Supplementary Figure 5 from MET and KRAS Gene Amplification Mediates Acquired Resistance to MET Tyrosine Kinase Inhibitors

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

Supplementary Figure 7 from MET and KRAS Gene Amplification Mediates Acquired Resistance to MET Tyrosine Kinase Inhibitors

Supplementary Figure 8 from MET and KRAS Gene Amplification Mediates Acquired Resistance to MET Tyrosine Kinase Inhibitors

Supplementary Figure Legends 1-9 from MET and KRAS Gene Amplification Mediates Acquired Resistance to MET Tyrosine Kinase Inhibitors

Supplementary Table 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 Figure 4 from MET and KRAS Gene Amplification Mediates Acquired Resistance to MET Tyrosine Kinase Inhibitors

Supplementary Figure 6 from MET and KRAS Gene Amplification Mediates Acquired Resistance to MET Tyrosine Kinase Inhibitors

Abstract Enhanced cell migration is a hallmark of metastatic cancer cells. The propensity of cancer cells to close an open wound in a cell monolayer is thought to predict this ability. Using our adenoviral shRNA knockdown library we have established a high-throughput wound healing assay to identify novel genes involved in cell migration. A 96-pin tool was designed to apply a constant mechanical scratch-wound in the cellular monolayer. We used transmitted light imaging for segmentation and quantification of the scratch wound that remained open. Genes whose knockdown inhibit cell migration can be identified by their effect on the open wound. We demonstrated that two knockdown constructs targeting a known player in motility, CXCR4, inhibit wound healing, thereby validating our approach. Using this wound healing assay we have identified a number of novel genes associated with cancer cell motility. These targets have consecutively been validated for their role in 3-D invasion using Boyden chambers. As our adenoviral knockdown libraries focus on druggable targets, these validated targets can quickly be employed to generate small-molecule compounds or antibody therapeutics targeting cancer metastasis. Citation Format: Remko de Pril, Annemarie Lekkerkerker, Desire van Steenhoven, Ilhem Maghrani, Tim Perera, Janine Arts, David Fischer, Richard Janssen. High-Content screen for inhibitors of cell migration in cancer metastasis using adenoviral knock-down [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr A15.

Abstract We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role. Mol Cancer Ther; 10(9); 1542–52. ©2011 AACR.

Abstract 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 < 42% in tumors of MKN-45, GTL-16, SNU-5 and Kato II met gene amplified models. Significant inhibition was also observed in K-ras mt (dependent) NCI-H441 NSCLC xenografts. Regression of U87 MG PTEN negative glioblastoma tumors (autocrine loop), grown either as subcutaneous or orthotopic models was also observed. 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.

Herceptin (trastuzumab) is used in patients with breast cancer who have HER2 (ErbB2)-positive tumours. However, its mechanisms of action and how acquired resistance to Herceptin occurs are still poorly understood. It was previously thought that the anti-HER2 monoclonal antibody Herceptin inhibits HER2 signalling, but recent studies have shown that Herceptin does not decrease HER2 phosphorylation. Its failure to abolish HER2 phosphorylation may be a key to why acquired resistance inevitably occurs for all responders if Herceptin is given as monotherapy. To date, no studies have explained why Herceptin does not abolish HER2 phosphorylation. The objective of this study was to investigate why Herceptin did not decrease HER2 phosphorylation despite being an anti-HER2 monoclonal antibody. We also investigated the effects of acute and chronic Herceptin treatment on HER3 and PKB phosphorylation in HER2-positive breast cancer cells. Using both Förster resonance energy transfer (FRET) methodology and conventional Western blot, we have found the molecular mechanisms whereby Herceptin fails to abolish HER2 phosphorylation. HER2 phosphorylation is maintained by ligand-mediated activation of EGFR, HER3, and HER4 receptors, resulting in their dimerisation with HER2. The release of HER ligands was mediated by ADAM17 through a PKB negative feedback loop. The feedback loop was activated because of the inhibition of PKB by Herceptin treatment since up-regulation of HER ligands and ADAM17 also occurred when PKB phosphorylation was inhibited by a PKB inhibitor (Akt inhibitor VIII, Akti-1/2). The combination of Herceptin with ADAM17 inhibitors or the panHER inhibitor JNJ-26483327 was able to abrogate the feedback loop and decrease HER2 phosphorylation. Furthermore, the combination of Herceptin with JNJ-26483327 was synergistic in tumour inhibition in a BT474 xenograft model. We have determined that a PKB negative feedback loop links ADAM17 and HER ligands in maintaining HER2 phosphorylation during Herceptin treatment. The activation of other HER receptors via ADAM17 may mediate acquired resistance to Herceptin in HER2-overexpressing breast cancer. This finding offers treatment opportunities for overcoming resistance in these patients. We propose that Herceptin should be combined with a panHER inhibitor or an ADAM inhibitor to overcome the acquired drug resistance for patients with HER2-positive breast cancer. Our results may also have implications for resistance to other therapies targeting HER receptors.

Abstract The establishment of the role of MET in human cancer has led to the development of small-molecule inhibitors, many of which are currently in clinical trials. Thus far, nothing is known about their therapeutic efficacy and the possible emergence of resistance to treatment, a problem that has been often observed with other receptor tyrosine kinase (RTK) inhibitors. To predict mechanisms of acquired resistance, we generated resistant cells by treating MET-addicted cells with increasing concentrations of the MET small-molecule inhibitors PHA-665752 or JNJ38877605. Resistant cells displayed MET gene amplification, leading to increased expression and constitutive phosphorylation of MET, followed by subsequent amplification and overexpression of wild-type (wt) KRAS. Cells harboring KRAS amplification progressively lost their MET dependence and acquired KRAS dependence. Our results suggest that MET and KRAS amplification is a general mechanism of resistance to specific MET inhibitors given that similar results were observed with two small inhibitors and in different cell lines of different histotypes. To our knowledge, this is the first report showing that overexpression of wt KRAS can overcome the inhibitory effect of a RTK inhibitor. In view of the fact that cellular models of resistance to inhibitors targeting other tyrosine kinases have predicted and corroborated clinical findings, our results provide insights into strategies for preventing and/or overcoming drug resistance. Cancer Res; 70(19); 7580–90. ©2010 AACR.

Abstract 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]

JNJ-26483327 is a novel oral Pan Her/Src/VEGFR-3 inhibitor which has previously been shown in preclinical models to cross the blood-brain barrier and to reach high levels in brain, solid tumor, and bone marrow sites. JNJ-26483327 is not an active substrate for P-glycoprotein pumps and has been well tolerated to date in an ongoing phase I trial. VEGF-C signaling through the VEGFR-3 (FLT-4) receptor has been shown to promote growth of acute myeloid leukemia (AML) cells and to mediate resistance to multiple chemotherapy drugs in vitro. Anti-VEGFR-3 antibody therapy decreased angiogenesis, increased hypoxia and necrosis, and reduced lymph node metastases in solid tumor xenografts. To date, however, VEGF-C/VEGFR-3 inhibition has not been actively been investigated for treatment of hematological malignancies. We hypothesized that JNJ- 26483327 treatment of VEGFR-3 expressing systemic AML would limit tumor growth and lymphatic spread via VEGF-C/VEGFR-3 mechanisms. An initial dose-finding pilot experiment was performed using SCID mice engrafted via tail vein with ten million HELluc cells, human acute myeloid leukemia cells with known expression of VEGF-A/C and VEGFR-2/3 and stably transfected with luciferase constructs to facilitate small animal imaging. Mice were treated with PBS, vehicle (200 mL by mouth twice daily), low dose JNJ-26483327 (75 mg/kg by mouth twice daily, total 150 mg/kg/day) and high dose JNJ- 26483327 (125 mg/kg by mouth daily, total 250 mg/kg/day) for 10 consecutive days. We found that low dose JNJ-26483327 therapy significantly improved the median survival of HELluc systemic xenografts by 46% (26 days longer than vehicle-treated controls) (p<0.05). Although high dose JNJ-26483327 prolonged median survival over vehicletreated controls, the difference was not statistically significant. Moreover, although JNJ- 26483327 improved survival, HELluc leukemia burden (as measured by bioluminescent imaging) was not significantly reduced or eradicated as compared to control, consistent with cytostatic but not cytotoxic anti-tumor effects. VEGFR-3 signaling has also been shown to mediate leukemia cell proliferation, survival, and resistance to chemotherapy. Based on preclinical and clinical data demonstrating improved anti-tumor activity of VEGF inhibitors when combined with chemotherapy, we hypothesized that combining JNJ-26483327 with chemotherapy used in conventional AML therapy may result in additive synergistic anti-tumor effects. To determine if JNJ-26483327 inhibition enhanced the effects of cytotoxic chemotherapy, systemic HELluc tumor bearing mice were treated with low dose JNJ-26483327 (150 mg/kg/day for 10 days) and a single maximally tolerated dose of doxorubicin (1.5mg/kg). Both single agent doxorubicin and single agent JNJ-26483327 treatment resulted in significant reduction of HELluc tumor burden. However, no significant decrease in leukemia burden was observed after combination JNJ-26483327+doxorubicin treatment when compared to single agent groups. Lastly we postulated that combination therapies of JNJ-26483327 with other anti-VEGF therapies directed at inhibition of VEGF-A, VEGFR-1, or VEGFR-2 would result in inhibition of all known VEGFR signaling pathways and result in improved anti-leukemic effects of JNJ-26483327 therapy. Systemic HELluc bearing mice were treated with PBS, vehicle, low dose JNJ-26483327, an anti-hVEGF-A antibody BV (bevacizumab, Genentech) or combination JNJ-26483327+ BV. Results showed that single agent low dose JNJ- 26483327 or single agent BV significantly reduced HELluc tumor burden up until day 20. Combination JNJ-26483327+BV treatment, however, did not result in additive/synergistic anti-leukemic effects as compared to single agent therapy and may in fact have resulted in possible antagonistic effects.

Dose-response studies are commonly used in experiments in pharmaceutical research in order to investigate the dependence of the response on dose, i.e., a trend of the response level toxicity with respect to dose. In this paper we focus on dose-response experiments within a microarray setting in which several microarrays are available for a sequence of increasing dose levels. A gene is called differentially expressed if there is a monotonic trend (with respect to dose) in the gene expression. We review several testing procedures which can be used in order to test equality among the gene expression means against ordered alternatives with respect to dose, namely Williams' (Williams 1971 and 1972), Marcus' (Marcus 1976), global likelihood ratio test (Bartholomew 1961, Barlow et al. 1972, and Robertson et al. 1988), and M (Hu et al. 2005) statistics. Additionally we introduce a modification to the standard error of the M statistic. We compare the performance of these five test statistics. Moreover, we discuss the issue of one-sided versus two-sided testing procedures. False Discovery Rate (Benjamni and Hochberg 1995, Ge et al. 2003), and resampling-based Familywise Error Rate (Westfall and Young 1993) are used to handle the multiple testing issue. The methods above are applied to a data set with 4 doses (3 arrays per dose) and 16,998 genes. Results on the number of significant genes from each statistic are discussed. A simulation study is conducted to investigate the power of each statistic. A R library IsoGene implementing the methods is available from the first author.