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.     

Hsp90 inhibition suppresses mutant EGFR-T790M signaling and overcomes kinase inhibitor resistance

The epidermal growth factor receptor (EGFR) secondary kinase domain T790M non-small cell lung cancer (NSCLC) mutation enhances receptor catalytic activity and confers resistance to the reversible tyrosine kinase inhibitors gefitinib and erlotinib. Currently, irreversible inhibitors represent the primary approach in clinical use to circumvent resistance. We show that higher concentrations of the irreversible EGFR inhibitor CL-387,785 are required to inhibit EGFR phosphorylation in T790M-expressing cells compared with EGFR mutant NSCLC cells without T790M. Additionally, CL-387,785 does not fully suppress phosphorylation of other activated receptor tyrosine kinases (RTK) in T790M-expressing cells. These deficiencies result in residual Akt and mammalian target of rapamycin (mTOR) activities. Full suppression of EGFR-mediated signaling in T790M-expressing cells requires the combination of CL-387,785 and rapamycin. In contrast, Hsp90 inhibition overcomes these limitations in vitro and depletes cells of EGFR, other RTKs, and phospho-Akt and inhibits mTOR signaling whether or not T790M is present. EGFR-T790M-expressing cells rendered resistant to CL-387,785 by a kinase switch mechanism retain sensitivity to Hsp90 inhibition. Finally, Hsp90 inhibition causes regression in murine lung adenocarcinomas driven by mutant EGFR (L858R) with or without T790M. However, efficacy in the L858R-T790M model requires a more intense treatment schedule and responses were transient. Nonetheless, these findings suggest that Hsp90 inhibitors may be effective in T790M-expressing cells and offer an alternative therapeutic strategy for this subset of 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.     

EGFR mutation status in pleural fluid predicts tumor responsiveness and resistance to gefitinib

It has been reported that the threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene is correlated with acquired resistance to gefitinib. We previously reported that there was some population that harbored the EGFR T790M mutation as a minor clone of tumor cells prior to drug treatment, may be causing resistance to gefitinib during treatment. This fact also suggests that the detection of the EGFR T790M mutation prior to treatment may predict the development of resistance. We also showed that pleural fluid is a useful specimen for detection of EGFR mutation using sensitive assays. In this study, we reported a female patient who was treated with gefitinib because an EGFR L858R mutation was found in her pleural fluid. Our patient showed partial response to gefitinib, but she had progressive disease only 4 months after the start of treatment. Furthermore, the EGFR T790M mutation was detected in the pleural fluid before gefitinib treatment by the mutant-enriched PCR assay. Our findings confirmed that the EGFR T790M mutation was occasionally present as a minor population in tumor cells before treatment and caused resistance after gefitinib administration. The detection of a small fraction of T790M-positive alleles may be useful to predict the clinical course of the gefitinib-treated non-small-cell lung cancer patients.     

吉非替尼在非小细胞肺癌治疗中的继发耐药

吉非替尼是一种表皮生长因子受体(EGFR)酪氨酸激酶抑制剂,对非小细胞肺癌(NSCLC)有良好的抗肿瘤活性,但大多数患者最终因继发耐药出现病情进展.在发生EGFR基因突变的肺腺癌患者中,约半数吉非替尼继发耐药与二次突变有关,这种二次突变导致EGFR 790位上的苏氨酸被甲硫氨酸所取代(T790M).其他导致耐药的机制包括EGFR受体的内化现象以及MET基因扩增.     

PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective treatments for a subset of non-small cell lung cancers. In particular, cancers with specific EGFR-activating mutations seem to be the most sensitive to these agents. However, despite their initial response, such cancers almost invariably develop resistance. In 50% of such cancers, a secondary EGFR mutation, T790M, has been identified that renders gefitinib and erlotinib ineffective inhibitors of EGFR kinase activity. Thus, there is a clinical need to develop novel EGFR inhibitors that can effectively inactivate T790M-containing EGFR proteins. In this study, we evaluate the effectiveness of a novel compound, PF00299804, an irreversible pan-ERBB inhibitor. The results from these studies show that PF00299804 is a potent inhibitor of EGFR-activating mutations as well as the EGFR T790M resistance mutation both in vitro and in vivo. Additionally, PF00299804 is a highly effective inhibitor of both the wild-type ERBB2 and the gefitinib-resistant oncogenic ERBB2 mutation identified in lung cancers. These preclinical evaluations support further clinical development of PF00299804 for cancers with mutations and/or amplifications of ERBB family members.     

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.     

Effects of Src inhibitors on cell growth and epidermal growth factor receptor and MET signaling in gefitinib-resistant non-small cell lung cancer cells with acquired MET amplification

The efficacy of epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitors such as gefitinib and erlotinib in non-small cell lung cancer (NSCLC) is often limited by the emergence of drug resistance conferred either by a secondary T790M mutation of EGFR or by acquired amplification of the MET gene. We now show that the extent of activation of the tyrosine kinase Src is markedly increased in gefitinib-resistant NSCLC (HCC827 GR) cells with MET amplification compared with that in the gefitinib-sensitive parental (HCC827) cells. In contrast, the extent of Src activation did not differ between gefitinib-resistant NSCLC (PC9/ZD) cells harboring the T790M mutation of EGFR and the corresponding gefitinib-sensitive parental (PC9) cells. This activation of Src in HCC827 GR cells was largely abolished by the MET-TKI PHA-665752 but was only partially inhibited by gefitinib, suggesting that Src activation is more dependent on MET signaling than on EGFR signaling in gefitinib-resistant NSCLC cells with MET amplification. Src inhibitors blocked Akt and Erk signaling pathways, resulting in both suppression of cell growth and induction of apoptosis, in HCC827 GR cells as effectively as did the combination of gefitinib and PHA-665752. Furthermore, Src inhibitor dasatinib inhibited tumor growth in HCC827 GR xenografts to a significantly greater extent than did treatment with gefitinib alone. These results provide a rationale for clinical targeting of Src in gefitinib-resistant NSCLC with MET amplification. ( Cancer Sci 2009)     

Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib.

PURPOSE: Non-small cell lung cancers carrying activating mutations in the gene for the epidermal growth factor receptor (EGFR) are highly sensitive to EGFR-specific tyrosine kinase inhibitors. However, most patients who initially respond subsequently experience disease progression while still on treatment. Part of this "acquired resistance" is attributable to a secondary mutation resulting in threonine to methionine at codon 790 (T790M) of EGFR. EXPERIMENTAL DESIGN: We sequenced exons 18 to 21 of the EGFR gene to look for secondary mutations in tumors with acquired resistance to gefitinib in 14 patients with adenocarcinomas. Subcloning or cycleave PCR was used in addition to normal sequencing to increase the sensitivity of the assay. We also looked for T790M in pretreatment samples from 52 patients who were treated with gefitinib. We also looked for secondary KRAS gene mutations because tumors with KRAS mutations are generally resistant to tyrosine kinase inhibitors. RESULTS: Seven of 14 tumors had a secondary T790M mutation. There were no other novel secondary mutations. We detected no T790M mutations in pretreatment specimens from available five tumors among these seven tumors. Patients with T790M tended to be women, never smokers, and carrying deletion mutations, but the T790M was not associated with the duration of gefitinib administration. None of the tumors had an acquired mutation in the KRAS gene. CONCLUSIONS: A secondary T790M mutation of EGFR accounted for half the tumors with acquired resistance to gefitinib in Japanese patients. Other drug-resistant secondary mutations are uncommon in the EGFR gene.     

Clinical and molecular evidences of epithelial to mesenchymal transition in acquired resistance to EGFR-TKIs

Background

Epithelial-to-mesenchymal transition (EMT), which was related with an acquired resistance to gefitinib, was found in the A549 lung cancer cell line. However, the clinical feasibility of this finding is still questionable. Here, we investigated whether EMT could be detected in a more clinically suitable situation using patient's tumor and cells with deletion mutation on exon 19 of EGFR gene.

Methods

HCC827 cell line was used to establish the subline resistant to EGFR-TKIs. The induction of EMT was analyzed by immunostainings and Western blots in resistant cells and biopsied tissue from a patient with acquired resistance to erlotinib. Migration and invasion assay was performed to characterize the resistant cells. EMT-related genes expression was evaluated by cDNA microarray. Phospho-receptor tyrosine kinase array analysis was carried out to find bypass activating signals such as MET.

Results

We found that EMT developed in a lung cancer patient who had an acquired resistance to erlotinib while there were no known resistant mechanisms such as T790M and MET amplification. CL-387,785-resistant cells (HCC827/CLR) were obtained by long-term exposure to increasing concentrations of CL-387,785 (an irreversible EGFR-TKI). The morphological and molecular maker changes compatible with EMT were also found in HCC827/CLR cells. However, there were also no secondary T790M mutation and MET amplification. Furthermore, the activity of most of tested RTKs including receptor HER family was decreased suggesting that there was no bypass activating signal leading to resistance. These cells showed an enhanced capability for migration (∼1.6-fold) and invasion (∼2.8-fold).

Conclusion

EMT should be considered as one of possible mechanisms for the acquired resistance to EGFR-TKIs in lung cancer cells.     

Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors.

PURPOSE: In patients whose lung adenocarcinomas harbor epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations, acquired resistance to the tyrosine kinase inhibitors (TKI) gefitinib (Iressa) and erlotinib (Tarceva) has been associated with a second-site EGFR mutation, which leads to substitution of methionine for threonine at position 790 (T790M). We aimed to elucidate the frequency and nature of secondary EGFR mutations in patients with acquired resistance to TKI monotherapy. EXPERIMENTAL DESIGN: Tumor cells from patients with acquired resistance were examined for secondary EGFR kinase domain mutations by molecular analyses. RESULTS: Eight of 16 patients (50% observed rate; 95% confidence interval, 25-75%) had tumor cells with second-site EGFR mutations. Seven mutations were T790M and one was a novel D761Y mutation found in a brain metastasis. When combined with a drug-sensitive L858R mutation, the D761Y mutation modestly reduced the sensitivity of mutant EGFR to TKIs in both surrogate kinase and cell viability assays. In an autopsy case, the T790M mutation was found in multiple visceral metastases but not in a brain lesion. CONCLUSIONS: The T790M mutation is common in patients with acquired resistance. The limited spectrum of TKI-resistant mutations in EGFR, which binds to erlotinib in the active conformation, contrasts with a wider range of second-site mutations seen with acquired resistance to imatinib, which binds to ABL and KIT, respectively, in closed conformations. Collectively, our data suggest that the type and nature of kinase inhibitor resistance mutations may be influenced by both anatomic site and mode of binding to the kinase target.     

Peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based detection test for gefitinib-refractory T790M epidermal growth factor receptor mutation

Mutations in the epidermal growth factor receptor ( EGFR ) are observed in a fraction of non-small-cell lung cancers (NSCLS). EGFR mutation-positive NSCLS responds to gefitinib. Secondary T790M mutation confers gefitinib resistance to NSCLS. A detection test for the T790M mutation was designed based on the peptide nucleic acid–locked nucleic acid polymerase chain reaction clamp method. The specificity and sensitivity of the test were both greater than 0.99. The test revealed that only a small population of the PC-13 cells carried the T790M mutation. The test also revealed that the T790M mutation was found in none of 151 NSCLC specimens obtained before gefitinib treatment, whereas it was found in four of four specimens obtained from NSCLS that had become refractory to gefitinib. In one patient in whom the L858R-positive EGFR allele was amplified to multiple copies, an L858R-T790M double-mutant allele emerged during the gefitinib therapy. This allele was expressed highly. The T790M mutation detection test based on the peptide nucleic acid–locked nucleic acid polymerase chain reaction clamp method is sensitive and specific, and is applicable to clinical practice. It detects T790M-positive cells in the course of gefitinib treatment, and thus will help to devise therapies effective for T790M-positive NSCLS. ( Cancer Sci 2008; 99: 595–600)     

Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer

The threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene has been reported in progressing lesions after gefitinib treatment in non-small cell lung cancer (NSCLC) that causes sensitive tumors to become resistant to gefitinib. Alternatively, the EGFR T790M mutation might be present in small fractions of tumor cells before drug treatment, and the tumor cells harboring the T790M mutation might be enriched during the proliferation after drug treatment. We developed a mutant-enriched PCR assay to detect small fractions of cells with T790M mutation and used this technique to detect mutations in 280 NSCLCs, including gefitinib-treated 95 cases. Although the direct sequencing detected only 1 T790M mutant case, the mutant-enriched PCR (confirmed to enrich one mutant out of 1 x 10(3) wild-type alleles) detected 9 additional cases among 280 cases. As linkage to clinicopathologic factors, the T790M mutation showed no bias for sex, smoking status, or histology but was significantly more frequent in advanced tumors (9 of 111 cases) than in early-stage tumors (1 of 169 cases; P = 0.0013). Among gefitinib-treated cases, gefitinib-sensitive mutations were found in 30 cases. The T790M mutation was present in 3 of 7 no-responders with the gefitinib-sensitive mutation and was not present in 19 responders (P = 0.014). Our results indicate that the T790M mutation is sometimes present in a minor population of tumor cells during the development of NSCLC and suggest that the detection of small fractions of T790M mutant alleles may be useful for predicting gefitinib resistance of NSCLCs with sensitive EGFR mutations.     

Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective therapies for non-small cell lung cancer patients whose tumors harbor somatic mutations in EGFR. All patients, however, ultimately develop resistance to these agents. Thus, there is a great need to understand how patients become resistant to develop effective therapies for these cancers. Studies over the last few years have identified two different EGFR tyrosine kinase inhibitor resistance mechanisms, a secondary mutation in EGFR, EGFR 790M, and amplification of the MET oncogene. These findings have led to clinical trials using newly designed targeted therapies that can overcome these resistance mechanisms and have shown promise in laboratory studies. Ongoing research efforts will likely continue to identify additional resistance mechanisms, and these findings will hopefully translate into effective therapies for non-small cell lung cancer patients.     

Lower gefitinib dose led to earlier resistance acquisition before emergence of T790M mutation in epidermal growth factor receptor‐mutated lung cancer model

Non‐small‐cell lung cancers with epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs); however, unlike cytotoxic agents, it is generally accepted that minimal doses of drugs inhibiting target molecules are sufficient when molecular‐targeted agents, including EGFR‐TKIs, are used. Thus, any utility of higher doses remains unclear. We compared low‐dose (15 mg/kg) gefitinib therapy with high‐dose (50 mg/kg) therapy using an EGFR‐mutated lung cancer xenograft model. Both gefitinib doses induced tumor shrinkage, but tumors regrew in the low‐dose group within 1 month, whereas tumors in the high‐dose group did not. Neither the T790M mutation nor MET amplification was apparent in regrown tumors. We also compared outcomes after two doses of gefitinib (5 and 25 mg/kg) in a transgenic EGFR‐mutated lung cancer mouse model. In line with the results obtained using the xenograft model, both gefitinib doses completely inhibited tumor growth, but tumors treated with the lower dose of gefitinib developed earlier drug resistance. In conclusion, a low gefitinib dose caused tumors to become drug‐resistant prior to acquisition of the T790M mutation or MET amplification in EGFR‐mutated models of lung cancer. This suggests that it is important to optimize the EGFR‐TKI dose for treatment of EGFR mutation‐associated lung cancer. Gefitinib may need to be given at a dose greater than the minimum required for inhibition of target molecules.     

The combination of irreversible EGFR TKIs and SAHA induces apoptosis and autophagy‐mediated cell death to overcome acquired resistance in EGFR T790M‐mutated lung cancer

To overcome T790M‐mediated acquired resistance of lung cancer cells to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), second generation TKIs such as BIBW2992 (afatinib) and third generation TKIs including WZ4002 have been developed. However, clinical data on their efficacy in treating T790M mutant tumors are lacking. Histone deacetylase (HDAC) inhibitors have been reported to arrest cell growth and to lead to differentiation and apoptosis of various cancer cells, both in vitro and in vivo. In the present study, we assessed whether the combination of suberoylanilide hydroxamic acid (SAHA, vorinostat), a potent HDAC inhibitor, and BIBW2992 or WZ4002 could overcome EGFR TKI resistance associated with T790M mutation in lung cancer cells. While treatment with BIBW2992 or WZ4002 alone slightly reduced the viability of PC‐9G and H1975 cells, which possess T790M mutation, combining them with SAHA resulted in significantly decreased cell viability through the activation of the apoptotic pathway. This combination also enhanced autophagy occurrence and inhibition of autophagy significantly reduced the apoptosis induced by the combination treatment, showing that autophagy is required for the enhanced apoptosis. Caspase‐independent autophagic cell death was also induced by the combination treatment with SAHA and either BIBW2992 or WZ4002. Finally, the combined treatment with SAHA and either BIBW2992 or WZ4002 showed an enhanced anti‐tumor effect on xenografts of H1975 cells in vivo. In conclusion, the combination of new generation EGFR TKIs and SAHA may be a new strategy to overcome the acquired resistance to EGFR TKIs in T790M mutant lung cancer.     

Mutations of the epidermal growth factor receptor gene in NSCLC patients

Mutations of the epidermal growth factor receptor (EGFR) in patients with non-small cell lung cancer (NSCLC) were identified by re-sequencing all exons of this gene to evaluate the frequencies of EGFR gene mutation and identify rare or novel EGFR mutations. A total of 55 NSCLC samples from 55 patients were included in the study. Genomic DNA was extracted and exons 1-28 of the EGFR gene were sequenced to identify mutations. The cDNA of the EGFR gene with P848L and T790M double mutants was constructed by introducing point mutations into the wild-type EGFR vector using a site-directed mutagenesis kit. Among the 55 patients with NSCLC, 8 patients carried mutations of the EGFR gene. Notably, of the mutation-harboring patients with a pathological type of adenocarcinoma, 6 were non-smokers. The in vitro study demonstrated that the P848L mutant had a similar response to that of the wild-type EGFR after gefitinib treatment, and the P848L and T790M double mutant exhibited high resistance to gefitinib. These EGFR mutations preferentially occurred in lung adenocarcinoma patients, most of whom were non-smokers. In the in vitro study, P848L mutant EGFR had a similar response as the wild-type EGFR to gefitinib treatment, suggesting that lung cancer patients with a rare mutation of EGFR, such as the P848L mutation, do not respond to gefitinib treatment.