Martin John Page

Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells

The mammalian endoglycosidase heparanase (Hpa1) is primarily responsible for cleaving heparan sulphate proteoglycans (HSPGs) present on the basement membrane of cells and its potential for remodelling the extracellular matrix (ECM) could be important in embryonic development and tumour metastasis. Elevated expression of this enzyme has been implicated in various pathological processes including tumour cell proliferation, metastasis, inflammation and angiogenesis. The enzyme therefore represents a potential therapeutic target. Hpa1 protein is initially synthesized as an inactive 65 kDa proenzyme that is then believed to be subsequently activated by proteolytic cleavage to generate an active heterodimer of 8 and 50 kDa polypeptides. By analysis of a series of Hpa1 deletion proteins we confirm that the 8 kDa subunit is essential for enzyme activity. We present here for the first time an insect cell expression system used for the generation of large amounts of recombinant protein of high specific activity. Individual subunits were cloned into baculoviral secretory vectors and co-expressed in insect cells. Active secreted heterodimer protein was recovered from the medium and isolated by a one-step heparin-Sepharose chromatography procedure to give protein of >90% purity. The recombinant enzyme behaved similarly to the native protein with respect to the size of HS fragments liberated on digestion, substrate cleavage specificity and its preference for acidic pH. A significant amount of activity, however, was also detectable at physiological pH values, as measured both by an in vitro assay and by in vivo degradation of cell-bound heparan sulphate.

JNJ-26481585, a Novel “Second-Generation” Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity

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)

New model of ErbB‐2 over‐expression in human mammary luminal epithelial cells

The ErbB‐2 receptor has been strongly implicated in the development of breast cancer. To establish a new model system to investigate the role of erbB‐2 in tumorigenesis of the breast, the conditionally immortalised human mammary luminal epithelial cell line HB4a was transfected with erbB‐2 cDNA. Biological and biochemical characterisation of the resulting cell lines demonstrated that high levels of ErbB‐2 expression were sufficient to cause transformation in vitro but did not cause tumours in vivo. Transformation by over‐expression of ErbB‐2 correlated with ligand‐independent tyrosine phosphorylation of ErbB‐2 and the adaptor protein Shc. Over‐expression of ErbB‐2 also resulted in the ligand‐independent constitutive association between Shc and another adaptor protein, Grb2, indicating that receptor activation was sufficient to activate downstream signalling pathways. Using the model described, it was found that elevation of ErbB‐2 expression levels caused marked quantitative and qualitative alterations in responses to the ligands epidermal growth factor and heregulin. Data indicate a central role for ErbB‐2 in mediating the responses induced by these ligands and suggest that these altered ligand‐dependent responses play an important role in tumorigenesis in vivo. Int. J. Cancer 80:477–484, 1999.

Cluster analysis of an extensive human breast cancer cell line protein expression map database

In the current study, the protein expression maps (PEMs) of 26 breast cancer cell lines and three cell lines derived from normal breast or benign disease tissue were visualised by high resolution two‐dimensional gel electrophoresis. Analysis of this data was performed with ChiClust and ChiMap, two analytical bioinformatics tools that are described here. These tools are designed to facilitate recognition of specific patterns shared by two or more (a series) PEMs. Both tools use PEMs that were matched by an image analysis program and locally written programs to create a match table that is saved in an object relational database. The ChiClust tool uses clustering and subclustering methods to extract statistically significant protein expression patterns from a large series of PEMs. The ChiMap tool calculates a differential value (either as percentage change or a fold change) and represents these graphically. All such differentials or just those identified using ChiClust can be submitted to ChiMap. These methods are not dependent on any particular commercial image analysis program, and the whole software package gives an integrated procedure for the comparison and analysis of a series of PEMs. The ChiClust tool was used here to order the breast cell lines into groups according to biological characteristics including morphology in vitro and tumour forming ability in vivo. ChiMap was then used to highlight eight major protein feature‐changes detected between breast cancer cell lines that either do or do not proliferate in nude mice. Mass spectrometry was used to identify the proteins. The possible role of these proteins in cancer is discussed.

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

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]

Identification of a series of substituted 2-piperazinyl-5-pyrimidylhydroxamic acids as potent histone deacetylase inhibitors

Pursuing our efforts in designing 5-pyrimidylhydroxamic acid anti-cancer agents, we have identified a new series of potent histone deacetylase (HDAC) inhibitors. These compounds exhibit enzymatic HDAC inhibiting properties with IC(50) values in the nanomolar range and inhibit tumor cell proliferation at similar levels. Good solubility, moderate bioavailability, and promising in vivo activity in xenograft model made this series of compounds interesting starting points to design new potent HDAC inhibitors.

Abstract 2054: Patient relevant preclinical in vivo models using image-guided small animal irradiation for drug discovery

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND: Radiotherapy is a primary, adjuvant or neoadjuvant treatment for a number of different cancers such as glioblastoma, breast, lung and prostate. Image-guided radiotherapy (IGRT) is widely used to treat cancer patients providing more accurate treatment plans and reduced side effects. However in the preclinical setting the use of IGRT is less common with traditional irradiation studies utilising whole body irradiation with lead shielding attempting to focus the radiation to specific area on the animal or simple single beam techniques. The development of the image-guided small animal radiation research platform (SARRP) allows the treatment of animal models of cancer more accurately and importantly, with planned protocols similar to those utilised in the clinic. In addition the high precision beam geometry allows the platform to be used in vitro in multi-well assays providing a more high-throughput system for screening. METHODS: We have established multiple cancer models which incorporate bioluminescence to track primary orthotopic and metastatic lesions in real-time using the Spectrum CT imaging system. Tumor growth was monitored longitudinally through quantification of the bioluminescent signal. Bioluminescence from established tumor was correlated to CT and co-registered with the integrated CT on the SARRP platform to allow estimation of tumor dimensions and position in order to deliver a more accurate dose of irradiation specifically to tumors whilst sparing the surrounding normal tissue. In addition we have established Caucasian non-small cell lung cancer (NSCLC) patient-derived xenograft (PDX) models which are admixed with human stroma and evaluated SARRP in these. Tumor specific protocols and dose calculation for irradiation were designed to deliver appropriate irradiation dose to immuno-deficient mice bearing xenografted tumor either alone or in combination with targeted agent or chemotherapeutic. RESULTS: Mice treated with SARRP tolerated irradiation doses in a range of different models either alone or in combination. Validation of different protocols will be reported as well as intrinsic resistant profiles for different models and outcome of any combination treatments to re-sensitize resistant cancer to radiotherapy. CONCLUSIONS: The SARRP platform allows the use of irradiation alone or in combination with anti-cancer agents in small animals with reduced side effects and improved clinical outcome allowing these novel preclinical models to be used effectively for drug discovery programmes to ultimately improve efficacy and treatment options for cancer patients using radiotherapy. Citation Format: Rajendra Kumari, Andrew McKenzie, Nektaria Papadopoulou, Yinfei Yin, Martin Page, Henry Li, Ian Wilson. Patient relevant preclinical in vivo models using image-guided small animal irradiation for drug discovery. [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 2054. doi:10.1158/1538-7445.AM2014-2054

Abstract 4875: In vivo and in vitro generation and characterisation of EGFR-TKI resistance in patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of NSCLC with activating EGFR mutations

Background: Non-small cell lung cancer (NSCLC) patients that have activating mutations in the epidermal growth factor receptor (EGFR) gene are treated with 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 c-MET kinase, Her-2 and EMT conversion; however, additional routes of resistance are poorly defined. Using a novel NSCLC patient-derived xenograft (PDX) 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 (exon 19 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 and for c-MET, Axl and Her2 over-expression and genomic amplification by quantitative PCR. Combination efficacy treatment (in vitro and in vivo) was carried out to verify documented resistance mechanisms. Results: Naive LU6422 and HCC827 tissue exhibited exquisite sensitivity to EGFR-TKIs (100% reduction in pre-treatment tumour volume, p Conclusions: EGFR-TKI resistant subtypes were generated in vivo and in vitro from a proprietary patient-derived xenograft model (LU6422) and the HCC827 cell line and were characterised for their resistance mechanisms. Pre-clinical modes of acquired 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, Nektaria Papadopoulou, Simon Jiang, Martin Page, Henry Li, Rajendra Kumari, Jane Wrigley. In vivo and in vitro generation and characterisation of EGFR-TKI resistance in patient-derived xenograft (PDX) and cell line-derived xenograft (CDX) models of NSCLC with activating EGFR mutations. [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 4875. doi:10.1158/1538-7445.AM2014-4875

Abstract 3628: JNJ-38877605: A selective oral Met inhibitor for the treatment of cancer

JNJ-38877605 is a potent and selective Met receptor tyrosine kinase inhibitor that has substantial pre-clinical antitumor activity. We recently described JNJ-38877605 as an ATP-competitive, inhibitor of the catalytic activity of Met kinase (IC50 4 nM) with a unique binding mode that leads to selectivity, exhibiting a 600-fold selectivity (vs cFMS IC50 2.6μM; the next potently inhibited kinase) for Met compared with a panel of 250 diverse tyrosine and serine-threonine kinases. Previously, JNJ-38877605 was shown to inhibit Met phosphorylation in tumor xenografts up to 16 h following a single oral dose, and that inhibition of receptor phosphorylation was associated with dose-dependent tumor growth inhibition. Herein we describe the activity of JNJ-38877605, on a broader range of preclinical solid tumor xenograft models, at doses below MTD and with different schedules of administration. In addition, we now also show data with primary patient-tumor derived xenografts where we have dosed JNJ-39977605 at the MTD using gastric, colorectal and liver metastatic (derived from primary CRC) samples. JNJ-38877605 was observed to induce significant tumor regression in large well established MET gene amplified gastric cancer models and in Met pathway activated (autocrine or paracrine) models. Significant growth inhibition was achieved when dosing JNJ-38877605 at MTD (and lower doses) with T/C Using primary patient derived xenograft models, we have shown that differential expression of the c-Met/HGF axis and epithelial-mesenchymal transition (EMT)-related gene markers between primary colorectal carcinomas and liver metastases in primary tissue, play a role in the activity of this compound in vivo. The strong preclinical properties of the JNJ-38877605 compound has resulted in the selection of this potent and uniquely selective Met inhibitor for clinical evaluation. JNJ-38877605 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 3628.

Abstract B111: Differential expression of c‐Met/HGF axis and epithelial‐mesenchymal transition (EMT)‐related gene markers between primary colorectal carcinomas and liver metastases in primary tissue, isolated cell types and in vivo

Introduction: EMT is frequently activated in a subset of tumor cells during disease progression. EMT is described as downregulation of E‐cadherin with a corresponding upregulation of mesenchymal markers such as Vimentin. EMT is linked to carcinoma progression stemming from increased tumor cell motility, growth, survival, and invasion. The c‐Met receptor and MACC1 (metastasis‐associated in colon cancer‐1) are often upregulated in CRC metastases, have been associated with increased metastasis, and represent potential drug targets. Here we assessed the expression of genes associated with EMT in human CRCs and liver metastases (LMs), in epithelial cells isolated from primary tissues and in mouse passaged primary tumor xenografts. Methods: Human primary CRC (n = 11) and LM (n = 21) samples, along with matched normal tissues, were obtained under full ethical approval from the Queen9s Medical Centre, Nottingham, UK. Real‐time quantitative PCR was used to determine expression levels of EMT‐related markers (growth factors, transcriptional repressors, mesenchymal markers) relative to the housekeeping gene hypoxanthine‐guanine phosphoribosyltransferase, from (i) primary tissues, (ii) epithelial cells and fibroblasts isolated from disaggregated primary tissues using cell type‐specific antibodies bound to magnetic beads, (iii) primary tumor tissues subcutaneously passaged in nude mice. A student9s t‐test was used for statistical analyses. Results: MACC1 and c‐Met were significantly upregulated, whereas E‐cadherin was significantly downregulated, in CRC and LM primary tissues compared to normal colonic mucosa, indicating that EMT had occurred. In LMs, hepatocyte growth factor (HGF), Snail, Slug, Twist, Vimentin, TGFβ1, and matrix metalloproteinase 9 were also significantly upregulated (P Conclusions: Phenotypic markers consistent with EMT were significantly increased in LMs compared to CRCs, and Snail, Twist, Vimentin, TGFβ1, and MMP9 provide the best markers for LM. In CRCs and LMs, paracrine signalling of HGF (mesenchymal) to the c‐Met receptor (epithelial) is likely to occur, and this signalling pathway appears to be lost during growth of the primary tumor in nude mice. This has important implications in therapy studies targeting the HGF‐MACC1‐cMet axis. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B111.