MET Kinase Inhibitor Research Articles

Amplification of the MET Receptor Drives Resistance to Anti-EGFR Therapies in Colorectal Cancer

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.

Frequency of MET amplification determined by comprehensive next-generation sequencing (NGS) in multiple solid tumors and implications for use of MET inhibitors.

11068 Background: MET expression has been shown to be prognostic and possibly predictive in several tumor types and both small molecule inhibitors of MET kinase as well as monoclonal antibodies are under study as therapies for this subset of patients. However, the methods to assess MET overexpression are not well standardized. In the clinic, NGS is an advanced diagnostic method for identifying known and unknown targeted-therapy options. We therefore sought to explore the ability of NGS to detect high level MET amplifications in a general oncology population. Methods: We review here the results from analysis of 2,221 FFPE tissue samples across a range of tumor types by an NGS assay in a CLIA-certified laboratory (Foundation Medicine). We specifically report base pair substitutions, small insertions/homozygous deletions (indels), high level (> 6 copies/nucleus) amplification (amp) and select rearrangements in 189 genes, including MET. Results: MET was altered in 28/2,223 (1.2%) of patient samples. MET alterations were limited to amplifications (median 15X, range 6X-40X); no base pair substitutions, indels, or rearrangements were found. MET amp was present in 3/48 (6.2%) primary bone sarcomas, 1/20 (5.0%) myogenic sarcomas, 2/58 (3.4%) gastric, 1/38 (2.6%) kidney, 2/78 (2.5%) hepatocellular, 1/41 (2.4%) uterine, 4/188 (2.1%) unknown primary, 2/99 (2.0%) ovarian, 8/386 (2.0%) lung, 3/157 (1.9%) colorectal, and 1/109 (0.9%) pancreatic carcinomas. Conclusions: Use of NGS to characterize MET alterations in a 2,000+ patient population provides evidence for the role of NGS in identifying the future use of MET-targeted therapy in a variety of common and uncommon solid tumors. Clinical follow-up of the subset of cases treated with a MET inhibitor is ongoing.

Abstract 3042: LY2801653: An orally bioavailable MET kinase inhibitor with inhibitory activity against the oncoproteins ROS1 and MKNK1/2.

Abstract Under normal conditions MET receptor activation, internalization, and degradation is a highly controlled process regulated by the binding of its ligand, HGF. In many cancers however, MET becomes highly dysregulated, and MET overexpression, with or without gene amplification, is associated with a poorer prognosis. Aberrant activation of MET can also occur through mutation, or increased autocrine or paracrine HGF production. MET activation leads to increased oncogenic activities including cell migration, proliferation, invasion, survival, tumor neovascularization and decreased apoptosis. Several inhibitors of MET, including type I and type II kinase inhibitors, are currently being investigated in clinical trials. LY2801653 is a type II MET kinase inhibitor being investigated in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037). In addition to MET, LY2801653 also inhibits ROS1 kinase, and the serine/threonine kinases MKNK1/2, with cell-based IC50 of 23nM and 7nM respectively. ROS1 is an orphan receptor that can be constitutively activated as a fusion protein either by genomic microdeletion or translocation. ROS1 fusion proteins are found in glioblastoma, NSCLC and cholangiocarcinoma. MKNK1/2 are kinases downstream of ERK and p38 and are capable of regulating translation through the direct phosphorylation of eIF4E. Increased MKNK1/2-dependent phosphorylation of the translation factor eIF4E is observed in head and neck squamous cell carcinoma and is correlated with a poor prognosis. This study evaluated the relative inhibitory effects of LY2801653, Crizotinib, XL184, XL880, and PF4217903 in a variety of cell lines against MET, ROS1, MKNK1/2, ERK, and other downstream molecules. In HCC78, a ROS1-fusion protein bearing cell line, and in NIH3T3 cells stably transfected with the FIG-ROS(s) fusion, LY2801653, XL184 and XL880 were more potent inhibitors of ROS1 compared with Crizotinib. Moreover, LY2801653 and Crizotinib were both efficacious in NIH3T3 FIG-ROS(s) xenografts, although LY2801653 was more potent in reducing p-ROS1 in these tumors than Crizotinib. In NCI-H2122 and Hs746T cells (mutated MET), A2780 cells (MET-negative), KP-4 cells (MET autocrine), H441 cells (HGF-independent; MET positive), HCT116 (mutated KRAS), and the cholangiocarcinoma cell lines EGI-1 and TFK-1, LY2801653 demonstrated more potent inhibitory activity against MKNK1/2 versus treatment with XL184 and XL880, while Crizotinib had no observed inhibitory activity against MKNK1/2. LY2801653 also showed inhibitory activity against MKNK1/2 in MET-amplified MKN45 cells. These findings suggests there may be clinical merits to testing LY2801653 in tumor types associated with increased MKNK1/2 activity or ROS1 fusion proteins alone, or in conjunction with aberrant MET activity, such as colorectal carcinoma, head and neck squamous cell carcinoma, and cholangiocarcinoma. Citation Format: Sau-Chi B. Yan, Megan N. Thobe, Bruce W. Konicek, Victoria L. Peek, Suzane L. Um, Richard A. Walgren, Huaxing Pei, Timothy P. Burkholder, Jeremy R. Graff. LY2801653: An orally bioavailable MET kinase inhibitor with inhibitory activity against the oncoproteins ROS1 and MKNK1/2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3042. doi:10.1158/1538-7445.AM2013-3042

Abstract 5637: A cellular model of acquired resistance to rilotumumab (AMG 102) in glioblastoma.

Abstract Acquired drug resistance is a long-standing problem of cancer therapeutics. The issue has become even more vexing with the development of highly selective agents; resistance to gefitinib or erlotinib is acquired frequently in lung adenocarcinomas. Thus, anticipating acquired resistance and understanding its basis may help us develop strategies to prevent or circumvent occurrence. Through its receptor tyrosine kinase Met, hepatocyte growth factor (HGF) regulates mitogenesis, motogenesis, and morphogenesis during development and adulthood. HGF/Met signaling also contributes to cancer progression in many malignancies, including glioblastoma. Rilotumumab is a fully human neutralizing monoclonal antibody against HGF tested in multiple Phase 2 clinical trials, including mono therapy in renal cell carcinoma and glioblastoma, as well as combination trials in gastric, colorectal, small cell lung cancers and castrate resistant prostate cancer. To generate a cellular model of acquired resistance to rilotumumab, an HGF/Met dependent human glioblastoma-derived cell line (U87-MG) was grown in rilotumumab (600 nM) for 120 days. Growth rate, HGF secretion, Met content and Met activation state were 10-fold, 10,000-fold, 10-fold and 80-fold higher than the parental cell values, respectively. The HGF and MET coding sequences were normal in both parental and resistant cells. Quantitative PCR studies to determine mRNA levels of all HGF isoforms revealed a dramatic increase in full-length HGF transcript. CGH array studies indicated amplification within both HGF and MET genes. Xenograft studies confirmed that tumor growth was resistant to rilotumumab, however the resistant cell line and tumors remained sensitive to a highly selective Met tyrosine kinase inhibitor, suggesting that resistance was achieved via increased HGF/Met signaling rather than mutation or activation of alternate pathways. Microarray expression analysis demonstrated transcript profiles that were consistent with HGF/Met pathway activation. Thus the molecular basis of acquired resistance in this model differs from those prevalent in: [1] lung cancers treated with EGFR inhibitors and medulloblastomas treated with hedgehog inhibitors, where most cases acquire secondary mutations in the targeted kinase; [2] breast cancers treated with HER2 inhibitors, where PTEN loss, p27 downregulation, and activation of other receptors are primary causes; or [3] malignant melanoma treated with BRAF inhibitors, where increased signaling via multiple pathways lead to PI3K- and/or MEK-mediated reactivation of the MAPK pathway. In addition to the importance of HGF/Met pathway activity in selecting glioblastoma patients for HGF-targeted therapeutics, our results suggest that monitoring Met pathway activity could provide early indications of acquired resistance to these agents, and that Met kinase inhibitors may still be efficacious when resistance occurs. Citation Format: Fabiola Cecchi, Karen Rex, Joanna Schmidt, Daniel Baker, Michael A. Damore, Angela Coxon, Teresa L. Burgess, Donald P. Bottaro. A cellular model of acquired resistance to rilotumumab (AMG 102) in glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5637. doi:10.1158/1538-7445.AM2013-5637

Abstract 3405: Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines.

Abstract Background: Resistance to targeted therapies represents a key hurdle in the treatment of cancer. Preclinical data suggest that resistance is frequently driven by redundant growth factor signaling. For example, HGF has been reported to mediate resistance to BRAF inhibitors in V600E BRAF mutant melanoma. Furthermore, clinical data support these findings as circulating and tumor HGF levels have been shown to correlate with outcome in BRAF mutant melanoma patients treated with vemurafenib. In this study, we evaluated the ability of HGF / MET signaling to attenuate the effects of BRAF and MEK inhibitors in the context of V600E BRAF and NRAS mutant cell lines. Our data suggest that combined blockade of MET and MAPK pathway signaling may be required for a robust and durable clinical response in melanoma. Methods: To confirm the ability of HGF to rescue V600E BRAF mutant melanoma cell lines from the effects of vemurafenib, G361 and SKMEL5 cells were treated with vemurafenib alone or in combination with HGF. Time lapse imaging, viability assays and cell cycle analysis were used to measure treatment effects. Analogous experiments were performed with the selective MEK inhibitor PD0325901. Combinations of a potent and selective MET inhibitor (Compound A) with vemurafenib or PD0325901 were tested to evaluate their ability to attenuate the HGF-mediated resistance. To identify other cell lines rescued by HGF, thirteen V600E BRAF mutant melanoma cell lines were treated with a dose response matrix of vemurafenib and HGF. The ability of six additional growth factors to rescue cells from the effects of vemurafenib was also tested. Three NRAS mutant melanoma cell lines were also screened for HGF-mediated resistance to PD0325901. Results: Nine of thirteen V600E BRAF mutant melanoma cell lines were rescued from the effects of vemurafenib by co-treatment with HGF. Detailed analysis of G361 and SKMEL5 cells revealed that HGF was also capable of rescuing the cells from the effects of PD0325901. This HGF-mediated rescue was attenuated by treatment with combinations of vemurafenib + compound A or PD0325901 + compound A. Mechanistic cell cycle analysis of the HGF-mediated rescue of vemurafenib in G361 cells revealed an increase in proliferating cells and a reduction in growth arrested cells, whereas rescue of PD0325901 treatment occurred through a reduction in the percentage of dead or dying cells. Of the seven growth factors tested, HGF was the predominate growth factor capable of conferring resistance to vemurafenib. Furthermore, HGF rescued one of three NRAS mutant melanoma cell lines from the effects of PD0325901, highlighting the breadth of HGF-mediated resistance in melanoma cell lines. Conclusion: These findings demonstrate the potential role for HGF / MET signaling in mediating resistance to BRAF and MEK inhibitors in melanoma, and support the clinical evaluation of MET kinase inhibitors and neutralizing antibodies to HGF in this setting. Citation Format: Sean Caenepeel, Elaina Cajulis, Rick Kendall, Angela Coxon, Paul Hughes. Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2013-3405

Pharmacological inhibition of beta-catenin in hepatoblastoma cells

The proto-oncogene beta-catenin is linked to an abnormal activation of the Wnt/beta-catenin-pathway and shows mutations in 50-90% of hepatoblastoma (HB). Corresponding, the recently published murine orthotopic HB model differs from the former subcutaneous model by nuclear beta-catenin distribution. As the nuclear localization of beta-catenin is considered to reflect a more aggressive tumor growth, the influence of beta-catenin inhibition on cell viability and drug-efficiency in HB cells was analyzed. Beta-catenin distribution in HB cells was analyzed by immunofluorescence. The influence of beta-catenin inhibitors Celecoxib, Etodolac, ICG001, and MET kinase inhibitor (SU11274) alone and in combination with cisplatin (CDDP) on HB cell lines (HuH6, HepT1) was evaluated by cell viability assays and BrdU incorporation. Celecoxib and ICG001 reduced dose-dependently HB cell viability and decreased nuclear beta-catenin in cultivated HB cells. Etodolac was without influence at concentrations up to 100μM. Combinations of Celecoxib or ICG001 with MET kinase inhibitor or CDDP resulted in additive reduction of cell viability. Pharmaceutical beta-catenin inhibitors can modulate the nuclear localization of beta-catenin and reduce cell viability of HB cells in vitro. These promising effects might optimize the outcome of high-risk HB. The orthotopic HB model is a suitable basis for further in vivo studies.

O1-076 - Met Kinase Inhibitor E7050 Reverses Three Different Mechanisms of Hepatocyte Growth Factor-Induced Tyrosine Kinase Inhibitor Resistance in EGFR Mutant Lung Cancer

ABSTRACT Purpose Hepatocyte growth factor (HGF) induces resistance to reversible and irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutant lung cancer cells by activating Met and the downstream PI3K/Akt pathway. Moreover, continuous exposure of HGF accelerates emergence of EGFR-TKI-resistant clones. The present study was carried out to determine whether a new Met kinase inhibitor, E7050, which is currently being evaluated in clinical trials, could overcome these three mechanisms of resistance to EGFR-TKIs. Experimental design The effects of E7050 on HGF-induced resistance to reversible (gefitinib), irreversible (BIBW2992) and mutant-selective (WZ4002) EGFR-TKIs were determined using the EGFR-mutant human lung cancer cell lines PC-9 and HCC827 with exon 19 deletion, and H1975 with T790M secondary mutation. As an in vivo model, PC-9 cells mixed with HGF-producing fibroblasts, MRC-5, were subcutaneously inoculated into SCID mice and the therapeutic effects of E7050 combined with gefitinib were examined. Results Our results indicated that E7050 circumvented resistance to all of the reversible, irreversible and mutant-selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking the Met/Gab1/PI3K/Akt pathway in vitro. E7050 also prevented emergence of gefitinib-resistant HCC827 cells induced by continuous exposure to HGF. In the in vivo model, E7050 combined with gefitinib-induced marked regression of tumor growth associated with inhibition of Akt phosphorylation in cancer cells. Conclusions A new Met kinase inhibitor, E7050, reverses three different mechanisms of gefitinib resistance induced by HGF, suggesting the usefulness of E7050 for overcoming HGF-induced resistance to gefitinib and next-generation EGFR-TKIs.

Abstract B42: Heat Shock Protein 27 (HSP27, HSPB1) is up-regulated by MET kinase inhibition and limits the effectiveness of inhibitors

Abstract The receptor for the hepatocyte growth factor (HGF) is encoded by the MET oncogene and its activation results in cell growth, survival, motility and invasiveness. Deregulated HGF/MET signaling is observed in many tumors and both HGF and MET are attractive candidates for targeted therapies. Unexpectedly, we found that in ovarian cancer cells the activation of the MET receptor downmodulated the expression of the small heat shock protein 27 (HSP27), which functions as a molecular chaperone and as an antiapoptotic protein. In MET addicted gastric carcinoma cells, where MET is constitutively active, MET kinase inhibition by JNJ-38877605 up-regulated HSP27 expression. More importantly, in these gastric carcinoma cells suppression of HSP27 was alone able to induce cell death and increased cell susceptibility to MET inhibition. HSP27 suppression did not affect MET stability and phosphorylation, but increased activation of caspase 3, which is a known HSP27 client protein. Regulation of HSP27 expression by MET signaling occurred at transcriptional level, as confirmed by the use of HSP27 promoter in a reporter assay. It was not due to the engagement of HSF1, the master key transcription factor regulating HSPs expression, but depended on MEK activation and resulting HIF 1alpha stabilization. MEK inhibition and HIF 1 alpha silencing resulted in the induction of HSP27, suggesting a direct or indirect transcriptional repression. Altogether data show that MET activation down-regulates and MET inhibition up-regulates HSP27 expression, and that HSP27 increased expression protects cells from death induced by MET inhibitors. Therefore, in therapies with MET targeting agents, HSP27 increase might impair the effectiveness of MET inhibitors. As it is known that HSP27 protects cells for chemotherapeutics, data suggest that HSP27 might strongly affect combinatorial treatments.

Abstract 845: In vitro and in vivo pharmacology of SAR125844, a potent and selective intravenous MET kinase inhibitor undergoing Phase I clinical trial

Abstract SAR125844 is a potent MET kinase inhibitor for intravenous (IV) administration, with nanomolar activity against the wild-type enzyme (IC50 = 4.2 nM) and some kinase domain mutants, such as M1250T and Y1235D. It is highly selective for MET kinase in a panel of 275 kinases tested, with only 5 other protein kinases inhibited at IC50 values below 300 nM. In cellular assays, SAR125844 inhibits MET autophosphorylation at the nanomolar range (IC50 from 1.4 to 5.1 nM), translating into antiproliferative activity selectively in MET-driven cell lines such as MET-amplified cell lines, with IC50 values in the low nanomolar range and induction of apoptosis. SAR125844 induces a G1 block in the two MET-amplified tumor cell lines tested. Moreover, the compound is also able to inhibit HGF-induced cell migration in PC-3 prostate tumor cell line. In two MET-amplified human gastric tumor xenograft models tested, SNU-5 and Hs 746T, SAR125844 intravenous treatment leads to potent impact on the MET signaling pathway in a dose and time dependent manner, with potent inhibition of MET autophosphorylation, as well as a significant effect on downstream PI3K/AKT and RAS/MAPK pathways. As a single agent, this translates into a dose-dependent antitumor activity in these two xenograft models, with tumor stasis at the lowest active dose and tumor regression at the highly active doses. High loading single dose administration of SAR125844 using a nanosuspension formulation allowed a sustained P-MET inhibition in tumors up to 7 days. In all models, antitumor activity was achieved at well tolerated doses without significant effect on body weight. The observed correlation between P-MET inhibition in the tumor and antitumor activity in preclinical settings suggests that P-MET evaluation in tumor biopsies could be a direct pharmacodynamic biomarker of SAR125844 activity in patients. In order to investigate whether a non-invasive biomarker could also be used as pharmacodynamic read-out, we measured shed-MET (soluble extracellular domain of the MET receptor) in the plasma of tumor-bearing mice and showed that its level correlates with tumor burden in 2 MET-amplified xenograft models. 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 845. doi:1538-7445.AM2012-845

Abstract 1789: Preclinical profile of novel and potent c-Met kinase inhibitors

Abstract Background: c-Met is a proto-oncogene that encodes the protein Met with intrinsic tyrosine kinase activity. Aberrant Met kinase activity triggers a series of unwarranted phosphorylation events and signalling processes that ultimately lead to the development of cancer. Alteration of the Met kinase signalling cascade represents an attractive approach aimed at blocking invasion and metastasis of cancer cells. Herein, we describe the biological and pharmacokinetic properties of representative molecules from a series of novel and small molecule inhibitors with scope to be further developed as clinical candidates for cancers mediated by dysregulated Met kinase activity. Methods: Met Kinase activity of test compounds was determined using an HTRF® KinEASE assay kit (Cisbio, Bedford, MA) with modifications. Met-dependent anti-proliferative effect was determined in MKN-45 cells. Inhibition of constitutive Met kinase phosphorylation in MKN-45 and NCI-H441 cells was measured in an ELISA assay. Subsequently, effect of the compounds on Akt phosphorylation, a downstream marker in the Met signalling cascade, was determined. Metabolic stability of the compounds was evaluated in microsomes obtained from mouse, rat, dog, monkey, and human. Results: Among the compounds evaluated, RP1269 and RP1316 demonstrated remarkable potency against the purified Met kinase enzyme (20.17 & 40.5 nM) as well as in an MKN-45 cell proliferation assay (18.0 & 2.3 nM). In addition, the compounds caused a significant reduction in constitutive Met kinase phosphorylation in MKN-45 (11.5 & 33.3 nM) and NCI-H441 (2.2 & 4.1 nM) cells. As a consequence, Akt phosphorylation was inhibited at half-maximally in MKN-45 and NCI-H441 cells at <100 nM and <50 nM respectively. Further, the compounds exhibited a favourable ADME profile across the species studied. Conclusions: Our findings demonstrate that RP1269 and RP1316 are potent Met kinase inhibitors with efficacy values comparable to existing Met kinase inhibitors in development. On lines with selective inhibitors, the compounds display anti-proliferative effect only in cells with amplification of the Met kinase gene. The compounds are currently being tested for efficacy and target inhibition in various xenograft models. Clinical candidate shall be nominated by 2012. 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 1789. doi:1538-7445.AM2012-1789

Abstract 1231: Differential roles of trans-phophorylated EGFR, HER2, HER3 and RET as heterodimerization partenrs of MET in lung cancer with MET amplification

Abstract Background: MET is a receptor tyrosine kinase (RTK) whose gene is amplified in various tumor types. We investigated the roles and mechanisms of RTK heterodimerization in lung cancer with MET amplification. Methods: With the use of an RTK array, we identified several phosphorylated RTKs in lung cancer cells with MET amplification. We examined the roles and mechanisms of action of these RTKs with immunoprecipitation, annexin V binding, and cell migration assays. Results: We identified EGFR, HER2, HER3, and RET in addition to MET as highly phosphorylated RTKs in lung cancer cells with MET amplification. Immunoprecipitation revealed that EGFR, HER2, HER3, and RET each formed a heterodimer exclusively with MET and that these associations were markedly reduced in extent by treatment with a MET kinase inhibitor, but not by treatment with their spcific inhibitors. RNA interference-mediated depletion of EGFR, HER2, or HER3 induced apoptosis in association with inhibition of AKT and ERK signaling pathways, whereas depletion of HER2 or RET inhibited both cell migration and STAT3 signaling. Conclusion: Our data suggest that heterodimers of MET with EGFR, HER2, HER3, or RET play differential roles in tumor development, and they provide new insight into the function of trans-phosphorylated RTKs as heterodimerization partners of MET in lung cancer with MET amplification. 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 1231. doi:1538-7445.AM2012-1231

Met Kinase Inhibitor E7050 Reverses Three Different Mechanisms of Hepatocyte Growth Factor–Induced Tyrosine Kinase Inhibitor Resistance in EGFR Mutant Lung Cancer

Hepatocyte growth factor (HGF) induces resistance to reversible and irreversible epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) in EGFR mutant lung cancer cells by activating Met and the downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Moreover, continuous exposure to HGF accelerates the emergence of EGFR-TKI-resistant clones. We assayed whether a new Met kinase inhibitor, E7050, which is currently being evaluated in clinical trials, could overcome these three mechanisms of resistance to EGFR-TKIs. The effects of E7050 on HGF-induced resistance to reversible (gefitinib), irreversible (BIBW2992), and mutant-selective (WZ4002) EGFR-TKIs were determined using the EGFR mutant human lung cancer cell lines PC-9 and HCC827 with an exon 19 deletion and H1975 with an T790M secondary mutation. PC-9 cells were mixed with HGF-producing fibroblasts, MRC-5 cells, and subcutaneously inoculated into severe combined immunodeficient mice, and the therapeutic effects of E7050 plus gefitinib were assayed. E7050 circumvented resistance to all of the reversible, irreversible, and mutant-selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking the Met/Gab1/PI3K/Akt pathway in vitro. E7050 also prevented the emergence of gefitinib-resistant HCC827 cells induced by continuous exposure to HGF. In the in vivo model, E7050 plus gefitinib resulted in marked regression of tumor growth associated with inhibition of Akt phosphorylation in cancer cells. A new Met kinase inhibitor, E7050, reverses the three HGF-induced mechanisms of gefitinib resistance, suggesting that E7050 may overcome HGF-induced resistance to gefitinib and next-generation EGFR-TKIs.

Migration induced by epidermal and hepatocyte growth factors in oral squamous carcinoma cells in vitro: role of MEK/ERK, p38 and PI‐3 kinase/Akt

Cell migration is a necessary part of malignant invasiveness. Oral squamous cell carcinomas (OSCC) have a great tendency for local invasive growth. We have investigated signalling pathways involved in cell migration induced by epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in OSCC cells and examined the effects of various experimental and clinically approved anti-tumour signal inhibitors on the migratory activity. Migration was studied in three human OSCC cell lines, using a scratch wound assay in vitro and time-lapse cinematography. Specific phosphorylation of signalling proteins was assessed by Western blotting. In the E10 cell line, EGF and HGF induced phosphorylation of EGF receptor (EGFR) and Met, respectively, phosphorylation of ERK1/2, p38 and Akt, and dose-dependent activation of cell migration. Addition of the EGFR-specific inhibitors cetuximab (antibody) or gefitinib (tyrosine kinase blocker) abolished cell migration elicited by EGF. Similarly, a Met kinase inhibitor (SU11274) blocked HGF-induced cell migration. Furthermore, when three cell lines were treated with blockers of the MEK/ERK, p38 or the PI-3 kinase/Akt pathways, the migratory response to both EGF and HGF was inhibited, but to varying degrees. Notably, in E10 and D12 cells, HGF-induced migration was particularly sensitive to PI-3 K-inhibition, while in C12 cells, both HGF- and EGF-induced migration were highly sensitive to p38-blockade. The results demonstrate that the MEK/ERK, p38 and PI-3 kinase pathways are all involved in mediating the increased migration in OSCC cell lines induced by EGF and HGF, but their relative importance and the effects of specific signal inhibitors differ.

Abstract B21: E7050, a Met kinase inhibitor, reverses three different mechanisms of hepatocyte growth factor-induced resistance to tyrosine kinase inhibitors in EGFR mutant lung cancer cells

Abstract Purpose: Hepatocyte growth factor (HGF) induces resistance to reversible and irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in EGFR mutant lung cancer cells by activating Met and downstream PI3K/Akt pathway. In addition, HGF accelerates expansion of minor clones with Met amplification and facilitates EGFR-TKI resistance. Purpose of this study is to determine whether E7050, a Met kinase inhibitor, could overcome these three resistant mechanisms to EGFR-TKIs. Experimental design: Effect of E7050 on HGF-induced resistance to reversible (gefitinib), irreversible (BIBW2992), and mutant selective (WZ4002) EGFR-TKIs was determined using EGFR-mutant human lung cancer cell lines, PC-9 and HCC827 with exon 19 deletion, and H1975 with T790M secondary mutation. As an in vivo models, PC-9 cells mixed with HGF producing fibroblasts, MRC-5, were subcutaneously inoculated into SCID mice and therapeutic effect of E7050 combined with gefitinib was examined. Results: We found that E7050 circumvented resistance to all of reversible, irreversible, and mutant selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking Met/Gab1/PI3K/Akt pathway in vitro. E7050 also prevented emergence of gefitinib resistant HCC827 cells induced by continuous exposure to HGF. In vivo model, E7050 combined with gefitinib dramatically regressed the tumor growth, associating with inhibiting Akt phosphorylation in cancer cells. Conclusions: E7050, a Met kinase inhibitor, reverses three different mechanisms of gefitinib resistance induced by HGF, suggesting usefulness of E7050 for overcoming HGF-induced resistance to gefitinib and new generation EGFR-TKIs.

Preclinical stereoselective disposition and toxicokinetics of two novel MET inhibitors

The R- and S-enantiomer of N-(4-(3-(1-ethyl-3,3-difluoropiperidin-4-ylamino)-1H-pyrazolo[3,4-b]pyridin-4-yloxy)-3-fluorophenyl)-2-(4-fluorophenyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide are novel MET kinase inhibitors that have been investigated as potential anticancer agents. The effect of the chirality of these compounds on preclinical in vivo pharmacokinetics and toxicity was studied.The plasma clearance for the S-enantiomer was low in mice and monkeys (23.7 and 7.8 mL min−1 kg−1, respectively) and high in rats (79.2 mL min−1 kg−1). The R/S enantiomer clearance ratio was 1.5 except in rats (0.49). After oral single-dose administration at 5 mg kg−1 the R/S enantiomer ratio of AUCinf was 0.95, 1.9 and 0.41 in mice, rats and monkeys, respectively.In an oral single-dose dose-ranging study at 200 and 500 mg kg−1 and multi-dose toxicity study in mice plasma AUC exposure was approximately 2- to 3-fold higher for the R-enantiomer compared to the S-enantiomer. Greater toxicity of the S-enantiomer was observed which appeared to be due to high plasma Cmin values and tissue concentrations approximately 24 h after the final dose.Both enantiomers showed low to moderate permeability in MDCKI cells with no significant efflux, no preferential distribution into red blood cells and similar plasma protein binding in vitro.Overall, the differences between the enantiomers with respect to low dose pharmacokinetics and in vitro properties were relatively modest. However, toxicity results warrant further development of the R-enantiomer over the S-enantiomer.

Abstract A204: A cellular model of acquired resistance to rilotumumab (AMG 102) in glioblastoma.

Abstract Acquired drug resistance is a long-standing problem of cancer therapeutics, for example chemoresistance to temozolomide occurs in >90% of recurrent gliomas. The issue has become even more vexing with the development of highly selective targeted agents; e.g. agents targeting the epidermal and hepatocyte growth factor (EGF and HGF, respectively) pathways; resistance to gefitinib and erlotinib has already been frequently demonstrated in lung adenocarcinomas. Thus, anticipating acquired resistance and understanding its basis may help us develop clinical strategies to prevent or circumvent its occurrence. HGF, through its receptor tyrosine kinase Met, regulates mitogenesis, motogenesis, and morphogenesis in a range of cellular targets during development and homeostasis. HGF/Met signaling also contributes to oncogenesis and tumor progression in many prevalent human malignancies, including glioblastoma. Rilotumumab (AMG102) is a fully human neutralizing monoclonal antibody against HGF tested in multiple Phase 2 clinical trials, including mono therapy in renal cell carcinoma and Glioblastoma, as well as combination trials in gastric, colorectal and small cell lung cancers and castrate resistant prostate cancer. To generate a cellular model of acquired resistance to rilotumumab, the HGF/Met dependent human glioblastoma-derived cell line U87-MG, was grown in continuous exposure to 600 nM rilotumumab for 120 days. Growth rate, HGF secretion, Met content and Met activation state were 10-fold, 10,000-fold, 10-fold and 80-fold higher than the parental cell values, respectively. The HGF and MET coding sequences and the HGF promoter DATE region, where truncation reportedly increases HGF expression level, were found to have normal sequence and length in both parental and resistant cell lines. Quantitative PCR studies to determine mRNA levels of all HGF isoforms revealed a dramatic increase in full-length HGF transcript. CGH array studies indicated amplification within both HGF and MET genes. Xenograft studies confirmed that tumor growth was resistant to rilotumumab, however the resistant cell line and tumors remained sensitive to a highly selective Met tyrosine kinase inhibitor suggesting that resistance was achieved via increased HGF/Met signaling rather than mutation or activation of alternate pathways. Microarray expression analysis demonstrated transcript profiles that were consistent with HGF/Met pathway activation. Thus the molecular basis of acquired resistance in this model differs from those prevalent in: [1] lung cancers treated with EGFR inhibitors and medulloblastomas treated with hedgehog inhibitors, where nearly all cases acquire secondary mutations in the targeted kinase; [2] breast cancers treated with HER2 inhibitors, where PTEN loss, p27 downregulation, and activation of other receptors are primary causes; or [3] malignant melanoma treated with BRAF inhibitors, where increased signaling by ARAF, CRAF, IGFR1, PDGFR, and other sources lead to PI3K- and/or MEK-mediated reactivation of the MAPK pathway. In addition to the importance of HGF/Met pathway activity in selecting glioblastoma patients for HGF-targeted therapeutics, our results suggest that monitoring Met pathway activity could provide early indications of acquired resistance to these agents, and that Met kinase inhibitors may still be efficacious when resistance occurs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A204.

Differential roles of trans-phosphorylated EGFR, HER2, HER3, and RET as heterodimerisation partners of MET in lung cancer with MET amplification

Background:MET is a receptor tyrosine kinase (RTK) whose gene is amplified in various tumour types. We investigated the roles and mechanisms of RTK heterodimerisation in lung cancer with MET amplification.Methods:With the use of an RTK array, we identified phosphorylated RTKs in lung cancer cells with MET amplification. We examined the roles and mechanisms of action of these RTKs with immunoprecipitation, annexin V binding, and cell migration assays.Results:We identified epidermal growth factor receptor (EGFR), human EGFR (HER)2, HER3, and RET in addition to MET as highly phosphorylated RTKs in lung cancer cells with MET amplification. Immunoprecipitation revealed that EGFR, HER2, HER3, and RET each formed a heterodimer exclusively with MET and that these associations were markedly reduced in extent by treatment with a MET kinase inhibitor. RNA interference-mediated depletion of EGFR, HER2, or HER3 induced apoptosis in association with inhibition of AKT and ERK signalling pathways, whereas depletion of HER2 or RET inhibited both cell migration and STAT3 signalling.Conclusion:Our data suggest that heterodimers of MET with EGFR, HER2, HER3, or RET have differential roles in tumour development, and they provide new insight into the function of trans-phosphorylated RTKs as heterodimerisation partners of MET in lung cancer with MET amplification.

MET-Independent Lung Cancer Cells Evading EGFR Kinase Inhibitors Are Therapeutically Susceptible to BH3 Mimetic Agents

Targeted therapies for cancer are inherently limited by the inevitable recurrence of resistant disease after initial responses. To define early molecular changes within residual tumor cells that persist after treatment, we analyzed drug-sensitive lung adenocarcinoma cell lines exposed to reversible or irreversible epidermal growth factor receptor (EGFR) inhibitors, alone or in combination with MET-kinase inhibitors, to characterize the adaptive response that engenders drug resistance. Tumor cells displaying early resistance exhibited dependence on MET-independent activation of BCL-2/BCL-XL survival signaling. Further, such cells displayed a quiescence-like state associated with greatly retarded cell proliferation and cytoskeletal functions that were readily reversed after withdrawal of targeted inhibitors. Findings were validated in a xenograft model, showing BCL-2 induction and p-STAT3[Y705] activation within the residual tumor cells surviving the initial antitumor response to targeted therapies. Disrupting the mitochondrial BCL-2/BCL-XL antiapoptotic machinery in early survivor cells using BCL-2 Homology Domain 3 (BH3) mimetic agents such as ABT-737, or by dual RNAi-mediated knockdown of BCL-2/BCL-XL, was sufficient to eradicate the early-resistant lung-tumor-cells evading targeted inhibitors. Similarly, in a xenograft model the preemptive cotreatment of lung tumor cells with an EGFR inhibitor and a BH3 mimetic eradicated early TKI-resistant evaders and ultimately achieved a more durable response with prolonged remission. Our findings prompt prospective clinical investigations using BH3-mimetics combined with targeted receptor kinase inhibitors to optimize and improve clinical outcomes in lung-cancer treatment.

Foretinib (GSK1363089), a multi-kinase inhibitor of MET and VEGFRs, inhibits growth of gastric cancer cell lines by blocking inter-receptor tyrosine kinase networks

To explore the mechanism of action of foretinib (GSK1363089), an oral multi-kinase inhibitor known to target MET, RON, AXL, and vascular endothelial growth factor receptors (VEGFRs), in gastric cancer, we evaluated the effects of the agent on cell growth and cell signaling in the following panel of gastric cancer cell lines: KATO-III, MKN-1, MKN-7, MKN-45, and MKN-74. Of these, only MKN-45 and KATO-III, which harbor MET and fibroblast growth factor receptor 2 (FGFR2) amplification, respectively, were highly sensitive to foretinib. In MKN-45, 1 μM of foretinib or PHA665752, another MET kinase inhibitor, inhibited phosphorylation of MET and downstream signaling molecules as expected. In KATO-III, however, PHA665752 inhibited phosphorylation of MET independently of downstream molecules. Further, 1 μM of foretinib or PD173074, a selective FGFR kinase inhibitor, inhibited phosphorylation of FGFR2 and downstream molecules, suggesting that foretinib targets FGFR2 in KATO-III. We confirmed this novel activity of foretinib against FGFR2 in OCUM-2M, another FGFR2-amplified gastric cancer cell line. Using a phospho-receptor tyrosine kinase array, we found that foretinib inhibits phosphorylation of epidermal growth factor receptor (EGFR), HER3 and FGFR3 via MET inhibition in MKN-45, and EGFR, HER3 and MET via FGFR2 inhibition in KATO-III. Knockdown of HER3 and FGFR3 in MKN-45 with siRNA resulted in the partial inhibition of cell signaling and cell growth. In conclusion, foretinib appears effective against gastric cancer cells harboring not only MET but also FGFR2 amplification, and exerts its inhibitory effects by blocking inter-RTK signaling networks with MET or FGFR2 at their core.

Abstract 578: Roles of mitochondrial protein kinases in targeted cancer therapy

Abstract Targeted cancer therapy is based on the curious dependence that some cancer cells have on a single oncogenic molecule, typically a protein kinase (the phenomenon of “oncogene addiction”). However, the molecular mechanisms of such dependence are incompletely understood. We have adopted a quantitative proteomic approach to investigate the phenomenon that MET-overexpressing cancer cells are paradoxically more sensitive to MET inhibition than cells with basal MET expression. Unexpectedly, we found that mitochondria are a direct target of MET kinase inhibition, in addition to inhibition of plasma membrane MET. Effects on activated MET in the mitochondria of cancer cells that are sensitive to MET inhibition could reflect a novel and critical non-canonical mechanism that contributes to the efficacy of MET-targeted therapeutics. We hypothesize that protein kinases which aberrantly translocate to mitochondria modulate mitochondrial proteins and functions. Here we have employed advanced LC-MS/MS-based stable isotope labeling with amino acids in cell culture (SILAC) quantitative phosphoproteomic approaches to identify, quantify and functionally annotate phosphorylated mitochondrial proteins of various cancer cell lines, including those that are sensitive or resistant to targeted agents. We identified several phosphorylated protein kinases e.g. MET, EGFR, HER2 and HER3, that were translocated to mitochondria of cancer cells. In addition to documenting the first kinome and phosphoproteome of mitochondria in cancer cells, we present evidence showing that the aberrant presence of protein kinases in mitochondria predicts sensitivity to specific kinase inhibitors. In conclusion, our data reveal a novel mitochondrial mechanism of oncogene addiction, and suggest that mitochondrial protein kinases might play decisive roles in molecularly-directed therapies. 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 578. doi:10.1158/1538-7445.AM2011-578