An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor

Mutations of the epidermal growth factor receptor (EGFR) gene have been identified in non-small cell lung cancer specimens from patients responding to anilinoquinazoline EGFR inhibitors. However, clinical resistance to EGFR inhibitor therapy is commonly observed. Previously, we showed that such resistance can be caused by a second mutation of the EGFR gene, leading to a T790M amino acid change in the EGFR tyrosine kinase domain and also found that CL-387,785, a specific and irreversible anilinoquinazoline EGFR inhibitor, was able to overcome this resistance on the biochemical level. Here, we present the successful establishment of a stable Ba/F3 cell line model system for the study of oncogenic EGFR signaling and the functional consequences of the EGFR T790M resistance mutation. We show the ability of gefitinib to induce growth arrest and apoptosis in cells transfected with wild-type or L858R EGFR, whereas the T790M mutation leads to high-level functional resistance against gefitinib and erlotinib. In addition, CL-387,785 is able to overcome resistance caused by the T790M mutation on a functional level, correlating with effective inhibition of downstream signaling pathways. Similar data was also obtained with the use of the gefitinib-resistant H1975 lung cancer cell line. The systems established by us should prove useful for the large-scale screening of alternative EGFR inhibitor compounds against the T790M or other EGFR mutations. These data also support the notion that clinical investigations of compounds similar to CL-387,785 may be useful as a treatment strategy for patients with resistance to EGFR inhibitor therapy caused by the T790M mutation.     

Resistance to an irreversible epidermal growth factor receptor (EGFR) inhibitor in EGFR-mutant lung cancer reveals novel treatment strategies

Patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer derive significant clinical benefit from treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Secondary EGFR mutations such as EGFR T790M commonly lead to resistance to these agents, limiting their long-term efficacy. Irreversible EGFR inhibitors such as CL-387,785 can overcome resistance and are in clinical development, yet acquired resistance against these agents is anticipated. We carried out a cell-based, in vitro random mutagenesis screen to identify EGFR mutations that confer resistance to CL-387,785 using T790M-mutant H1975 lung adenocarcinoma cells. Mutations at several residues occurred repeatedly leading to functional resistance to CL-387,785. These variants showed uninhibited cell growth, reduced apoptosis, and persistent EGFR activation in the presence of CL-387,785 as compared with parental H1975 cells, thus confirming their role in resistance. A screen of alternative agents showed that both an alternative EGFR inhibitor and a cyclin-dependent kinase 4 inhibitor led to significant inhibition of cell growth of the resistant mutants, suggestive of potential alternative treatment strategies. These results identify novel mutations mediating resistance to irreversible EGFR inhibitors and reveal alternative strategies to overcome or prevent the development of resistance in EGFR-mutant non-small cell lung cancers.     

Conversion from the "oncogene addiction" to "drug addiction" by intensive inhibition of the EGFR and MET in lung cancer with activating EGFR mutation

Emergence of acquired resistance is virtually inevitable in patients with a mutation in the epidermal growth factor receptor gene (EGFR) treated with EGFR tyrosine kinase inhibitors (TKIs). Several novel TKIs that may prevent or overcome the resistance mechanisms are now under clinical development. However, it is unknown how tumor cells will respond to intensive treatment using these novel TKIs. We previously established HCC827EPR cells, which are T790M positive, through combined treatment with erlotinib and a MET-TKI from erlotinib-hypersensitive HCC827 cells. In this study, we treated HCC827EPR cells sequentially with an irreversible EGFR-TKI, CL-387,785, to establish resistant cells (HCC827CLR), and we analyzed the mechanisms responsible for resistance. In HCC827CLR cells, PTEN expression was downregulated and Akt phosphorylation persisted in the presence of CL-387,785. Akt inhibition restored CL-387,785 sensitivity. In addition, withdrawal of CL-387,785 reduced cell viability in HCC827CLR cells, indicating that these cells were "addicted" to CL-387,785. HCC827CLR cells overexpressed the EGFR, and inhibition of the EGFR or MEK-ERK was needed to maintain cell proliferation. Increased senescence was observed in HCC827CLR cells in the drug-free condition. Through long-term culture of HCC827CLR cells without CL-387,785, we established HCC827-CL-387,785-independent cells, which exhibited decreased EGFR expression and a mesenchymal phenotype. In conclusion, PTEN downregulation is a newly identified mechanism underlying the acquired resistance to irreversible EGFR-TKIs after acquisition of T790M against erlotinib. This series of experiments highlights the flexibility of cancer cells that have adapted to environmental stresses induced by intensive treatment with TKIs.     

Antitumor activity of HM781-36B, a highly effective pan-HER inhibitor in erlotinib-resistant NSCLC and other EGFR-dependent cancer models

The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases has been implicated in a variety of cancers. In particular, activating mutations such as the L858R point mutation in exon 21 and the small in-frame deletions in exon 19 of the EGFR tyrosine kinase domain are correlated with sensitivity to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) patients. Clinical treatment of patients is limited by the development of drug resistance resulting mainly from a gatekeeper mutation (T790M). In this study, we evaluated the therapeutic potential of a novel, irreversible pan-HER inhibitor, HM781-36B. The results from this study show that HM781-36B is a potent inhibitor of EGFR in vitro, including the EGFR-acquired resistance mutation (T790M), as well as HER-2 and HER-4, compared with other EGFR tyrosine kinases inhibitors (erlotinib, lapatinib and BIBW2992). HM781-36B treatment of EGFR DelE746_A750-harboring erlotinib-sensitive HCC827 and EGFR L858R/T790M-harboring erlotinib-resistant NCI-H1975 NSCLC cells results in the inhibition of EGFR phosphorylation and the subsequent deactivation of downstream signaling proteins. Additionally, HM781-36B shows an excellent efficacy in a variety of EGFR- and HER-2-dependent tumor xenograft models, including erlotinib-sensitive HCC827 NSCLC cells, erlotinib-resistant NCI-H1975 NSCLC cells, HER-2 overexpressing Calu-3 NSCLC cells, NCI-N87 gastric cancer cells, SK-Ov3 ovarian cancer cells and EGFR-overexpressing A431 epidermoid carcinoma cancer cells. On the basis of these preclinical results, HM781-36B is the most potent pan-HER inhibitor, which will be advantageous for the treatment of patients with NSCLC including clinical limitation caused by acquired mutation (EGFR T790M), breast cancer and gastric cancer.     

Epidermal growth factor receptors harboring kinase domain mutations associate with the heat shock protein 90 chaperone and are destabilized following exposure to geldanamycins

Somatic mutations in the kinase domain of the epidermal growth factor receptor (EGFR), including L858R and exon 19 deletions, underlie responsiveness to gefitinib and erlotinib in non-small cell lung cancer (NSCLC). Acquired resistance to these tyrosine kinase inhibitors is in some cases mediated by a second mutation, T790M. Ansamycin antibiotics, such as geldanamycin, potently inhibit heat shock protein 90 (Hsp90), promoting ubiquitin-mediated degradation of oncogenic kinases that require the chaperone for proper conformational folding. Here, we show that L858R and deletion mutant EGFR proteins found in NSCLC interact with the chaperone and are sensitive to degradation following Hsp90 inhibition. In NIH/3T3 cells expressing either wild-type or mutant EGFR, diminution of expression of both L858R and EGFR delL747-S752, P753S occurred following exposure to 50 nmol/L geldanamycin over 24 hours, whereas partial diminution of wild-type EGFR required a minimum of 200 nmol/L drug. In time course experiments, mutant EGFR expression was depleted after only 4 hours of exposure to 1 micromol/L geldanamycin, whereas diminution of wild-type EGFR was less substantial and seen only following 12 hours. Similarly, EGFR proteins in NSCLC cell lines harboring EGFR mutations, including NCI-H1650, NCI-H3255, and NCI-H1975, were also more sensitive to geldanamycin-induced degradation compared with the protein in wild-type cells. Exposure of EGFR-mutant cell lines to geldanamycin induced marked depletion of phospho-Akt and cyclin D1 as well as apoptosis. These data suggest mutational activation of EGFR is associated with dependence on Hsp90 for stability and that Hsp90 inhibition may represent a novel strategy for the treatment of EGFR-mutant NSCLC.     

244-MPT overcomes gefitinib resistance in non-small cell lung cancer cells

The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer(NSCLC). Several EGFR tyrosine kinase inhibitors(TKIs), such as gefitinib, have been used as effective clinical therapies for patients with NSCLC. Unfortunately, acquired resistance to gefitinib commonly occurs after 6–12 months of treatment. The resistance is associated with the appearance of the L858R/T790M double mutation of the EGFR. In our present study, we discovered a compound,referred to as 244-MPT, which could suppress either gefitinib-sensitive or -resistant lung cancer cell growth and colony formation, and also suppressed the kinase activity of both wildtype and double mutant (L858R/T790M) EGFR. The underlying mechanism reveals that 244-MPT could interact with either the wildtype or double-mutant EGFR in an ATP-competitive manner and inhibit activity. Treatment with 244-MPT could substantially reduce the phosphorylation of EGFR and its downstream signaling pathways, including Akt and ERK1/2 in gefitinib-sensitive and -resistant cell lines. It was equally effective in suppressing EGFR phosphorylation and downstream signaling in NL20 cells transfected with wildtype, single-mutant (L858R) or mutant (L858R/T790M) EGFR. 244-MPT could also induce apoptosis in a gefitinib-resistant cell line and strongly suppress gefitinib-resistant NSCLC tumor growth in a xenograft mouse model. In addition, 244-MPT could effectively reduce the size of tumors in a gefitinib-resistant NSCLC patient-derived xenograft (PDX) SCID mouse model. Overall, 244-MPT could overcome gefitinib-resistance by directly targeting the EGFR.     

Bronchial and peripheral murine lung carcinomas induced by T790M-L858R mutant EGFR respond to HKI-272 and rapamycin combination therapy

The EGFR T790M mutation has been identified in tumors from lung cancer patients that eventually develop resistance to erlotinib. In this study, we generated a mouse model with doxycycline-inducible expression of a mutant EGFR containing both L858R, an erlotinib-sensitizing mutation, and the T790M resistance mutation (EGFR TL). Expression of EGFR TL led to development of peripheral adenocarcinomas with bronchioloalveolar features in alveoli as well as papillary adenocarcinomas in bronchioles. Treatment with an irreversible EGFR tyrosine kinase inhibitor (TKI), HKI-272, shrunk only peripheral tumors but not bronchial tumors. However, the combination of HKI-272 and rapamycin resulted in significant regression of both types of lung tumors. This combination therapy may potentially benefit lung cancer patients with the EGFR T790M mutation.     

Anti‐tumor activity of WK88‐1, a novel geldanamycin derivative,in gefitinib‐resistant non‐small cell lung cancers with Met amplification

Although epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) have been introduced for the treatment of non‐small cell lung cancer (NSCLC), the emergence of secondary T790M mutation in EGFR or amplification of the Met proto‐oncogene restrain the clinical success of EGFR‐TKIs. Since heat shock protein‐90 (Hsp90) stabilizes various oncoproteins including EGFR and c‐Met, the inhibition of Hsp90 activity appears as a rational strategy to develop anticancer drugs. Despite preclinical efficacy of geldanamycin‐anasamycin (GA)‐derivatives containing benzoquinone moiety as Hsp90 inhibitors, the hepatotoxicity of these GA‐derivatives restricts their therapeutic benefit. We have prepared WK‐88 series of GA‐derivatives, which lack the benzoquinone moiety. In this study, we have examined the anticancer effects of WK88‐1 in Met‐amplified‐ and gefitinib‐resistant (HCC827GR) NSCLC cells and its parental HCC827 cells. Treatment with WK88‐1 reduced the cell viability in both HCC827 and HCC827GR cells, which was associated with marked decrease in the constitutive expression of Hsp90 client proteins, such as EGFR, ErbB2, ErbB3, Met and Akt. Moreover, WK88‐1 attenuated phosphorylation of these Hsp90 client proteins and reduced the anchorage‐independent growth of HCC827GR cells. Administration of WK88‐1 did not cause hepatotoxicity in animals and significantly reduced the growth of HCC827GR cells xenograft tumors in nude mice. Our study provides evidence that ErbB3 might be a client for Hsp90 in Met‐amplified NSCLCs. In conclusion, we demonstrate that inhibition of Hsp90 dampens the activation of EGFR‐ or c‐Met‐mediated survival of Met‐amplified NSCLCs and that WK88‐1 as a Hsp90 inhibitor alleviates gefitinib resistance in HCC827GR cells.     

Searching for a magic bullet in NSCLC: the role of epidermal growth factor receptor mutations and tyrosine kinase inhibitors

The epidermal growth factor receptor (EGFR) has been implicated in the pathophysiology of various cancers, including non-small cell lung cancer (NSCLC). Inhibitors targeting the tyrosine kinase domain of this receptor have been seen to elicit favourable responses in a subset of NSCLC patients. Mutational analysis of the EGFR has revealed that the response to reversible tyrosine kinase inhibitors (TKIs) is a result of the presence of activating mutations present between exons 18 and 21, most frequently the exon 19 deletion and the L858R mutations. After a prolonged treatment with reversible TKIs, patients have been seen to develop resistance that results, in part, from the presence of the secondary T790M mutation in exon 20. Preclinical data suggest that second-generation TKIs may be able to overcome T790M resistance by virtue of their irreversible mode of binding. In addition to the predominant mutations in the EGFR gene, alternative genetic changes between exons 18 and 21 have been observed. Experimental models have demonstrated that TKIs exhibit differential efficacy depending on which mutations are present. Such information may result in the design of highly specific agents that target specific mutations, which could result in more efficacious treatments.     

Afatinib increases sensitivity to radiation in non-small cell lung cancer cells with acquired EGFR T790M mutation

Afatinib is a second-generation of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor and has shown a significant clinical benefit in non-small cell lung cancer (NSCLC) patients with EGFR-activating mutations. However, the potential therapeutic effects of afatinib combining with other modalities, including ionizing radiation (IR), are not well understood. In this study, we developed a gefitinib-resistant cell subline (PC-9-GR) with a secondary EGFR mutation (T790M) from NSCLC PC-9 cells after chronic exposures to increasing doses of gefitinib. The presence of afatinib significantly increases the cell killing effect of radiation in PC-9-GR cells harboring acquired T790M, but not in H1975 cells with de novo T790M or in H460 cells that express wild-type EGFR. In PC-9-GR cells, afatinib remarkable blocks baseline of EGFR and ERK phosphorylations, and causes delay of IR-induced AKT phosphorylation. Afatinib treatment also leads to increased apoptosis and suppressed DNA damage repair in irradiated PC-9-GR cells, and enhanced tumor growth inhibition when combined with IR in PC-9-GR xenografts. Our findings suggest a potential therapeutic impact of afatinib as a radiation sensitizer in lung cancer cells harboring acquired T790M mutation, providing a rationale for a clinical trial with combination of afatinib and radiation in NSCLCs with EGFR T790M mutation.     

Inhibiting proliferation of gefitinib-resistant, non-small cell lung cancer

Purpose

Sensitivity to a tyrosine kinase inhibitor (TKI) is correlated with the presence of somatic mutations that affect the kinase domain of epidermal growth factor receptor (EGFR). Development of resistance to TKI is a major therapeutic problem in non-small cell lung cancer (NSCLC). Aim of this study is to identify agents that can overcome TKI resistance in NSCLC.

Methods

We used a carefully selected panel of 12 NSCLC cell lines to address this clinical problem. Initially, the cell lines were treated with a variety of 10 compounds. Cellular proliferation was measured via MTT assay. We then focused on the gefitinib-resistant, EGFR mutant cell lines [H1650: exon 19 and PTEN mutations; and H1975: exons 20 (T790M) and 21 (L858R)] to identify agents that could overcome TKI resistance.

Results

Both 17-DMAG (Hsp90 inhibitor) and belinostat (histone deacetylase inhibitor, HDACi) effectively decreased the growth of almost all NSCLC lines. Also, belinostat markedly decreased the expression of EGFR and phospho-Akt in the cells. Combination of 17-DMAG and belinostat synergistically inhibited in vitro proliferation of these cells. Furthermore, both agents and their combination almost completely prevented TKI-resistant tumor formation (EGFR T790M mutation) in a xenograft model.

Conclusion

These results suggest that the combination of 17-DMAG and belinostat should be examined in a clinical trial for TKI-resistant NSCLC cell.     

Double mutation and gene copy number of EGFR in gefitinib refractory non-small-cell lung cancer

Mutations of the epidermal growth factor receptor (EGFR) gene have been reported in non-small-cell lung cancer (NSCLC), especially in patients with adenocarcinoma and never smokers. Some common somatic mutations in EGFR, including deletion mutations in exon 19 and leucine-to-arginine substitution at amino acid position 858 (L858R) in exon 21, have been examined for their ability to predict sensitivity to gefitinib or erlotinib, which are selective EGFR tyrosine kinase inhibitors (EGFR-TKIs). On the other hand, reports have shown that the threonine-to-methionine substitution at amino acid position 790 (T790M) in exon 20 is related to gefitinib resistance. Some studies have indicated that high copy numbers of the EGFR gene may be a more effective molecular predictor to responsiveness and prolonged survival in patients treated with EGFR-TKIs. Here, we describe two NSCLC patients with the L858R mutation who did not respond to gefitinib. Case 1 harbored both the T790M and L858R mutations, and fluorescence in situ hybridization showed EGFR gene amplification. Case 2 harbored both the L858R and aspartic acid-to-tyrosine substitution at amino acid position 761 in exon 19 of EGFR mutations and had a high polysomy status for EGFR. In these two cases, tumors showed resistance to gefitinib treatment despite the presence of EGFR L858R mutation and increased copy number. Our findings encourage further molecular analysis to elucidate the relationship between the EGFR status, including mutations and amplifications, and the responsiveness of NSCLC to gefitinib.     

EGFR mutants found in non-small cell lung cancer show different levels of sensitivity to suppression of Src: implications in targeting therapy

Mutations in epidermal growth factor receptor (EGFR) kinase domain associate with clinical responses to EGFR inhibitors and are frequently observed in non-small cell lung cancer (NSCLC) patients in East Asian populations. Clinically identified EGFR mutations cause constitutive receptor activation. The activating mechanisms were unclear but appeared to be different among EGFR mutants. We found that EGFR mutants had different sensitivity to an Src inhibitor PP2. S768I and L861Q mutants were less sensitive to Src suppression than others. Mutation at tyrosine 869 (845) residue, an Src phosphorylation site, decreased the phosphorylation levels of wild-type EGFR and other mutants, but not that of S768I and L861Q mutants, suggesting that S768I and L861Q mutants became Src independent for their activation and biological functions. In contrast, cells expressing EGFR-L858R or exon 19 deletion mutants were more sensitive to PP2 than cells expressing wild-type EGFR. Interestingly, EGFR with exon 19-deletion/T790M double mutations, which was resistant to gefitinib, remained sensitive to PP2. Taken together, our data indicate that Src inhibitors might be effective in treating NSCLC harboring specific types of EGFR mutations.     

Characterization of epidermal growth factor receptor mutations in non-small-cell lung cancer patients of African-American ancestry

Epidermal growth factor receptor (EGFR) mutations are predictive markers for response to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). The most common mutations, exon 19 short deletions and exon 20 point mutation (L858R), activate the tyrosine kinase and confer sensitivity to EGFR-TKIs. However, the function and sensitivity of rare mutations to EGFR-TKIs are unknown. In this study, we found five EGFR mutations out of 16 patients with NSCLC of African-American descent. The frequency of such mutations in this patient population appears to be significantly higher than previously reported. Two of them (N771GY and A767-V769dup) are rare insertion mutations located in exon 20. Using YFP-tagged EGFR mutants, we demonstrated that the mutations confer increased kinase activity, but no sensitivity to erlotinib at clinically available concentrations. In addition, we examined efficacy of PF00299804, an irreversible EGFR-TKI. Although the drug failed to show efficacy to T790M and S768N mutations, the exon 20 insertion mutations were sensitive to PF00299804. These data suggest that rare mutations in exon 20 are resistant to erlotinib but may be sensitive to irreversible inhibitors.     

Combined EGFR/MET or EGFR/HSP90 inhibition is effective in the treatment of lung cancers codriven by mutant EGFR containing T790M and MET

Tyrosine kinase inhibitors (TKI) that target the EGF receptor (EGFR) are effective in most non-small cell lung carcinoma (NSCLC) patients whose tumors harbor activating EGFR kinase domain mutations. Unfortunately, acquired resistance eventually emerges in these chronically treated cancers. Two of the most common mechanisms of acquired resistance to TKIs seen clinically are the acquisition of a secondary "gatekeeper" T790M EGFR mutation that increases the affinity of mutant EGFR for ATP and activation of MET to offset the loss of EGFR signaling. Although up to one-third of patient tumors resistant to reversible EGFR TKIs harbor concurrent T790M mutation and MET amplification, potential therapies for these tumors have not been modeled in vivo. In this study, we developed a preclinical platform to evaluate potential therapies by generating transgenic mouse lung cancer models expressing EGFR-mutant Del19-T790M or L858R-T790M, each with concurrent MET overexpression. We found that monotherapy targeting EGFR or MET alone did not produce significant tumor regression. In contrast, combination therapies targeting EGFR and MET simultaneously were highly efficacious against EGFR TKI-resistant tumors codriven by Del19-T790M or L858R-T790M and MET. Our findings therefore provide an in vivo model of intrinsic resistance to reversible TKIs and offer preclinical proof-of-principle that combination targeting of EGFR and MET may benefit patients with NSCLC.     

Dual MET�CEGFR combinatorial inhibition against T790M-EGFR-mediated erlotinib-resistant lung cancer

Despite clinical approval of erlotinib, most advanced lung cancer patients are primary non-responders. Initial responders invariably develop secondary resistance, which can be accounted for by T790M-EGFR mutation in half of the relapses. We show that MET is highly expressed in lung cancer, often concomitantly with epidermal growth factor receptor (EGFR), including H1975 cell line. The erlotinib-resistant lung cancer cell line H1975, which expresses L858R/T790M-EGFR in-cis, was used to test for the effect of MET inhibition using the small molecule inhibitor SU11274. H1975 cells express wild-type MET, without genomic amplification (CNV=1.1). At 2 μM, SU11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P=0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells. In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay. Using small-animal microPET/MRI, SU11274 treatment was found to induce an early tumour metabolic response in H1975 tumour xenografts. MET and EGFR pathways were found to exhibit collaborative signalling with receptor cross-activation, which had different patterns between wild type (A549) and L858R/T790M-EGFR (H1975). SU11274 plus erlotinib/CL-387,785 potentiated MET inhibition of downstream cell proliferative survival signalling. Knockdown studies in H1975 cells using siRNA against MET alone, EGFR alone, or both, confirmed the enhanced downstream inhibition with dual MET–EGFR signal path inhibition. Finally, in our time-lapse video-microscopy and in vivo multimodal molecular imaging studies, dual SU11274-erlotinib concurrent treatment effectively inhibited H1975 cells with enhanced abrogation of cytoskeletal functions and complete regression of the xenograft growth. Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T790M-EGFR-mediated erlotinib-resistant non-small-cell lung cancer. Furthermore, optimised inhibition may be further achieved with MET inhibition in combination with erlotinib or an irreversible EGFR-TKI.     

Impact of common epidermal growth factor receptor and HER2 variants on receptor activity and inhibition by lapatinib

The goal of this study was to characterize the effects of non-small cell lung carcinoma (NSCLC)-associated mutations in epidermal growth factor receptor (EGFR/ErbB1) and HER2 (ErbB2) on interactions with the dual tyrosine kinase inhibitor lapatinib. Biochemical studies show that commonly observed variants of EGFR [G719C, G719S, L858R, L861Q, and Delta746-750 (del15)] are enzyme activating, increasing the tyrosine kinase V(max) and increasing the K(m)((app)) for ATP. The point mutations G719C and L861Q had minor effects on lapatinib K(i)s, whereas EGFR mutations L858R and del15 had a higher K(i) for lapatinib than wild-type EGFR. Structural analysis of wild-type EGFR-lapatinib complexes and modeling of the EGFR mutants were consistent with these data, suggesting that loss of structural flexibility and possible stabilization of the active-like conformation could interfere with lapatinib binding, particularly to the EGFR deletion mutants. Furthermore, EGFR deletion mutants were relatively resistant to lapatinib-mediated inhibition of receptor autophosphorylation in recombinant cells expressing the variants, whereas EGFR point mutations had a modest or no effect. Of note, EGFR T790M, a receptor variant found in patients with gefitinib-resistant NSCLC, was also resistant to lapatinib-mediated inhibition of receptor autophosphorylation. Two HER2 insertional variants found in NSCLC were less sensitive to lapatinib inhibition than two HER2 point mutants. The effects of lapatinib on the proliferation of human NSCLC tumor cell lines expressing wild-type or variant EGFR and HER2 cannot be explained solely on the basis of the biochemical activity or receptor autophosphorylation in recombinant cells. These data suggest that cell line genetic heterogeneity and/or multiple determinants modulate the role played by EGFR/HER2 in regulating cell proliferation.     

Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation

Activating mutations in the epidermal growth factor receptor (EGFR) characterize a subset of non-small cell lung cancers (NSCLC) with extraordinary sensitivity to targeted tyrosine kinase inhibitors (TKI). A single secondary EGFR mutation, T790M, arising in cis with the primary activating mutation, confers acquired resistance to these drugs. However, the T790M mutation is also detected in the absence of drug selection, suggesting that it may provide a growth advantage. We show here that although T790M alone has only a modest effect on EGFR function, when combined with the characteristic activating mutations L858R or del746-750, it results in a dramatic enhancement of EGFR activity. The double mutants show potent ligand-independent receptor autophosphorylation associated with altered cellular phenotypes, soft agar colony formation, and tumorigenesis in nude mice. The significant gain-of-function properties of these double mutants may explain their initial presence before drug selection and their rapid selection as the single drug resistance mutation during therapy with gefitinib/erlotinib, and suggests that they may contribute to the adverse clinical course of TKI-resistant NSCLC.