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Afatinib, a second-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), has demonstrated a significant survival benefit over platinum-based chemotherapy in a first-line setting in advanced non–small-cell lung cancer (NSCLC) harboring EGFR exon 19 deletion. In addition, we and other groups have shown there to be favorable progression-free survival (PFS) outcomes, with acceptable toxicity profiles, with bevacizumab and first-generation EGFR-TKI combination therapy. On the basis of the above, we hypothesized that a combination of bevacizumab and afatinib could potentially improve efficacy. In our phase 1 study, a daily 30 mg dose of afatinib and 15 mg/kg intravenous bevacizumab every 3 weeks was well tolerated and was defined as the recommended dose. We have initiated a randomized phase 2 trial comparing afatinib (30 mg daily) and bevacizumab (15 mg/kg every 3 weeks) with afatinib (40 mg daily) alone for nonsquamous NSCLC harboring EGFR common mutations as a first-line therapy. A total of 100 patients will be enrolled onto this study and randomized in a 1:1 ratio. Patients will continue to receive treatment until disease progression or unacceptable toxicity. The primary end point is PFS, and the secondary end points are overall survival, tumor response, and time to treatment failure. The power is greater than 50% under the assumptions of a median PFS of 12 months for the afatinib group and a hazard ratio of 0.6 for the combination group (2-sided α = 0.05). We hypothesize that the combination therapy will be more efficacious than standard therapies for EGFR-mutant NSCLC patients.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, such as gefitinib and erlotinib, are effective for non-small cell lung cancer with activating EGFR mutations. However, even in patients with an initial dramatic response to such a drug, acquired resistance develops after 612 similar to months. A secondary mutation of T790M in EGFR and amplification of the MET gene account for this resistance; however, the mechanism(s) of approximately 30% of acquired resistance cases remain unknown. We established an erlotinib-resistant lung cancer cell line named PC-9/ER3 that harbors an EGFR mutation after continuously exposing PC-9 cells to erlotinib. PC-9/ER3 cells were 136-fold more resistant to erlotinib than the parental cells. Although the PC-9/ER3 cells did not carry the T790M mutation or MET amplification and had similar levels of phosphorylated (p) STAT3, pJAK2 increased in the resistant cells. It was found in the present study that 312 similar to h of exposure to erlotinib in both cell lines did not affect pJAK2 expression, but did result in increased pSTAT3 expression. pAkt in PC-9/ER3 cells was less suppressed than in PC-9 cells, although pEGFR and pMAPK were markedly suppressed in both cell lines. The combined treatment of erlotinib plus a JAK2 inhibitor (JSI-124) suppressed pAkt in PC-9/ER3 cells. Similarly, the combination of erlotinib plus JSI-124 or siRNA against JAK2 restored sensitivity to erlotinib in PC-9/ER3 cells. The combination of erlotinib plus JSI-124 was also effective for reducing PC-9/ER3 tumors in a murine xenograft model. Our results suggest that the activation of JAK2 partially accounts for acquired erlotinib resistance.(Cancer Sci, doi: 10.1111/j.1349-7006.2012.02363.x, 2012)

Crizotinib is the standard of care for advanced non–small cell lung cancer (NSCLC) patients harboring the anaplastic lymphoma kinase (ALK) fusion gene, but resistance invariably develops. Unlike crizotinib, alectinib is a selective ALK tyrosine kinase inhibitor (TKI) with more potent antitumor effects and a favorable toxicity profile, even in crizotinib-resistant cases. However, acquired resistance to alectinib, as for other TKIs, remains a limitation of its efficacy. Therefore, we investigated the mechanisms by which human NSCLC cells acquire resistance to alectinib. We established two alectinib-resistant cell lines that did not harbor the secondary ALK mutations frequently occurring in crizotinib-resistant cells. One cell line lost the EML4-ALK fusion gene, but exhibited increased activation of insulin-like growth factor-1 receptor (IGF1R) and human epidermal growth factor receptor 3 (HER3), and overexpressed the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF1R and HER3 signaling overcame resistance to alectinib in this cell line. The second alectinib-resistant cell line displayed stimulated HGF autocrine signaling that promoted MET activation and remained sensitive to crizotinib treatment. Taken together, our findings reveal two novel mechanisms underlying alectinib resistance that are caused by the activation of alternative tyrosine kinase receptors rather than by secondary ALK mutations. These studies may guide the development of comprehensive treatment strategies that take into consideration the various approaches ALK-positive lung tumors use to withstand therapeutic insult. Cancer Res; 76(6); 1506–16. ©2015 AACR.

Everolimus is an orally administered mTOR inhibitor. The effect, and mechanism of action, of everolimus on lung cancers with an epidermal growth factor receptor (EGFR) mutation remain unclear. Four gefitinib-sensitive and -resistant cell lines were used in the present work. Growth inhibition was determined using the MTT assay. Transgenic mice carrying the EGFR L858R mutation were treated with everolimus (10 mg/kg/day), or vehicle alone, from 5 to 20 weeks of age, and were then sacrificed. To evaluate the efficacy of everolimus in prolonging survival, everolimus (10 mg/kg/day) or vehicle was administered from 5 weeks of age. The four cell lines were similarly sensitive to everolimus. Expression of phosphorylated (p) mTOR and pS6 were suppressed upon treatment with everolimus in vitro, whereas the pAKT level increased. The numbers of lung tumors with a long axis exceeding 1 mm in the everolimus-treated and control groups were 1.9 +/- 0.9 and 9.4 +/- 3.2 (t-test, p

To study epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance mechanisms, we established a novel gefitinib-resistant lung cancer cell line derived from an EGFR-mutant non-small cell lung cancer cell line (PC-9) pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (designated PC9-GR). We found that gefitinib substantially suppressed the EGFR signaling pathway, whereas ERK was reactivated after several hours in PC9-GR but not in PC-9. The combination of gefitinib with ERK inhibition (by U0126) restored gefitinib susceptibility in PC9-GR, but PI3K-Akt inhibition with LY294002 did not. Although the levels of phosphorylated Src were up-regulated simultaneously with ERK reactivation, neither ERK suppression using U0126 nor an ERK-specific siRNA induced Src phosphorylation. Furthermore, dual inhibition of EGFR and Src restored gefitinib sensitivity in PC9-GR in vitro and in vivo. In conclusion, our results indicate that Src-mediated ERK reactivation may play a role in a novel gefitinib resistance mechanism, and that the combined use of gefitinib with a Src inhibitor may be a potent strategy to overcome this resistance.

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.

Background The second-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI), alectinib, demonstrated high response rate, long response duration and a favorable toxic profile in patients with ALK-rearranged advanced non-small cell lung cancer in a phase II study (Lancet Oncol 14:590-8, 2013). However, even this promising drug is predicted to develop acquired resistance. Therefore, we investigated the mechanisms of resistance using two alectinib-resistant cell lines. Methods We established alectinib-resistant cell lines, H2228/CHR and ABC-11/CHR, from H2228 (EML4-ALK fusion genes variant 3a/b E6) and ABC-11 (EML4-ALK fusion genes variant 3b E6) respectively, by continuous exposure to alectinib. They were characterized using MTT assay, Western blotting, receptor tyrosine kinase array, ELISA, FISH, RT-PCR, and xenograft models. Results H2228/CHR and ABC-11/CHR cells were 117- and 40-fold more resistant than the parental lines, respectively, and maintained downstream AKT and ERK phosphorylation even in the presence of 10 μM alectinib. There were no ALK secondary mutations in those resistant cell lines. H2228/CHR lost the EML4-ALK fusion gene, and exhibited increased activation of insulin-like growth factor-1 receptor (IGF-1R) and human epidermal growth factor receptor 3 (HER3) with overexpression of the HER3 ligand neuregulin 1. Accordingly, pharmacologic inhibition of IGF-1R and HER3 signaling overcame the resistance. In ABC-11/CHR, MET was activated by stimulated hepatocyte growth factor (HGF) autocrine signaling. We found HGF gene translocation underlying the HGF autocrine system. Anti-HGF antibody suppressed the MET activation and combined treatment with alectinib and anti-HGF antibody or a MET inhibitor suppressed downstream signaling in ABC-11/CHR cells. Finally, crizotinib, which targets both ALK and MET, most effectively inhibited the growth of ABC-11/CHR both in vitro and in vivo. Conclusions We identified novel alectinib resistance mechanisms caused by the activation of alternative tyrosine kinase receptors. Our findings provide new insights into constructing a therapeutic strategy for ALK-positive lung cancer. Citation Format: Hideko Isozaki, Eiki Ichihara, Masayuki Yasugi, Nagio Takigawa, Kadoaki Ohashi, Toshio Kubo, Takashi Ninomiya, Nobuaki Ochi, Daisuke Minami, Kenichiro Kudo, Yuka Kato, Hiroe Kayatani, Tomoki Tamura, Kiichiro Ninomiya, Toshio Higo, Tsuyoshi Makimoto, Akiko Sato, Katsuyuki Hotta, Kunio Matsumoto, Toshiaki Sendo, Mitsune Tanimoto, Katsuyuki Kiura. Activating alternative receptor tyrosine kinases induced alectinib-resistance in ALK rearranged non-small cell lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2103.

Dramatic clinical responses to a first generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) crizotinib have been observed in patients with advanced non-small cell lung cancer (NSCLC) haboring ALK fusion gene. Alectinib (CH5424802) (Chugai Pharmaceutical Co., Ltd.) is a second generation ALK-TKI could partially overcome acquired resistance to crizotinib in vitro (Cancer Cell 19, 2011: 679-90), and showed promising result in phase I/II clinical trials with high response rate (93.5 %) and less toxicity than crizotinib. However, even this promising ALK-TKI, the problem of acquired resistance is inevitable. To elucidate resistant mechanisms, we established an alectinib-resistant cell line H2228/CHR from H2228 that have echinoderm microtubule associated protein like 4 (EML4)-ALK fusion gene, by continuous exposure to this agent. ALK fusion gene disappeared in H2228/CHR, which was confirmed Western blotting, IHC, qRT-PCR and FISH. H2228/CHR cells presented higher phosphorylated levels of epidermal growth factor receptor (EGFR) than the parental H2228 cells and became more sensitive to EGFR-TKI erlotinib. Next, we established alectinib-resistant ABC-11/CHR cells from ABC-11, a cell line established from pleural effusion of EML4-ALK positive ALK-TKI naïve patient. In ABC-11/CHR cells, phosphorylated levels of ALK was markedly decreased and phosphorylated levels of MET was increased.In addition, autocrine of HGF was increased in ABC-11/CHR cells. ABC-11/CHR showed sensitivity to ALK/MET tyrosine kinase inhibitor, crizotinib in vitro and in vivo model. In conclusion, inactivation of ALK signaling induced acquired resistance to alectinib in NSCLC harboring ALK fusion gene. Signaling switch from ALK to EGFR or MET was observed in the alectinib-resistant cells. We revealed to overcome such acquired resistance using erlotinib or crizotinib. Citation Format: Hideko Isozaki, Eiki Ichihara, Masayuki Yasugi, Ochi Nobuaki, Katsuyuki Hotta, Nagio Takigawa, Toshiaki Sendo, Mitsune Tanimoto, Katsuyuki Kiura. ALK inactivation induced acquired resistance to alectinib in lung cancer harboring EML4-ALK fusion gene. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3721. doi:10.1158/1538-7445.AM2014-3721

Aim: Alectinib (CH/RO5424802) is a second generation anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI). It has been reported to overcome acquired resistance to the first generation ALK-TKI, crizotinib, in a preclinical study (Cancer Cell 19, 2011: 679-90). A phase I/II clinical trial of alectinib for ALK positive non-small cell lung cancer (NSCLC) showed an unprecedented response rate (93.5 % in phase II portion) and the drug seemed to lead to less toxicities than crizotinib (Lancet Oncol 14, 2013: 590-98). However, subsequent resistance to alectinib is expected after its approval. Thus, we need to consider the therapeutic strategy to overcome the resistance to alectinib in advance. Methods: Two alectinib-resistant H2228/CHR and ABC-11/CHR NSCLC cell lines from H2228 and ABC-11, respectively, harboring EML4-ALK fusion genes were established by continuous exposure to alectinib. We characterized the resistant cell lines using MTT assay, western blotting, immunohistochemistry, polymerase chain reaction, fluorescent in situ hybridization, phospho-receptor tyrosine kinase array, comparative genomic hybridization, RNA array, ELISA and xenograft models. Results: H2228/CHR and ABC-11/CHR cells showed 130-fold and 17-fold higher resistance to alectinib than their respective parent cells in vitro. H2228/CHR cells had lost EML4-ALK fusion gene and ALK protein expression. In addition, the resistant cells overexpressed IGF1R, and were sensitive to IGF1R-TKI (TAE684) as a result. While ABC-11/CHR cells reduced phospho-ALK in spite of preservation of total ALK protein, they activated MET with increment of its ligand (hepatocyte growth factor) as an autocrine action. Because crizotinib originally functions as MET-TKI along with ALK-TKI, ABC-11/CHR was sensitive to the drug both in vitro and in vivo. Conclusions: Loss or inactivation of ALK leads to the acquired resistance to alectinib in the two cell lines we established. In addition, signaling switch from ALK to IGF1R or MET was observed. IGF1R inhibitor or MET inhibitor was effective on such cases. Thus, ALK positive NSCLC patients refractory to alectinib might benefit from IGF1R or MET inhibitors. Disclosure: K. Hotta: Honoraria from speaker's bureau from Chugai pharmaceutical CO., LTD.; N. Takigawa: Honoraria from speaker's bureau from Pfizer Inc. Japan; K. Kiura: Honoraria from speaker's bureau from Chugai pharmaceutical CO., LTD., Pfizer Inc. Japan and Novartis Pharma K.K. All other authors have declared no conflicts of interest.

There has been considerable evidence that (-)-epigallocatechin-3-gallate (EGCG) inhibited enzyme activities and signal transduction pathways, resulting in the suppression of cell proliferation and enhancement of apoptosis, as well as the inhibition of cell invasion, angiogenesis and metastasis. EGCG has been shown to bind directly to several receptors including epidermal growth factor receptor (EGFR) and to inhibit the functions. EGFR tyrosine kinase inhibitors such as gefitinib and ALK inhibitors such as crizotinib are effective for non-small cell lung cancer harboring activating EGFR mutations and ALK fusion genes, respectively. However, even in the patients with an initial response to the drug, acquired resistance develops after 6-12 months. In this study, we investigated effects of EGCG on EGFR or ALK driven lung cancer models. We used four lung cancer cell lines: PC-9, RPC-9, H1975 and H2228. RPC-9 cells that acquired EGFR T790M mutation were established from parental PC-9 cells harboring EGFR exon 19 deletion mutation in our laboratory. RPC-9 cells that showed a 400-fold resistance to gefitinib compared with parental PC-9 cells. H1975 cells have L858R point mutation in exon 21 with T790M. H2228 cells harbor EML4-ALK fusion genes. Growth inhibition was measured using MTT assay. The drug concentration required to inhibit the growth of tumor cells by 50% (IC50) for 96 h exposure was used to evaluate the effectiveness of EGCG. Protein expression was determined by Western blotting. The IC50s (mean + standard deviation) were 46 + 7.0 μM for PC-9 cells, 37 + 3.6 μM for RPC-9 cells, 32 + 0.6 μM for H1975 cells and 57 + 7.6 μM for H2228 cells: the four cell lines have similar sensitivity to EGCG. Phosphorylated (p)EGFR, pAKT and pERK in RPC-9 and H1975 cells were suppressed by EGCG (50 or 100 μM). pALK, pAKT and pERK in H2228 cells were also inhibited by EGCG (50 or 100μM). The antitumor effect of EGCG was examined using mouse xenograft model. PC-9, RPC-9, H1975, and H2228 cells (2 × 106) were injected s.c. into the backs of the athymic mice at 7 wk of age. At about 1 week after injection, mice harboring about 5 - 10 mm tumor size were randomly assigned into one of two groups that received either EGCG or vehicle. The tumor sizes (mm3; mean + standard deviation) of PC-9 on day 42 were 1114 + 478 (n = 10) in EGCG group and 2803 + 973 (n = 9) in control group (p < 0.05). Similarly, the xenograft tumors of RPC-9, H1975 and H2228 cells in EGCG-treated groups were significantly smaller than those in vehicle-treated groups, respectively. The numbers of tumor blood vessels of xenograft tissues, which were evaluated by immunohistochemistry using CD31 antibody, in EGCG-treated mice were significantly reduced than those in vehicle-treated mice. In conclusion, these preclinical data suggest that EGCG may be effective for both EGFR-driven in spite of T790M presence and ALK-driven lung tumors. Citation Format: Yoshihiro Honda, Nagio Takigawa, Eiki Ichihara, Takashi Ninomiya, Toshio Kubo, Nobuaki Ochi, Masayuki Yasugi, Toshi Murakami, Mitsune Tanimoto, Katsuyuki Kiura. Effects of (-)-epigallocatechin-3-gallate on EGFR or ALK driven lung cancer models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2031. doi:10.1158/1538-7445.AM2013-2031

We experienced dramatic responses to crizotinib, a multitargeted tyrosine kinase inhibitor including anaplastic lymphoma kinase (ALK), MET, and ROS1 in patients with non-small cell lung cancer (NSCLC) harboring ALK fusion gene; however, majority of the patients relapsed until around 10 months. The emergence of resistance to crizotinib is reportedly conferred by genetic alterations, such as ALK kinase domain mutation, ALK copy number gain or activation of the bypass pathways. CH5424802 (Chugai Pharmaceutical Co., Ltd.) is a new, highly selective ALK tyrosine kinase inhibitor that partially overcome acquired resistance to crizotinib, thereby inhibiting crizotinib resistant mutations of ALK in a preclinical experiment (Cancer Cell 19, 2011: 679-90) A phase II study of CH5424802 demonstrated 93.5% response rate (JLCS 2012); however, we also encountered acquired resistance phenomenon. To elucidate the acquired resistant mechanisms, we established CH5424802-resistant H2228 cells harboring EML4-ALK fusion gene under the step-wise continuous exposures of CH5424802. Six sublines that could grow in the presence of 1 μmol/L of CH5424802 (designated H2228CHR clones -1, -2, -3, -5, -6, and -9) were obtained. All sublines revealed more than 30-fold resistance to CH5424802 (50% inhibitory concentration [IC50]: 1.0-4.8 μmol/L) compared with the parental H2228 cells (IC50: 0.03 μmol/L). In addition, the sublines similarly indicated resistance to Crizotinib. H2228CHR clones showed the constitutive phosphorylation of Akt and Erk1/2 even in the presence of CH5424802, whereas phosphorylation of Akt and Erk1/2 was markedly decreased in the parental H2228 cells. Surprisingly, ALK fusion gene disappeared in all H2228 CHR clones, which were confirmed immunostaining, reverse transcription PCR, FISH and Western blotting. H2228 CHR clones -1 and -2 presented higher phosphorylated levels of epidermal growth factor receptor (EGFR) than the parental H2228 cells and showed sensitivity to EGFR tyrosine kinase inhibitors, gefitinib and erlotinib. Furthermore, we also established CH5424802-resistant ABC-11CHR cells derived from ABC-11 cells from pleural effusion of an ALK inhibitor naïve patient. In ABC-11CHR cells, phosphorylated ALK was markedly decreased and phosphorylated MET was increased. In conclusion, signal switching from ALK to other tyrosine kinase receptors induced resistance to a highly selective ALK tyrosine kinase inhibitor CH5424802 in NSCLC cell lines harboring ALK fusion gene. Citation Format: Hideko Isozaki, Eiki Ichihara, Masayuki Yasugi, Takashi Ninomiya, Yoshihiro Honda, Toshi Murakami, Daisuke Minami, Yuka Kato, Kenitiro Kudo, Akiko Sato, Katsuyuki Hotta, Nagio Takigawa, Toshiaki Sendo, Mitsune Tanimoto, Katsuyuki Kiura. Switching oncogene signaling in a highly selective ALK tyrosine kinase inhibitor CH5424802 resistant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4444. doi:10.1158/1538-7445.AM2013-4444

A 72-year-old woman was referred to our hospital with Stage IV non-small-cell lung cancer (NSCLC). Chest computed tomography revealed a mass in the upper lobe of the right lung, with pleural effusion. Cytologic examination identified adenocarcinoma cells in the right pleural effusion. Furthermore, both a deletion mutation in exon 19 and a threonine-methionine substitution mutation at position 790 in exon 20 (T790M) were detected in the epidermal growth factor receptors (EGFR) in the malignant cells. As systemic chemotherapy consisting of car- boplatin and pemetrexed or erlotinib proved ineffective, docetaxel monotherapy was initiated as a third-line treatment. Following salvage chemotherapy, her Eastern Cooperative Oncology Group performance status improved from 3 to 1, with tumor regression over 5 months. To the best of our knowledge, this is the first report of successful docetaxel treatment for a patient with NSCLC harboring the T790M EGFR-activating mutation identified before treatment with EGFR tyrosine kinase inhibitors.

Afatinib (BIBW2992) is an irreversible pan-HER inhibitor. Kinase inhibitors have different dissociation constant for binding deletion mutation and point mutation in EGFR. We previously reported that afatinib with short-term (4 weeks) administration tended to be more effective for treatment of lung cancer model induced by the deletion mutation in exon 19 than gefitinib, although similar effect of both drugs was observed in L858R mutated mice (#3566, AACR 2011). Our present study was carried out to investigate the preclinical efficacy of long-term treatment of afatinib in lung cancer harboring deletion mutation in EGFR exon 19. 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 lung adenocarcinomas of various sizes at 5 weeks of age and died of lung cancer, if left untreated. To evaluate the efficacy of afatinib on survival in the mice, afatinib, gefitinib (5 mg/kg/day, n=15 each) or vehicle alone (n=13) was administered orally from 7 weeks of age until inability to take due to toxicity or death. All mice in the control group died and the median survival time (MST) was 17 weeks. At the time, 12 gefitinib-treated mice and 6 afatinib-treated mice died, and the afatinib-treated mice survived longer significantly than gefitinib-treated mice (MST: not reached vs 54 weeks; p < 0.05 by logrank test). No toxic death was observed in all mice. After the initiation of treatment at week 4, the body weight of the mice in the control group was significantly lower than that in gefitinib or afatinib group. The gefitinib-treated mice significantly lost weight compared to afatinib-treated mice after 34 weeks. To understand the efficacy of afatinib in the mice, EGFR/HER2 protein expression was determined by immunoblotting assay. The mice of 14 weeks of age were sacrificed at 2, 6, 10, 14, and 18 hours after single administration of each drug. In gefitinib-treated mice, phosphorylated EGFR (pEGFR) was suppressed at 2 and 6 hours but was recovered at 10 hours. On the other hand, in afatinib-treated mice, pEGFR was suppressed at 2, 6, and 10 hours and was partially recovered at 14 and 18 hours. At 14 hours, the expressions of pEGFR in gefitinib-treated mice and in afatinib-treated mice were more and less than those in mice without treatment, respectively. In gefitinib-treated mice, phosphorylated HER2 (pHER2) was recovered at 10 hours after decreasing at 2 and 6 hours. Presumably, EGFR/HER2 heterodimer might be suppressed temporally by gefitinib. In afatinib-treated mice, pHER2 was suppressed from 2 to 18 hours. The results suggest that afatinib is more effective than gefitinib because it suppresses pEGFR for longer time than gefitinib. In conclusion, our data suggest that afatinib can well control lung cancer driven by EGFR exon19 deletion mutation longer than gefitinib. 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 LB-360. doi:1538-7445.AM2012-LB-360

The mammalian target of rapamycin (mTOR) plays a major role in the regulation of protein translation, cell growth, and metabolism. Alterations of the mTOR signaling pathway are common in cancer, and thus mTOR is being actively pursued as a therapeutic target. Everolimus is an orally available rapamycin analogue showing anticancer effects on renal cell carcinoma and used in clinical. Furthermore, in preclinical studies, everolimus shows anticancer effects in various cancer cell lines and xenograft models, including lung cancer. However, the effect of mTOR inhibitors on non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutation remains unclear. Our present study was carried out to investigate the preclinical efficacy of everolimus in NSCLC harboring EGFR mutation. Growth inhibition was measured by MTT assays. RPC-9 cells that acquired T790M mutation of EGFR gene 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 effectiveness of everolimus. Protein expression was determined by Western blotting. The IC50s were 2 μM for PC-9 cells and 4 μM for RPC-9 cells. RPC-9 cells that showed a 400-fold resistance to gefitinib compared with parental PC-9 cells remained the sensitivity to everolimus. Phosphorylated mTOR and phosphorylated S6 were suppressed by treatment with everolimus (2 μM), while phosphorylated AKT was increased presumably due to negative feedback of mTOR inhibition. We generated transgenic mice expressing the L858R mutation of EGFR driven by the SP-C promoter (Cancer Res 69, 2009). In the absence of treatment, the transgenic mice developed atypical adenomatous hyperplasia at age 4 weeks and adenocarcinoma at 6 to 7 weeks of age, and died due to tumor progression at 25 to 40 weeks. To investigate the effect of everolimus on lung tumors induced by an activating EGFR mutation in vivo, the transgenic mice were treated with oral everolimus (10 mg/kg/day, n = 13) or vehicle alone (n = 14) from 5 to 20 weeks of age. The number of lung tumors (long axis exceeding 1 mm) in the everolimus-treated group and control group was 1.9 ± 0.9 and 9.4 ± 3.2 (log-rank test, p 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 4487. doi:10.1158/1538-7445.AM2011-4487

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 < 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

Background: More than 80% of all patients with small cell lung cancer (SCLC) receiving chemotherapy achieve an objective response; however, most responders eventually relapse because of drug resistance. Tumors are presumed to contain a small population of cancer stem cells (CSCs) that initiate tumor growth and promote tumor spreading. Multidrug resistance in CSCs is thought to allow the tumor to evade conventional therapy. This study focused on the expression of CD133 and CD87, because CD133 is a putative marker of CSCs in some cancers including brain, colon and lung and CD87 is associated with stem cell like properties in SCLC. Material and methods: This study used six SCLC cell lines (SBC-3, 4, 5, 6, 7, 9) established in the laboratory. The sensitivities of these cell lines to cisplatin, etoposide, paclitaxel, SN-38 (active metabolite of irinotecan), and amrubicinol (active metabolite of amrubicin) were determined by an MTT assay. The expression levels of CD133 and CD87 were analyzed by real-time quantitative reverse transcriptase-PCR assay and flow cytometry. CD133+/− and CD87+/− cells were isolated by flow cytometry and were subsequently cloned twice. Cell cycle traverse perturbations were analyzed by flow cytometry using propidium iodide and Pyronin Y. NOD/SCID mice were used for the tumor formation assay. Results: The SBC-7 cells showed the highest expression levels of both CD133 and CD87 among the six cell lines. CD133-/CD87-, CD133+/CD87-, and CD133-/CD87+ cells were successfully isolated from SBC-7 cells; however, double positive cells could not be isolated. Both CD133+/CD87- and CD133-/CD87+ sub-populations showed higher resistance to chemotherapeutic drugs and repopulating ability, and contained more G0 quiescent cells than the CD133-/CD87- sub-population in vitro. Contrary to expectations, the CD133-/CD87- as well as the CD133+/CD87- sub-populations showed higher tumorigenic potential than the CD133-/CD87+ sub-population in vivo. When 5000 (or 2000) CD133+/CD87-, CD133-/CD87+, and CD133-/CD87- cells were inoculated into NOD/SCID mice, the tumors were detected in 100% (or 50%), 67% (or 0%) and 100% (or 100%), respectively. The drugs were screened for their ability to overcome the high resistance of CD133+/CD87- cells to chemotherapy and zoledronic acid was thus found to restore the sensitivity of CD133+/CD87- cells to amrubicinol in vitro. Conclusions: Both CD133 and CD87 proved to be inadequate markers for CSCs, however, they were able to predict resistance to chemotherapy. Amrubicin in combination with zoledronic acid might be beneficial for the treatment of SCLC patients who are refractory to conventional chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3370.