Hepatocyte Growth Factor Receptor Met Research Articles

Spatiotemporal regulation of the hepatocyte growth factor receptor MET activity by sorting nexins 1/2 in HCT116 colorectal cancer cells.

Cumulative research findings support the idea that endocytic trafficking is crucial in regulating receptor signaling and associated diseases. Specifically, strong evidence points to the involvement of sorting nexins (SNXs), particularly SNX1 and SNX2, in the signaling and trafficking of the receptor tyrosine kinase (RTK) MET in colorectal cancer (CRC). Activation of hepatocyte growth factor (HGF) receptor MET is a key driver of CRC progression. In the present study, we utilized human HCT116 CRC cells with SNX1 and SNX2 genes knocked out to demonstrate that their absence leads to a delay in MET entering early endosomes. This delay results in increased phosphorylation of both MET and AKT upon HGF stimulation, while ERK1/2 (extracellular signal-regulated kinases 1 and 2) phosphorylation remains unaffected. Despite these changes, HGF-induced cell proliferation, scattering, and migration remain similar between the parental and the SNX1/2 knockout cells. However, in the absence of SNX1 and SNX2, these cells exhibit increased resistance to TRAIL-induced apoptosis. This research underscores the intricate relationship between intracellular trafficking, receptor signaling, and cellular responses and demonstrates for the first time that the modulation of MET trafficking by SNX1 and SNX2 is critical for receptor signaling that may exacerbate the disease.

A Randomized, Open-Label Phase II Study Evaluating Emibetuzumab Plus Erlotinib and Emibetuzumab Monotherapy in MET Immunohistochemistry Positive NSCLC Patients with Acquired Resistance to Erlotinib

IntroductionThe hepatocyte growth factor receptor MET represents a resistance mechanism to epidermal growth factor receptor (EGFR) inhibition in EGFR mutant (mt) non–small cell lung cancer (NSCLC). This Phase 2 study tested whether acquired resistance to erlotinib in MET protein positive NSCLC patients enriched for EGFRmt can be overcome by emibetuzumab plus erlotinib. Patient and MethodsPatients with Stage IV NSCLC with acquired resistance to erlotinib and MET diagnostic (+) (≥ 10% of cells expressing MET at ≥ 2+ IHC staining intensity at any time) were randomized (3:1) to receive emibetuzumab 750 mg every 2 weeks with or without erlotinib 150 mg once daily. The primary objective was to evaluate the overall response rate (ORR) relative to historic control, with a co-primary objective of ORR in patients with MET expression in ≥ 60% of cells ≥ 2+ (MET ≥ 60%). ResultsOne hundred and eleven MET+ patients received emibetuzumab plus erlotinib (N = 83) or emibetuzumab monotherapy (N = 28). 89 of 111 MET+ samples were post-erlotinib. ORR was 3.0% for emibetuzumab plus erlotinib (95% CI: 0.4, 10.5) and 4.3% for emibetuzumab (95% CI: 0.1, 21.9), in patients with post-erlotinib progression biopsies available (n = 89). Similar results were observed in patients with MET ≥ 60% expression (n = 74). Disease control rate and progression-free survival were higher for emibetuzumab plus erlotinib (50%/3.3 months) than for emibetuzumab (26%/1.6 months). No unexpected safety signals emerged. Partial responses were observed in patients with and without EGFRmt or MET amplification. EGFR sensitizing mutations were identified retrospectively in 84.2% of those with available tissue (85/101). ConclusionAcquired resistance to erlotinib in MET diagnostic (+) patients was not reversed by emibetuzumab plus erlotinib or emibetuzumab monotherapy, although a subset of patients obtained clinical benefit.

De novo peptide grafting to a self-assembling nanocapsule yields a hepatocyte growth factor receptor agonist

SummaryLasso-grafting (LG) technology is a method for generating de novo biologics (neobiologics) by genetically implanting macrocyclic peptide pharmacophores, which are selected in vitro against a protein of interest, into loops of arbitrary protein scaffolds. In this study, we have generated a neo-capsid that potently binds the hepatocyte growth factor receptor MET by LG of anti-MET peptide pharmacophores into a circularly permuted variant of Aquifex aeolicus lumazine synthase (AaLS), a self-assembling protein nanocapsule. By virtue of displaying multiple-pharmacophores on its surface, the neo-capsid can induce dimerization (or multimerization) of MET, resulting in phosphorylation and endosomal internalization of the MET-capsid complex. This work demonstrates the potential of the LG technology as a synthetic biology approach for generating capsid-based neobiologics capable of activating signaling receptors.

CDCP1 promotes compensatory renal growth by integrating Src and Met signaling.

Compensatory growth of organs after loss of their mass and/or function is controlled by hepatocyte growth factor (HGF), but the underlying regulatory mechanisms remain elusive. Here, we show that CUB domain-containing protein 1 (CDCP1) promotes HGF-induced compensatory renal growth. Using canine kidney cells as a model of renal tubules, we found that HGF-induced temporal up-regulation of Src activity and its scaffold protein, CDCP1, and that the ablation of CDCP1 robustly abrogated HGF-induced phenotypic changes, such as morphological changes and cell growth/proliferation. Mechanistic analyses revealed that up-regulated CDCP1 recruits Src into lipid rafts to activate STAT3 associated with the HGF receptor Met, and activated STAT3 induces the expression of matrix metalloproteinases and mitogenic factors. After unilateral nephrectomy in mice, the Met-STAT3 signaling is transiently up-regulated in the renal tubules of the remaining kidney, whereas CDCP1 ablation attenuates regenerative signaling and significantly suppresses compensatory growth. These findings demonstrate that CDCP1 plays a crucial role in controlling compensatory renal growth by focally and temporally integrating Src and Met signaling.

Generation of the heterogeneity of extracellular vesicles by membrane organization and sorting machineries

BackgroundCells secrete heterogeneous populations of extracellular vesicles (EVs) via unknown mechanisms. EV biogenesis has been postulated to involve lipid-protein clusters, also known as membrane microdomains. MethodsMembrane properties and heterogeneity of melanoma-derived EVs were analyzed by a detergent solubilization assay, sucrose density gradient ultracentrifugation and immunoprecipitation. EV secretion was modulated by RNA interference and pharmacological treatments. ResultsWe identified two EV membranes (low-density exosomal detergent-insoluble membranes [EV-DIMs]; EV detergent-soluble membranes [EV-DSMs]) and discovered an abundant, novel type of high-density EV-DIMs. The high-density EV-DIMs accumulated the microdomain-resident protein flotillin-1, as well as a disintegrin and metalloproteinase domain containing protein 10 (Adam10), the hepatocyte growth factor receptor Met and its proteolytic fragments. Low-density EV-DIMs also contained flotillin-1. EV-DSMs were enriched with tetraspanin CD81, melanogenic enzymes and proteolytic fragments of Adam10. Intact and fragmented forms of Adam10, which resided in distinct membrane types, were secreted by different EVs. The fragmented form of Met was associated with DIMs much more efficiently than the intact form and they were secreted by distinct EVs. We identified that the endosomal sorting complexes required for transport machinery was indispensable for EV secretion of both mature and fragmented forms of Adam10 and Met. ConclusionThe findings of this study reveal the role of the interplay between membrane organization and sorting machineries in generating the heterogeneity of EVs. General significanceThis study provides novel insights into important aspects of EV biogenesis.

Abstract 1830: A clinical paired resistant model elucidated novel dual salvage signaling that confers alectinib resistance in ALK-rearranged lung cancer

Abstract Conventional drug-resistant cancer cell line models have contributed to the elucidation of drug-resistant mechanisms. However, whether or not these models reflect patients in clinical settings is often controversial. We herein report a patient-derived drug resistant model system named the “clinical paired resistant model”. This model consists of 2 cell lines derived from a treatment-naïve patient (drug sensitive cell line model) and again after disease progression (drug resistant cell line model). The clinical paired resistant model of alectinib, the second generation ALK inhibitor, revealed the properties that cancer cells acquired during treatment using phosphoproteome and immunoblotting analyses; the proto-oncogene protein tyrosine kinase Src and hepatocyte growth factor receptor MET were activated after the development of alectinib resistance. No secondary mutations were detected in the coding region of ALK tyrosine kinase in the alectinib resistant model. In this alectinib-resistant model, the inhibition of Src and MET using saracatinib and PHA-665752 significantly restored alectinib sensitivity in vitro (17.2-fold change in IC50). Downstream signaling molecules for proliferation and survival, phosphorylation of Akt and ERK1/2, were inhibited and caspase 3/7 activity was significantly increased when the cells were treated with all three inhibitors (saracatinib, PHA-665752, and alectinib). Combined knockdown of SRC and MET restored alectinib sensitivity and inhibited downstream signaling in combination with ALK inhibition using alectinib, suggesting that the dual salvage signaling of MET and Src conferred alectinib resistance. A xenograft generated from our paired resistant model (N=5-6, in each group) indicated that combination therapy with a saracatinib and crizotinib, the first generation ALK inhibitor which also inhibits MET, significantly decreased tumor size in vivo as compared with saracatinib or crizotinib monotherapy. We also established a conventional alectinib resistant cell line model in vitro by exposing NCI-H2228 cells (EML4-ALK variant 3a/b) to 300 nM of alectinib for 3 months and found MET and Src were also activated in the model. Our clinical paired resistant model permits the detection of drug-resistant mechanisms without exploring the common characteristics of numerous drug-resistant patients. Our results demonstrate that MET and Src are potential therapeutic targets in patients with alectinib resistance and that the clinical paired resistant model may be a new strategy to elucidate drug-resistant mechanisms in relatively rare cancers. Citation Format: Takahiro Tsuji, Hiroaki Ozasa, Takashi Nomizo, Tomoko Funazo, Yuto Yasuda, Yuichi Sakamori, Hironori Yoshida, Kiyoshi Uemasu, Hitomi Ajimizu, Ryoko Okutani, Shunsuke Aburaya, Wataru Aoki, Mitsuyoshi Ueda, Koh Furugaki, Yasushi Yoshimura, Toyohiro Hirai, Young Hak Kim. A clinical paired resistant model elucidated novel dual salvage signaling that confers alectinib resistance in ALK-rearranged lung cancer [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 1830.

Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells

A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with potential as drug targets in BRAFi refractory melanoma.

The MET/Vascular Endothelial Growth Factor Receptor (VEGFR)-targeted Tyrosine Kinase Inhibitor Also Attenuates FMS-dependent Osteoclast Differentiation and Bone Destruction Induced by Prostate Cancer

The tyrosine kinase inhibitor TAS-115 that blocks VEGF receptor and hepatocyte growth factor receptor MET signaling exhibits antitumor properties in xenografts of human gastric carcinoma. In this study, we have evaluated the efficacy of TAS-115 in preventing prostate cancer metastasis to the bone and bone destruction using the PC3 cell line. When PC3 cells were injected into proximal tibiae in nude mouse, severe trabecular and cortical bone destruction and subsequent tumor growths were detected. Oral administration of TAS-115 almost completely inhibited both PC3-induced bone loss and PC3 cell proliferation by suppressing osteoclastic bone resorption. In an ex vivo bone organ culture, PC3 cells induced osteoclastic bone resorption when co-cultured with calvarial bone, but TAS-115 effectively suppressed the PC3-induced bone destruction. We found that macrophage colony-stimulating factor-dependent macrophage differentiation and subsequent receptor activator of NF-κB ligand-induced osteoclast formation were largely suppressed by adding TAS-115. The phosphorylation of the macrophage colony-stimulating factor receptor FMS and osteoclast related kinases such as ERK and Akt were also suppressed by the presence of TAS-115. Gene expression profiling showed that FMS expression was only seen in macrophage and in the osteoclast cell lineage. Our study indicates that tyrosine kinase signaling in host pre-osteoclasts/osteoclasts is critical for bone destruction induced by tumor cells and that targeting of MET/VEGF receptor/FMS activity makes it a promising therapeutic candidate for the treatment of prostate cancer patients with bone metastasis.

MET expression during prostate cancer progression.

Tyrosine-kinase inhibitors of the hepatocyte growth factor receptor MET are under investigation for the treatment of hormone-refractory prostate cancer (HRPC) metastasis. Analysis of MET protein expression and genetic alterations might contribute to therapeutic stratification of prostate cancer patients. Our objective was to investigate MET on protein, DNA and RNA level in clinical prostate cancer at various stages of progression.Expression of MET was analyzed in hormone-naive primary prostate cancers (N=481), lymph node (N=40) and bone (N=8) metastases, as well as HRPC (N=54) and bone metastases (N=15). MET protein expression was analyzed by immunohistochemistry (D1C2 C-terminal antibody). MET mRNA levels and MET DNA copy numbers were determined by in situ hybridization.None of the hormone-naive primary prostate cancer or lymph node metastases demonstrated MET protein or mRNA expression. In contrast, MET protein was expressed in 12/52 (23%) evaluable HRPC resections. RNA in situ demonstrated cytoplasmic signals in 14/54 (26%) of the HRPC patients, and was associated with MET protein expression (p=0.025, χ2), in absence of MET amplification or polysomy. MET protein expression was present in 7/8 (88%) hormone-naive and 10/15 (67%) HRPC bone metastases, without association of HRPC (p=0.37; χ2), with MET polysomy in 8/13 (61%) evaluable cases.In conclusion, MET was almost exclusively expressed in HRPC and prostate cancer bone metastasis, but was not related to MET amplification or polysomy. Evaluation of MET status could be relevant for therapeutic stratification of late stage prostate cancer.

Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury.

The enteric nervous system has many neuronal subtypes that coordinate and control intestinal activity. Trophic factors that support these neuron types and enhance neurite growth after fetal development are not well understood. We show that a subset of adult calcitonin gene-related peptide (CGRP)-expressing myenteric neurons produce MET, the receptor for hepatocyte growth factor, and that loss of MET activity affects peristalsis in response to mucosal stroking, reduces MET-immunoreactive neurites, and increases susceptibility to dextran sodium sulfate-induced bowel injury. These observations may be relevant for understanding and treating intestinal motility disorders and also suggest that enhancing the activity of MET-expressing CGRP neurons might be a useful strategy to reduce bowel inflammation.

Activated Platelets Interfere with Recruitment of Mesenchymal Stem Cells to Apoptotic Cardiac Cells via High Mobility Group Box 1/Toll-like Receptor 4-mediated Down-regulation of Hepatocyte Growth Factor Receptor MET

Recruitment of mesenchymal stem cells (MSC) following cardiac injury, such as myocardial infarction, plays a critical role in tissue repair and may contribute to myocardial recovery. However, the mechanisms that regulate migration of MSC to the site of tissue damage remain elusive. Here, we demonstrate in vitro that activated platelets substantially inhibit recruitment of MSC toward apoptotic cardiac myocytes and fibroblasts. The alarmin high mobility group box 1 (HMGB1) was released by platelets upon activation and mediated inhibition of the cell death-dependent migratory response through Toll-like receptor (TLR)-4 expressed on the MSC. Migration of MSC to apoptotic cardiac myocytes and fibroblasts was driven by hepatocyte growth factor (HGF), and platelet activation was followed by HMGB1/TLR-4-dependent down-regulation of HGF receptor MET on MSC, thereby impairing HGF-driven MSC recruitment. We identify a novel mechanism by which platelets, upon activation, interfere with MSC recruitment to apoptotic cardiac cells, a process that may be of particular relevance for myocardial repair and regeneration.

Multikinase Inhibitors for the Treatment of Progressive, Metastatic Medullary Thyroid Cancer - An Evolving Paradigm.

Medullary thyroid cancer (MTC) is an uncommon type of thyroid cancer, representing around 4 % of the all thyroid cancers, and is a challenging malignancy. So far, surgery has been the only curative treatment and until recently there have been no effective medications. Within the past 5 years, multi-targeted kinase inhibitors have emerged that have shown convincing efficacy against such tumours. These drugs have changed the landscape in MTC treatment by providing effective medication for the first time. The modes of action of these drugs differ, but most target RET, a tyrosine kinase shown to play an important role in the pathobiology of MTC, as well as other receptors including vascular endothelial growth factor receptors (VEGFRs), epidermal growth factor receptor (EGFR) and the hepatocyte growth factor receptor MET. Two agents in this class, vandetanib and cabozantinib, have demonstrated efficacy and safety in phase III trials and have consequently received regulatory approval. Other therapies for MTC treatment, including some with similar modes of action, are also in early development.

The Novel ATP-Competitive Inhibitor of the MET Hepatocyte Growth Factor Receptor EMD1214063 Displays Inhibitory Activity against Selected MET-Mutated Variants

The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results show a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five of the eight cell lines (IC50 2-43 nmol/L). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell-cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biologic functions, such as cellular morphology, MET-dependent cell motility, and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET-mutated variants. Animals were randomized for the treatment with EMD1214063 (50 mg/kg/d) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, whereas tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small-molecule inhibitor with selective activity towards mutated MET variants.

Met Activation Is Required for Early Cytoprotection after Ischemic Kidney Injury

Renal proximal tubule epithelial cells express high levels of the hepatocyte growth factor receptor Met, and both the receptor and ligand are upregulated after ischemic injury. Activation of the Met receptor after hepatocyte growth factor stimulation in vitro promotes activities involved in kidney repair, including cell survival, migration, and proliferation. However, characterizing the in vivo role of these signaling events in proximal tubule responses to kidney injury has been difficult because global Met knockout results in embryonic lethality due to placental and liver abnormalities. Here, we used γGT-Cre to knockout Met receptor expression selectively in the proximal tubules of mice (γGT-Cre;Met(fl/fl)). The kidneys of these mice developed normally, but exhibited increased initial tubular injury, tubular cell apoptosis, and serum creatinine after ischemia/reperfusion compared with γGT-Cre;Met(+/+) kidneys. These changes in γGT-Cre;Met(fl/fl) mice correlated with a selective reduction in PI3K/Akt activation in response to injury and subsequent decreases in inhibitory phosphorylation of the proapoptotic factor Bad and activating phosphorylation of the ribosomal regulatory protein p70-S6 kinase. Moreover, tubular cell proliferation after ischemia/reperfusion was delayed in γGT-Cre;Met(fl/fl) mice. In conclusion, this study identifies Met-dependent phosphoinositide 3-kinase activation in proximal tubules as a critical determinant of initial tubular cell survival and reparative proliferation after ischemic injury.

Cell Surface Receptor Targeted Biomimetic Apatite Nanocrystals for Cancer Therapy

Nanosized drug carriers functionalized with moieties specifically targeting tumor cells are promising tools in cancer therapy, due to their ability to circulate in the bloodstream for longer periods and their selectivity for tumor cells, enabling the sparing of healthy tissues. Because of its biocompatibility, high bioresorbability, and responsiveness to pH changes, synthetic biomimetic nanocrystalline apatites are used as nanocarriers to produce multifunctional nanoparticles, by coupling them with the chemotherapeutic drug doxorubicin (DOXO) and the DO-24 monoclonal antibody (mAb) directed against the Met/Hepatocyte Growth Factor receptor (Met/HGFR), which is over-expressed on different types of carcinomas and thus represents a useful tumor target. The chemical-physical features of the nanoparticles are fully investigated and their interaction with cells expressing (GTL-16 gastric carcinoma line) or not expressing (NIH-3T3 fibroblasts) the Met/HGFR is analyzed. Functionalized nanoparticles specifically bind to and are internalized in cells expressing the receptor (GTL-16) but not in the ones that do not express it (NIH-3T3). Moreover they discharge DOXO in the targeted GTL-16 cells that reach the nucleus and display cytotoxicity as assessed in an MTT assay. Two different types of ternary nanoparticles are prepared, differing for the sequence of the functionalization steps (adsorption of DOXO first and then mAb or vice versa), and it is found that the ones in which mAb is adsorbed first are more efficient under all the examined aspects (binding, internalization, cytotoxicity), possibly because of a better mAb orientation on the nanoparticle surface. These multifunctional nanoparticles could thus be useful instruments for targeted local or systemic drug delivery, allowing a reduction in the therapeutic dose of the drug and thus adverse side effects. Moreover, this work opens new perspectives in the use of nanocrystalline apatites as a new platform for theranostic applications in nanomedicine.

Trametinib for patients with advanced melanoma – Authors' reply

Gerald Falchook and colleagues show clinical activity of the MEK inhibitor trametinib in patients with either BRAF-mutant or wild-type melanoma. Recently, two essential topics for melanoma have been reported: hepatocyte growth factor (HGF)-dependent resistance to therapy and new driver mutations in melanoma. Stroma-mediated resistance to treatment of BRAF-mutant melanoma is common and HGF secretion from stromal cells seems to be responsible for this drug resistance. Analysis of biopsy samples from patients with BRAF-mutant melanoma suggests that patients with abundant HGF from stromal cells have poor responses to treatment. Furthermore, an inverse relation has been reported between plasma HGF concentration and response to treatment in patients with BRAF-mutant melanoma. HGF secretion leads to activation of the HGF receptor MET, and dual inhibition of RAF and either HGF or MET results in reversal of drug resistance in BRAFmutant melanoma. In Falchook and colleagues’ study, examination by immuno histochemistry of whether HGF expression in melanoma sections correlated with poor responses to the MEK inhibitor trametinib would have been informative, not only in patients with the BRAF mutation, but also in patients with wild-type BRAF. These results would clarify the possibility that a combination of the MEK inhibitor and MET inhibitors have a synergistic eff ect. Moreover, although EGF is less able than HGF to reactivate ERK in most melanoma cell lines, EGF and EGFR mediated resistance might be of interest in melanoma and in colon cancers. For driver mutations, six genes related to melanoma have been newly identifi ed (PPP6C, RAC1, SNX31, TACC1, STK19, and ARID2), with RAC1 mutations also reported. The Illumina platform was used in Falchook and colleagues’ study to analyse the mutational and copy number status of 78 diff erent genes commonly implicated in tumorigenesis. Within these data, is there any information about the six new melanoma genes? If these driver mutations are present, examination of whether they correlate with reduced responses to therapy with trametinib would be interesting, because such examination would not only contribute to drug response predictions but also might help select other treatment options, including combination therapies. Furthermore, data on amplifi cation of BRAF, either with or without mutations, could provide information on whether copy number of this gene plays a role in response to therapy? In the context of treatment with the MEK inhibitor trametinib, an integrated examination including HGF secretion and other gene mutations would open up the possibility of prediction of drug response and combination therapy.

Abstract 1786: Characterization of the inhibitory capacity of EMD1214063, a novel small molecule inhibitor of the MET hepatocyte growth factor receptor on a panel of MET mutated variants

Abstract The receptor tyrosine kinase MET is a prime target in clinical oncology because of its aberrant activation in a broad spectrum of malignancies. Here, we tested the anti tumor activity of a novel MET inhibitor, EMD1214063, on cancer cells overexpressing MET and in eight transfected NIH3T3 cell lines expressing different activating mutants of the MET kinase. Our results demonstrate a dose-dependent decrease in MET autophosphorylation in both MET overexpressing cell lines (IC50 of 20nM and 30nM for GTL-16 and H1993, respectively) and in six of the eight cell lines transfected with MET activating mutants (IC50 5-18nM). Blockade of MET by EMD1214063 resulted in reduced downstream activation of AKT, ERK and PLCγ. In contrast, lack of MET inhibition by EMD1214063 in the resistant Y1248H and L1213V MET mutants was associated with high levels of AKT, ERK and PLCγ activation. In all sensitive mutants, EMD1214063 attenuated MET-dependent cellular proliferation and significantly altered cell cycle distribution profiles (increase in G1 and concomitant decrease of S phase). Furthermore, EMD1214063 strongly affected MET-driven biological functions, such as cellular morphology, motility and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we established tumor transplantation models exploiting NIH3T3 cells expressing the H1112L or L1213V MET mutants, respectively sensitive and resistant, to in vitro treatment with EMD1214063. Animals bearing H1112L or L1213V tumors were randomized for the treatment with EMD1214063 (50mg/kg/day) or vehicle only. Remarkably, 5 days of treatment with EMD1214063 induced complete regression of sensitive H1112L tumors, while tumor growth remained unaffected in mice injected with resistant L1213V tumor cells and in vehicle-treated mice. Taken together, these results underscore the concordance between in vitro and in vivo effects of EMD1214063 and strongly support its efficacy in inhibiting MET-driven tumor cell growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1786. doi:1538-7445.AM2012-1786

Abstract 4837: Tumor microenvironment contributes to RAF-inhibitor resistance through HGF secretion

Abstract Drug resistance remains a vexing problem in the treatment of cancer patients. While many studies have focused on cell autonomous mechanisms of drug resistance, we hypothesized that the tumor microenvironment may confer innate resistance to therapy. We employed a novel co-culture assay to systematically assay the ability of 23 stromal cell types to influence the sensitivity of 45 cancer cell lines to 35 anti-cancer drugs testing more than 15,000 cancer-stroma-drug combinations. We found that stroma-mediated resistance is surprisingly common - particularly to targeted chemotherapies. We decided to further characterize the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibition because most patients exhibit some degree of innate resistance. Proteomic analysis showed that stromal secretion of the growth factor hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the MAPK and PI3K/AKT pathways, and immediate resistance to RAF inhibition. Immunohistochemistry confirmed tumor stromal HGF expression in 11 out of 15 patients with BRAF-mutant melanoma. Among these patients, only one experienced a complete response, and that patient was negative for stromal HGF, consistent with our prediction of HGF-mediated RAF-inhibitor resistance. In contrast, of 6 evaluable patients with stable disease 4 had particularly high levels of stromal HGF expression. Dual inhibition of RAF and MET in BRAF-mutant melanoma cell lines resulted in reversal of drug resistance, suggesting RAF/MET combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. Interestingly, the activation of MET in these cell lines was cell autonomous, caused, for example, by autocrine secretion of HGF and resulting in complete resistance to RAF inhibitors even in the absence of stromal cells. Treatment with a MET inhibitor re-sensitized these cell lines to RAF inhibitors suggesting the same RAF/MET combination therapy for non-melanoma BRAF mutant cancers as well. More generally, these studies indicate that the systematic dissection of tumor-microenvironment interactions may reveal important mechanisms underlying drug resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4837. doi:1538-7445.AM2012-4837

Abstract 1731: TAK-701, a humanized monoclonal antibody to HGF, reverses gefitinib resistance induced by tumor-derived HGF in non-small cell lung cancer with an EGFR mutation

Abstract Most non-small cell lung cancer (NSCLC) tumors with an activating mutation of the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib but ultimately develop resistance to these drugs. Hepatocyte growth factor (HGF) induces EGFR-TKI resistance in NSCLC cells with such a mutation. We investigated strategies to overcome gefitinib resistance induced by HGF. Human NSCLC cells with an activating EGFR mutation (HCC827 cells) were engineered to stably express HGF (HCC827-HGF cells). HCC827-HGF cells secreted large amounts of HGF and exhibited resistance to gefitinib in vitro to an extent similar to that of HCC827 GR cells, in which the gene for the HGF receptor MET is amplified. A MET-TKI reversed gefitinib resistance in HCC827-HGF cells as well as in HCC827 GR cells, suggesting that MET activation induces gefitinib resistance in both cell lines. TAK-701, a humanized monoclonal antibody to HGF, in combination with gefitinib inhibited the phosphorylation of MET, EGFR, ERK, and AKT in HCC827-HGF cells, resulting in suppression of cell growth and indicating that autocrine HGF-MET signaling contributes to gefitinib resistance in these cells. Combination therapy with TAK-701 and gefitinib also markedly inhibited the growth of HCC827-HGF tumors in vivo. The addition of TAK-701 to gefitinib is a promising strategy to overcome EGFR-TKI resistance induced by HGF in NSCLC with an activating EGFR mutation. 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 1731. doi:10.1158/1538-7445.AM2011-1731

Primary resistance to cetuximab in a panel of patient-derived tumour xenograft models: Activation of MET as one mechanism for drug resistance

Cetuximab (Erbitux®) targets the epidermal growth factor receptor (EGFR) and is approved for treatment of colorectal and head and neck cancer. Despite wide expression of EGFR, only a subgroup of cancer patients responds to cetuximab therapy. In the present study we assessed the cetuximab response in vivo of 79 human patient-derived xenografts originating from five tumour histotypes. We analysed basic tumour characteristics including EGFR expression and activation, mutational status of KRAS, BRAF and NRAS, the expression of EGFR ligands and the activation of HER3 (ErbB3) and the hepatocyte growth factor receptor MET. Based on these results, a cetuximab response score including positive and negative factors affecting therapeutic response is proposed. Positive factors are high expression and activation of EGFR and its ligands epiregulin or amphiregulin, negative factors are markers for downstream pathway activation independent of EGFR. In cetuximab resistant NSCL adenocarcinoma LXFA 526 and LXFA 1647, overexpression due to gene amplification and strong activation of MET was identified. Knock-down of MET by siRNA in the corresponding cell lines showed that anchorage-independent growth and migration are dependent on MET. MET knock down sensitized LXFA 526L and LXFA 1647L to EGF. Combined treatments of a MET inhibitor and cetuximab were additive. Therefore, combination therapy of cetuximab and a MET inhibitor in selected lung cancer patients could be of high clinical significance.