Tatsushi Kodama

CH5424802, a Selective ALK Inhibitor Capable of Blocking the Resistant Gatekeeper Mutant

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers, following gene alterations such as chromosomal translocation, amplification, or point mutation. Here, we identified CH5424802, a potent, selective, and orally available ALK inhibitor with a unique chemical scaffold, showing preferential antitumor activity against cancers with gene alterations of ALK, such as nonsmall cell lung cancer (NSCLC) cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion in vitro and in vivo. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven cell growth. Our results support the potential for clinical evaluation of CH5424802 for the treatment of patients with ALK-driven tumors.

Antitumor activity of the selective ALK inhibitor alectinib in models of intracranial metastases

PurposeThe clinical efficacy of the anaplastic lymphoma kinase (ALK) inhibitor crizotinib has been demonstrated in ALK fusion-positive non-small cell lung cancer (NSCLC); however, brain metastases are frequent sites of initial failure in patients due to poor penetration of the central nervous system by crizotinib. Here, we examined the efficacy of a selective ALK inhibitor alectinib/CH5424802 in preclinical models of intracranial tumors.MethodsWe established intracranial tumor implantation mouse models of EML4–ALK-positive NSCLC NCI-H2228 and examined the antitumor activity of alectinib in this model. Plasma distribution and brain distribution of alectinib were examined by quantitative whole-body autoradiography administrating a single oral dose of 14C-labeled alectinib to rats. The drug permeability of alectinib was evaluated in Caco-2 cell.ResultsAlectinib resulted in regression of NCI-H2228 tumor in mouse brain and provided a survival benefit. In a pharmacokinetic study using rats, alectinib showed a high brain-to-plasma ratio, and in an in vitro drug permeability study using Caco-2 cells, alectinib was not transported by P-glycoprotein efflux transporter that is a key factor in blood–brain barrier penetration.ConclusionsWe established intracranial tumor implantation models of EML4–ALK-positive NSCLC. Alectinib showed potent efficacy against intracranial EML4–ALK-positive tumor. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with brain metastases.

Selective ALK inhibitor alectinib with potent antitumor activity in models of crizotinib resistance

The clinical efficacy of the ALK inhibitor crizotinib has been demonstrated in ALK fusion-positive NSCLC; however, resistance to crizotinib certainly occurs through ALK secondary mutations in clinical use. Here we examined the efficacy of a selective ALK inhibitor alectinib/CH5424802 in models of crizotinib resistance. Alectinib led to tumor size reduction in EML4-ALK-positive xenograft tumors that failed to regress fully during the treatment with crizotinib. In addition, alectinib inhibited the growth of some EML4-ALK mutant-driven tumors, including the G1269A model. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with ALK secondary mutations.

Alectinib Shows Potent Antitumor Activity against RET-Rearranged Non–Small Cell Lung Cancer

Alectinib/CH5424802 is a known inhibitor of anaplastic lymphoma kinase (ALK) and is being evaluated in clinical trials for the treatment of ALK fusion–positive non–small cell lung cancer (NSCLC). Recently, some RET and ROS1 fusion genes have been implicated as driver oncogenes in NSCLC and have become molecular targets for antitumor agents. This study aims to explore additional target indications of alectinib by testing its ability to inhibit the activity of kinases other than ALK. We newly verified that alectinib inhibited RET kinase activity and the growth of RET fusion–positive cells by suppressing RET phosphorylation. In contrast, alectinib hardly inhibited ROS1 kinase activity unlike other ALK/ROS1 inhibitors such as crizotinib and LDK378. It also showed antitumor activity in mouse models of tumors driven by the RET fusion. In addition, alectinib showed kinase inhibitory activity against RET gatekeeper mutations (RET V804L and V804M) and blocked cell growth driven by the KIF5B-RET V804L and V804M. Our results suggest that alectinib is effective against RET fusion–positive tumors. Thus, alectinib might be a therapeutic option for patients with RET fusion–positive NSCLC. Mol Cancer Ther; 13(12); 2910–8. ©2014 AACR.

A Novel Mechanism of EML4-ALK Rearrangement Mediated by Chromothripsis in a Patient-Derived Cell Line

Introduction: EML4-ALK is a driver oncogene in non–small-cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of this gene fusion remain unknown. This study aimed to examine the mechanism of EML4-ALK rearrangement using a novel cell line with the EML4-ALK fusion gene. Methods: An EML4-ALK-positive cell line, termed JFCR-LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization and comparative genomic hybridization (CGH). Alectinib/CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against JFCR-LC649 in vitro and in vivo xenograft model. Results: We established an EML4-ALK-positive cell line, termed JFCR-LC649, derived from a patient with NSCLC and revealed that the JFCR-LC649 cells harbor variant 3 of the EML4-ALK fusion with twofold copy number gain. Interestingly, comparative genomic hybridization and metaphase-fluorescence in situ hybridization analysis showed that in addition to two normal chromosome 2, JFCR-LC649 cells contained two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in JFCR-LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed chromothripsis. Furthermore, a selective ALK inhibitor alectinib/CH5424802 suppressed tumor growth of the JFCR-LC649 cells through inhibition of phospho-ALK in vitro and in vivo in a xenograft model. Conclusion: Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. In addition, alectinib was effective against EML4-ALK-positive tumors with ALK copy number gain mediated by chromothripsis.

Abstract 754: Selective ALK inhibitor alectinib (CH5424802/RO5424802) with potent antitumor activity in models of crizotinib resistance, including intracranial metastases

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: EML4-ALK has been implicated as a driver oncogene and a therapeutic target in non-small cell lung cancer (NSCLC). The clinical efficacy of the ALK inhibitor crizotinib has been demonstrated in ALK fusion-positive NSCLC; however, resistance to crizotinib certainly occurs through ALK secondary mutations in clinical use. In addition, brain metastases are frequent sites of initial failure in patients because crizotinib has poor penetration of the central nervous system. This study aimed to clarify the efficacy of ALK inhibitor alectinib (CH5424802/RO5424802) in models of crizotinib-resistant ALK mutant tumors and intracranial tumors. Experimental Design: The antitumor activity of alectinib was evaluated in subcutaneous xenograft tumor models of Ba/F3 cells expressing mutated EML4-ALK, and in intracranial tumor implantation models of EML4-ALK-positive NSCLC NCI-H2228. Results: Alectinib had kinase inhibitory activity against some ALK mutations, including G1269A, and blocked tumor growth driven by a mutant of EML4-ALK in vitro and in vivo. In intracranial tumor implantation mouse models, alectinib resulted in regression of NCI-H2228 tumor in brain and provided a survival benefit. Alectinib is not a substrate for P-glycoprotein efflux transporter in vitro and showed a high brain-to-plasma ratio in rat. Additionally, alectinib led to tumor size reduction in the subcutaneous NCI-H2228 xenograft tumor that had failed to regress fully during treatment with crizotinib. Conclusions: Alectinib was effective against most EML4-ALK mutations and showed potent efficacy against intracranial ALK-positive tumor. Thus, alectinib might provide therapeutic opportunities for crizotinib-treated patients with ALK secondary mutations and brain metastases. Citation Format: Tatsushi Kodama, Toshiyuki Tsukaguchi, Masami Hasegawa, Miyuki Yoshida, Kenji Takanashi, Osamu Kondoh, Hiroshi Sakamoto. Selective ALK inhibitor alectinib (CH5424802/RO5424802) with potent antitumor activity in models of crizotinib resistance, including intracranial metastases. [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 754. doi:10.1158/1538-7445.AM2014-754

Abstract 773: Alectinib shows potent antitumor activity against both ALK- and RET-rearranged non-small cell lung cancers

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We have shown that alectinib is a potent ALK inhibitor and is being evaluated in clinical trials for the treatment of ALK fusion-positive non-small cell lung cancer (NSCLC). Recently, RET and ROS1 fusion genes have been implicated as driver oncogenes in 1-2% of NSCLC and have been developed into promising molecular targets for antitumor agents. Here, we investigate the additional target indication of alectinib by testing its ability to inhibit the activity of RET and ROS1 kinases. In enzyme assay, alectinib showed kinase inhibitory activity against RET as wells as ALK but did not show against ROS1. Alectinib inhibited the growth of LC-2/ad cells harboring CCDC6-RET and Ba/F3 cells expressing KIF5B-RET by suppressing RET phosphorylation. Alectinib also showed antitumor activity in mouse models of RET fusion-positive tumors (LC-2/ad cells and Ba/F3 cells expressing KIF5B-RET) and of ALK fusion-positive tumors (NCI-H2228 cells harboring EML4-ALK). In addition, alectinib showed kinase inhibitory activity against RET gatekeeper mutations (RET V804L and V804M) and blocked KIF5B-RET gatekeeper mutation-driven cell growth. Our results suggest that alectinib is effective against RET fusion-positive tumors, as observed in ALK fusion-positive tumors. Thus, alectinib might be a therapeutic option for patients with RET fusion-positive NSCLC. Citation Format: Tatsushi Kodama, Toshiyuki Tsukaguchi, Yasuko Satoh, Miyuki Yoshida, Yoshiaki Watanabe, Osamu Kondoh, Hiroshi Sakamoto. Alectinib shows potent antitumor activity against both ALK- and RET-rearranged non-small cell lung cancers. [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 773. doi:10.1158/1538-7445.AM2015-773

Abstract 3853: Unique pattern of copy number changes including chromothripsis in pulmonary adenocarcinoma with EML4-ALK fusion

Background: A subset of lung adenocarcinomas harbors an ALK fusion gene resulting in dominant oncogenic activity. Gene inversion may play a main role in forming the fusion, not producing copy number changes. But its detailed mechanism leading to gene fusion remains unsolved. MATERIAL AND METHODS: To elucidate the underlying mechanisms resulting in gene fusion, two cancer cell lines positive for EML4-ALK fusion and surgically resected frozen specimens (33 ALK fusion-positive and 95 ALK fusion-negative adenocarcinomas) were analyzed using Affymetrix CytoScan HD Array with Chromosome Analysis Suite (ChAS) software for cell lines (JFCR-LC649 and JFCR-LC654) and Affymetrix GeneChip Mapping 250K arrays for surgical specimens. RESULTS: One (JFCR-LC649) of the two cell lines with gene fusion showed an extremely frequent change of copy number, or “copy number oscillation”, which we thought was chromothripsis. In surgical materials, 30% (10/33) of adenocarcinomas with ALK fusion harbored frequent copy number changes at various degrees, but not like the “oscillation” pattern. None of the 95 resected cases without ALK fusion showed similar genomic aberrations. These results from the JFCR-LC649 line and the 10 surgical cases indicated that genomic changes resulting in copy number alterations (including chromothripsis) as well as a simple inversion may cause gene fusion. CONCLUSION: As well as genome inversion, copy number alterations such as chromothripsis contribute to gene rearrangements resulting in the EML4-ALK fusion. Citation Format: Hironori Ninomiya, Motohiro Kato, Seishi Ogawa, Noriko Motoi, Kengo Takeuchi, Tatsushi Kodama, Hiroshi Sakamoto, Nobuya Ishii, Mutsunori Fujiwara, Yuichi Ishikawa. Unique pattern of copy number changes including chromothripsis in pulmonary adenocarcinoma with EML4-ALK fusion. [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 3853. doi:10.1158/1538-7445.AM2015-3853

Abstract 2192: A novel mechanism of EML4-ALK rearrangement in a patient-derived cell line and its tumor growth inhibition by an ALK inhibitor CH5424802.

Purpose: EML4-ALK is a driver oncogene in non-small cell lung cancer (NSCLC) and has been developed into a promising molecular target for antitumor agents. Although EML4-ALK is reported to be formed by inversion of chromosome 2, other mechanisms of gene fusion remain unknown. Here we characterize the EML4-ALK fusion gene of a new cell line derived from a patient of NSCLC. In addition, the efficacy of ALK inhibitors against this new cell line was examined. Experimental Design: An EML4-ALK-positive cell line, termed LC649, was established from pleomorphic carcinoma, a rare subtype of NSCLC. We investigated the chromosomal aberrations using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). CH5424802, a selective ALK inhibitor, was evaluated in the antitumor activity against LC649 in vitro and in vivo xenograft model. Results: Our initial characterization revealed that LC649 harbors variant 3 of EML4-ALK fusion with 2-fold copy number gain. Interestingly, CGH and metaphase-FISH analysis showed that LC649 contained, in addition to two normal chromosome 2, two aberrant chromosome 2 that were fragmented and scattered. These observations provided the first evidence that EML4-ALK fusion in LC649 cells was formed in chromosome 2 by a distinct mechanism of genomic rearrangement, termed “chromothripsis”. Additionally, CH5424802 inhibited phospho-ALK and suppressed tumor growth of the LC649 cells in vitro and in vivo in a xenograft model. Conclusions: Our results suggested that chromothripsis may be a mechanism of oncogenic rearrangement of EML4-ALK. CH5424802 was effective against EML4-ALK positive tumors with ALK copy number gain mediated by chromothripsis. Citation Format: Tatsushi Kodama, Noriko Motoi, Hironori Ninomiya, Hiroshi Sakamoto, Kunio Kitada, Toshiyuki Tsukaguchi, Yasuko Sato, Nobuya Ishii, Yasuhito Terui, Kiyohiko Hatake, Yuichi Ishikawa. A novel mechanism of EML4-ALK rearrangement in a patient-derived cell line and its tumor growth inhibition by an ALK inhibitor CH5424802. [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 2192. doi:10.1158/1538-7445.AM2013-2192

Abstract 3559: A new selective ALK inhibitor CH5424802 shows potent efficacy against ALK-positive cancers including the gatekeeper mutant-driven tumors

The recent development of targeted protein kinase inhibitors has provided new opportunity in cancer treatment. However, certain factors limit the efficacy of cancer therapies, such as a narrow therapeutic index caused by inhibition of multiple kinases, and the emergence of resistant mutants. Thus, kinase inhibitors with more potent and selective properties and effectiveness against resistant mutants need to be developed. Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in certain cancers following gene alterations such as chromosomal translocation, amplification, or point mutation. Here, we identified CH5424802, a potent, selective, and orally available ALK inhibitor with a new chemical scaffold, showing preferential antitumor activity against cancers with gene alterations of ALK, such as non-small cell lung cancer (NSCLC) cells expressing EML4-ALK fusion and anaplastic large-cell lymphoma (ALCL) cells expressing NPM-ALK fusion in vitro and in vivo. CH5424802 inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4-ALK L1196M-driven tumors. Our results support the potential for the clinical evaluation of CH54242802 for long-term treatment of patients with ALK-driven tumors. CH5424802 is currently being investigated in Phase I/II clinical trials for patients with ALK-positive NSCLC. 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 3559. doi:10.1158/1538-7445.AM2011-3559