Ichiro Kawada

Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

BackgroundAlterations in the processing of the genetic information in carcinogenesis result from stable genetic mutations or epigenetic modifications. It is becoming clear that nucleosomal histones are central to proper gene expression and that aberrant DNA methylation of genes and histone methylation plays important roles in tumor progression. To date, several histone lysine methyltransferases (HKMTs) have been identified and histone lysine methylation is now considered to be a critical regulator of transcription. However, still relatively little is known about the role of HKMTs in tumorigenesis.ResultsWe observed differential HKMT expression in a lung cancer model in which normal human bronchial epithelial (NHBE) cells expressing telomerase, SV40 large T antigen, and Ras were immortal, formed colonies in soft agar, and expressed specific HKMTs for H3 lysine 9 and 27 residues but not for H3 lysine 4 residue. Modifications in the H3 tails affect the binding of proteins to the histone tails and regulate protein function and the position of lysine methylation marks a gene to be either activated or repressed. In the present study, suppression by siRNA of HKMTs (EZH2, G9A, SETDB1 and SUV39H1) that are over-expressed in immortalized and transformed cells lead to reduced cell proliferation and much less anchorage-independent colony growth. We also found that the suppression of H3-K9, G9A and SUV39H1 induced apoptosis and the suppression of H3-K27, EZH2 caused G1 arrest.ConclusionOur results indicate the potential of these HKMTs in addition to the other targets for epigenetics such as DNMTs and HDACs to be interesting therapeutic targets.

An alternative method for screening EGFR mutation using RFLP in non-small cell lung cancer patients

Introduction: Epidermal growth factor receptor (EGFR) mutations are strong determinants of tumor response to EGFR tyrosine kinase inhibitors in non-small cell lung cancers (NSCLCs). Currently available methods of EGFR mutation detection rely on direct sequencing. Here, we describe the use of an alternative way to screen EGFR mutations. Methods: A total of 109 frozen tumor specimens from NSCLC patients were obtained. For mutational analysis of EGFR exons 18, 19, and 21, reverse transcription-polymerase chain reaction was performed on the cDNA using original primers designed for restriction fragment length polymorphism (RFLP). Results: EGFR mutations were detected in 37 patients (34%) by both RFLP and direct sequencing except one case in which it was detected only by RFLP. EGFR mutations were more frequently observed to be significant by multivariate analysis in patients with adenocarcinoma (OR = 5.56), no-smoking history (OR = 4.34), and 65-year-old or younger (OR = 2.64), but not in women (OR = 1.14). Among 37 patients, 18 were treated with gefitinib and 9 responded to the treatment. One patient without any mutation responded. Conclusion: RFLP is a useful method for screening EGFR mutations and can also be applied to predicting the sensitivity of NSCLC patients to EGFR-tyrosine kinase inhibitors.

The Combination of Multiple Receptor Tyrosine Kinase Inhibitor and Mammalian Target of Rapamycin Inhibitor Overcomes Erlotinib Resistance in Lung Cancer Cell Lines through c-Met Inhibition

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) show antitumor activity in a subset of non–small cell lung cancer (NSCLC) patients. However, the initial tumor response is followed by recurrence. Several studies have suggested the importance of other receptor tyrosine kinases (RTK) and downstream kinases as potential targets in the treatment of NSCLC. We used the multiple-RTK inhibitor AEE788, which inhibits EGFR, vascular endothelial growth factor receptor, and human epidermal growth factor receptor 2, with and without the downstream kinase inhibitor RAD001 (an inhibitor of mammalian target of rapamycin). AEE788 inhibited cell growth more effectively than did erlotinib in three NSCLC cell lines examined (A549, H1650, and H1975). However, in the EGFR-TKI–resistant cell line H1975 harboring T790M resistance mutation, cell growth inhibition by AEE788 was only mild, and the phosphorylation of its leading targets such as EGFR and vascular endothelial growth factor receptor 2 was not inhibited. In H1975, AEE788 induced significantly greater cell growth inhibition when combined with RAD001 than when used alone. This cooperative effect was not seen with the combination of erlotinib and RAD001. We found that c-Met was highly phosphorylated in this cell line, and the phosphorylated c-Met was inhibited effectively by AEE788. Using a phospho-RTK array, the phosphorylation of c-Met and insulin-like growth factor-I receptor was inhibited by AEE788. These results suggest that upstream RTK inhibitor overcomes the acquired resistance to EGFR-TKI when combined with downstream kinase inhibitor. Thus, the combined inhibition of upstream and downstream RTKs is a promising strategy for the treatment of NSCLC. Mol Cancer Res; 8(8); 1142–51. ©2010 AACR.

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.

Bronchoscopic Microsampling is a Useful Complementary Diagnostic Tool for Detecting Lung Cancer

PURPOSE Bronchoscopic microsampling (BMS) is a novel and direct method with which to obtain epithelial lining fluid (ELF) from the lungs. Analysis of DNA hypermethylation of tumor suppressor genes (TSGs) is expected to be a sensitive tool for the early detection of lung cancer. It has been reported that the existence of EGFR mutations and EML4-ALK gene rearrangements are related to the sensitivity of corresponding kinase inhibitors. We aimed to evaluate the suitability of ELF as a sample for analyzing molecular changes specific for lung cancer. PATIENTS AND METHODS We collected ELF from 61 lung cancer patients by BMS from the airway close to the peripheral lung nodule and purified the nucleic acids. We performed methylation specific PCR in each ELF as well as matched serum and tumor tissue for TSGs for DNA methylation analysis. We also examined EGFR mutations and EML4-ALK rearrangement. RESULTS The sensitivity for detecting DNA hypermethylation in ELF vs serum was 74.1% vs 18.5%. We found 60.1% of patients had at least one hypermethylation in ELF, while only 27.9% had it in serum. Of note, DNA hypermethylation was detected even in stage I patients (60.0%) and the detection rate was almost the same level in each stage. We also found the sensitivity for detecting EGFR mutation in ELF vs serum was 58.3% vs 8.3%. We detected an EML4-ALK fusion gene using ELF in one patient. CONCLUSIONS BMS is an alternative method to detect cancer specific genetic and epigenetic alterations and will be a useful complementary diagnostic tool for lung cancer. SUMMARY Investigation of genetic and epigenetic changes associated with lung cancer has clinical importance for its diagnosis and management. The clinical usefulness of bronchoscopic microsampling (BMS) in lung cancer has not yet been evaluated. This study demonstrates that BMS could be useful for detecting lung cancer specific molecular changes and valuable for early diagnosis and determination of treatment options for lung cancer.

Amplification of EGFR Wild-Type Alleles in Non–Small Cell Lung Cancer Cells Confers Acquired Resistance to Mutation-Selective EGFR Tyrosine Kinase Inhibitors

EGFR-mutated lung cancers account for a significant subgroup of non-small cell lung cancers overall. Third-generation EGFR tyrosine kinase inhibitors (TKI) are mutation-selective inhibitors with minimal effects on wild-type EGFR. Acquired resistance develops to these agents, however, the mechanisms are as yet uncharacterized. In this study, we report that the Src-AKT pathway contributes to acquired resistance to these TKI. In addition, amplification of EGFR wild-type alleles but not mutant alleles was sufficient to confer acquired resistance. These findings underscore the importance of signals from wild-type EGFR alleles in acquiring resistance to mutant-selective EGFR-TKI. Our data provide evidence of wild-type allele-mediated resistance, a novel concept of acquired resistance in response to mutation-selective inhibitor therapy in cancer treatment. Cancer Res; 77(8); 2078-89. ©2017 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.

Unexpected recalcitrant course of drug-induced erythema multiforme-like eruption and interstitial pneumonia sequentially occurring after nivolumab therapy

Vemurafenib improves survival of melanoma patients. However, cutaneous side‐effects commonly occur in them. Nivolumab and ipilimumab are monoclonal antibodies against programmed death 1 and cytotoxic T‐lymphocyte‐associated antigen 4, both of which regulate excessive T‐cell activation. Although these agents induce antitumor immunity against melanoma, the modified immune condition may result in an unexpected adverse reaction which has not been observed previously. Herein, we report a case who manifested severe erythema multiforme‐like eruption with mucosal involvement associated with vemurafenib following nivolumab. The patient also subsequently suffered from ipilimumab‐induced interstitial pneumonia with refractory course. Such a case has never been reported. This case suggested that dermatologists should pay special attention to unexpected adverse events of these drugs, and carefully observe cutaneous and respiratory status of patients during the treatment of melanoma.

Dry pleurisy complicating solitary pulmonary nodules caused by Mycobacterium avium: a case report

IntroductionMycobacterium avium complex (MAC) lung disease presenting as a solitary pulmonary nodule (MAC-SPN) is often asymptomatic, is more common in middle to old age, and mimics lung cancer or tuberculoma. We report herein a case of MAC-SPN in an immunocompetent young adult patient, presenting with persistent chest pain and a subacutely progressive nodule with high intense 18F-fluorodeoxyglucose uptake. Histological examination of resected specimens revealed pleurisy, which is a rare finding of MAC-SPN.Case presentationA 36-year-old Japanese male presented with chest pain and a subacutely progressive pulmonary nodule. Positron emission tomography-computed tomography showed high intense 18F-fluorodeoxyglucose uptake in the nodule. Owing to his continuous chest pain and subacutely progressive nodules, wedge resection was performed using video-assisted thoracoscopic surgery. Histological examination revealed an epithelioid granuloma and pleurisy, and the lung tissue culture was positive for mycobacteria identified as M. avium.ConclusionThis is the first report of MAC-SPN occurring with persistent chest pain, suggesting that MAC should be considered in the differential diagnosis of a solitary pulmonary nodule, even for patients who experience persistent chest pain. As in the present case, surgical resection with video-assisted thoracoscopic surgery is a reasonable approach to the diagnosis and treatment of MAC-SPN with possible malignancy, especially as MAC can be diagnosed using resected lung tissue culture with histological confirmation.