Tetsuo Tani

In vitro modeling to determine mutation specificity of EGFR tyrosine kinase inhibitors against clinically relevant EGFR mutants in non-small-cell lung cancer

EGFR mutated lung cancer accounts for a significant subgroup of non-small-cell lung cancer (NSCLC). Over the last decade, multiple EGFR tyrosine kinase inhibitors (EGFR-TKIs) have been developed to target mutated EGFR. However, there is little information regarding mutation specific potency of EGFR-TKIs against various types of EGFR mutations. The purpose of this study is to establish an in vitro model to determine the “therapeutic window” of EGFR-TKIs against various types of EGFR mutations, including EGFR exon 20 insertion mutations. The potency of 1st (erlotinib), 2nd (afatinib) and 3rd (osimertinib and rociletinib) generation EGFR-TKIs was compared in vitro for human lung cancer cell lines and Ba/F3 cells, which exogenously express mutated or wild type EGFR. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFR. The in vitro model identified a wide therapeutic window of afatinib for exon 19 deletions and L858R and of osimertinib and rociletinib for T790M positive mutations. The results obtained with our models matched well with previously reported preclinical and clinical data. Interestingly, for EGFR exon 20 insertion mutations, most of which are known to be resistant to 1st and 2nd generation EGFR-TKIS, osimertinib was potent and presented a wide therapeutic window. To our knowledge, this is the first report that has identified the therapeutic window of osimertinib for EGFR exon 20 insertion mutations. In conclusion, this model will provide a preclinical rationale for proper selection of EGFR-TKIs against clinically-relevant EGFR mutations.

Activation of EGFR Bypass Signaling by TGFα Overexpression Induces Acquired Resistance to Alectinib in ALK-Translocated Lung Cancer Cells

Alectinib is a highly selective ALK inhibitor and shows promising efficacy in non–small cell lung cancers (NSCLC) harboring the EML4-ALK gene rearrangement. The precise mechanism of acquired resistance to alectinib is not well defined. The purpose of this study was to clarify the mechanism of acquired resistance to alectinib in ALK-translocated lung cancer cells. We established alectinib-resistant cells (H3122-AR) from the H3122 NSCLC cell line, harboring the EML4-ALK gene rearrangement, by long-term exposure to alectinib. The mechanism of acquired resistance to alectinib in H3122-AR cells was evaluated by phospho-receptor tyrosine kinase (phospho-RTK) array screening and Western blotting. No mutation of the ALK-TK domain was found. Phospho-RTK array analysis revealed that the phosphorylation level of EGFR was increased in H3122-AR cells compared with H3122. Expression of TGFα, one of the EGFR ligands, was significantly increased and knockdown of TGFα restored the sensitivity to alectinib in H3122-AR cells. We found combination therapy targeting ALK and EGFR with alectinib and afatinib showed efficacy both in vitro and in a mouse xenograft model. We propose a preclinical rationale to use the combination therapy with alectinib and afatinib in NSCLC that acquired resistance to alectinib by the activation of EGFR bypass signaling. Mol Cancer Ther; 15(1); 162–71. ©2015 AACR.

Overcoming EGFR bypass signal-induced acquired resistance to ALK tyrosine kinase inhibitors in ALK-translocated lung cancer

Activation of the EGFR pathway is one of the mechanisms inducing acquired resistance to anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI) such as crizotinib and alectinib. Ceritinib is a highly selective ALK inhibitor and shows promising efficacy in non–small cell lung cancers (NSCLC) harboring the ALK gene rearrangement. However, the precise mechanism underlying acquired resistance to ceritinib is not well-defined. This study set out to clarify the mechanism in ALK-translocated lung cancer and to find the preclinical rationale overcoming EGFR pathway–induced acquired resistance to ALK-TKIs. To this end, ceritinib-resistant cells (H3122-CER) were established from the H3122 NSCLC cell line harboring the ALK gene rearrangement via long-term exposure to ceritinib. H3122-CER cells acquired resistance to ceritinib through EGFR bypass pathway activation. Furthermore, H3122 cells that became resistant to ceritinib or alectinib through EGFR pathway activation showed cross-resistance to other ALK-TKIs. Ceritinib and afatinib combination treatment partially restored the sensitivity to ceritinib. Implications: This study proposes a preclinical rationale to use ALK-TKIs and afatinib combination therapy for ALK-translocated lung cancers that have acquired resistance to ALK-TKIs through EGFR pathway activation. Mol Cancer Res; 15(1); 106–14. ©2016 AACR.

Erlotinib as second‑ or third‑line treatment in elderly patients with advanced non‑small cell lung cancer: Keio Lung Oncology Group Study 001 (KLOG001)

The aim of this study was to assess the efficacy and safety of erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), as second- or third-line treatment for elderly Japanese patients with non-small-cell lung cancer (NSCLC). The patients eligible for this phase II trial were aged ≥70 years, had stage III/IV or recurrent NSCLC, and had previously received 1 or 2 chemotherapy regimens that did not include EGFR-TKIs. The patients received erlotinib at a dose of 150 mg/day. The primary endpoint was overall response rate (ORR), and the secondary endpoints were progression-free survival (PFS), overall survival (OS) and toxicity. A total of 38 patients with a median age of 76 years were enrolled. The majority of the patients were men (66%), had an Eastern Cooperative Oncology Group performance status of 1 (58%), stage IV disease (66%) and adenocarcinoma (74%). Of the 35 patients, 13 (34%) had tumors with EGFR mutations. The ORR was 26.3% (95% confidence interval: 12.1-40.5%) and the disease control rate was 47.4%. The median PFS was 3.7 months and the median OS was 17.3 months. The grade 3 adverse events observed included rash (13%), diarrhea (5%), interstitial pneumonitis (5%), anorexia (3%) and gastrointestinal bleeding (3%). Grade 4 or 5 adverse events were not observed. The median OS did not differ significantly between patients aged <75 years (14.9 months) and those aged ≥75 years (19.0 months; P=0.226). Therefore, erlotinib was found to be effective and well-tolerated in elderly patients with previously treated NSCLC.

A Case of Disseminated Cryptococcal Infection and Concurrent Lung Tuberculosis in a Patient under Steroid Therapy for Interstitial Pneumonia

Both disseminated cryptococcal infection and tuberculosis occur in hosts with impaired cell-mediated immunity, but there have been few reports about the concurrent infections in patients without human immunodeficiency virus infection. A 64-year-old man, who had been taking corticosteroids for interstitial pneumonia, was diagnosed with disseminated cryptococcal infection. While the patient was receiving anticryptococcal therapy, pulmonary tuberculosis also emerged. The patient developed acute exacerbation of interstitial pneumonia and passed away. Based on the patients clinical course, serial computed tomography images, and autopsy results, we believe that the preceding several months of corticosteroid treatment might have contributed to these coinfections in the lungs already vulnerable due to underlying fibrosis.

Abstract 4: ABT-263 is effective in a subset of non-small cell lung cancer cell lines

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Rationale: ABT-263 (Navitoclax) is one of the BH3 mimetics targeting anti-apoptotic B-cell lymphoma-2 (Bcl-2) family proteins such as Bcl-2, Bcl-XL, and Bcl-w, thereby inducing apoptosis. It has been reported that the response to ABT-263 is associated with expressions of myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic protein. Given its effectiveness as a single agent in preclinical studies, ABT-263 is currently being evaluated in clinical trials for small cell lung cancer (SCLC) and leukemia. However, the efficacy of ABT-263 in non-small cell lung cancer (NSCLC) has not been fully evaluated. We examined the effect of ABT-263 on cell proliferation of NSCLC cell lines and investigated the underlying mechanisms. Methods: The following 9 NSCLC cell lines were examined: SK-LU-1, A549, H358, Calu3, H3122, H1975, H460, H441, and BID007. The effects of ABT-263 in NSCLC cell lines were evaluated by MTS assay. Apoptosis was examined by flowcytometry using staining for annexin V and propidium iodide (PI), and also western blotting for cleaved PARP. Quantitative RT-PCR was carried out to assess the mRNA expression levels of anti-apoptotic genes and pro-apoptotic genes. Immunoprecipitation and western blotting were performed to compare the levels of anti-apoptotic and pro-apoptotic proteins between the sensitive and resistant cell lines. In addition, knockdown of Mcl-1 was performed by siRNA. Results: By screening 9 NSCLC cell lines using MTS assay, we found Calu3 and BID007were sensitive to ABT-263. We also confirmed that apoptosis was induced only in the ABT-263 sensitive lines but not in the ABT-263 resistant cell lines after ABT-263 treatment. However, the expression levels of Bcl-2 family proteins, including Mcl-1, did not differ significantly among the ABT-263 sensitive and resistant cell lines. Unlike the results in previous reports regarding SCLC, Mcl-1 was not decreased in the sensitive cell lines. The ABT-263 resistant cell lines became sensitive to ABT-263 after knockdown of Mcl-1 by siRNA, while the ABT-263 sensitive cell lines maintained the same sensitivity. Conclusion: We found that Calu3 and BID007 were sensitive to ABT-263. In the sensitive NSCLC cell lines, ABT-263 induces apoptosis irrespective of Mcl-1 expression levels. Citation Format: Aoi Kuroda, Keiko Ohgino, Hiroyuki Yasuda, Junko Hamamoto, Daisuke Arai, Kota Ishioka, Tetsuo Tani, Shigenari Nukaga, Ichiro Kawada, Katsuhiko Naoki, Kenzo Soejima, Tomoko Betsuyaku. ABT-263 is effective in a subset of non-small cell lung cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4. doi:10.1158/1538-7445.AM2015-4

Abstract 3006: Hyperoxia may be a treatment option for NSCLC

Introduction: Many anti-tumor drugs have been developed, however, prognosis of NSCLC remains poor and new approaches for NSCLC treatment are expected. Lung is a unique organ whose epithelial cells are directly exposed to oxygen. Hyperoxia produces reactive oxygen species (ROS) and induces cell damage. Therefore we attempted to investigate the potential of hyperoxia as an anti-tumor treatment in NSCLC cells in this study. Methods: We used oxygen-concentration-adjustable incubators and validated effect of hyperoxia on various lung cancer cell lines in vitro. The effect of hyperoxia on cell proliferation, apoptosis and gene expression was evaluated using MTS assay, flowcytometry and cDNA microarray, respectively. Results: We found that the growth of lung cancer cell lines (A549, NCI-H1975) was inhibited by 50% of hyperoxia in a dose-dependent manner, while that of normal airway epithelial cells (NHBE, BEAS-2B) was not. Two independent pathway analysis using cDNA microarray revealed the activation of NF-kB and AMPK pathways, and the production of nitric oxide and ROS in hyperoxia (50%) treated lung cancer cell lines compared to untreated ones. The combined treatment with ABT-263, Bcl-2 inhibitor, and hyperoxia (50%) induced synergistic growth inhibition and apoptosis in NCI-H1975 and SK-MES-1 cells. Moreover, the combination of ABT-263 and activator of either ROS, NF-kB or AMPK also showed synergistic growth inhibition in those cells. Conclusions: Hyperoxia (50%) showed anti-tumor effect in NSCLC cell lines through the activation of NF-kB and AMPK pathways, and the production of ROS. The effect was augumented in combination with Bcl-2 inhibitor. Hyperoxia may be a treatment option for NSCLC patients. Citation Format: Junko Hamamoto, Hiroyuki Yasuda, Ichiro Nakachi, Michael G. Edwards, Masayoshi Miyawaki, Makoto Nishino, Aoi Kuroda, Tetsuo Tani, Daisuke Arai, Kota Ishioka, Ichiro Kawada, Katsuhiko Naoki, Tomoko Betsuyaku, Kenzo Soejima. Hyperoxia may be a treatment option for NSCLC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3006. doi:10.1158/1538-7445.AM2015-3006

Abstract 5195: Claudin-1, a novel target of miR-375 in non-small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA MicroRNAs, the small and noncoding RNAs, regulate the translation of specific protein-coding genes. Accumulated evidence strongly suggests that microRNAs play important and complex roles in human cancers, including lung cancer. We previously reported that miR-375 expression was low in squamous cell carcinoma (SCC) and high in adenocarcinoma (AC) of lung cancer. The target gene of miR-375 in non-small cell lung cancer (NSCLC) has not been elucidated. The purpose of this study was to identify a target of miR-375 and clarify the function of miR-375 in NSCLC. Candidate genes of miR-375 targets were determined using the prediction databases and also previous findings about the different gene expression between SCC and AC. We focused on claudin-1 (CLDN1), which has four putative target sites of miR-375 in its 3′-untranslated region (UTR). CLDN1 was reported to express high in SCC and low in AC opposite to miR-375. We evaluated miR-375 and CLDN1 expression levels by quantitative polymerase chain reaction (qPCR) and Western blotting in 12 NSCLC cell lines. The effect of miR-375 overexpression upon the CLDN1 expression was evaluated in 5 NSCLC cell lines by transfecting miR-375 precursor. It showed that the expression of CLDN1 messenger RNA and protein were attenuated by miR-375 overexpression. Luciferase reporter assay was performed to confirm direct interaction between miR-375 and CLDN1. We cloned 3′-UTR of CLDN1 cDNA into the downstream of a luciferase reporter gene and co-transfected this vector into A549 cells with miR-375 precursor. MiR-375 overexpression resulted in a 3-fold repression of luciferase activity (p < 0.001). To ascertain the clinical validity, we analyzed the relationship between miR-375 and CLDN1 expression in 63 clinical samples of NSCLC. There was a negative correlation between miR-375 and CLDN1 expression (r = -0.35, p = 0.005). In addition, we analyzed the correlation between miR-375 expression and overall survival in the same samples. High miR-375 expression correlated with poor survival in NSCLC (p = 0.043). To investigate the reason why high miR-375 expression lead to poor survival, wound healing assay was performed to evaluate the effect of miR-375 overexpression on the cell migration in SK-MES-1 cells. The cell migration was promoted by miR-375 overexpression, suggesting the high potential of invasion and metastasis in NSCLC expressing high level of miR375. In conclusion, we found that CLDN1 is a novel target of miR-375, and high miR-375 expression leads to poor survival in NSCLC. Citation Format: Satoshi Yoda, Kenzo Soejima, Hiroyuki Yasuda, Takashi Sato, Daisuke Arai, Keiko Ohgino, Kota Ishioka, Tetsuo Tani, Ayano Oashi, Aoi Kuroda, Makoto Nishino, Masayoshi Miyawaki, Junko Hamamoto, Katsuhiko Naoki, Tomoko Betsuyaku. Claudin-1, a novel target of miR-375 in non-small cell lung cancer. [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 5195. doi:10.1158/1538-7445.AM2014-5195

Abstract 414: Aberrant DNA methylation and expression of mRNA in EGFR-mutant lung cancer cell line with long-term exposure to gefitinib

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Many of the patients with non-small cell lung cancer (NSCLC) who initially responded well to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) will eventually relapse. The mechanisms of resistance to EGFR-TKIs is not yet fully clarified. The suppression of genes by DNA methylation is reported to be involved in the mechanism of tolerance to cytotoxic drugs. The purpose of this study is to identify epigenetically regulated genes and to clarify the contribution of epigenetic alteration to the acquired resistance to EGFR-TKI. We established gefitinib-resistant PC-9, which was originally gefitinib-sensitive lung cancer cell line, by serial long term exposure to gefitinib. We collected RNA and DNA from both gefitinib-sensitive and -resistant PC-9 cells and performed comprehensive analysis for DNA methylation and mRNA expression using infinium array and cDNA microarray, respectively. We identified 640 genes those DNA methylations were increased in gefitinib-resistant cell line compared to parental cell line. Then, we selected 29 candidate genes those mRNA expression were decreased in resistant PC-9. We are currently underway to elucidate the specific function of each gene and updated data will be presented at the meeting. Citation Format: Hideki Terai, Kenzo Soejima, Katsuhiko Naoki, Hiroyuki Yasuda, Takashi Sato, Daisuke Arai, Keiko Ohgino, Kota Ishioka, Aoi Kuroda, Tetsuo Tani, Ayano Ohashi, Makoto Nishino, Masayoshi Miyawaki, Junko Hamamoto, Tomoko Betsuyaku. Aberrant DNA methylation and expression of mRNA in EGFR-mutant lung cancer cell line with long-term exposure to gefitinib. [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 414. doi:10.1158/1538-7445.AM2014-414

Abstract 5652: Activation of FGF2-FGFR1 pathway in EGFR-mutant lung cancer cell line with long-term gefitinib exposure.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Many of the patients with non-small cell lung cancer (NSCLC) with sensitive epidermal growth factor receptor (EGFR)-mutation who initially responded well to EGFR-tyrosine kinase inhibitors (TKIs) eventually relapse. In spite of many studies over the last few years to elucidate this mechanism of acquired resistance to EGFR-TKIs, approximately 30% of the mechanisms of acquired resistance are still unknown. Recently autocrine signaling of fibroblast growth factors (FGFs) and their receptors (FGFRs) has been demonstrated in NSCLC cell lines. And several studies suggest that the FGF-FGFR autocrine growth pathway could be an important mechanism for intrinsic resistance to EGFR-TKIs in NSCLC cell lines with wild-type EGFR. But until now, no report has clarified the role of FGF-FGFR pathway in acquired resistance to EGFR-TKIs in NSCLC cell lines with sensitive EGFR mutations. We have established a gefitinib-resistant cell line (PC9 GR), by serial exposure of gefitinib to PC9, an originally gefitinib-sensitive lung cancer cell line (PC9 na). We confirmed that these cell lines did not harbor two well-known EGFR-TKI resistance mechanisms, the second mutation in the EGFR gene itself, EGFR T790 and the amplification of the MET oncogene. We collected total RNA from both PC9 na and PC9 GR and examined mRNA expression profile, by using cDNA microarray analysis. We found the expressions of FGFR1 and FGF2 were increased in PC9 GR compared to in PC9 na. The growth of PC9 GR cells was inhibited either by PD173074 (inhibitors of FGFRs) or knocking down of FGFR1 or FGF2 by siRNA in combination with gefitinib. FACS analysis revealed that the combination treatment with PD173074 and gefitinib induced apoptosis more efficiently in PC9 GR cells compared to gefitinib alone. PC9 na cells and PC9 GR cells did not show any change in the proportion of apoptotic cells after treatment with PD173074 alone. To further investigate how FGF2-FGFR1 pathway affects resistance to gefitinib in these cell lines, the downstream targets of EGFR signaling including the MEK-ERK and PI3K-AKT pathways were examined. In PC9 na cells, the phosphorylation of EGFR, ERK, and AKT was efficiently inhibited by gefitinib alone. On the other hand, in PC9 GR cells, the phosphorylation of ERK and AKT was not efficiently inhibited by gefitinib alone. However, the inhibition of phosphorylation of ERK was completely and AKT was less efficiently rescued by gefitinib and PD173074 combination therapy. In conclusion, these data suggest the activation of FGF2-FGFR1 signaling pathway contributes to the gefitinib resistance in PC9 GR. FGF2-FGFR1 pathway will be a therapeutic target for a subset of NSCLC that acquires EGFR-TKI resistance. Citation Format: Hideki Terai, Kenzo Soejima, Katsuhiko Naoki, Hiroyuki Yasuda, Ryosuke Satomi, Sohei Nakayama, Satoshi Yoda, Shinnosuke Ikemura, Takashi Sato, Kota Ishioka, Daisuke Arai, Keiko Ohgino, Tetsuo Tani, Aoi Kuroda, Junko Hamamoto, Tomoko Betsuyaku. Activation of FGF2-FGFR1 pathway in EGFR-mutant lung cancer cell line with long-term gefitinib exposure. [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 5652. doi:10.1158/1538-7445.AM2013-5652