Shigenari Nukaga

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.

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.

Blue-Black Trachea as a Result of Minocycline-induced Hyperpigmentation

A 61-year-old woman presented with a blue–black discoloration of the skin, ears, and nails that had slowly progressed over the last three years. Five years previously, she was diagnosed with Mycobacterium abscessus pulmonary disease, and she had been receiving long-term multiple antimicrobial therapies, including minocycline (100 mg, twice a day). On admission, a physical examination indicated blue–black hyperpigmentation of the nail beds, gingiva, teeth, and back portion of the pinna of the ears (Figure 1). Bronchoscopy was performed to evaluate disease activity, which indicated the change in the color of visible tracheal cartilage to blue–black (Figure 2A indicates condition 5 yr previously, Figure 2B indicates current condition). Minocycline administration was discontinued, as the disease was presumed to be stable. The hyperpigmentation improved after 6 months. To our knowledge, this is the first report describing the hyperpigmentation of tracheal cartilages because of minocycline administration. Various etiologies have been reported for hyperpigmentation of the airway, including congenital disease, inborn errors of

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

Pharmacological and Structural Characterizations of Naquotinib, a Novel Third-Generation EGFR Tyrosine Kinase Inhibitor, in EGFR-Mutated Non–Small Cell Lung Cancer

Multiple epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI) have been developed to effectively inhibit EGFR-derived signals in non–small cell lung cancer (NSCLC). In this study, we assessed the efficacy of EGFR-TKIs, including a novel third-generation inhibitor naquotinib (ASP8273), in clinically relevant EGFR mutations, including L858R, exon 19 deletion, L858R+T790M, exon 19 deletion+T790M with or without a C797S mutation, and several exon 20 insertion mutations. Using structural analyses, we also elucidated the mechanism of activation and sensitivity/resistance to EGFR-TKIs in EGFR exon 20 insertion mutations. The efficacy of naquotinib in cells with L858R, exon 19 deletion and exon 19 deletion+T790M was comparable with that of osimertinib. Interestingly, naquotinib was more potent than osimertinib for L858R+T790M. Additionally, naquotinib and osimertinib had comparable efficacy and a wide therapeutic window for cells with EGFR exon 20 insertions. Structural modeling partly elucidated the mechanism of activation and sensitivity/resistance to EGFR-TKIs in two EGFR exon 20 insertion mutants, A767_V769dupASV and Y764_V765insHH. In summary, we have characterized the efficacy of EGFR-TKIs for NSCLC using in vitro and structural analyses and suggested the mechanism of activation and resistance to EGFR-TKIs of EGFR exon 20 insertion mutations. Our findings should guide the selection of appropriate EGFR-TKIs for the treatment of NSCLC with EGFR mutations and help clarify the biology of EGFR exon 20 insertion mutations. Mol Cancer Ther; 17(4); 740–50. ©2018 AACR.

Secondary brain neoplasm after stereotactic radiosurgery in patients with metastatic non-small cell lung cancer

Stereotactic radiosurgery (SRS) using the Gamma Knife (GK) is now being increasingly utilized for the treatment of brain metastases. However, there are a few reported cases of SRS-induced brain neoplasms. We herein report the case of a Japanese woman with metastatic non-small cell lung cancer (NSCLC) harboring epidermal growth factor (EGFR)-mutations who was treated four times with a GK for brain metastases. She developed glioblastoma 5.7 years after the initial GK surgery. Radiation-induced secondary neoplasms generally appear after a latency period of several years. Advances in cancer therapy have improved the survival of patients with NSCLC, providing enough time for secondary neoplasms to appear after SRS.

Characterization of the efficacies of osimertinib and nazartinib against cells expressing clinically relevant epidermal growth factor receptor mutations

Third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) were developed to overcome EGFR T790M-mediated resistance to first- and second-generation EGFR-TKIs. Third-generation EGFR-TKIs, such as osimertinib and nazartinib, are effective for patients with the EGFR T790M mutation. However, there are no direct comparison data to guide the selection of a third-generation EGFR-TKI for patients with different EGFR mutations. We previously established an in vitro model to estimate the therapeutic windows of EGFR-TKIs by comparing their relative efficacies against cells expressing mutant or wild type EGFRs. The present study used this approach to characterize the efficacy of third-generation EGFR-TKIs and compare them with that of other EGFR-TKIs. Treatment efficacy was examined using human lung cancer-derived cell lines and Ba/F3 cells, which were transduced with clinically relevant mutant EGFRs. Interestingly, mutation-related differences in EGFR-TKI sensitivity were observed. For classic EGFR mutations (exon 19 deletion and L858R, with or without T790M), osimertinib showed lower IC50 values and wider therapeutic windows than nazartinib. For less common EGFR mutations (G719S or L861Q), afatinib showed the lowest IC50 values. For G719S+T790M or L861Q+T790M, the IC50 values of osimertinib and nazartinib were around 100 nM, which was 10- to 100-fold higher than those for classic+T790M mutations. On the contrary, osimertinib and nazartinib showed similar efficacies in cells expressing EGFR exon 20 insertions. The findings highlight the diverse mutation-related sensitivity pattern of EGFR-TKIs. These data may help in the selection of EGFR-TKIs for non-small cell lung cancer patients harboring EGFR mutations.

Non-small cell lung cancer PC-9 cells exhibit increased sensitivity to gemcitabine and vinorelbine upon acquiring resistance to EGFR-tyrosine kinase inhibitors

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (EGFR-TKIs) are widely used for the treatment of non-small cell lung cancers (NSCLCs) harboring EGFR-activating mutations. However, lung cancer cells inevitably acquire resistance to these EGFR-TKIs. The majority of patients whose lung cancer acquires resistance to EGFR-TKIs are subjected to treatment using cytotoxic agents. The present study aimed to determine if lung cancer cells acquiring resistance to EGFR-TKIs also develop altered sensitivity to cytotoxic agents. It was revealed that lung cancer cells that had developed resistance to EGFR-TKIs had increased sensitivity to gemcitabine and vinorelbine compared with EGFR-TKI naïve cells. The expression levels of ATP-binding cassette (ABC) transporter genes, including ABCC3, ABCC5 and ABCG2, were observed to be commonly repressed in EGFR-TKI-resistant cells. In addition, two cases were identified in which gemcitabine and vinorelbine exerted marked responses to lung cancers that had acquired resistance to EGFR-TKIs, even with late-line treatment. Therefore, it was proposed that gemcitabine and vinorelbine may be effective agents for patients with lung cancer previously treated with EGFR-TKIs.

Abstract 2099: In vitro characterization of the effect of nazartinib against non-small cell lung cancer activating clinically relevant EGFR mutants

Purpose: Multiple EGFR-TKIs are available and under development to treat patients with lung cancer harboring EGFR mutations. Nazartinib is one of the 3rd generation EGFR-TKIs targeting EGFR T790M as well as common mutations such as L858R and exon 19 deletions. Today, there is no clear guideline regarding which EGFR-TKIs should be used for which mutations. The purpose of this study is to clarify which EGFR-TKIs including nazartinib are best for each EGFR mutation, especially exon 20 insertion mutations using in vitro modeling. Experimental design: We evaluated drug sensitivity and downstream signaling of human lung cancer cell lines (PC9, H3255, H1975, PC9ER, BID007) and Ba/F3 cells harboring multiple types of EGFR mutations for 1st- (erlotinib), 2nd- (afatinib) and 3rd- (osimertinib and nazartinib) generation EGFR-TKIs by MTS assay and western blotting. An in vitro model of mutation specificity was created by calculating the ratio of IC50 values between mutated and wild type EGFRs. Results: The model of mutation specificity identified a wide therapeutic window of each EGFR-TKIs for exon 19 deletions and L858R. On the other hand, osimertinib and nazartinib have wide therapeutic window for T790M positive mutations in human cell lines and Ba/F3 cells. In human cell lines and Ba/F3 cells harboring exon 19 deletions or L858R, afatinib dramatically inhibited the phosphorylation of EGFR, AKT, and ERK. Afatinib and 3rd generation EGFR-TKIs, osimertinib and nazartinib, effectively inhibited the phosphorylation of EGFR, AKT, and ERK in T790M positive cells. For EGFR exon 20 insertion mutations, althogh afatinib, osimertinib and nazartinib effectively inhibited the phosphorylation of EGFR, AKT, and ERK in several exon 20 insertion mutations, osimertinib and nazartinib were potent and presented a wide therapeutic window. Conclusion: Nazartinib as well as osimertinib has wide therapeutic windows for exon 19 deletions, L858R, T790M and some exon 20 insertions . Citation Format: Keita Masuzawa, Hiroyuki Yasuda, Toshiyuki Hirano, Shigenari Nukaga, Hanako Hasegawa, Junko Hamamoto, Katsuhiko Naoki, Kenzo Soejima, Tomoko Betsuyaku. In vitro characterization of the effect of nazartinib against non-small cell lung cancer activating clinically relevant EGFR mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2099. doi:10.1158/1538-7445.AM2017-2099