The epidermal growth factor receptor directed antibody, cetuximab, is an effective clinical therapy for patients with colorectal, head and neck and non-small cell lung cancer patients particularly for those with KRAS and BRAF wild type cancers. Treatment in all patients is limited eventually by the development of acquired resistance but little is known about the underlying mechanism. Here we show, that activation of ERBB2 signaling, either through ERBB2 amplification or through heregulin upregulation, leads to persistent ERK 1/2 signaling and consequently cetuximab resistance. Inhibition of ERBB2 or disruption of ERBB2/ERBB3 heterodimerization restores cetuximab sensitivity in vitro and in vivo. A subset of colorectal cancer patients that exhibit either de novo or acquired resistance to cetuximab based therapy possess ERBB2 amplification or high levels of circulating heregulin. Collectively, these findings identify two distinct resistance mechanisms, both of which promote aberrant ERBB2 signaling, that mediate cetuximab resistance. Moreover, these results suggest that ERBB2 inhibitors, in combination with cetuximanb, represent a rational therapeutic strategy that should be assessed in cetuximab-resistant cancers.

The identification of somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) in patients with non-small cell lung cancer (NSCLC) and the association of such mutations with the clinical response to EGFR tyrosine kinase inhibitors (TKI), such as gefitinib and erlotinib, have had a substantial effect on the treatment of this disease. EGFR gene amplification has also been associated with an increased therapeutic response to EGFR-TKIs. The effects of these two types of EGFR alteration on EGFR function have remained unclear, however. We have now examined 16 NSCLC cell lines, including eight newly established lines from Japanese NSCLC patients, for the presence of EGFR mutations and amplification. Four of the six cell lines that harbor EGFR mutations were found to be positive for EGFR amplification, whereas none of the 10 cell lines negative for EGFR mutation manifested EGFR amplification, suggesting that these two types of EGFR alteration are closely associated. Endogenous EGFRs expressed in NSCLC cell lines positive for both EGFR mutation and amplification were found to be constitutively activated as a result of ligand-independent dimerization. Furthermore, the patterns of both EGFR amplification and EGFR autophosphorylation were shown to differ between cell lines harboring the two most common types of EGFR mutation (exon 19 deletion and L858R point mutation in exon 21). These results reveal distinct biochemical properties of endogenous mutant forms of EGFR expressed in NSCLC cell lines and may have implications for treatment of this condition. [Cancer Res 2007;67(5):2046-53]

Kinase domain mutations of the epidermal growth factor receptor (EGFR) are common oncogenic events in lung adenocarcinoma. Here we explore the dependency upon asymmetric dimerization of the kinase domain for activation of lung cancer-derived EGFR mutants. We show that while wild-type EGFR and the L858R mutant require dimerization for activation and oncogenic transformation, the exon 19 deletion, exon 20 insertion, and L858R/T790M EGFR mutants do not require dimerization. In addition, treatment with the monoclonal antibody, cetuximab, shrinks mouse lung tumors induced by the dimerization-dependent L858R mutant, but exerts only a modest effect on tumors driven by dimerization-independent EGFR mutants. These data imply that different EGFR mutants show differential requirements for dimerization, and that disruption of dimerization may be among the antitumor mechanisms of cetuximab.

Purpose: Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib show marked efficacy in patients with non-small cell lung cancer positive for the echinoderm microtubuleassociated protein-like 4 (EML4)-ALK fusion protein. However, acquired resistance to these agents has already been described in treated patients, and the mechanisms of such resistance remain largely unknown.Experimental Design: We established lines of EML4-ALK-positive H3122 lung cancer cells that are resistant to the ALK inhibitor TAE684 (H3122/TR cells) and investigated their resistance mechanism with the use of immunoblot analysis, ELISA, reverse transcription and real-time PCR analysis, and an annexin V binding assay. We isolated EML4-ALK-positive lung cancer cells (K-3) from a patient who developed resistance to crizotinib and investigated their characteristics.Results: The expression of EML4-ALK was reduced at the transcriptional level, whereas phosphorylation of epidermal growth factor receptor (EGFR), HER2, and HER3 was upregulated, in H3122/TR cells compared with those in H3122 cells. This activation of HER family proteins was accompanied by increased secretion of EGF. Treatment with an EGFR-TKI induced apoptosis in H3122/TR cells, but not in H3122 cells. The TAE684-induced inhibition of extracellular signal-regulated kinase (ERK) and STAT3 phosphorylation observed in parental cells was prevented by exposure of these cells to exogenous EGF, resulting in a reduced sensitivity of cell growth to TAE684. K-3 cells also manifested HER family activation accompanied by increased EGF secretion.Conclusions: EGF-mediated activation of HER family signaling is associated with ALK-TKI resistance in lung cancer positive for EML4-ALK.

Anti-programmed-death-1 (PD-1) immunotherapy improves survival in non-small cell lung cancer (NSCLC), but some cases are refractory to treatment, thereby requiring alternative strategies. B7-H3, an immune-checkpoint molecule, is expressed in various malignancies. To our knowledge, this study is the first to evaluate B7-H3 expression in NSCLCs treated with anti-PD-1 therapy and the therapeutic potential of a combination of anti-PD-1 therapy and B7-H3 targeting. B7-H3 expression was evaluated immunohistochemically in patients with NSCLC ( = 82), and its relationship with responsiveness to anti-PD-1 therapy and CD8 tumor-infiltrating lymphocytes (TILs) was analyzed. The antitumor efficacy of dual anti-B7-H3 and anti-programmed death ligand-1 (PD-L1) antibody therapy was evaluated using a syngeneic murine cancer model. T-cell numbers and functions were analyzed by flow cytometry. B7-H3 expression was evident in 74% of NSCLCs and was correlated critically with nonresponsiveness to anti-PD-1 immunotherapy. A small number of CD8 TILs was observed as a subpopulation with PD-L1 tumor proportion score less than 50%, whereas CD8 TILs were still abundant in tumors not expressing B7-H3. Anti-B7-H3 blockade showed antitumor efficacy accompanied with an increased number of CD8 TILs and recovery of effector function. CD8 T-cell depletion negated antitumor efficacy induced by B7-H3 blockade, indicating that improved antitumor immunity is mediated by CD8 T cells. Compared with a single blocking antibody, dual blockade of B7-H3 and PD-L1 enhanced the antitumor reaction. B7-H3 expressed on tumor cells potentially circumvents CD8-T-cell-mediated immune surveillance. Anti-B7-H3 immunotherapy combined with anti-PD-1/PD-L1 antibody therapy is a promising approach for B7-H3-expressing NSCLCs. .

Purpose Irreversible EGFR-tyrosine kinase inhibitors (TKIs) are thought to be one strategy to overcome EGFR-TKI resistance induced by T790M gate-keeper mutations in non-small cell lung cancer (NSCLC), yet they display limited clinical efficacy. We hypothesized that additional resistance mechanisms that cooperate with T790M could be identified by profiling tyrosine phosphorylation in NSCLC cells with acquired resistance to reversible EGFR-TKI and harboring T790M. Experimental Design We profiled PC9 cells with TKI-sensitive EGFR mutation and paired EGFR-TKI-resistant PC9GR (gefitinib-resistant) cells with T790M using immunoaffinity purification of tyrosine-phosphorylated peptides and mass-spectrometry-based identification/quantification. Profiles of erlotinib perturbations were examined. Results We observed a large fraction of the tyrosine phosphoproteome was more abundant in PC9- and PC9GR-erlotinib treated cells, including phosphopeptides corresponding to MET, IGF, and AXL signaling. Activation of these receptor tyrosine kinases by growth factors could protect PC9GR cells against the irreversible EGFR-TKI afatinib. We identified a Src-family kinase (SFK) network as EGFR-independent and confirmed that neither erlotinib nor afatinib affected Src phosphorylation at the activation site. The SFK-inhibitor dasatinib plus afatinib abolished Src phosphorylation and completely suppressed downstream phosphorylated Akt and Erk. Dasatinib further enhanced anti-tumor activity of afatinib or T790M-selective EGFR-TKI (WZ4006) in proliferation and apoptosis assays in multiple NSCLC cell lines with T790M mediated resistance. This translated into tumor regression in PC9GR xenograft studies with combined afatinib and dasatinib. Conclusions Our results identified both co-drivers of resistance along with T790M and support further studies of irreversible or T790M-selective EGFR inhibitors combined with dasatinib in NSCLC patients with acquired T790M.

Molecular inhibition of the epidermal growth factor receptor (EGFR) is a promising anticancer strategy, and monoclonal antibodies (mAbs) to EGFR are undergoing extensive evaluation in preclinical and clinical trials. However, the effects of anti-EGFR mAbs on EGFR signaling have remained unclear. We have now examined the effects of 2 anti-EGFR mAbs, matuzumab (EMD72000) and cetuximab (Erbitux), both of which are currently under assessment for treatment of various cancers, on EGFR signal transduction and cell survival in nonsmall cell lung cancer cell lines. Similar to EGF, matuzumab and cetuximab each induced phosphorylation of EGFR at several tyrosine phosphorylation sites as a result of receptor dimerization and activation of the receptor tyrosine kinase. In contrast to the effects of EGF, however, EGFR activation induced by these antibodies was not accompanied by receptor turnover or by activation of downstream signaling pathways that are mediated by Akt and Erk and are important for regulation of cell proliferation and survival. In addition, clonogenic survival assays revealed that matuzumab and cetuximab reduced the survival rate of H292 cells, in which they also inhibited the EGF-induced activation of Akt and Erk. Although we have examined only a few cell lines, our results indicate that the antitumor effects of matuzumab and cetuximab depend on inhibition of EGFR downstream signaling mediated by Akt or Erk rather than on inhibition of EGFR itself. ' 2007 Wiley-Liss, Inc.

Somatic mutations in the epidermal growth factor receptor (EGFR) gene are associated with the therapeutic response to EGFR tyrosine kinase inhibitors (TKI) in patients with advanced non-small cell lung cancer (NSCLC). The response rate to these drugs remains low, however, in NSCLC patients with wild-type EGFR alleles. Combination therapies with EGFR-TKIs and cytotoxic agents are considered a therapeutic option for patients with NSCLC expressing wild-type EGFR. We investigated the antiproliferative effect of the combination of the oral fluorouracil S-1 and the EGFR-TKI gefitinib in NSCLC cells of differing EGFR status. The combination of 5-fluorouracil and gefitinib showed a synergistic antiproliferative effect in vitro in all NSCLC cell lines tested. Combination chemotherapy with S-1 and gefitinib in vivo also had a synergistic antitumor effect on NSCLC xenografts regardless of the absence or presence of EGFR mutations. Gefitinib inhibited the expression of the transcription factor E2F-1, resulting in the down-regulation of thymidylate synthase at the mRNA and protein levels. These observations suggest that gefitinib-induced down-regulation of thymidylate synthase is responsible, at least in part, for the synergistic antitumor effect of combined treatment with S-1 and gefitinib and provide a basis for clinical evaluation of combination chemotherapy with S-1 and EGFR-TKIs in patients with solid tumors. [Mol Cancer Ther 2008;7(3):599 -606]