragment with a binding affinity in the micromolar range. Structure-guided medicinal chemistry led to the identification of a novel quinoxaline-based chemical series with nanomolar affinity for FGFR1, 2, 3, and 4, activity in cells, and selectivity with respect to VEGFR-2. Further optimisation resulted in the generation of JNJ-42756493, a compound with favourable drug-like properties that demonstrated strong anti-tumoral activity in a FGFR2-dependent SNU-16 human gastric carcinoma xenograft model. This report represents the first disclosure of the structure-activity relationships as well as the chemical synthesis pathway of the JNJ-42756493 series and illustrates how a fragment-based drug discovery approach has been efficiently used to discover FGFR1-4 inhibitors with nanomolar affinity. aTurner, N. and Grose, R. Nat. Rev. Cancer, 2010, 10, 116-129. Citation Format: Patrick R. Angibaud, Laurence Mevellec, Gordon Saxty, Christophe Adelinet, Rhalid Akkari, Valerio Berdini, Pascal Bonnet, Marine Bourgeois, Xavier Bourdrez, Anne Cleasby, Helene Colombel, Imre Csoka, Werner Embrechts, Eddy Freyne, Ronaldus Gilissen, Eleonora Jovcheva, Peter King, Jean Lacrampe, Delphine Lardeau, Yannick Ligny, Steve Mcclue, Lieven Meerpoel, David R. Newell, Martin Page, Alexandra Papanikos, Elisabeth Pasquier, Isabelle Pilatte, Virginie Poncelet, Olivier Querolle, David C. Rees, Sharna Rich, Bruno Roux, Elodie Sement, Yvan Simonnet, Matthew Squires, Virginie Tronel, Tinne Verhulst, Jorge Vialard, Marc Willems, Steven J. Woodhead, Berthold Wroblowski, Christopher W. Murray, Timothy Perera. Discovery of JNJ-42756493, a potent fibroblast growth factor receptor (FGFR) inhibitor using a fragment based approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2014-4748

Previously we have described several types of charge division electronic image readouts for microchannel plate based imaging detectors developed at MSSL, primarily for space astronomy applications. These have included the wedge and strip anode1 (WSA), the Vernier anode2 - a high resolution readout, capable of exploiting the limiting spatial resolution offered by the microchannel plate, and FIRE3 - an imaging device operating at event rates in excess of 10 MHz. MSSL and Photek have now joined in collaboration to develop an intensifier based imaging system designed to employ this range of readout systems for general laboratory use. The image intensifier uses the image charge technique4,5 whereby the event charge is used to induce electrical signals on the capacitively coupled readout pattern, obviating the requirement for the readout to be inside the vacuum enclosure. The image readout is manufactured as a separate component, and can be interchanged to suit the specific application requirements. The intensifier tube design can be generic enabling it to be used with a variety if image readouts designs. We describe the image intensifier and electronic design, including the common charge amplifier, event timing and computer interface. We discuss the anticipated performance of the various readout systems - Wedge and Strip, Vernier and FIRE in terms of spatial resolution, maximum count rate, and timing resolution.

Introduction: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, carries a poor prognosis and remains an area of high unmet need. Despite recent advances in treating defined subsets of NSCLC adenocarcinomas with targeted agents such as erlotinib and crizotinib few advances have been made in the treatment of squamous cell carcinoma (SCC), which accounts for 25% of all NSCLC. Although the incidence of fibroblast growth factor receptor (FGFR) mutations in SCC is modest, amplification of FGFR is well documented and there are several first generation FGFR tyrosine kinase inhibitors (TKI) currently in clinical trials. It is therefore essential to develop relevant in vivo patient-derived xenograft (PDX) models for the development and characterisation of new FGFR agents and/or combination strategies which may prolong benefit and delay the emergence of resistance. Methods: LU6429 is a novel Caucasian NSCLC SCC PDX model which is maintained subcutaneously in vivo admixed with a human stromal cell component. Cohorts of mice were dosed with AZD4547, which is an FGFR TKI undergoing evaluation in the clinic, alone or in combination with chemotherapy (CTx; parallel or serial dosing regimens). The extent of inhibition was measured over several in vivo passages and following cryopreservation/resuscitation. CTx and FGFR TKI resistant models of LU6429 were generated in vivo through repeated dosing and the resulting models characterized for their treatment response. Tumor material was characterized for 50 key oncogenes and mutations by Ion Torrent sequencing, for FGFR1-3 expression and genomic amplification by quantitative PCR and FGFR1-2 by FISH analysis. Immunohistochemistry (IHC) was used to confirm NSCLC subtype and EMT status. Results: LU6429 PDX tumours (SCC confirmed by IHC) exhibited reproducible sensitivity to AZD4547 alone (P Conclusions: LU6429 is a novel patient-derived NSCLC SCC xenograft model with reproducible FGFR TKI sensitivity, which is directly relevant to the clinical setting. This model could be invaluable in assessing novel agents targeting the FGFR pathway and in the development of new combination strategies which may prevent or overcome resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A142. Citation Format: Andrew J. McKenzie, Aaron N. Cranston, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Yinfei Yin, Henry Li, Martin Page, Rajendra Kumari. In vivo response and molecular characterization of a Caucasian NSCLC squamous cell carcinoma PDX sensitive to FGFR inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A142.

Introduction: Most non-small cell lung cancer (NSCLC) patients that have activating mutations in the EGFR gene will respond to treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) e.g. erlotinib and gefitinib. However, in about 50% of these cases a secondary mutation in EGFR (T790M) subsequently occurs, which results in resistance to treatment. Other mechanisms of clinical resistance can also occur such as amplification of c-MET kinase, HER2 and epithelial-mesenchymal transition (EMT) conversion; however, additional routes of resistance are poorly defined. Using a novel Caucasian NSCLC patient-derived xenograft (PDX) model and a cell line derived model, driven by the L858R EGFR mutation, we set out to recapitulate the reported clinical routes of resistance to EGFR inhibitors and to evaluate if additional mechanism could also be identified. Methods: LU6422 is a Caucasian NSCLC adenocarcinoma PDX model with an activating EGFR mutation (L858R) which is maintained subcutaneously in vivo admixed with a human stromal cell component. HCC827 is NSCLC adenocarcinoma cell line with an activating EGFR mutation (del E746-A750). Resistant models of LU6422 and HCC827 were generated in vivo and in vitro (respectively) through repeated dosing or exposure to EGFR TKIs. Resistant tumour material was characterised for further mutations in the EGFR gene by direct sequencing as well as for c-MET, AXL and HER2 over-expression and genomic amplification by quantitative PCR. Results: Naïve LU6422 and HCC827 tissue exhibited exquisite sensitivity to EGFR TKIs (100% & 30% reduction respectively in pre-treatment tumor volume, p Conclusions: EGFR TKI resistant subtypes were generated in vivo and in vitro from a proprietary Caucasian NSCLC PDX model (LU6422) and the HCC827 NSCLC cell line and were characterized for their resistance mechanisms. Models of resistance will be invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent or overcome resistance to EGFR TKIs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A145. Citation Format: Andrew J. McKenzie, Aaron N. Cranston, Nektaria Papadopoulou, Simon Jiang, Jane Wrigley, Yinfei Yin, Henry Li, Martin Page, Rajendra Kumari. In vivo and in vitro generation and characterization of EGFR TKI resistance in a patient-derived and a cell line-derived xenograft model of NSCLC with activating EGFR mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A145.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients that have activating mutations in the EGFR gene are treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) e.g. Erlotinib (Tarceva®) and Gefitinib (Iressa®). Most NSCLC patients with activating EGFR mutations will respond to EGFR-TKIs; however, in about 50% of these cases a secondary mutation in EGFR (T790M) subsequently occurs which results in resistance to treatment. Other mechanisms of clinical resistance can also occur such as amplification of MET kinase and EMT conversion; however, additional routes of resistance are poorly defined. Using a novel patient-derived NSCLC xenograft model, driven by the L858R EGFR mutation, we set out to recapitulate the reported clinical routes of resistance to EGFR inhibitors and to evaluate if additional mechanism could also be identified. METHODS: LION102 is a NSCLC adenocarcinoma patient-derived xenograft (PDX) model with an activating EGFR mutation (L858R) which is maintained subcutaneously in vivo admixed with a human stromal cell component. Resistant models were generated in vivo through repeated cycling of treatment for up to 10 weeks with EGFR-TKI alone or in combination with a MET inhibitor. Resistant tumour material was characterised for further mutations in the EGFR gene by direct sequencing as well as for MET, AXL and HER2 over-expression and genomic amplification by quantitative PCR; Epithelial-to-Mesenchymal transition (EMT) was assessed by immunohistochemistry (IHC). RESULTS: Naïve LION102 PDX tumours exhibited exquisite sensitivity to EGFR-TKIs (100% reduction in pre-treatment tumour volume, p CONCLUSIONS: EGFR-TKI resistant subtypes were generated in vivo from a proprietary patient-derived xenograft model (LION102) bearing an activating EGFR mutation (L858R) and characterised for their resistance mechanism. PDX models of resistance will be invaluable in assessing novel agents targeting the EGFR pathway and the development of new combination strategies which seek to prevent or overcome resistance to EGFR-TKIs. Citation Format: Andrew Mckenzie, Aaron Cranston, Phil Mallinder, Nektaria Papadopoulou, Simon Jiang, Kerry Moakes, Yinfei Yin, David Onion, Anna Grabowska, Martin Page, Rajendra Kumari. In vivo generation of EGFR-TKI resistance in a patient-derived xenograft (PDX) with an activating EGFR mutation (L858R), and molecular characterisation of resistance mechanisms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5651. doi:10.1158/1538-7445.AM2013-5651

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, carries a poor prognosis and remains an area of high unmet need. Despite recent advances in treating NSCLC adenocarcinomas with targeted agents such as Erlotinib (Tarceva©) and Crizotinib (Xalkori©) few advances have been made in the treatment of squamous cell carcinoma (SCC), which accounts for 25% of all NSCLC. Although the incidence of fibroblast growth factor receptor (FGFR) mutations in SCC is modest, amplification of FGFR is well documented and there are several first generation FGFR inhibitors currently in clinical trial. It is therefore essential to develop relevant in vivo patient-derived xenograft (PDX) models for the development and characterisation of new FGFR agents and/or combination strategies which may prolong benefit and delay the emergence of resistance. METHODS: LION137 is a novel NSCLC SCC patient-derived xenograft (PDX) model which is maintained subcutaneously in vivo admixed with a human stromal cell component. Cohorts of mice were dosed with AZD4547 alone or in combination with chemotherapy (CTx; parallel or serial dosing regimens). The extent of inhibition was measured over several in vivo passages and following cryopreservation/resuscitation. Response to treatment was evaluated by tumour growth inhibition and waterfall plots. Tumour material was characterised for 50 key oncogenes and mutations by Ion Torrent sequencing and FGFR1-4 expression and genomic amplification by quantitative PCR. Immunohistochemistry (IHC) was used to confirm SCC subtype, EMT status and FGFR expression. RESULTS: LION137 PDX tumours (SCC confirmed by IHC) exhibited reproducible sensitivity to AZD4547 alone (P CONCLUSIONS: LION137 is a novel patient-derived NSCLC SCC xenograft model with reproducible FGFRi-sensitivity which is directly relevant to the clinical setting. This model could be invaluable in assessing novel agents targeting the FGFR pathway and in the development of new combination strategies which may prevent or overcome resistance. Citation Format: Andrew Mckenzie, Aaron Cranston, Phil Mallinder, Nektaria Papadopoulou, Simon Jiang, Kerry Moakes, Yinfei Yin, David Onion, Anna Grabowska, Martin Page, Rajendra Kumari. In vivo response and molecular characterisation of a NSCLC squamous cell carcinoma PDX model exhibiting reproducible sensitivity to FGFR inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2013-342

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer cases, carries a poor prognosis and remains an area of high unmet need. Despite recent advances in treating NSCLC adenocarcinomas with targeted epidermal growth factor receptor (EGFR) kinase inhibitors, few advances have been made in the treatment of squamous cell carcinoma (SCC), which accounts for 25% of NSCLC. Although the incidence of EGFR mutations in SCC is low, a retrospective clinical study of erlotinib in SCC suggested that a proportion of SCC patients would benefit from treatment with an EGFR inhibitor (Tseng et al., 2012). METHODS: NSCLC samples obtained from untreated patients undergoing surgery were collected with ethical consent, disaggregated and implanted subcutaneously in MF-1 nude mice (Harlan UK) admixed with a human stromal cell component to generate patient-derived xenograft (PDX) models (LION PDX models). Tumour material was diagnosed on the basis of operative histology and immunohistochemistry (IHC) used to confirm histological subtype. IHC was also used to evaluate EGFR expression and tumours characterised for mutations in p53, LKB1, EGFR and K-RAS by direct DNA sequencing. Patient-derived xenografts were maintained in vivo and study cohorts of mice dosed when tumours reached 100-200mm3 with either erlotinib or gefitinib or vehicle placebo. Response to treatment was evaluated by tumour growth inhibition and waterfall plots. RESULTS: We have identified a subset of tumours with squamous histology in our LION panel of patient-derived NSCLC xenografts that respond to treatment with a targeted EGFR inhibitor. Four out of 11 SCC models tested (36%) responded to treatment with either erlotinib or gefitinib with greater than 50% growth inhibition and two SCC models responded with ∼30% growth inhibition. Responses correlated well with EGFR expression. Work to characterise a further 8 LION SCC PDX models is also underway. CONCLUSIONS: These data support the proposition that a significant subset of NSCLC patients with squamous histology may benefit from treatment with targeted EGFR inhibitors. Our LION SCC models could be valuable for biomarker studies to identify the basis of the EGFR inhibitor responder subset and for evaluating new agents in a more clinically relevant setting. Citation Format: Aaron Cranston, Andrew Mckenzie, Phil Mallinder, Simon Jiang, Kerry Moakes, Yinfei Yin, Alex Reece-Smith, Anna Grabowska, Martin Page, Rajendra Kumari. Patient-derived xenograft models reveal a subset of clinically relevant squamous non-small cell lung cancers that respond to targeted EGFR inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2796. doi:10.1158/1538-7445.AM2013-2796

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. A fragment screening campaign was conducted against the tyrosine kinase domain of FGFR1 to detect low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment inhibitors (molecular weight The poster will focus on the description of previously undescribed compounds bearing an imidazo[1,2-a]pyridine core scaffold where selectivity versus other protein kinases, for example FLT3, is obtained using the X-ray crystal structure and structure-based design. In summary we will illustrate how X-ray crystallography and fragment-based drug design (FBDD) can be used to discover compounds with activity in an FGFR driven xenograft model when dosed by the oral route. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2011-1361

Mouse c-Abl type IV and human BCR/ABL proteins have been expressed in insect cells using the baculovirus system. The proteins were expressed as full-length polypeptides as judged by electrophoresis in denaturing gels. They were identified by immunoprecipitation and immunoblotting with antibodies against ABL peptides and, for BCR/ABL, against a BCR peptide. In these immunoprecipitates both proteins gave autophosphorylation principally on tyrosine. Both proteins were active tyrosine kinases, phosphorylating a variety of tyrosine-containing substrates. In fresh extracts both proteins contained phosphotyrosine as shown by Western blots with antiphosphotyrosine antibodies. Partial purification could be achieved readily using ion exchange columns, and the BCR/ABL protein, p210BCR/ABL, could be further purified to near-homogeneity using an antiphosphotyrosine column. Both enzymes required a divalent metal ion for activity. At low concentrations of ATP (2 microM) and with angiotensin II as substrate both enzymes were activated by Mn2+ or by Mg2+. No major differences in catalytic properties were found between the two isolated enzymes in solution. The oncogenic properties of p210BCR/ABL may be due to its different subcellular location, or to the presence of an intracellular inhibitor of c-Abl that does not inhibit BCR/ABL, or to altered substrate-specificity such that it can phosphorylate a unique substrate which is not recognised by c-Abl.

We have recently reported the identification and characterization of JNJ-26481585, as a “second generation” oral pan-HDAC inhibitor. The compound has shown potent in vitro activity against solid and hematological tumor cell lines and significant induction of apoptosis was observed, consistent with the potency of JNJ-26481585 for Class I HDACs. In dose response to JNJ-26481585, western blot analysis of A2780 cell extracts confirmed modulation of HDAC1-3 substrates and also HDAC6 substrates. Inhibition of HDAC6, the deacetylase for tubulin and Hsp90, results in depletion of oncogenic Hsp90 client proteins, decreased cell motility, and potentiation of therapeutics such as taxanes. We hypothesized that rational combinations with taxanes and JNJ-26481585 could result in enhanced tumor cell killing and describe our observations from the combination of JNJ-26481585 in PC3-M prostate tumors. Combinations of JNJ-26481585 and taxanes were performed by co-administration or 24h separation of agents in A549, NCI-H460, NCI-H1373, NCI-H1703, NCI-H322 and NCI H-1650 lung cell lines; breast cell lines, MDA-MB-231, T47D, BT474 and MCF-7 cells; and prostate cell lines PC3M, DU-145, 22Rv1 and LnCap. We will present data on the outcome of these combinations, including additive and synergistic effects. The in vitro data encouraged us to explore the effects of the compound in vivo and in prostate models where we already had excellent single agent activity for JNJ-26481585 in DU-145, PC-3 and the PC-3M orthotopic model (significant T/C values in each model and at doses substantially below MTD). In PC3-M xenograft models, the combination of 1.25, 5 or 10mpk JNJ-26481585 (Q2D) and taxotere (Q7D), was more effective than either agent alone and resulted in dose dependent tumor regression and substantial extension of time to relapse. We then selected the more challenging PC-3M orthotopic mouse model to further define the contribution to efficacy from the excellent tissue distribution of our agent. Comparing 5mpk taxotere (Q7D), with 1 or 3mpk of JNJ-26481585 (QD) or the combination of agents, the activity of taxotere alone was surpassed by either dose of HDAC inhibitor alone and substantial tumor growth delay was observed from the combination of 3mpk JNJ-26481585 and taxotere; activity greater than predicted from the in vitro experiments. Finally, although HDAC6 inhibition could directly explain potentiation of taxotere by JNJ-26481585 in vitro, we believe the substantial effects in vivo may be due to the anti-angiogenic effect observed for our agent. We present also our data showing anti-angiogenic effects of JNJ-26481585 in PC3-M tumors, consistent with this hypothesis. Our data support the further use of more complex models, reflective of human disease in preclinical assessment and also support further exploration of this agent in rational combinations with SOC agents. JNJ-26481585 is currently in Phase I clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5443.

I am not an academic, just a foot soldier: I help out with the Children's University and as a Schools Science Ambassador, giving talks and demonstrations in physics. The recent £40m cuts in the budget of the UK's Science and Technology Facilities Council (January p6) have created quite a stir in the academic community, with talk of a "brain drain". Those in the penthouse should come down to the basement, where they would see just how thin and fragile the scientific foundation is.

The cellular gene c-abl is the normal homologue of the transforming gene (v-abl) within the genome of the Abelson leukaemia virus. The cDNA sequence coding for the cellular form of the murine abl gene (c-abl type IV) has been inserted into the baculovirus transfer vector, pAc36C, so that the c-abl gene is under the control of the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus (AcNPV). Spodoptera frugiperda cells infected with the recombinant transfer vector in the presence of wild type AcNPV DNA yielded recombinant, polyhedrin negative virus that expressed moderate levesl of the c-Abl protein (representing approx. 0.5–1% of the stained cellular proteins as determined by densitometric scanning). The insect derived c-Abl protein was compared to the P210-BCR/ABL protein from K562 cells, a cell line derived from a patient with chronic myelogenous leukaemia. Antibodies raised againts synthetic peptides based on c-abl encoded peptides react with the insect derived c-Abl. In addition, the baculovirus derived c-Abl protein has a tyrosine kinase activity as demonstrated by phosphorylation of a synthetic polypeptide and also by autophosphorylation. Phosphoamino acid analysis of immunoprecipitated, autophosphorylated baculovirus derived c-Abl protein indicates that the majority of label incorporated is on the tyrosine residues. Immunofluorescence microscopy has been used to show that the majority of the c-Abl protein expressed in cells infected with recombinant virus is located in the nuclear and plasma membranes.

People in education are more and more interested in using the technologies of information and communication for providing educational content. In particular, the internet infrastructure can facilitate that task in many ways, such as distance learning or web-based learning. All such software artefacts, which we call internet-based learning environments, have as a common goal to offer "good" and "easy" products for learning. We think that discount usability engineering, a usability engineering approach, can provide some guidance for building such environments. In this paper, we describe the concepts and methods of usability engineering and in particular its discounted version. Then we promote the exploitation of discount usability engineering by presenting arguments to apply it to the construction and assessment of internet-based learning environments, with respect to educational goals, budget constraints, time schedule, and tight human resources availability. Finally, we close with an example where usability engineering is applied in an educational context.

The ability to introduce DNA into eucaryotic cells has provided a means to analyse the expression of cloned genes. By manipulating the genes in vitro using recombinant DNA techniques it is possible to identify regulatory DNA sequences which are important for individual steps in gene expression. This review will summarize how these techniques have been applied to study steroid-responsive genes and what we have learned about steroid hormone action.

The mechanisms whereby androgens modulate mRNA levels for the C1, C2 and C3 prostatic binding protein genes in the rat ventral prostate have been investigated. Transcription rates for the 3 genes were lowered 2-3-fold upon castration but found to increase again upon re-administration of testosterone. Although this demonstrated a direct effect of androgens at the transcriptional level for these genes, the rates of mRNA synthesis were insufficient to account for previously observed steady-state measurements. This was confirmed by analysis of the nuclear steady-state levels for each mRNA following hormonal manipulation of the rats in vivo. It is concluded that besides direct transcriptional regulation, androgens must also exert a major effect of the half-life of these mRNAs in the rat ventral prostate.

Prostatic steroid binding protein, whose expression is stimulated by androgens, consists of two subunits: one containing the polypeptides C1 and C3 and the other containing C2 and C3. We have characterised genomic clones containing the C1 and C2 genes by restriction enzyme analysis and DNA sequencing. Both genes are 3.2 Kb, have similar exon/intron arrangements and share considerable DNA sequence homologies in their coding regions, intervening sequences and 5' upstream DNA sequence which suggests that they have probably arisen from the duplication of an ancestral gene. The 5' termini of C1 and C2 mRNA have been mapped; the sequence TATAAA appears 30 nucleotides upstream but a CAAT-like sequence at -60 - -80 is absent. Finally, homologous human genes have not been detected.

The expression of the rat prostatic steroid binding protein gene C3 is androgen-responsive in vivo in the rat prostate. Recombinant transfection vectors were constructed containing a cloned C3 rat gene ligated directly into the dominant acting selection vector pSV2-gpt, and were used to transfect androgen-responsive mouse mammary tumor cells (S115 cells). Transformants containing intact copies of the C3 gene were isolated and shown by primer extension analysis to produce C3 transcripts that had authentic 5' termini. The steady state level of the transcripts was under androgen control in three cell lines tested which had low copy numbers of the intact C3 gene, but was not regulated in a cell line which had a very high C3 gene copy number. Despite this difference in the androgenic regulation of integrated C3 rat genes, both type of cell lines still demonstrated androgen-regulated growth characteristics.

The function of regulatory regions of DNA, which flank the genes for prostatic steroid binding protein, is being analysed by introducing the cloned genes into heterologous cells. Two non-allelic genes for the C3 polypeptide, C3(1) and C3(2) have been transfected into androgen-responsive S115 cells using SV2-gpt vectors. The genes have been introduced either intact or as so-called fusion genes consisting of putative C3 promoters plus human beta-interferon cDNA. Both genes for C3 were accurately transcribed and their expression was stimulated 5-10-fold with 10(-8) M testosterone. Thus we should be able to define at least one region of DNA which confers androgen sensitivity to the genes. Although both genes were expressed similarly in S115 cells only C3(1) is responsible for C3 in vivo and C3(2) is transcribed poorly, if at all. The most likely explanation for the difference is that in vivo the C3(2) gene remains hypermethylated whereas the DNA which was introduced into S115 cells was unmethylated. This result supports the notion that the state of DNA methylation is important for controlling the transcription of genes.

We have cloned the genes for prostatic steroid binding protein to study the mechanism whereby their expression is regulated by testosterone. The genes for the C1 and C2 polypeptides are probably unique whereas there are two genes C3(1) and C3(2) for the C3 polypeptide of which only the former is transcribed in vivo. The state of DNA methylation associated with the two genes for C3 also differ, insofar as C3(1) is demethylated in ventral prostate from 14-28 days of age, whereas the C3(2) gene remains hypermethylated. The organization of all four genes is similar and appreciable DNA sequence homologies suggest that they may have arisen from a single ancestral gene. To study C3 expression and its hormonal regulation we have introduced the cloned genes into mouse S115 cells, an androgen-responsive cell line. Both genes were accurately transcribed and their expression was stimulated up to fivefold by 10(-8) M testosterone in approximately one third of the clones tested. To delineate the site of action of the hormone we have constructed chimeric genes consisting of putative C3 promoters and regulatory sequences together with a marker gene, interferon. This chimeric gene resulted in interferon production but its expression was stimulated by less than twofold in all clones tested. Therefore, these results indicate that, in mouse cells, testosterone does not interact directly with the rat C3 promoter but, in certain clones, may act post-transcriptionally.