Supplementary Methods from Lung Cancer Cell Lines Harboring MET Gene Amplification Are Dependent on Met for Growth and Survival

Supplementary Information from Lung Cancer Cell Lines Harboring MET Gene Amplification Are Dependent on Met for Growth and Survival

Supplementary Figures 1-10 from FGFR2-Amplified Gastric Cancer Cell Lines Require FGFR2 and Erbb3 Signaling for Growth and Survival

Supplementary Figures 1-10 from FGFR2-Amplified Gastric Cancer Cell Lines Require FGFR2 and Erbb3 Signaling for Growth and Survival

Supplementary Figure 1 Legend from Lung Cancer Cell Lines Harboring MET Gene Amplification Are Dependent on Met for Growth and Survival

Aberrant kinase activation resulting from mutation, amplification, or translocation can drive growth and survival in a subset of human cancer. FGFR2 is amplified in breast and gastric cancer, and we report here the first characterization of FGFR2 gene amplification in colorectal cancer in the NCI-H716 colorectal cancer cell line. FGFR2 is highly expressed and activated in NCI-H716 cells, and FGFR selective small molecule inhibitors or FGFR2 shRNA strongly inhibited cell viability in vitro, indicating "addiction'' of NCI-H716 cells to FGFR2. NCI-H716 growth in a xenograft model was also inhibited by an FGFR small molecule inhibitor. FGFR2 was required for activation of multiple downstream signaling proteins including AKT, ERK, S6RP and NFKB. Inhibition of downstream kinases such as AKT or ERK alone had modest effects on proliferation, whereas combined inhibition of AKT and ERK signaling resulted in a loss of viability similar to FGFR2 inhibition. We identified elevated FGFR2 expression in a small subset of primary colorectal cancer, however FGFR2 amplification was not observed. Although FGFR2 amplification is not common in primary colon cancer or lymph node and liver metastases, other subsets of colorectal cancer such as ascites, from which the NCI-H716 cell line was derived, have yet to be tested. These results suggest that emerging FGFR inhibitor therapeutics may have efficacy in a subset of colon cancer driven by FGFR2 amplification.

This report documents the first example of a specific inhibitor of protein kinases with preferential binding to the activated kinase conformation: 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one 11r (MK-8033), a dual c-Met/Ron inhibitor under investigation as a treatment for cancer. The design of 11r was based on the desire to reduce time-dependent inhibition of CYP3A4 (TDI) by members of this structural class. A novel two-step protocol for the synthesis of benzylic sulfonamides was developed to access 11r and analogues. We provide a rationale for the observed selectivity based on X-ray crystallographic evidence and discuss selectivity trends with additional examples. Importantly, 11r provides full inhibition of tumor growth in a c-Met amplified (GTL-16) subcutaneous tumor xenograft model and may have an advantage over inactive form kinase inhibitors due to equal potency against a panel of oncogenic activating mutations of c-Met in contrast to c-Met inhibitors without preferential binding to the active kinase conformation.

BRAF and MEK inhibitors induce striking tumor regressions in BRAF V600E melanoma patients. However, relapse occurs in the majority of patients within several months. Reported resistance mechanisms include acquisition of NRAS mutations, PDGFR or IGF1R activation, Cot amplification, or truncated BRAF upregulation. We report here that hepatocyte growth factor (HGF), neuregulin-1 (NRG1), and fibroblast growth factor-2 (FGF2) cause robust resistance in vitro to BRAF or MEK inhibitors in BRAF mutant melanoma cell lines. NRAS mutant lines were similarly resistant to MEK inhibition in the presence of these growth factors. By contrast other mitogenic factors such as PDGF, EGF, NGF, BDNF, MSP, MDK, GRO, and IGF1 did not maintain cell viability or growth. HGF, NRG1 and FGF2 prevented growth arrest and cell death and in some cell lines maintained cells in progressive growth. In addition, HGF and NRG1 could prevent growth arrest after dual treatment with BRAF and MEK inhibitors. Western analysis revealed that HGF and NRG1 reactivated Erk signaling and activated S6RP and AKT phosphorylation. Combined pharmacological inhibition mTor signaling and Akt signaling was required to fully block growth factor-mediated proliferation. In addition, small molecule Met inhibitors and an anti-HGF antibody blocked HGF-mediated resistance, while lapatinib and an anti-NRG1 antibody blocked NRG1-mediated resistance. While combined Akt and mTor inhibition potently inhibited cell line growth independent of BRAF or MEK inhibition, lapatinib and Met inhibitors had minimal effect on growth, suggesting potential for less adverse events with receptor inhibitors. In vitro co-culture experiments revealed that primary cells present in normal skin can cause resistance, and we have identified growth factors produced by these cells that contribute to resistance. We are analyzing receptor activation (Met and Erbb3) and FGF2 expression in wildtype, NRAS, and BRAF mutant primary melanoma samples. In future experiments we plan to test clinical samples from patients treated with BRAF/MEK inhibitors, and we hypothesize that HGF, NRG1, and FGF2 signaling is elevated in patients with poor initial responses or relapse to BRAF or MEK inhibitors. 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 1065. doi:1538-7445.AM2012-1065

c-Met is a transmembrane tyrosine kinase that mediates activation of several signaling pathways implicated in aggressive cancer phenotypes. In recent years, research into this area has highlighted c-Met as an attractive cancer drug target, triggering a number of approaches to disrupt aberrant c-Met signaling. Screening efforts identified a unique class of 5H-benzo[4,5]cyclohepta[1,2-b]pyridin-5-one kinase inhibitors, exemplified by 1. Subsequent SAR studies led to the development of 81 (MK-2461), a potent inhibitor of c-Met that was efficacious in preclinical animal models of tumor suppression. In addition, biochemical studies and X-ray analysis have revealed that this unique class of kinase inhibitors binds preferentially to the activated (phosphorylated) form of the kinase. This report details the development of 81 and provides a description of its unique biochemical properties.

Most non-small cell lung cancer (NSCLC) patients harboring activating epidermal growth factor receptor (EGFR) mutations respond to tyrosine kinase inhibitor (TKI) therapy. However, about 30% exhibit primary resistance to EGFR TKI therapy. Here we report that Met protein expression and phosphorylation were associated with primary resistance to EGFR TKI therapy in NSCLC patients harboring EGFR mutations, implicating Met as a de novo mechanism of resistance. In a separate patient cohort, Met expression and phosphorylation were also associated with development of NSCLC brain metastasis and were selectively enriched in brain metastases relative to paired primary lung tumors. A similar metastasis-specific activation of Met occurred in vitro in the isogenous cell lines H2073 and H1993, which are derived from the primary lung tumor and a metastasis, respectively, from the same patient. We conclude that Met activation is found in NSCLC before EGFR-targeted therapy and is associated with both primary resistance to EGFR inhibitor therapy and with the development of metastases. If confirmed in larger cohorts, our analysis suggests that patient tumors harboring both Met activation and EGFR mutation could potentially benefit from early intervention with a combination of EGFR and Met inhibitors.

There has been increasing interest in developing cancer therapies targeting PI3K pathway nodes. Unfortunately, the number of tumor types responding to PI3K pathway inhibitors as monotherapy has been limited, since most cancer cells accumulate genetic alterations that result in the constitutive activation of a complex network of proliferation and survival signals. As such, the inhibition of a single survival/proliferation pathway or node within this broader network may be circumvented by constitutive activation (or feedback activation) of an alternative survival pathway, and thus be insufficient for inducing a clinical response. Therefore, in most tumor types, dual pathway inhibition, guided by an in-depth understanding of feedback and cross-talk between pathways, may be required to induce tumor regression. Here we present a nonclinical study exploring mutual feedback activation of MAPK and PI3K pathways. Inhibition of the PI3K pathway by MK-2206 (AKT inhibitor) or MK-8669 (mTOR inhibitor) resulted in feedback activation of MAPK pathway nodes in subsets of lung cell lines. Similarly, inhibition of the MAPK pathway by a MEK inhibitor resulted in feedback activation of the PI3K pathway. Notably, a lack of such feedback activation was associated with sensitivity to the mono inhibitors. The strength of the feedback activation correlated with changes of several receptor tyrosine kinases, suggesting them as a part of the feedback mechanism. Finally, combined inhibition of both MAPK and PI3K pathways synergistically inhibited the feedback activation of both pathways and led to improved in vitro efficacy. Taken together, our findings identify mutual feedback activation of the PI3K and MAPK pathways in response to specific inhibitors and underscore the importance of combined therapeutic approaches with inhibitors to both pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4151.

Abstract FGFR2 amplification has been reported in subsets of gastric cancer and breast cancer. We describe FGFR2 amplification in a cell line derived from a colorectal carcinoma arising in a young patient with ulcerative colitis. The cell line has features of endocrine differentiation, representing a rare (< 1%) subset of colorectal cancer. FGFR2 amplification in this cell line results in an overexpressed and highly activated FGFR2 kinase. The FGFR selective inhibitor PD173074 blocked FGFR2 phosphorylation and inhibited Erk, Akt, and S6 ribosomal protein phosphorylation. FGFR2 inhibition resulted in profound cell death as measured by robust accumulation of cells in subG1 by flow cytometry and PARP cleavage by western analysis. A structurally unrelated FGFR2 inhibitor, MK2461, also caused cell death and inhibited downstream signaling pathways. RTK arrays revealed coactivation of EGFR family kinases, and a functional role for these kinases is being investigated. Interestingly, an FGFR2 isoform in this cell line migrated at 160kD, in contrast to the 140kD isoform observed in SNU16 and other cell lines. However, C-terminal FGFR2 antibodies did not react with the 160kD FGFR2 isoform, suggesting that it has a C-terminal deletion similar to the FGFR2 isoform in OCUM2M and KatoIII cell lines. We are currently defining the frequency of FGFR2 overexpression and activation in colon cancer tissue microarrays. The strong growth inhibition and induction of cell death observed in vitro suggests that colorectal cancer tumor cells harboring FGFR2 amplification / activation may show similar response to FGFR inhibitor therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 284.

Aberrant activation of the serine/threonine kinase Akt, a central node of the PI3K pathway, has been found in a significant proportion of human solid tumors, making Akt an attractive target for therapeutic intervention. MK-2206 is a potent inhibitor of Akt isozymes 1, 2, and 3 with in vitro IC50 values of 8, 12, and 65 nM, respectively. The compound is an allosteric inhibitor of Akt, requiring the presence of the Pleckstrin homology domain for activity. As a consequence, MK-2206 is highly selective against Akt, exhibiting no inhibitory activities against over 250 protein kinases when tested at 1\#956;M. In several cancer cell lines, MK-2206 potently inhibited Akt1 kinase activity (IC50 \#8776; 20 nM), and blocked Akt2 and Akt3 activities 2- to 6-fold less potently. MK-2206 potently inhibited phosphorylation of T308 and S473 of Akt in these cell lines and prevented Akt-mediated phosphorylation of down-stream signaling molecules, including TSC2, PRAS40 and ribosomal S6 proteins. MK-2206 exhibited potent anti-proliferative activity against a number of cancer cell lines harboring one or more of the following genetic defects: 1) constitutive activation of receptor tyrosine kinases such as HER2; 2) PTEN mutation, 3) PI3KCA mutation, and 4) Akt2 amplification. In nude mice bearing A2780 ovarian cancer xenografts, a single oral dose of MK-2206 at 240 mg/kg caused sustained inhibition (>70 %) of phospho-Akt1/2 (T308 and S473) in the tumors. In the same tumor model, MK-2206 inhibited tumor growth by \#8776; 60% when administrated orally at 240 mg/kg per day three times a week. These preclinical results support further clinical development of MK-2206. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3714.

Background> The Met (Hepatocyte Growth Factor Receptor, HGFR) tyrosine kinase can stimulate cancer cell migration and invasion as well as proliferation and survival: these biological functions are consistent with a role for Met in the metastatic process. Methods> We investigated the role of Met in a lymph node-derived lung cancer cell line (H1993) and a line derived from the primary lung tumor of the same patient (H2073), as well as in tissue samples from 18 primary NSCLCs and their paired brain metastases and from primary 22 NSCLCs without metastases. The two groups of patients had matched clinical-pathological features, were never treated with epidermal growth factor receptor (EGFR) inhibitors and were followed-up for an average of 35 months. Met was investigated by immunohistochemistry (IHC) and FISH. IHC for EGFR and Erbb2 and wide screen mutation analysis by Oncomap were also performed in all the cases. Results> While Met was found to be active and amplified in the metastatic line H1993, the H2073 line did not harbor Met activation or amplification. The inhibition of Met by shRNA as well as by specific small molecule inhibitors resulted in the arrest of both migration and growth of H1993 but had not effect on H2073. In contrast, EGFR was activated in primary tumor-derived H2073 cells and not in H1993 cells, and EGFR inhibition severely limited the proliferation only of H2073 cells. The analysis of the metastatic NSCLCs and their paired brain metastases showed that both phosphorylation and expression of Met were heterogeneous and focal in primary tumors but were markedly enriched in the brain metastases (p=0.031, p=0.002 respectively). In contrast, neither EGFR not Erbb2 expression were up-regulated in the metastases. Met activation and expression were significantly associated with shorter metastasis-free survival (p=0.041 and p=0.011 respectively). High Met copy number gain was detected in a subset of brain metastases (27%) and paired primary lung tumors (13%), but bona fide Met amplification as well as Met mutations were never observed. Finally we observed that 70% of the samples with activated Met also carried KRAS mutations. Conclusions>(1) Met activation in metastatic NSCLC never treated with EGFR inhibitors is not driven by genetic mutations and/or amplification of Met; (2) treatment with Met inhibitors of NSCLC patients harboring activated Met may prevent metastatic spread; (3) the presence of RAS mutations in a significant number of Met-activated cases may affect the strategy of pathway inhibition. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4808.

Aberrant c-Met activity has been implicated in the pathogenesis of a variety of human tumors and is therefore an attractive target for therapeutic intervention. We have identified MK-8033, a highly selective inhibitor of c-Met and Ron kinases. MK-8033 potently inhibits the kinase activity of c-Met (IC50 1.3 nM) and Ron (IC50 14 nM) while exhibiting no inhibitory activities against over 220 protein kinases when tested at 1\#956;M. The compound is a novel ATP-competitive inhibitor that is equally potent against wild type c-Met and naturally occurring oncogenic c-Met mutants, including activation loop mutants. MK-8033 inhibits both ligand stimulated and constitutive c-Met phosphosphorylation. As a result c-Met driven in vitro cell proliferation and scattering are inhibited by MK-8033. Genomic amplification and constitutive activation of c-Met are predictors of tumor cell line response to treatment in vitro. In a gastric cancer xenograft model driven by MET amplification, MK-8033 inhibits autophosphorylation of c-Met docking site tyrosine residues and suppresses downstream signaling events such as ERK1/2 and Akt phosphorylation. Tumor growth is inhibited at well tolerated oral doses of the compound in MET amplified gastric and NSCLC xenograft models. These preclinical data provide support for ongoing clinical evaluation of MK-8033 as a potential targeted therapy for patients with c-Met driven tumors. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1751.

AbstractWe have identified a critical role for amplified FGFR2 in gastric cancer cell proliferation and survival. In a panel of gastric cancer cell lines, fibroblast growth factor receptor 2 (FGFR2) was overexpressed and tyrosine phosphorylated selectively in FGFR2-amplified cell lines KatoIII, Snu16, and OCUM-2M. FGFR2 kinase inhibition by a specific small-molecule inhibitor resulted in selective and potent growth inhibition in FGFR2-amplified cell lines, resulting in growth arrest in KatoIII cells and prominent induction of apoptosis in both Snu16 and OCUM-2M cells. FGFR2-amplified cell lines also contained elevated phosphotyrosine in EGFR, Her2, and Erbb3, but the elevated phosphorylation in EGFR could not be inhibited by gefitinib or erlotinib. We show that the elevated EGFR, Her2, and Erbb3 phosphotyrosine is dependent on FGFR2, revealing EGFR family kinases to be downstream targets of amplified FGFR2. Moreover, shRNA to Erbb3 resulted in a loss of proliferation, confirming a functional role for the activated EGFR signaling pathway. These results reveal that both the FGFR2 and EGFR family signaling pathways are activated in FGFR2-amplified gastric cancer cell lines to drive cell proliferation and survival. Inhibitors of FGFR2 or Erbb3 signaling may have therapeutic efficacy in the subset of gastric cancers containing FGFR2 amplification. [Cancer Res 2008;68(7):2340–8]