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Supplementary Figure S4 - PDF file 19K, Ectopic expression of MET in cetuximab sensitive DIFI and LIM1215 cell lines

XLSX file 75K, This is an excel file containing 9 sheets, one for each of the 9 supplementary tables (S1 to S9) mentioned in the main text

PDF file 234K, This is a file containing the list of supplementary tables, the list of supplementary figures, as well as the 7 supplementary figures (S1 to S7) and their correspondent legends, mentioned in the main text

We discovered a novel somatic gene fusion, CD74–NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74–NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2–ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74–NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K–AKT pathway, and led to increased colony formation in soft agar. Thus, CD74–NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment.Significance: CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease. Cancer Discov; 4(4); 415–22. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 377

Purpose: We determined the gene copy numbers for MET, for its transcriptional activator MACC1 and for its ligand hepatocyte growth factor (HGF) in liver metastases from colorectal carcinoma (mCRC). We correlated copy numbers with mRNA levels and explored whether gain and/or overexpression of MET and MACC1 predict response to anti-Met therapies. Finally, we assessed whether their genomic or transcriptional deregulation correlates with pathologic and molecular parameters of aggressive disease.Experimental Design: One hundred three mCRCs were analyzed. Copy numbers and mRNA were determined by quantitative PCR (qPCR). Thirty nine samples were implanted and expanded in NOD (nonobese diabetic)/SCID (severe combined immunodeficient) mice to generate cohorts that were treated with the Met inhibitor JNJ-38877605. In silico analysis of MACC1 targets relied on genome-wide mapping of promoter regions and on expression data from two CRC datasets.Results: No focal, high-grade amplifications of MET, MACC1, or HGF were detected. Chromosome 7 polysomy and gain of the p-arm were observed in 21% and 8% of cases, respectively, and significantly correlated with higher expression of both Met and MACC1. Met inhibition in patient-derived xenografts did not modify tumor growth. Copy number gain and overexpression of MACC1 correlated with unfavorable pathologic features better than overexpression of Met. Bioinformatic analysis of putative MACC1 targets identified elements besides Met, whose overexpression cosegregated with aggressive forms of colorectal cancer.Conclusions: Experiments in patient-derived xenografts suggest that mCRCs do not rely on Met genomic gain and/or overexpression for growth. On the basis of pathologic correlations and bioinformatic analysis, MACC1 could contribute to CRC progression through mechanisms other than or additional to Met transcriptional upregulation. Clin Cancer Res; 17(10); 3146–56. ©2011 AACR.

In vivo rebound MET activation

Supplementary Table S1 from Genetic and Expression Analysis of MET, MACC1, and HGF in Metastatic Colorectal Cancer: Response to Met Inhibition in Patient Xenografts and Pathologic Correlations

Supplementary Figure S1 from Genetic and Expression Analysis of MET, MACC1, and HGF in Metastatic Colorectal Cancer: Response to Met Inhibition in Patient Xenografts and Pathologic Correlations

MET oncogene amplification is emerging as a major mechanism of acquired resistance to EGFR-directed therapy in lung and colorectal cancers. Furthermore, MET amplification predicts responsiveness to MET inhibitors currently in clinical trials. Among the anti-MET drugs available, ATP-competitive small-molecule kinase inhibitors abrogate receptor autophosphorylation and downstream activation of ERK1/2 and AKT, resulting in cell-cycle arrest. However, this antiproliferative effect allows persistence of a pool of cancer cells that are quiescent but alive. Once the inhibition is removed, rebound activation of MET-driven cell proliferative pathways and tumor growth may occur, an adverse event observed frequently in clinical settings after drug discontinuation. Here we show that inhibitor withdrawal prompts receptor phosphorylation to levels higher than those displayed at steady-state and generates a rebound effect pushing quiescent cancer cells back into the cell cycle, both in vitro and in experimental tumor models in vivo. Mechanistically, we found that inhibitor treatment blocks MET endocytosis, causing a local increase in the number of receptors at the plasma membrane. Upon inhibitor washout, the receptor is readily rephosphorylated. The initial phosphorylation is not only increased but also prolonged in duration due to downmodulation of a phosphatase-mediated MET-negative feedback loop, which accompanies receptor internalization. Notably, treatment with a MET therapeutic antibody that induces proteolytic cleavage of the receptor at the cell surface substantially prevents this rebound effect, providing a rationale to combine or alternate these mechanistically different types of MET-targeted therapy. Cancer Res; 76(17); 5019–29. ©2016 AACR.

Supplementary Materials and Methods from Genetic and Expression Analysis of MET, MACC1, and HGF in Metastatic Colorectal Cancer: Response to Met Inhibition in Patient Xenografts and Pathologic Correlations

Legends to Supplementary Figures S1-S6

Supplementary Fig. 1. HGF protects MET-addicted tumor cells against MET TKI-induced growth inhibition.

Metastatic colorectal cancer remains largely incurable, although in a subset of patients, survival is prolonged by new targeting agents such as anti-EGF receptor (anti-EGFR) antibodies. This disease is believed to be supported by a subpopulation of stem-like cells termed colon cancer–initiating cell (CCIC), which may also confer therapeutic resistance. However, how CCICs respond to EGFR inhibition has not been fully characterized. To explore this question, we systematically generated CCICs through spheroid cultures of patient-derived xenografts of metastatic colorectal cancer. These cultures, termed “xenospheres,” were capable of long-term self-propagation in vitro and phenocopied the original patient tumors in vivo, thus operationally defining CCICs. Xenosphere CCICs retained the genetic determinants for EGFR therapeutic response in vitro and in xenografts; like the original tumors, xenospheres harboring a mutated KRAS gene were resistant to EGFR therapy, whereas those harboring wild-type RAS pathway genes (RASwt) were sensitive. Notably, the effects of EGFR inhibition in sensitive CCICs could be counteracted by cytokines secreted by cancer-associated fibroblasts. In particular, we found that the MET receptor ligand hepatocyte growth factor (HGF) was especially active in supporting in vitro CCIC proliferation and resistance to EGFR inhibition. Ectopic production of human HGF in CCIC xenografts rendered the xenografts susceptible to MET inhibition, which sensitized the response to EGFR therapy. By showing that RASwt CCICs rely on both EGFR and MET signaling, our results offer a strong preclinical proof-of-concept for concurrent targeting of these two pathways in the clinical setting. Cancer Res; 74(6); 1857–69. ©2014 AACR.

Supplementary Fig. 3. HGF restores physiological, GAB-1-mediated MET signaling in DN30 Fab-treated MET-addicted cells.

PDF file - 121K

Supplementary Fig. 6. HGF-induced activation of the PI3K pathway inversely correlates with response to MET-targeted drugs.

Supplementary Methods. Supplementary information on drugs and chemicals, human HGF knock-in mice, in vivo signal transduction analysis, study approval, and statistical analysis.

PDF file - 8193K, Supplementary Figure S1. Xensophere characterization. Supplementary Figure S2. EGF in Raswt xenosphere proliferation. Supplementary Figure S3. MET inhibition in xenospheres treated with FCM. Supplementary Figure S4. HGF dose-response in RASwt xenosphere viability. Supplementary Figure S5. M049 spheropatient treatment and HGF autocrine loop in M049 xenosphere. Supplementary Figure S6. CC-IC marker expression in M049 xenospheres and spheropatients treated with inhibitors.

Supplementary Fig. 5. Human HGF knock-in SCID mice display a physiological HGF expression pattern.

PDF file - 733K, Supplementary Table S1. Gene alterations in xenospheres. Supplementary Table S2. Cetuximab response in Raswt xenopatients and spheropatients. Supplementary Table S3. Tumor volumes and regression of M049-HGF spheropatients. Supplementary Table S4. List of primers used for gDNA sequencing and qPCR.

Supplementary Fig. 4. Low concentrations of HGF are sufficient to fully antagonize DN30 Fab-induced EBC-1 lung carcinoma cell growth inhibition.

Supplementary Fig. 2. HGF sustains HER3-independent PI3K signaling in c-MET-amplified cells exposed to MET tyrosine kinase inhibitors.

Erdafitinib (JNJ-42756493, BALVERSA) is a tyrosine kinase inhibitor indicated for the treatment of advanced urothelial carcinoma. In this work, a translational model-based approach to inform the choice of the doses in phase 1 trials is illustrated. A pharmacokinetic (PK) model was developed to describe the time course of erdafitinib plasma concentrations in mice and rats. Data from multiple xenograft studies in mice and rats were analyzed using the Simeoni tumor growth inhibition (TGI) model. The model parameters were used to derive a range of erdafitinib exposures that might inform the choice of the doses in oncology phase 1 trials. Conversion of exposures to doses was based on preliminary PK assessments from the first-in human (FIH) study. A one-compartment PK disposition model, with linear absorption and dose-dependent clearance, adequately described the PK data in both mice and rats via an allometric scaling approach. The TGI model was able to describe tumor growth dynamics, providing quantitative measurements of erdafitinib antitumor potency in mice and rats. Based on these estimates, ranges of efficacious unbound concentration were identified for erdafitinib in mice (0.642–5.364 μg/L) and rats (0.782–2.565 μg/L). Based on the FIH data, it was possible to transpose exposures into doses and doses of above 4 mg/day provided erdafitinib exposures associated with significant TGI in animals. The findings were in agreement with the results of the FIH trial, in which the first hints of clinical activities were observed at 6 mg. The successful modeling exercise of erdafitinib preclinical data showed how translational PK-PD modeling might be a tool to help to inform the choice of the doses in FIH studies.

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: Interferon-induced expression of programmed cell death ligands (PD-L1/PD-L2) may sustain tumour immuneevasion. Patients featuring MET amplification, a genetic lesion driving transformation, may benefit from anti-MET treatment. We explored if MET-targeted therapy interferes with Interferon-gamma modulation of PD-L1/PD-L2 in MET-amplified tumours.METHODS: PD-L1/PD-L2 expression and signalling pathways downstream of MET or Interferon-gamma were analysed in MET-amplified tumour cell lines and in patient-derived tumour organoids, in basal condition, upon Interferon-gamma stimulation, and after anti-MET therapy.RESULTS: PD-L1 and PD-L2 were upregulated in MET-amplified tumour cells upon Interferon-gamma treatment. This induction was impaired by JNJ-605, a selective inhibitor of MET kinase activity, and MvDN30, an antibody inducing MET proteolytic cleavage. We found that activation of JAKs/STAT1, signal transducers downstream of the Interferon-gamma receptor, was neutralised by MET inhibitors. Moreover, JAK2 and MET associated in the same signalling complex depending on MET phosphorylation. Results were confirmed in MET-amplified organoids derived from human colorectal tumours, where JNJ-605 treatment revoked Interferon-gamma induced PD-L1 expression.CONCLUSIONS: These data show that in MET-amplified cancers, treatment with MET inhibitors counteracts the induction of PD-1 ligands by Interferon-gamma. Thus, therapeutic use of anti-MET drugs may provide additional clinical benefit over and above the intended inhibition of the target oncogene.

// Alessia Mira 1 , Virginia Morello 1,2 , Maria Virtudes Cespedes 3,4 , Timothy Perera 5 , Paolo M. Comoglio 1 , Ramon Mangues 3,4 and Paolo Michieli 1,2 1 Candiolo Cancer Institute, FPO, IRCCS, Candiolo, Turin, Italy 2 Department of Oncology, University of Torino Medical School, Candiolo, Turin, Italy 3 Biomedical Research Institute Sant Pau, Hospital de Sant Pau, Barcelona, Spain 4 Centro de Investigacion Biomedica en Red en Bioingenieria, Biomateriales y Nanomedicina, Barcelona, Spain 5 OCTIMET Oncology Ltd., Oxford, United Kingdom Correspondence to: Paolo Michieli, email: // Keywords : colorectal cancer, HGF, anti-angiogenic therapy, resistance, tumor metabolism Received : March 03, 2017 Accepted : March 28, 2017 Published : April 07, 2017 Abstract The role of paracrine Hepatocyte Growth Factor (HGF) in the resistance to angiogenesis inhibitors (AIs) is hidden in xenograft models because mouse HGF fails to fully activate human MET. To uncover it, we compared the efficacy of AIs in wild-type and human HGF knock-in SCID mice bearing orthotopic human colorectal tumors. Species-specific HGF/MET signaling dramatically impaired the response to anti-angiogenic agents and boosted metastatic dissemination. In cell-based assays mimicking the consequences of anti-angiogenic therapy, colorectal cancer cells were completely resistant to hypoxia but extremely sensitive to nutrient deprivation. Starvation-induced apoptosis could be prevented by HGF, which promoted GLUT1-mediated glucose uptake, sustained glycolysis and activated autophagy. Pharmacological inhibition of GLUT1 in the presence of glucose killed tumor cells as effectively as glucose deprivation, and this effect was antagonized by HGF. Concomitant targeting of GLUT1 and HGF potently suppressed growth and dissemination of AI-resistant human tumors in human HGF knock-in SCID mice without exacerbating tumor hypoxia. These data suggest that stroma-derived HGF protects CRC cells against glucose starvation-induced apoptosis, promoting resistance to both AIs and anti-glycolytic agents. Combined inhibition of glucose metabolism and HGF/MET signaling (‘anti-METabolic therapy’) may represent a more effective CRC treatment compared to utterly blocking tumor blood supply.

The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.—Musiani, D., Konda, J. D., Pavan, S., Torchiaro, E., Sassi, F., Noghero, A., Erriquez, J., Perera, T., Olivero, M., Di Renzo, M. F. Heat-shock protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibitors and confers resistance to MET-targeted therapy.

We discovered a novel somatic gene fusion, CD74–NRG1, by transcriptome sequencing of 25 lung adenocarcinomas of never smokers. By screening 102 lung adenocarcinomas negative for known oncogenic alterations, we found four additional fusion-positive tumors, all of which were of the invasive mucinous subtype. Mechanistically, CD74–NRG1 leads to extracellular expression of the EGF-like domain of NRG1 III-β3, thereby providing the ligand for ERBB2–ERBB3 receptor complexes. Accordingly, ERBB2 and ERBB3 expression was high in the index case, and expression of phospho-ERBB3 was specifically found in tumors bearing the fusion (P < 0.0001). Ectopic expression of CD74–NRG1 in lung cancer cell lines expressing ERBB2 and ERBB3 activated ERBB3 and the PI3K–AKT pathway, and led to increased colony formation in soft agar. Thus, CD74–NRG1 gene fusions are activating genomic alterations in invasive mucinous adenocarcinomas and may offer a therapeutic opportunity for a lung tumor subtype with, so far, no effective treatment. Significance: CD74–NRG1 fusions may represent a therapeutic opportunity for invasive mucinous lung adenocarcinomas, a tumor with no effective treatment that frequently presents with multifocal unresectable disease. Cancer Discov; 4(4); 415–22. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 377

3062 Background: MET kinase is a therapeutic target in a range of cancer indications; it is a primary oncogenic driver and a mechanism of therapy resistance. OMO-1 is a highly potent, selective oral inhibitor of MET kinase and Organic Cation Transporter 2 (OCT2). Methods: This study assesses the safety, tolerability, pharmacokinetics (PK) and preliminary activity of OMO-1 in patients (pts) with advanced malignancies (NCT03138083). Module 1 data, evaluating ascending doses of OMO-1 monotherapy, are reported here. Results: As of January 16, 2019, 34 pts were enrolled at 5 twice-daily (BD) dose levels of OMO-1: 100, 200, 250, 350, and 400 mg, including 10 with MET gene amplified or mutated tumours. OMO-1 was generally well tolerated between 100 - 250 mg BD; pts were in the study for an average of 94 days (range: 15-291 days) and 20/34 pts discontinued due to disease progression. Most frequently-reported AEs were nausea (17/34), vomiting (14/34) and fatigue (14/34), mainly G1-2. Notably, no peripheral oedema, cardiovascular events or non-malignancy related LFT abnormalities were observed. A total of 36 SAEs were reported: 17 in 11 subjects were considered related to OMO-1, and included nausea (3/17), vomiting (4/17), chills, diarrhoea, influenza-like illness (2/17), increased blood bilirubin, blood creatinine (3/17) and neutrophil count, and sepsis. A dose of 250 mg BD was determined as the recommended Phase 2 dose (RP2D); doses ≥350mg BD were not in keeping with optimum long-term dosing: at 400 mg BD, 2/3 subjects experienced influenza-like illness (G2 and G3) and at 350 mg BD 2/5 subjects had G2 fatigue and nausea/vomiting. OMO-1 has a half-life of 2.5-3 hrs and plasma exposure is dose-proportional without accumulation. Elevated creatinine was observed across all dose levels, consistent with OCT2 inhibition. IHC analysis on paired tumour biopsies from a MET-mutated NSCLC pt dosed at 200 mg BD showed near-complete inhibition of phosphorylated MET, without affecting total MET. Conclusions: OMO-1 has a favourable safety profile at a RP2D of 250mg BD. Expansion cohorts for MET mutated/amplified tumour types are enrolling. Clinical trial information: NCT03138083.

e14674 Background: The MET kinase is an established therapeutic target in a range of cancer indications, being a primary oncogenic and therapy resistance driver. Methods: OMO-1 is a highly potent, selective oral inhibitor of MET kinase and Organic Cation Transporter 2 (OCT2) that is currently being explored in a Phase I/II study (NCT03138083). Results: Pre-clinical data for OMO-1 indicates that anti-tumor efficacy against MET driven tumor models does not require 24/7 exposure in the plasma. PK/PD assessments indicate that OMO-1 efficacy is AUC driven. Exposures above a trough concentration of ~600ng/ml for a period of 6-8hr and an AUC of 3000ng.h/ml result in complete target inhibition sufficient to induce regression or stasis of MET driven in-vivo models. Dosing of OMO-1 at 12.5mg/kg BD 4hr apart, or split over 4 doses 2hr apart, with a dosing-free period of 18-20hr was sufficient to obtain the effects preclinically. The ongoing Phase I/II study in patients with solid tumours utilised learnings from the pre-clinical and FIH study (NCT01964872) where ascending single doses and multiple doses were evaluated. The selected starting dose for the patient study (100mg BD, 4 hr apart) achieved exposures above the predicted MBAD. Paired tumor biopsies were obtained from a higher dose level (200mg BD). A MET exon 14 skipping mutation NSCLC patient at this dosed demonstrated near-complete inhibition of phospho MET accompanied by signs of clinical benefit and lesion shrinkage, thereby matching the preclinical data. Certain ‘class effect’ adverse events, such as edema, were not observed. Conclusions: OMO-1 is an oral, potent MET TKI with a novel dosing regime, identified during preclinical optimization (BD 4hr apart), that demonstrates encouraging signs of clinical activity without certain ‘class-specific’ adverse events. Further evaluation of this differentiated and efficacious agent is warranted and ongoing.

MET oncogene amplification is emerging as a major mechanism of acquired resistance to EGFR-directed therapy in lung and colorectal cancers. Furthermore, MET amplification predicts responsiveness to MET inhibitors currently in clinical trials. Among the anti-MET drugs available, ATP-competitive small-molecule kinase inhibitors abrogate receptor autophosphorylation and downstream activation of ERK1/2 and AKT, resulting in cell-cycle arrest. However, this antiproliferative effect allows persistence of a pool of cancer cells that are quiescent but alive. Once the inhibition is removed, rebound activation of MET-driven cell proliferative pathways and tumor growth may occur, an adverse event observed frequently in clinical settings after drug discontinuation. Here we show that inhibitor withdrawal prompts receptor phosphorylation to levels higher than those displayed at steady-state and generates a rebound effect pushing quiescent cancer cells back into the cell cycle, both in vitro and in experimental tumor models in vivo. Mechanistically, we found that inhibitor treatment blocks MET endocytosis, causing a local increase in the number of receptors at the plasma membrane. Upon inhibitor washout, the receptor is readily rephosphorylated. The initial phosphorylation is not only increased but also prolonged in duration due to downmodulation of a phosphatase-mediated MET-negative feedback loop, which accompanies receptor internalization. Notably, treatment with a MET therapeutic antibody that induces proteolytic cleavage of the receptor at the cell surface substantially prevents this rebound effect, providing a rationale to combine or alternate these mechanistically different types of MET-targeted therapy. Cancer Res; 76(17); 5019–29. ©2016 AACR.

// Lucia D’Amico 1, 2 , Dimas Belisario 2 , Giorgia Migliardi 3 , Cristina Grange 4 , Benedetta Bussolati 5 , Patrizia D’Amelio 6 , Timothy Perera 7 , Ettore Dalmasso 8 , Luca Dalle Carbonare 9 , Laura Godio 10 , Paolo Comoglio 3 , Livio Trusolino 3 , Riccardo Ferracini 11 , Ilaria Roato 2 1 Department of Biomedicine, Cancer Immunology, University of Basel, Basel, Switzerland 2 CeRMS, A.O. Citta della Salute e della Scienza, Torino, Italy 3 IRCC, Institute for Cancer Research and Treatment, Candiolo, Torino, Italy 4 Department of Medical Sciences, University of Turin, Torino, Italy 5 Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Center, University of Turin, Torino, Italy 6 Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy 7 Janssen Research and Development, Beerse, Belgium 8 Urology Section, A.O. Citta della Salute e della Scienza, Torino, Italy 9 Clinic of Internal Medicine, Section D, Policlinico G.B. Rossi Verona, Verona, Italy 10 Department of Pathology, A.O. Citta della Salute e della Scienza, Torino, Italy 11 Department of Orthopaedic Oncology, CTO Hospital, Torino, Italy Correspondence to: Ilaria Roato, email: ilaria.roato@unito.it Keywords: bone metastasis, renal cancer, cancer stem cells, c-MET, CCL20 Received: May 01, 2016 Accepted: May 29, 2016 Published: June 14, 2016 ABSTRACT Cancer stem cells (CSCs) are key players in bone metastasis. In some renal tumors CSCs overexpress the HGF receptor c-MET, speculating that c-MET targeting could lead to bone metastasis inhibition. To address this hypothesis we isolated renal CD105+/CD24−CSCs, expressing c-MET receptor from a primary renal carcinoma. Then, to study their ability to metastasize to bone, we injected renal CSCs in NOD/SCID mice implanted with a human bone and we tested the effect of a c-MET inhibitor (JNJ-38877605) on bone metastasis development. JNJ-38877605 inhibited the formation of metastases at bone implant site. We showed that JNJ-38877605 inhibited the activation of osteoclasts induced by RCC stem cells and it stimulated osteoblast activity, finally resulting in a reduction of bone turnover consistent with the inhibition of bone metastases. We measured the circulating levels of osteotropic factors induced by RCC stem cells in the sera of mice treated with c-Met inhibitor, showing that IL-11 and CCL20 were reduced in mice treated with JNJ-38877605, strongly supporting the involvement of c-MET in the regulation of this process. To address the clinical relevance of c-MET upregulation during tumor progression, we analysed c-MET in renal cancer patients detecting an increased expression in the bone metastatic lesions by IHC. Then, we dosed CCL20 serum levels resulting significantly increased in patients with bone metastases compared to non-metastatic ones. Collectively, our data highlight the importance of the c-MET pathway in the pathogenesis of bone metastases induced by RCC stem cells in mice and humans.

Purpose: We determined the gene copy numbers for MET, for its transcriptional activator MACC1 and for its ligand hepatocyte growth factor (HGF) in liver metastases from colorectal carcinoma (mCRC). We correlated copy numbers with mRNA levels and explored whether gain and/or overexpression of MET and MACC1 predict response to anti-Met therapies. Finally, we assessed whether their genomic or transcriptional deregulation correlates with pathologic and molecular parameters of aggressive disease. Experimental Design: One hundred three mCRCs were analyzed. Copy numbers and mRNA were determined by quantitative PCR (qPCR). Thirty nine samples were implanted and expanded in NOD (nonobese diabetic)/SCID (severe combined immunodeficient) mice to generate cohorts that were treated with the Met inhibitor JNJ-38877605. In silico analysis of MACC1 targets relied on genome-wide mapping of promoter regions and on expression data from two CRC datasets. Results: No focal, high-grade amplifications of MET, MACC1, or HGF were detected. Chromosome 7 polysomy and gain of the p-arm were observed in 21% and 8% of cases, respectively, and significantly correlated with higher expression of both Met and MACC1. Met inhibition in patient-derived xenografts did not modify tumor growth. Copy number gain and overexpression of MACC1 correlated with unfavorable pathologic features better than overexpression of Met. Bioinformatic analysis of putative MACC1 targets identified elements besides Met, whose overexpression cosegregated with aggressive forms of colorectal cancer. Conclusions: Experiments in patient-derived xenografts suggest that mCRCs do not rely on Met genomic gain and/or overexpression for growth. On the basis of pathologic correlations and bioinformatic analysis, MACC1 could contribute to CRC progression through mechanisms other than or additional to Met transcriptional upregulation. Clin Cancer Res; 17(10); 3146–56. ©2011 AACR.

Background Ionizing radiation (IR) is effectively used in cancer therapy. However, in subsets of patients, a few radioresistant cancer cells survive and cause disease relapse with metastatic progression. The MET oncogene encodes the hepatocyte growth factor (HGF) receptor and is known to drive "invasive growth", a regenerative and prosurvival program unduly activated in metastasis. Methods Human tumor cell lines (MDA-MB-231, MDA-MB-435S, U251) were subjected to therapeutic doses of IR. MET mRNA, and protein expression and signal transduction were compared in treated and untreated cells, and the involvement of the DNA-damage sensor ataxia telangiectasia mutated (ATM) and the transcription factor nuclear factor kappa B (NF-κB) in activating MET transcription were analyzed by immunoblotting, chromatin immunoprecipitation, and use of NF-κB silencing RNA (siRNA). Cell invasiveness was measured in wound healing and transwell assays, and cell survival was measured in viability and clonogenic assays. MET was inhibited by siRNA or small-molecule kinase inhibitors (PHA665752 or JNJ-38877605). Combinations of MET-targeted therapy and radiotherapy were assessed in MDA-MB-231 and U251 xenografts (n = 5-6 mice per group). All P values were from two-sided tests. Results After irradiation, MET expression in cell lines was increased up to fivefold via activation of ATM and NF-κB. MET overexpression increased ligand-independent MET phosphorylation and signal transduction, and rendered cells more sensitive to HGF. Irradiated cells became more invasive via a MET-dependent mechanism that was further enhanced in the presence of HGF. MET silencing by siRNA or inhibition of its kinase activity by treatment with PHA665752 or JNJ-38877605 counteracted radiation-induced invasiveness, promoted apoptosis, and prevented cells from resuming proliferation after irradiation in vitro. Treatment with MET inhibitors enhanced the efficacy of IR to stop the growth of or to induce the regression of xenografts (eg, at day 13, U251 xenografts, mean volume increase relative to mean tumor volume at day 0: vehicle = 438%, 5 Gy IR = 151%, 5 Gy IR + JNJ-38877605 = 76%; difference, IR vs JNJ-38877604 + IR = 75%, 95% CI = 59% to 91%, P = .01). Conclusion IR induces overexpression and activity of the MET oncogene through the ATM-NF-κB signaling pathway; MET, in turn, promotes cell invasion and protects cells from apoptosis, thus supporting radioresistance. Drugs targeting MET increase tumor cell radiosensitivity and prevent radiation-induced invasiveness.

Activation of the MET/HGF pathway has been linked to tumor initiation, metastasis, angiogenesis and resistance to therapeutic agents. Here we present pharmacological characterization of OMO-1 (formerly JNJ-38877618), a potent, highly selective, orally bioavailable MET kinase inhibitor with nM binding affinity (Kd=1.4 nM) and enzyme inhibitory activity against wt and M1268T mutant MET (2 and 3 nM IC50). MET inhibitory effects were assessed in proliferation, colony formation and motility assays. OMO-1 displayed nM potency against MET Ampl/mutant and therapy resistant models. In vivo, OMO-1 induced complete inhibition of tumor growth in 3 models: the SNU5 MET amp gastric, U87-MG HGF autocrine glioblastoma and Hs746T MET exon 14 skipping mutant gastric cancer. OMO-1 induced regression of large MET amplified EBC-1 SqNSCLC where OMO-1 led to dose- and time-dependent inhibition of MET kinase activation, with the duration of target shut down considerably exceeding plasma exposure times. Combination treatments were well tolerated and improved EGFR targeted therapy. Although single agent OMO-1 had no effect on NSCLC HCC827 EGFR, combination with Erlotinib led to delayed onset of tumor recurrence. The acquired EGFR inhibitor resistant model HCC827-ER1 was determined to be MET amplified. OMO-1 and erlotinib both inhibited tumor growth of this model whilst combination induced tumor regression. In an EGFR inhibitor resistant PDX having MET amplification, single agent OMO-1 caused tumour stasis whereas MetMab/erlotinib only led to tumor growth delay. The potent preclinical activity we have observed, supports ongoing clinical development of OMO-1 in patients with MET pathway-driven tumors. Citation Format: Marion Libouban, Eleonora Jovcehva, Desiree De Lange, Boudewijn Janssens, Tinne Verhulst, Souichi Ogata, Berthold Wroblowski, Laurence Mevellec, Tianbao Lu, Glen Clack, Timothy Perera. OMO-1, a potent, highly selective, orally bioavailable, MET kinase inhibitor with a favorable preclinical toxicity profile, shows both monotherapy activity, against MET pathway-driven tumors, and EGFR TKI combination activity in acquired resistance 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 4791.

Cell-based drug screenings indicate that tumors displaying c-MET gene amplification are “addicted” to MET signaling and therefore are very sensitive to MET-targeted agents. However, these screenings were conducted in the absence of the MET ligand, hepatocyte growth factor (HGF), which is abundant in the tumor microenvironment. Sensitivity of six MET-addicted human tumor cells to three MET kinase inhibitors (JNJ-38877605, PHA-665752, crizotinib) and one antagonistic anti-MET antibody (DN30 Fab) was analyzed in the absence or presence of HGF, in a stroma–tumor coculture system, and by combining anti-MET drugs with an HGF neutralizing antibody (ficlatuzumab) in human HGF knock-in mice bearing c-MET–amplified tumors. In all models examined, HGF promoted resistance to MET-targeted agents, affecting both their potency and efficacy. HGF-induced resistance was due to restoration of physiologic GAB1–mediated PI3K activation that compensated for loss of aberrant HER3-dependent PI3K signaling. Ficlatuzumab restored sensitivity to MET-targeted agents in coculture systems and overcame resistance to JNJ-38877605, crizotinib, and DN30 Fab in human HGF knock-in mice. These data suggest that c-MET–amplified tumor cells—which normally exhibit ligand-independent, constitutive MET activation—become dependent on HGF for survival upon pharmacologic MET inhibition. Because HGF is frequently overexpressed in human cancer, this mechanism may represent a major cause of resistance to anti-MET therapies. The ability of ficlatuzumab to overcome HGF-mediated resistance generates proof of principle that vertical inhibition of both a tyrosine kinase receptor and its ligand can be therapeutically beneficial and opens new perspectives for the treatment of MET-dependent tumors. Cancer Res; 74(22); 6598–609. ©2014 AACR.

Metastatic colorectal cancer remains largely incurable, although in a subset of patients, survival is prolonged by new targeting agents such as anti-EGF receptor (anti-EGFR) antibodies. This disease is believed to be supported by a subpopulation of stem-like cells termed colon cancer–initiating cell (CCIC), which may also confer therapeutic resistance. However, how CCICs respond to EGFR inhibition has not been fully characterized. To explore this question, we systematically generated CCICs through spheroid cultures of patient-derived xenografts of metastatic colorectal cancer. These cultures, termed “xenospheres,” were capable of long-term self-propagation in vitro and phenocopied the original patient tumors in vivo, thus operationally defining CCICs. Xenosphere CCICs retained the genetic determinants for EGFR therapeutic response in vitro and in xenografts; like the original tumors, xenospheres harboring a mutated KRAS gene were resistant to EGFR therapy, whereas those harboring wild-type RAS pathway genes (RASwt) were sensitive. Notably, the effects of EGFR inhibition in sensitive CCICs could be counteracted by cytokines secreted by cancer-associated fibroblasts. In particular, we found that the MET receptor ligand hepatocyte growth factor (HGF) was especially active in supporting in vitro CCIC proliferation and resistance to EGFR inhibition. Ectopic production of human HGF in CCIC xenografts rendered the xenografts susceptible to MET inhibition, which sensitized the response to EGFR therapy. By showing that RASwt CCICs rely on both EGFR and MET signaling, our results offer a strong preclinical proof-of-concept for concurrent targeting of these two pathways in the clinical setting. Cancer Res; 74(6); 1857–69. ©2014 AACR.

A protein (SNP70) has been isolated that binds to the Src homology domain 3 of p47phox, p85α, and c-src. Cloning and sequencing of the polypeptide revealed it to be a 70-kDa protein that has a number of potential domains, including Src homology 3 binding motifs and several nuclear localization signals. Immunofluorescence using anti-peptide antibodies revealed SNP70 to be primarily concentrated in the nucleus but excluded from nucleoli, in interphase cells. However, it was distributed throughout the cytoplasm in dividing cells. Extraction and subfractionation experiments indicated that SNP70 did not bind directly to DNA but did bind to poly(G)-rich oligonucleotides and was resistant to extraction with non-ionic detergents but was solubilized by treatment with RNase, high salt, or ammonium sulfate. Double-immunofluorescence experiments showed that SNP70 co-localized with two pre-mRNA splicing factors SC35 and U2B" within the nucleus. A population of SNP70 was found outside the nucleus, and double-immunofluorescence and immunoelectron microscopy demonstrated that it associated with vimentin-containing intermediate filaments, particularly those surrounding the nucleus. The data suggest that SNP70 associates with nuclear or perinuclear filaments and may play a role in the regulation of pre-mRNA processing.

EGF receptor (EGFR)-targeted monoclonal antibodies are effective in a subset of metastatic colorectal cancers. Inevitably, all patients develop resistance, which occurs through emergence of KRAS mutations in approximately 50% of the cases. We show that amplification of the MET proto-oncogene is associated with acquired resistance in tumors that do not develop KRAS mutations during anti-EGFR therapy. Amplification of the MET locus was present in circulating tumor DNA before relapse was clinically evident. Functional studies show that MET activation confers resistance to anti-EGFR therapy both in vitro and in vivo. Notably, in patient-derived colorectal cancer xenografts, MET amplification correlated with resistance to EGFR blockade, which could be overcome by MET kinase inhibitors. These results highlight the role of MET in mediating primary and secondary resistance to anti-EGFR therapies in colorectal cancer and encourage the use of MET inhibitors in patients displaying resistance as a result of MET amplification. Significance: Amplification of the MET proto-oncogene is responsible for de novo and acquired resistance to anti-EGFR therapy in a subset of colorectal cancers. As multiple anti-MET therapeutic strategies are available, these findings offer immediate novel opportunities to design clinical studies. Cancer Discov; 3(6); 658–73. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 591

Basal-like breast cancer is an aggressive subtype of mammary carcinoma. Despite expressing basal markers, typical of mammary stem cells, this tumor has been proposed to originate from luminal progenitors, which are downstream of stem cells along the mammary epithelial hierarchy. This suggests that committed luminal progenitors may reacquire basal, stem-like characteristics, but the mechanisms that regulate this transition remain unclear. Using mouse models, we found that luminal progenitors express high levels of the Met receptor for hepatocyte growth factor (HGF), as compared with the other mammary epithelial sub-populations. Constitutive activation of Met led luminal progenitors to attain stem cell properties, including enhanced clonogenic activity in vitro and de novo ability to reconstitute mammary glands in repopulation assays in vivo. Moreover, in response to Met signaling, luminal progenitors gave rise to hyperplastic ductal morphogenesis and preferentially underwent basal lineage commitment at the expense of luminal cell-fate specification. Opposite and symmetric results were produced by systemic pharmacological inhibition of Met. Hence, Met signaling targets luminal progenitors for expansion, impairs their differentiation toward the mature luminal phenotype and enables their commitment toward the basal lineage. These results emphasize a critical role for Met in promoting deregulated proliferation and basal plasticity of normal luminal progenitors in the mammary gland, a complex of events that may be required for sustaining the functional and phenotypic properties of basal-like breast tumors.

The MET oncogene is amplified in a fraction of human gastric carcinoma cell lines, with consequent overexpression and constitutive activation of the corresponding protein product, the Met tyrosine kinase receptor. This genetically driven hyperactivation of Met is necessary for cancer cell growth and survival, so that Met pharmacological blockade results in cell-cycle arrest or apoptosis (oncogene addiction). MET gene amplification also occurs in vivo in a number of human gastric carcinomas, and clinical trials are now ongoing to assess the therapeutic efficacy of Met inhibitors in this type of malignancy. The aim of our study was to identify a preclinical algorithm of soluble surrogate biomarkers indicative of response to Met inhibition in gastric tumors, as a potential tool to integrate imaging criteria during patient follow-up. We started from a survey of candidate molecules based on antibody proteomics and gene expression profiling; after ELISA validation and analytical quantification, four biomarkers were identified that appeared to be strongly and consistently modulated by Met inhibition in a panel of Met-addicted gastric carcinoma cell lines, but not in Met-independent cell lines. Pharmacologic blockade of Met using specific small-molecule inhibitors led to reduced secretion of IL-8, GROα and the soluble form of uPAR and to increased production of IL-6 both in vitro (in culture supernatants) and in vivo (in the plasma of xenografted mice). If confirmed in patients, this information might prove useful to monitor clinical response to Met-targeted therapies in MET-amplified gastric carcinomas.

Background Tau protein is the principal component of the neurofibrillary tangles found in Alzheimer's disease, where it is hyperphosphorylated on serine and threonine residues, and recently phosphotyrosine has been demonstrated. The Src-family kinase Fyn has been linked circumstantially to the pathology of Alzheimer's disease, and shown to phosphorylate Tyr18. Recently another Src-family kinase, Lck, has been identified as a genetic risk factor for this disease. Results In this study we show that Lck is a tau kinase. In vitro, comparison of Lck and Fyn showed that while both kinases phosphorylated Tyr18 preferentially, Lck phosphorylated other tyrosines somewhat better than Fyn. In co-transfected COS-7 cells, mutating any one of the five tyrosines in tau to phenylalanine reduced the apparent level of tau tyrosine phosphorylation to 25-40% of that given by wild-type tau. Consistent with this, tau mutants with only one remaining tyrosine gave poor phosphorylation; however, Tyr18 was phosphorylated better than the others. Conclusions Fyn and Lck have subtle differences in their properties as tau kinases, and the phosphorylation of tau is one mechanism by which the genetic risk associated with Lck might be expressed pathogenically.

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: JNJ-42756493 is an FGFR 1, 2, 3, and 4 inhibitor with nanomolar affinity, orally bioavailable, and has demonstrated a broad spectrum antitumor activity in cell line, xenograft and patient-derived explant models with abnormality in FGFR signaling pathway such as FGFR gene amplification, mutation and translocation. Methods: A multipart phase 1 first in human study of JNJ-42756493 was initiated in advanced solid tumor patients ([NCT01962532][1]) including, Part 1 dose-escalation to determine the recommended phase 2 dose (RP2D) based on safety, pharmacokinetic, and pharmacodynamic data; Part 2 biopsy cohort to confirm the RP2D and Part 3 expansion phase to evaluate anti-tumor activity in NSCLC, SCLC, Breast cancer and other solid tumor patients with FGFR gene amplification, mutation or translocation at RP2D. Multiple biomarkers from tissue (including phospho-FGFR, phospho-Erk and phospho-S6) and serum (phosphate, calcium, Vitamin D, PTH and FGF23) were also assessed in the study. Results: At data cut-off (22 October 2013), total of 28 patients had been treated at 5 dose levels (0.5, 2, 4, 6 and 9 mg daily continuously) in Part 1 of the study. JNJ-42756493 appeared safe, did not generate any dose-limiting toxicities or any drug related severe adverse events. One patient from 4 mg cohort died after receiving 6 doses in Cycle 1, autopsy indicated a serious adverse event (SAE) of pulmonary edema which not related to the study drug. The most common adverse events (AEs) were hyperphosphatemia (57%), asthenia (46%), dry mouth (32%), abdominal pain (29%), diarrhea (25%), vomiting (25%), decreased appetite (21%) and constipation (21%). Grade 1 or 2 hyperphosphatemia was managed by co-administration of phosphate lowering therapy and by treatment interruption, other AEs were generally mild to moderate. No clinically significant cardiac safety findings were observed. Pharmacokinetics was linear and predictable with a half-life of ∼50 hours. Dose levels ≥ 6mg achieved plasma concentrations predicted to be efficacious from preclinical experiments. Dose dependent changes in biomarkers including increases in phosphate, FGF23 and calcium and decreases in PTH were observed in blood, evaluation of biomarker response in tissue is ongoing. A bladder cancer patient with lung metastasis harboring a FGFR3-TACC3 translocation treated at 9 mg had a confirmed PR. 9 mg was declared as the first RP2D, but safety evaluation of JNJ-42756493 at higher doses is ongoing. Conclusions: JNJ-42756493 has excellent pharmaceutical properties and appeared safe with manageable side effects at dose levels that elicit anti-tumor activity. Citation Format: Rodrigo Dienstmann, Rastilav Bahleda, Barbara Adamo, Jordi Rodon, Andrea Varga, Anas Gazzah, Suso Platero, Hans Smit, Timothy Perera, Bob Zhong, Kim Stuyckens, Yusri Elsayed, Chris Takimoto, Vijay Peddareddigari, Josep Tabernero, Feng Roger Luo, Jean-Charles Soria. First in human study of JNJ-42756493, a potent pan fibroblast growth factor receptor (FGFR) inhibitor in patients with advanced solid tumors. [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 CT325. doi:10.1158/1538-7445.AM2014-CT325 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01962532&atom=%2Fcanres%2F74%2F19_Supplement%2FCT325.atom