Andrew Rogers

MET Amplification Leads to Gefitinib Resistance in Lung Cancer by Activating ERBB3 Signaling

The epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib are effective treatments for lung cancers with EGFR activating mutations, but these tumors invariably develop drug resistance. Here, we describe a gefitinib-sensitive lung cancer cell line that developed resistance to gefitinib as a result of focal amplification of the MET proto-oncogene. inhibition of MET signaling in these cells restored their sensitivity to gefitinib. MET amplification was detected in 4 of 18 (22%) lung cancer specimens that had developed resistance to gefitinib or erlotinib. We find that amplification of MET causes gefitinib resistance by driving ERBB3 (HER3)–dependent activation of PI3K, a pathway thought to be specific to EGFR/ERBB family receptors. Thus, we propose that MET amplification may promote drug resistance in other ERBB-driven cancers as well.

Preexistence and Clonal Selection of MET Amplification in EGFR Mutant NSCLC

MET amplification activates ERBB3/PI3K/AKT signaling in EGFR mutant lung cancers and causes resistance to EGFR kinase inhibitors. We demonstrate that MET activation by its ligand, HGF, also induces drug resistance, but through GAB1 signaling. Using high-throughput FISH analyses in both cell lines and in patients with lung cancer, we identify subpopulations of cells with MET amplification prior to drug exposure. Surprisingly, HGF accelerates the development of MET amplification both in vitro and in vivo. EGFR kinase inhibitor resistance, due to either MET amplification or autocrine HGF production, was cured in vivo by combined EGFR and MET inhibition. These findings highlight the potential to prospectively identify treatment naive, patients with EGFR-mutant lung cancer who will benefit from initial combination therapy.

Activation of ERBB2 Signaling Causes Resistance to the EGFR-Directed Therapeutic Antibody Cetuximab

Several cancers become resistant to cetuximab by activating a bypass signaling pathway and preventing cetuximab inhibition of ERK1/2-stimulated growth. Combating Resistance to an EGF Receptor Inhibitor Many promising anticancer drugs are effective only for a limited time, because the tumor cells develop resistance. Cetuximab, directed against the epidermal growth factor receptor (EGFR), is no exception, and patients with colorectal, head and neck, or non–small cell lung cancer eventually cease to respond to the drug. Yonesaka and colleagues have determined that cetuximab-resistant cancer cells—both in culture and in patients—can up-regulate signaling through the ERBB2 growth factor receptor in several ways, permanently turning on extracellular signal–regulated kinase 1/2 (ERK1/2)–mediated growth, differentiation, and survival. They further show that interference with the ERBB2 pathway restores the ability of cetuximab to control these cancers, pointing to a promising resistance-fighting approach. The authors generated clones of cetuximab-resistant non–small cell lung and colorectal cancer cell lines by exposing the cells to increasing concentration of the drug. In some of these resistant clones, the ERBB2 receptor oncogene was genetically amplified, resulting in activated ERK1/2 signaling. Down-regulation of ERBB2 with a small interfering RNA or antibody restored sensitivity. Other clones did not have amplified ERBB2 genes but did make excess heregulin, an activating ligand for the ERBB2 receptor. Heregulin depletion or ERBB2 inhibition restored cetuximab sensitivity. After replicating these studies in xenografts in mice, the authors also looked for evidence that these resistance-associated alterations pertain to human tumors. In several groups of patients with colorectal cancer, they saw decreased survival or decreased sensitivity to cetuximab in those who exhibited amplified ERBB2 gene or higher heregulin concentrations. The concordance of their cellular data with patient experience improves confidence that concomitant treatment of certain lung, head and neck, or colorectal cancers with cetuximab and an anti-ERBB2 drug may prevent or delay the development of drug resistance. These studies add to other successes for this approach, which has also been used for analysis of other molecular targeted therapies, including EGFR kinase inhibitors. Cetuximab, an antibody directed against the epidermal growth factor receptor, is an effective clinical therapy for patients with colorectal, head and neck, and non–small cell lung cancer, 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 in cell lines, either through ERBB2 amplification or through heregulin up-regulation, leads to persistent extracellular signal–regulated kinase 1/2 signaling and consequently to 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 who exhibit either de novo or acquired resistance to cetuximab-based therapy has 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 cetuximab, represent a rational therapeutic strategy that should be assessed in patients with cetuximab-resistant cancers.

A Rapid and Sensitive Enzymatic Method for Epidermal Growth Factor Receptor Mutation Screening

Purpose: Mutations in the epidermal growth factor receptor (EGFR) are associated with clinical and radiographic responses to EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Currently available methods of EGFR mutation detection rely on direct DNA sequencing, which requires isolation of DNA from a relatively pure population of tumor cells, cannot be done on small diagnostic specimens, and lack sensitivity. Here we describe the use of a sensitive screening method that overcomes many of these limitations. Experimental Design: We screened 178 non–small cell lung cancer specimens for mutations in exons 18 to 21 of EGFR using a DNA endonuclease, SURVEYOR, which cleaves mismatched heteroduplexed DNA. Samples were analyzed by high-performance liquid chromatography on the Transgenomic WAVE HS system. Selected specimens that produced digestion products using SURVEYOR were subsequently reanalyzed by size separation or under partially denaturing conditions, followed by fractionation and sequencing. The specimens included DNA isolated from frozen tumor specimens, dissected formalin-fixed, paraffin-embedded tumor specimens undergoing clinical sequencing, and undissected formalin-fixed, paraffin-embedded specimens. One hundred sixty specimens were independently analyzed using direct DNA sequencing in a blinded fashion. Results: EGFR mutations were detected in 16 of 61 fresh frozen tumor specimens, 24 of 91 dissected formalin-fixed, paraffin-embedded tumor specimens, and 11 of 26 undissected formalin-fixed, paraffin-embedded tumor specimens. Compared with sequencing, the sensitivity and specificity of the present method were 100% and 87%. The positive and negative predictive values were 74% and 100%, respectively. SURVEYOR analysis detected 7 (4%) mutations that were not previously detected by direct sequencing. Conclusions: SURVEYOR analysis provides a rapid method for EGFR mutation screening with 100% sensitivity and negative predictive value. This unbiased scanning technique is superior to direct sequencing when used with undissected formalin-fixed, paraffin-embedded specimens.

Amplification of EGFR T790M causes resistance to an irreversible EGFR inhibitor

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib are effective therapies against mutant non-small cell lung cancers (NSCLCs). Treatment is limited by the development of resistance in part explained by the gain of a secondary EGFR mutation, T790M, at the gatekeeper residue. Irreversible EGFR inhibitors, including PF00299804, are effective in vitro and in vivo against EGFR mutant tumors that contain EGFR T790M and are currently under clinical development. In this study, we generate models of resistance to PF00299804, using cell lines with EGFR T790M and show that the PF00299804-resistant models develop focal amplification of EGFR that preferentially involves the T790M-containing allele. These PF00299804-resistant cell lines remain dependent on EGFR for growth as downregulation of EGFR by shRNA compromises their viability. We show that resistance to PF00299804 arises, at least in part, through selection of a pre-existing EGFR T790M-amplified clone both in vitro and using a xenograft model in vivo. Our findings show that EGFR T790M is a common resistance mechanism to both reversible, and when amplified, the irreversible EGFR kinase inhibitors further emphasizing the need to develop more potent therapies against EGFR T790M. These findings can be used to guide studies of patient tumor specimens from ongoing clinical trials of irreversible EGFR kinase inhibitors.

Reactivation of ERK Signaling Causes Resistance to EGFR Kinase Inhibitors

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors is limited by the development of drug resistance. The irreversible EGFR kinase inhibitor WZ4002 is effective against the most common mechanism of drug resistance mediated by the EGFR T790M mutation. Here, we show, in multiple complementary models, that resistance to WZ4002 develops through aberrant activation of extracellular signal-regulated kinase (ERK) signaling caused by either an amplification of mitogen-activated protein kinase 1 (MAPK1) or by downregulation of negative regulators of ERK signaling. Inhibition of MAP-ERK kinase (MEK) or ERK restores sensitivity to WZ4002 and prevents the emergence of drug resistance. We further identify MAPK1 amplification in an erlotinib-resistant EGFR-mutant non-small cell lung carcinoma patient. In addition, the WZ4002-resistant MAPK1-amplified cells also show an increase both in EGFR internalization and a decrease in sensitivity to cytotoxic chemotherapy. Our findings provide insights into mechanisms of drug resistance to EGFR kinase inhibitors and highlight rational combination therapies that should be evaluated in clinical trials.

Noninvasive Detection of EGFR T790M in Gefitinib or Erlotinib Resistant Non-Small Cell Lung Cancer

Purpose: Tumors from 50% of epidermal growth factor receptor (EGFR) mutant non–small cell lung cancer patients that develop resistance to gefitinib or erlotinib will contain a secondary EGFR T790M mutation. As most patients do not undergo repeated tumor biopsies we evaluated whether EGFR T790M could be detected using plasma DNA. Experimental Design: DNA from plasma of 54 patients with known clinical response to gefitinib or erlotinib was extracted and used to detect both EGFR-activating and EGFR T790M mutations. Forty-three (80%) of patients had tumor EGFR sequencing (EGFR mutant/wild type: 30/13) and seven patients also had EGFR T790M gefitinib/erlotinib-resistant tumors. EGFR mutations were detected using two methods, the Scorpion Amplification Refractory Mutation System and the WAVE/Surveyor, combined with whole genome amplification. Results: Both EGFR-activating and EGFR T790M were identified in 70% of patients with known tumor EGFR-activating (21 of 30) or T790M (5 of 7) mutations. EGFR T790M was identified from plasma DNA in 54% (15 of 28) of patients with prior clinical response to gefitinib/erlotinib, 29% (4 of 14) with prior stable disease, and in 0% (0 of 12) that had primary progressive disease or were untreated with gefitinib/erlotinib. Conclusions:EGFR T790M can be detected using plasma DNA from gefitinib- or erlotinib-resistant patients. This noninvasive method may aid in monitoring drug resistance and in directing the course of subsequent therapy.

Multiple Mutations and Bypass Mechanisms Can Contribute to Development of Acquired Resistance to MET Inhibitors

Therapies targeting receptor tyrosine kinases have shown efficacy in molecularly defined subsets of cancers. Unfortunately, cancers invariably develop resistance, and overcoming or preventing resistance will ultimately be key to unleashing their full therapeutic potential. In this study, we examined how cancers become resistant to MET inhibitors, a class of drugs currently under clinical development. We utilized the highly sensitive gastric carcinoma cell line, SNU638, and two related MET inhibitors PHA-665752 and PF-2341066. To our surprise, we observed at least two mechanisms of resistance that arose simultaneously. Both resulted in maintenance of downstream PI3K (phosphoinositide 3-kinase)-AKT and MEK (MAP/ERK kinase)-ERK signaling in the presence of inhibitor. One mechanism, observed by modeling resistance both in vitro and in vivo, involved the acquisition of a mutation in the MET activation loop (Y1230). Structural analysis indicates that this mutation destabilizes the autoinhibitory conformation of MET and abrogates an important aromatic stacking interaction with the inhibitor. The other cause of resistance was activation of the epidermal growth factor receptor (EGFR) pathway due to increased expression of transforming growth factor α. Activation of EGFR bypassed the need for MET signaling to activate downstream signaling in these cells. This resistance could be overcome by combined EGFR and MET inhibition. Thus, therapeutic strategies that combine MET inhibitors capable of inhibiting Y1230 mutant MET in combination with anti-EGFR-based therapies may enhance clinical benefit for patients with MET-addicted cancers. Importantly, these results also underscore the notion that a single cancer can simultaneously develop resistance induced by several mechanisms and highlight the daunting challenges associated with preventing or overcoming resistance.

Autocrine Production of Amphiregulin Predicts Sensitivity to Both Gefitinib and Cetuximab in EGFR Wild-type Cancers

Purpose: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, gefitinib and erlotinib, lead to significant tumor regressions in 10% to 15% of non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. However, 30% to 40% of NSCLC patients, majority of whom are EGFR wild-type, develop stable disease following EGFR tyrosine kinase inhibitor therapy. EGFR-directed antibodies (cetuximab) are effective treatments for head and neck squamous cell carcinomas, which seldom contain EGFR mutations. The determinant(s) of efficacy of EGFR-targeted therapies in EGFR wild-type cancers is not well defined. Experimental Design: We examined the relationship of EGFR ligands, EGF, transforming growth factor-α,and amphiregulin and the efficacy of gefitinib and cetuximab in EGFR wild-type NSCLC (n = 10) and head and neck squamous cell carcinoma (n = 4) cell lines. We compared amphiregulin expression using immunohistochemistry in EGFR wild-type NSCLC patients (n = 24) that developed either stable or progressive disease following erlotinib or gefitinib treatment. Results: Cell lines which produced ≥20 pmol/L amphiregulin, as detected by an ELISA, were significantly more likely to be growth inhibited by both gefitinib and cetuximab than those that produced minimal or no amphiregulin. In these cell lines, both cetuximab and gefitinib led to cell cycle arrest at the G1-S boundary and was associated with preferential inhibition of extracellular signal-regulated kinase 1/2 but not Akt signaling. Amphiregulin expression was significantly higher in NSCLC patients that developed stable disease compared with those that developed disease progression following gefitinib or erlotinib treatment. Conclusions: Amphiregulin expression may help select EGFR wild-type patients who are likely to develop stable disease from EGFR-targeted therapies.

Mutations in the LKB1 tumour suppressor are frequently detected in tumours from Caucasian but not Asian lung cancer patients.

Somatic mutations of LKB1 tumour suppressor gene have been detected in human cancers including non-small cell lung cancer (NSCLC). The relationship between LKB1 mutations and clinicopathological characteristics and other common oncogene mutations in NSCLC is inadequately described. In this study we evaluated tumour specimens from 310 patients with NSCLC including those with adenocarcinoma, adenosquamous carcinoma, and squamous cell carcinoma histologies. Tumours were obtained from patients of US (n=143) and Korean (n=167) origin and screened for LKB1, KRAS, BRAF, and EGFR mutations using RT—PCR-based SURVEYOR-WAVE method followed by Sanger sequencing. We detected mutations in the LKB1 gene in 34 tumours (11%). LKB1 mutation frequency was higher in NSCLC tumours of US origin (17%) compared with 5% in NSCLCs of Korean origin (P=0.001). They tended to occur more commonly in adenocarcinomas (13%) than in squamous cell carcinomas (5%) (P=0.066). LKB1 mutations associated with smoking history (P=0.007) and KRAS mutations (P=0.042) were almost mutually exclusive with EGFR mutations (P=0.002). The outcome of stages I and II NSCLC patients treated with surgery alone did not significantly differ based on LKB1 mutation status. Our study provides clinical and molecular characteristics of NSCLC, which harbour LKB1 mutations.