Irreversible EGFR Inhibitor Research Articles

793: Effective inhibition of glioblastoma growth with dacomitinib: an irreversible EGFR inhibitor

793: Effective inhibition of glioblastoma growth with dacomitinib: an irreversible EGFR inhibitor

A phase II and biomarker study of an irreversible pan-human EGF receptor (HER) tyrosine kinase inhibitor dacomitinib in patients with recurrent and/or metastatic squamous cell carcinoma of esophagus.

4083 Background: Dacomitinib is an oral irreversible tyrosine kinase inhibitor of EGFR, HER2 and HER4 signaling. The goals of this study were to investigate the clinical activity, safety and predic...

Combination of BIBW2992 and ARQ 197 is effective against erlotinib-resistant human lung cancer cells with the EGFR T790M mutation.

Although the EGFR tyrosine kinase inhibitors (EGFR-TKI) erlotinib and gefitinib have shown marked effects against EGFR-mutated lung cancer, patients acquire resistance by various mechanisms, including the EGFR T790M mutation and Met induction, consequently suffering relapse. Thus, novel agents to overcome EGFR-TKI resistance are urgently needed. We aimed to investigate the inhibitory effects of a combination of BIBW2992 (irreversible EGFR inhibitor)/ARQ 197 (MET inhibitor) on the human lung adenocarcinoma cell line H1975. H1975 cells (harboring a T790M mutation in EGFR) were treated with erlotinib, BIBW2992 or ARQ 197 separately or with combinations of erlotinib/ARQ 197 or BIBW2992/ARQ 197. Cell growth, apoptosis and cell cycle distribution were evaluated by MTT assay, Annexin V/propidium iodide (PI) double staining and flow cytometry, respectively. EGFR, MET, AKT, ERK and the respective phosphorylated counterparts were detected by western blot analysis. Pathway-specific knockdown of MET and/or EGFR kinase signaling was achieved by siRNA interference. H1975 cells displayed EGFR and MET activation, and were resistant to erlotinib. The BIBW2992/ARQ 197 combination significantly inhibited growth, induced cell cycle arrest and apoptosis, and altered the phosphorylation of EGFR, MET, AKT and ERK1/2 in the H1975 cells. Phosphorylation of AKT and ERK1/2, downstream effectors of the EGFR and MET pathways, was not affected by the other tested treatments. Finally, knockdown of MET and/or EGFR in the H1975 cells confirmed the enhanced downstream inhibition of both MET and EGFR pathways. Combination of an irreversible EGFR inhibitor and MET inhibitor is effective in controlling H1975 cells with acquired resistance to erlotinib, by a mechanism involving the downregulation of PI3K/AKT and MEK/ERK signaling pathways.

Abstract B278: PR610: A novel hypoxia-selective tyrosine kinase inhibitor in phase I clinical trial.

Abstract PR610 is a hypoxia-selective irreversible Human Epidermal Growth Factor Receptor (HER) family inhibitor currently in phase I clinical trials in New Zealand and the USA (clinical trial ID NCT01631279). The prodrug PR610 releases the TKI (Tyrosine Kinase Inhibitor) PR610E, a picomolar irreversible inhibitor of EGFR (HER1), under oxygen-limiting conditions typically found in solid tumors. Human neoplastic cell lines exposed to PR610 show an anoxia-selective anti-proliferative response that is associated with G1 arrest and induction of apoptosis arising from inhibition of EGFR auto-phosphorylation and downstream silencing of associated signal transduction pathways. PR610 is optimized for long tumor residency (T½ >2 days); in a preclinical model of erlotinib-resistant NSCLC, a single injection of PR610 produces profound, global shutdown of signal transduction via EGFRT790M/L858R. Tumor PR610E concentrations were above cellular anti-proliferative IC50 concentrations for over 5 days, being more than sufficient to induce apoptosis via “oncogenic shock”. Pharmacokinetic (PK) studies of PR610 show significant differences in species toxicokinetics. Both the rat and dog preclinical toxicology models display considerable systemic conversion of PR610 to PR610E (6% - 30% and 24% - 29% of prodrug AUC, respectively) with attendant symptoms of EGFR inhibition including acneiform skin rash and diarrhea. In contrast NIH-III nude mice display minimal circulating TKI (PR610E) relative to PR610 (1.5% - 1.7% of AUC) consistent with a substantially improved tolerance as judged by PR610 plasma AUCinf at the maximum tolerated dose (MTD). Human subjects from the phase I clinical trial consistently experience the lowest systemic levels of PR610E (Mean 1.06% ± 0.69%; range 0.34% - 2.7%; n=20) across dose levels ranging from 10 - 150 mg/m2. Notably, a dose of 150 mg/m2 in human subjects produces a PR610 plasma AUCinf equivalent to that measured for 30 mg/kg PR610 in NIH-III mice, a dose that is active in several HER-dependent subcutaneous tumor xenograft models. Collectively, these data indicate that PR610 has the desirable characteristics of a deactivated prodrug in human subjects and preclinical models predict that an active dose range has been reached in the phase I trial. PR610 is a first-in-class hypoxia-selective EGFR/HER2 inhibitor with exciting clinical potential. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B278. Citation Format: Adam V. Patterson, Jagdish Jaiswal, Kendall Carlin, Maria R. Abbattista, Christopher P. Guise, Shevan Silva, Ho Lee, Guo-Liang Lu, Robert F. Anderson, Teresa J. Melink, John C. Gutheil, Jeff B. Smaill. PR610: A novel hypoxia-selective tyrosine kinase inhibitor in phase I clinical trial. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B278.

Design, Synthesis, and Biological Evaluation of 2-Oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl Derivatives as New Irreversible Epidermal Growth Factor Receptor Inhibitors with Improved Pharmacokinetic Properties

Structural optimization of a series of 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl compounds, potential new irreversible EGFR inhibitors, was performed to improve pharmacokinetic properties of the compounds. This led to compound 2v with improved aqueous solubility and good pharmacokinetic properties which at the nanomolar level potently inhibits gefitinib-resistant EGFR(L858R/T790M) kinase and displays strong antiproliferative activity against H1975 nonsmall cell lung cancer cells. The new inhibitor also shows promising antitumor efficacy in a murine EGFR(L858R/T790M)-driven H1975 xenograft model without effect on body weight. These studies provide new lead compounds for further development of drugs for treatment of gefitinib-resistant nonsmall cell lung cancer patients.

Long-lasting inhibition of EGFR autophosphorylation in A549 tumor cells by intracellular accumulation of non-covalent inhibitors

In the present study, a small set of reversible or irreversible 4-anilinoquinazoline EGFR inhibitors was tested in A549 cells at early (1h) and late (8h) time points after inhibitor removal from culture medium. A combination of assays was employed to explain the observed long-lasting inhibition of EGFR autophosphorylation. We found that EGFR inhibition at 8h can be due, besides to the covalent interaction of the inhibitor with Cys797, as for PD168393 (2) and its prodrug 4, to the intracellular accumulation of non-covalent inhibitors by means of an active cell uptake, as for 5 and 6. Compounds 5–6 showed similar potency and duration of inhibition of EGFR autophosphorylation as the covalent inhibitor 2, while being devoid of reactive groups forming covalent bonds with protein thiols.

Assessment of neratinib-resistance and cross-resistance in HER2-overexpressing breast cancer cells.

e11520 Background: While current therapies approved for HER2-overexpressing breast cancer (BC) treatment has led to advances in treatment of this BC subtype, issues of innate/acquired drug resistance are evolving. Neratinib (N) is an irreversible EGFR, HER2 and HER4 tyrosine kinase inhibitor currently in phase III clinical trials for BC treatment. We aimed to investigate the ability of BC cells to acquire resistance to N and the implications of this in terms of potential cross-resistance to other drugs and phenotypic changes in cells. Methods: Neratinib-resistant (NR) cell line variants were developed by exposing HER2-overexpressing BC cell lines, HCC1954 and SKBR3, to increasing concentrations of N over several months. Proliferation assays were used to establish IC50 values for N in parent and NR variants; as well as to assess possible cross-resistance to lapatinib (L), afatinib (A) and docetaxel (D). Phenotypic changes in NR cell variants were examined using invasion, migration and anoikis assays. IGF1R expression was determined using immunoblots. Results: HCC1954-NR and SKBR3-NR cell variants were 6.5±0.4 and 194±47 fold more resistant to neratinib than parent cells, respectively. Furthermore, HCC1954-NR variants were cross-resistant to afatinib (37±7.23 fold) and lapatinib (10±0.8 fold). SKBR3-NR variants were cross-resistant to afatinib by >163.3±22.7 fold (IC50 not reached at 9µM afatinib where, on average, 65.5 % cell growth is maintained) and lapatinib (162.3±22 fold). No cross-resistance to docetaxel was observed. Cells that have acquired N resistance are more invasive, migratory and resistant to anoikis than their parent controls. IGF1R expression is increased in NR cell variants compared to parent cells. Conclusions: Chronic exposure of BC cells to N results in acquired resistance to the drug. N resistance conferred cross-resistance to other HER2-targeting drugs and induced a more aggressive phenotype. Overexpression of IGF1R in NR variants suggested its role in the mechanism of N resistance.

MIS4-1 - Molecular Networks of the EGFR-TKI Resistant Non-Small Cell Lung Cancer: How to Apply New Technologies to Bench-to-Bed Research

ABSTRACT Background A secondary mutation of EGFR (T790M) accounts for 50% of all cases of acquired resistance to EGFR-TKIs in NSCLC patients with EGFR mutation. Although irreversible EGFR-TKIs are thought to be one of the strategies to overcome T790M, the efficacy of these drugs appears limited. Methods We generated EGFR-TKI resistant PC9GR cells with T790M by long-term gefitinib exposure of PC9 cells with exon 19 deletion. To visualize their molecular networks related to T790M, we carried out immunoaffinity purification of tyrosine-phosphorylated peptides by anti-phospho tyrosine antibodies (pTyr100) and subsequent LC-MS/MS identification in PC9 and PC9GR cells with or without erlotinib treatment. Results Our phosphoproteomics detected a total of 403 unique phosphosites on 266 proteins. Compared with non-treated PC9 cells, 32 pY sites (24 proteins) are decreased and 45 pY sites (33 proteins) are increased in non-treated PC9GR cells. In parental PC9 cells, 24 pY sites (19 proteins) are decreased and 12 pY sites (12 proteins) are increased by erlotinib treatment. In resistant PC9GR cells, 5 pY sites (5 proteins) are decreased and 6 pY sites (6 proteins) are increased by erlotinib treatment. The detailed analyses including different response to erlotinib between PC9 and PC9GR cells will be presented at the meeting. As a preliminary validation, we found that PC9GR cells have persistent Src kinase levels even with erlotinib treatment in our phosphoproteomics. Western blot assays also demonstrated that the irreversible EGFR-TKI BIBW-2992 (afatinib) did not alter the activation of Src family kinases in PC9GR cells. Src inhibitor dasatinib combined with afatinib abolished this Src phosphorylation along with complete suppression of downstream phospho Akt and Erk. Dasatinib enhances anti-tumor activity of afatinib in both cell proliferation assay and caspase-3 apoptosis assay in these cells. This combination showed significant in vivo tumor regression in PC9GR xenograft studies. Conclusions Applying global phosphoproteomics effectively visualizes the molecular networks of EGFR-TKI-resistant NSCLC related to T790M gate keeper mutation. Our preliminary validation supports further studies of irreversible EGFR inhibitors combined with dasatinib in NSCLC patients who acquired resistance to EGFR-TKI by T790M.

Irreversible and Reversible EGFR Inhibitors Are Directly Compared in NSCLC

Abstract Dacomitinib significantly improved PFS compared with erlotinib in patients with advanced NSCLC.

Abstract 4832: Novel EGFR mutations that cause drug resistance to irreversible pyrimidine but not quinazoline based EGFR inhibitors

Abstract Background: Oncogenic EGFR T790M causes drug resistance to quinazoline based EGFR kinase inhibitors by increasing ATP affinity (Yun PNAS 2008). Mutant selective irreversible pyrimidine EGFR kinase inhibitor, WZ4002, is effective in non-small cell lung cancer (NSCLC) models harboring EGFR T790M (Zhou Nature 2009). We aimed to determine potential mechanisms of resistance to WZ4002 and explore alternative strategies to overcome acquired resistance to pyrimidine based EGFR inhibitors. Methods and Results: We performed an ENU mutagenesis screen in Ba/F3 cells expressing EGFR L858R, L858R/T790M, Del E746_A750 and Del E746_A750/T790M followed by culture in the presence of WZ4002 (100 nM or 1 μM). Using RT-PCR, we sequenced resistant clones for secondary EGFR mutations. No EGFR T790M mutations were identified. We detected novel secondary EGFR L718Q (9/27; 33%) or L844V (1/27; 3%) mutations in the drug resistant cells. We also recovered the EGFR C797S (1/27; 3%) mutation previously known to prevent covalent binding and decrease potency of WZ4002. Unlike EGFR T790M, EGFR L718Q and EGFR L844V did not lead to constitutive EGFR phosphorylation, were not transforming in Ba/F3 cells and required EGF for proliferation and survival. The EGFR L858R/L844V Ba/F3 cells were resistant to WZ4002 (IC50 0.7 μM) but sensitive to irreversible quinazoline EGFR inhibitors CL-387,785, HKI-272 (neratinib) and BIBW2992 (afatinib) (IC50 values all < 10 nM). Similar findings were observed with the EGFR L858R/L718Q cells although the IC50 values were slightly higher (100 nM or less) and with the Del E746_A750/L718Q and Del E746_A750/L844V cells. All triple mutants harboring EGFR T790M (e.g. L858R/T790M/L844V) were resistant to WZ4002 and irreversible quinazoline EGFR inhibitors. EGFR L858R/L844V and L858R/L718Q Ba/F3 cells were growth inhibited by clinical concentrations (1 μM) of gefitinib and the combination of 1 μM gefitinib and 100 nM WZ4002 completely prevented the emergence of resistant clones in our ENU assay. Using structural modeling, both the L718Q and L844V mutations likely lead to steric hindrance and could impact WZ4002 binding. To verify this hypothesis, we developed a biotinylated-WZ4002 compound and used it to assay binding to different EGFR mutant proteins. This agent effectively bound to EGFR L858R and DelE746_A750 (with or without T790M). However, in the presence of a concurrent L718Q or L844V mutation, protein binding was significantly reduced, consistent with the reduced in vitro efficacy in the Ba/F3 cells. Conclusions: We identify novel EGFR mutations that confer drug resistance to irreversible pyrimidine but not quinazoline EGFR kinase inhibitors. Our findings have implications for understanding drug resistance mechanisms and for the development of combinations of EGFR kinase inhibitors as therapies for cancer patients. 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 4832. doi:1538-7445.AM2012-4832

SKLB1206, a Novel Orally Available Multikinase Inhibitor Targeting EGFR Activating and T790M Mutants, ErbB2, ErbB4, and VEGFR2, Displays Potent Antitumor Activity Both In Vitro and In Vivo

Anti-epidermal growth factor receptor (EGFR) treatment has been successfully applied in clinical cancer therapy. However, the clinical efficacy of first-generation reversible EGFR inhibitors, such as gefitinib and erlotinib, is limited by the development of drug-resistant mutations, including the gatekeeper T790M mutation and upregulation of alternative signaling pathways. Second-generation irreversible EGFR inhibitors that were designed to overcome the drug resistance due to the T790M mutation have thus far had limited success. Here, we report a novel reversible EGFR inhibitor, SKLB1206, which has potent activity against EGFR with gefitinib-sensitive and -resistant (T790M) mutations. In addition, SKLB1206 has also considerable inhibition potency against some other related oncokinases, including ErbB2, ErbB4, and VEGF receptor 2 (VEGFR2). SKLB1206 exhibited highly antiproliferative activity against a range of EGFR-mutant cell lines, including gefitinib-sensitive and -resistant cell lines, and EGFR or ErbB2-overexpressing cell lines. SKLB1206 also showed a potent antiangiogenesis effect in vitro, in a zebrafish embryonic angiogenesis assay, and in an alginate-encapsulate tumor cell assay. In vivo, oral administration of SKLB1206 showed complete tumor regression in gefitinib-sensitive HCC827 and PC-9 xenograft models and showed a considerable antitumor effect on the gefitinib-resistant H1975 model as well as other EGFR/ErbB2-overexpressing or -dependent tumor models including A431, LoVo, and N87 established in athymic mice. SKLB1206 also showed a very good oral bioavailability (50.1%). Collectively, these preclinical evaluations may support clinical development of SKLB1206 for cancers with EGFR-activating/resistance mutations or EGFR/ErbB2 overexpressed.

Irreversible EGFR inhibitors in advanced non-small-cell lung carcinoma: rationale and clinical evidence

The EGFR has become an important target in lung cancer treatment. Reversible inhibitors of this receptor have been shown to improve outcomes for patients whose tumors harbor EGFR mutations, as well as for those with wild-type EGFR. The development of resistance (either primary or acquired) to EGFR tyrosine kinase inhibitors is a common occurrence and irreversible EGFR inhibitors have been developed to overcome this problem. There are now emerging clinical data for the two major irreversible inhibitors; afatinib and dacomitinib. The question of how and when we use these irreversible inhibitors still needs to be answered.

Design, Synthesis, and Biological Evaluation of Novel Conformationally Constrained Inhibitors Targeting Epidermal Growth Factor Receptor Threonine790 → Methionine790 Mutant

The EGFR(T790M) mutant contributes approximately 50% to clinically acquired resistance against gefitinib or erlotinib. However, almost all the single agent clinical trials of the second generation irreversible EGFR inhibitors appear inadequate to overcome the EGFR(T790M)-related resistance. We have designed and synthesized a series of 2-oxo-3,4-dihydropyrimido[4,5-d]pyrimidinyl derivatives as novel EGFR inhibitors. The most potent compounds, 2q and 2s, inhibited the enzymatic activities of wild-type and mutated EGFRs, with IC(50) values in subnanomolar ranges, including the T790M mutants. The kinase inhibitory efficiencies of the compounds were further validated by Western blot analysis of the activation of EGFR and downstream signaling in cancer cells harboring different mutants of EGFR. The compounds also strongly inhibited the proliferation of H1975 non small cell lung cancer cells bearing EGFR(L858R/T790M), while being significantly less toxic to normal cells. Moreover, 2s displayed promising anticancer efficacy in a human NSCLC (H1975) xenograft nude mouse model.

Superiority of a novel EGFR targeted covalent inhibitor over its reversible counterpart in overcoming drug resistance

Recently, the importance of targeted covalent inhibitors in addressing potency, selectivity and drug resistance has become of great interest, especially in the area of non-small cell lung cancer (NSCLC). Although several covalent EGFR TKIs that are advancing in NSCLC clinical development are active against mutations which are refractory to the reversible TKI drugs Tarceva and Iressa, limited chemical diversity has been explored; all of the irreversible and reversible clinical compounds share the same quinazoline scaffold. We describe the design of a novel pyrimidine-based irreversible inhibitor of EGFR (CNX17) which is active against both the WT EGFR as well as the resistance mutation L858R/T790M in biochemical assays. The inhibitor is also a potent inhibitor of EGFR signaling, including the L858R/T790M resistance mutation in cells (H1975 cell line, EC50 441 nM). Importantly, it also potently inhibits proliferation in both HCC827 (EGFRΔ746–750 EC50 < 5 nM) and H1975 (EC50 134 nM). This novel chemical scaffold may be an important addition to the armamentarium in overcoming drug resistance to current EGFR therapies.

Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway

BackgroundEpidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. MethodsIn this study, using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. ResultsAmong the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. ConclusionOur studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.

Advances in malignant glioma drug discovery

Introduction: Outcome for patients with glioblastoma (GBM), the most common malignant primary brain tumor among adults, remains poor. However, two key treatment options have recently generated meaningful improvements in outcome for GBM patients. The addition of temozolomide (a methylating chemotherapeutic agent) to surgical resection and radiation therapy increases survival and is the first evidence that systemic chemotherapy can benefit GBM patients. Also, bevacizumab (a humanized mAb against VEGF) has significant antitumor activity among recurrent GBM patients. Additional areas of ongoing research are generating more therapeutic options that offer exciting potential to build on these results and further improve the outcome for malignant glioma patients.Areas covered: This review describes three foci of advanced clinical research aimed at improving the outcome of GBM patients: protracted temozolomide dosing, VEGF-inhibiting agents and integrin inhibitors. This review also discusses potential clinical trial strategies to evaluate irreversible EGFR inhibitors as well as therapeutics targeting PI3K and the hedgehog signaling pathway.Expert opinion: Several factors limit the efficacy of therapeutics targeting GBM. However, significant advances from basic science laboratories have recently generated important insights into the pathophysiology and molecular genetic abnormalities of these tumors. Efforts to translate these findings into innovative treatment strategies offer substantial promise to overcome therapeutic hurdles and treat individual patients more effectively. Improved understanding of malignant glioma biology and factors associated with treatment response will probably lead to improved therapeutic options and a better patient outcome.

Abstract 4736: Amplification of ERK2 mediates resistance to the novel irreversible EGFR inhibitor WZ4002

Abstract Background: EGFR T790M causes clinical resistance to the quinazoline EGFR kinase inhibitors gefitinib and erlotinib in non-small cell lung cancer. Amplification of EGFR T790M causes resistance to the irreversible quinazoline EGFR inhibitor PF00299804 (Ercan etl al. Oncogene 2010). A novel T790M selective irreversible pyrimidine EGFR kinase inhibitor, WZ4002, is effective in NSCLC models harboring EGFR T790M (Zhou, W, Ercan, D et al., Nature 2009). The mechanism(s) of resistance to this class of irreversible EGFR inhibitors have not been explored. Methods and Results: We exposed the Gefitinib resistant PC9 GR cells (EGFR del E746_A750/T790M; IC50 to WZ4002: ∼30nM) to increasing concentrations of WZ4002 to generate PC9 GW resistant cells (IC50 to WZ4002 ∼10µM) and isolated several resistant clones. The PC9 GWR cells contain EGFR del E746_A750/T790M but do not harbor additional EGFR mutations and are cross resistant to the irreversible quinazoline EGFR inhibitor BIBW2992. The PC9 GWR cells demonstrate an increase in basal phosphorylation of ERK2 but not ERK1 as well as total protein compared to parental PC9 GR cells. WZ4002 effectively inhibits EGFR phosphorylation but not ERK 2 phosphorylation in the PC9 GWR cells whereas in the PC9 GR cells WZ4002 inhibits both EGFR and ERK 1/2 phosphorylation. Furthermore, unlike in the PC9 GR cells, EGFR inhibition does not lead to BIM induction or apoptosis in the PC9 GWR cells. Genomic analyses of the PC9 GWR compared with the PC9 GR cells, using single nucleotide polymorphism (SNP) arrays, identify a focal amplification in the PC9 GWR cells on chromosome 22 which includes ERK2 (MAP2K). Using fluorescence in situ hybridization (FISH), we confirm amplified intra-chromosomal regions mapping to MAP2K. Inhibition of ERK signaling by either an ERK2 specific shRNA or by the MEK inhibitor CI-1040, restores sensitivity to WZ4002 and BIBW2992. In addition, combination of WZ4002 and CI-1040 results in BIM induction and apoptosis in the PC9 GWR cells. In contrast inhibition of PI3K signaling using the PI3K inhibitor PI-103 does not restore WZ4002 sensitivity. Ectopic activation of ERK signaling using an activated MEK1 allele (MEK K57N) found in lung cancer, in different lung cancer cell lines (PC9 (EGFR del E746_A750), PC9 GR (EGFR del E746_A750), and H1975 (L858R/T790M), is sufficient to cause resistance to WZ4002 and block WZ4002 mediated apoptosis. Conclusions: We identify amplification of ERK2, a component downstream component of EGFR signaling, as a mechanism of resistance to structurally distinct irreversible EGFR kinase inhibitors. The combination of an EGFR inhibitor and a MEK inhibitor is effective in against these drug resistant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4736. doi:10.1158/1538-7445.AM2011-4736

Abstract 1736: Src inhibitor dasatinib enhances anti-tumor activity of irreversible or T790M-selective epidermal growth factor receptor-tyrosine kinase inhibitors in non-small cell lung cancer cells with T790M gate-keeper mutation

Abstract Despite the therapeutic benefit of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in non-small cell lung cancer (NSCLC) patients with EGFR mutation, most if not all eventually develop resistance to these drugs. A secondary mutation of EGFR (T790M) accounts for 50% of all cases of acquired resistance to EGFR-TKIs. Although irreversible EGFR-TKIs are thought to be one of the strategies to overcome T790M, the efficacy of these drugs appears limited. To investigate how signal transduction is altered in NSCLC cells with T790M, we generated PC9GR cells with T790M by long term gefitinib exposure of PC9 cells with exon 19 deletion. Global phospho-proteomics approach revealed that Src family kinases were phosphorylated by erlotinib treatment in PC9GR cells, suggesting that Src family kinases compensate EGFR inhibition in NSCLC cells with T790M. Western blot assays demonstrated that BIBW2992, an irreversible EGFR-TKI currently in several clinical trials, also activated Src family kinases in PC9GR and H1975 NSCLC cells with T790M. The Src inhibitor dasatinib combined with BIBW2992 abolished this Src phosphorylation along with complete suppression of phospho Akt and Erk. In addition, significant PARP cleavage was observed in these cells suggesting apoptotic cell death. Consistent with the effects on cell signaling and apoptosis, dasatinib enhances anti-tumor activity of BIBW2992 in both GLO cell proliferation assay and caspase-3 apoptosis assay in PC9GR and H1975 cells. Additional experiments also demonstrated that dasatinib enhanced the effects of T790M selective EGFR-TKI WZ4002, which was recently reported to be another possible strategy to overcome T790M, in PC9GR and H1975 cells. Finally, the combination of BIBW2992 and dasatinib showed significant in vivo tumor suppression (including tumor regression) in PC9GR xenograft studies. These results suggest a role for Src in maintaining downstream signaling despite irreversible EGFR inhibitors and support further studies of irreversible EGFR combined with dasatinib in NSCLC patients who acquired resistance to EGFR-TKI by T790M. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1736. doi:10.1158/1538-7445.AM2011-1736

Abstract 3566: Effect of afatinib on lung cancer burden induced by an exon 19 EGFR mutation in transgenic mice

Abstract Afatinib (BIBW2992) is an irreversible dual inhibitor of EGFR and HER2. Afatinib was reported to have anti-tumor effect against L858R/T790M EGFR lung cancer model (Oncogene 27, 2008). Kinase inhibitors had different dissociation constant for binding deletion mutation and point mutation (Nat Biotechnol 26, 2008). Thus, the effect of afatinib on lung cancer harboring deletion or deletion/T790M EGFR double mutations should be clarified because it is possible that afatinib may affect differently tumors with deletion mutations compared to tumors with L858R or L858R/T790M point mutations. Our present study was carried out to investigate the preclinical efficacy of afatinib in lung cancer harboring exon19 EGFR del mutations. Growth inhibition was measured by MTT assays. RPC-9 cells that acquired T790M mutation of EGFR were established from parental PC-9 cells harboring exon 19 del mutation (delE746-A750) in our laboratory (Cancer Res 67, 2007). The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96 h exposure was used to evaluate the effect of afatinib and gefitinib. Protein expression was determined by Western blotting. The mean IC50s of afatinib were 0.00032 µM for PC-9 cells and 0.164 µM for RPC-9 cells and were less than those of gefitinib for PC-9 (0.0033 µM) and RPC-9 (11.45 µM), respectively. Phosphorylated EGFR, AKT and ERK of PC-9 were suppressed by treatments with afatinib (1 nM) and with gefitinib (100 nM). In RPC-9 cells, afatinib was 100-fold less active (100 nM) compared to parental cells while gefitinib was ineffective (10000 nM). In addition, 1 nM and 100 nM of afatinib completely inhibited phosphorylation of HER2 in PC-9 and RPC-9, respectively. We generated transgenic mice expressing the delE748-A752 mutant of mouse EGFR, which is equivalent to the delE746-A750 mutant, driven by the SP-C promoter (Cancer Sci 99, 2008). The mice invariably developed multi-focal pulmonary adenocarcinomas of various sizes at 5 to 6 weeks of age and died of lung cancer about 2 months later, if left untreated. To investigate the effect of afatinib on lung tumors induced by the activating EGFR mutation, the transgenic mice were treated with oral afatinib (5 mg/kg/day), gefitinib (5 mg/kg/day) or vehicle alone from 11 to 15 weeks of age. The number of lung tumors (long axis exceeding 1 mm) in the afatinib-treated group, gefitinib-treated group and vehicle group was 0.12 ± 0.13, 1.25 ± 0.60 and 9.00 ± 1.5 respectively. There were significant differences of the tumor numbers between afatinib-treated group and vehicle group (P &amp;lt; 0.01), while afatinib tended to reduce the tumors compared to gefitinib (P = 0.06). In addition, afatinib was more effective than gefitinib in pathological specimens as the tumors in afatinib-treated group almost disappeared. In conclusion, our data suggest that afatinib may be more effective for treatment of lung cancer induced by the EGFR mutation in exon 19 than gefitinib. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3566. doi:10.1158/1538-7445.AM2011-3566

Phase I Trial of the Irreversible EGFR and HER2 Kinase Inhibitor BIBW 2992 in Patients With Advanced Solid Tumors

Preclinical data have demonstrated that BIBW 2992 is a potent irreversible inhibitor of ErbB1 (EGFR/HER1) and mutated ErbB1 receptors including the T790M variant, as well as ErbB2 (HER2). A phase I study of continuous once-daily oral BIBW 2992 was conducted to determine safety, maximum-tolerated dose, pharmacokinetics (PK), food effect, and preliminary antitumor efficacy. Patients with advanced solid tumors were treated. PK evaluation was performed after the first dose and at steady-state. Fifty-three patients received BIBW 2992 at 10 to 50 mg/d. BIBW 2992 was generally well-tolerated. The most common adverse effects included diarrhea, nausea, vomiting, rash, and fatigue. Dose-limiting toxicities included grade 3 rash (n = 2) and reversible dyspnea secondary to pneumonitis (n = 1). The recommended phase II dose was 50 mg/d. PK was dose proportional with a terminal elimination half-life ranging between 21.3 and 27.7 hours on day 1 and between 22.3 and 67.0 hours on day 27; BIBW 2992 exposure decreased after food intake. Three patients with non-small-cell lung carcinoma (NSCLC; two with in-frame exon 19 mutation deletions) experienced confirmed partial responses (PR) sustained for 24, 18, and 34 months, respectively. Two other patients (esophageal carcinoma and NSCLC) had nonconfirmed PRs. A patient with a PR at 10 mg/d progressed and developed symptomatic brain metastases, which subsequently regressed with an increased dose of 40 mg/d of BIBW 2992. A further seven patients had disease stabilization lasting > or = 6 months. Continuous, daily, oral BIBW 2992 is safe and has durable antitumor activity. It is currently being evaluated in phase III trials.