Yasuko Satoh

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.

Mechanism of Oncogenic Signal Activation by the Novel Fusion Kinase FGFR3-BAIAP2L1

Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding FGFR family members. However, most fusion genes are not functionally characterized, and their potentials in targeted therapy are unclear. We investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). We identified 4 patients with bladder cancer and 2 patients with lung cancer harboring the FGFR3–BAIAP2L1 fusion through PCR and FISH assay screens. To investigate the oncogenic potential of the fusion gene, we established an FGFR3–BAIAP2L1 transfectant with Rat-2 fibroblast cells (Rat-2_F3-B). The FGFR3–BAIAP2L1 fusion had transforming activity in Rat2 cells, and Rat-2_F3-B cells were highly tumorigenic in mice. Rat-2_F3-B cells showed in vitro and in vivo sensitivity in the selective FGFR inhibitor CH5183284/Debio 1347, indicating that FGFR3 kinase activity is critical for tumorigenesis. Gene signature analysis revealed that FGFR3–BAIAP2L1 activates growth signals, such as the MAPK pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. We also established Rat-2_F3-B-ΔBAR cells expressing an FGFR3–BAIAP2L1 variant lacking the Bin–Amphiphysin–Rvs (BAR) dimerization domain of BAIAP2L1, which exhibited decreased tumorigenic activity, FGFR3 phosphorylation, and F3-B-ΔBAR dimerization, compared with Rat-2_F3-B cells. Collectively, these data suggest that constitutive dimerization through the BAR domain promotes constitutive FGFR3 kinase activation and is essential for its potent oncogenic activity. Mol Cancer Ther; 14(3); 704–12. ©2015 AACR.

Combining Onartuzumab with Erlotinib Inhibits Growth of Non-Small Cell Lung Cancer with Activating EGFR Mutations and HGF Overexpression

Erlotinib, a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR-TKI), benefits survival of patients with non–small cell lung cancer (NSCLC) who harbor activating EGFR mutations. However, elevated expression of hepatocyte growth factor (HGF), a ligand of the MET receptor tyrosine kinase, causes erlotinib resistance. Because onartuzumab, a monovalent antibody to MET, blocks HGF-induced MET activation, the addition of onartuzumab to erlotinib may improve therapeutic efficacy. We engineered the human NSCLC cell line PC-9 (MET-positive cells harboring an exon 19 deletion of EGFR) to overexpress hHGF and evaluated the effects of an onartuzumab and erlotinib combination in vitro and in vivo in xenograft models. A stable clone of PC-9/hHGF was less sensitive to erlotinib than the parental PC-9, and the addition of onartuzumab to erlotinib suppressed the proliferation of these cells in vitro. In PC-9/hHGF xenograft tumors, onartuzumab or erlotinib alone minimally inhibited tumor growth; however, combining onartuzumab and erlotinib markedly suppressed tumor growth. The total MET protein level was decreased in PC-9/hHGF cells, because MET is constitutively phosphorylated by autocrine HGF, leading to its ubiquitination and degradation. Onartuzumab reduced phospho-MET levels, inhibited MET ubiquitination, and consequently restored MET protein levels. Moreover, in NSCLC clinical specimens harboring activating EGFR mutations, more than 30% of patients expressed high levels of HGF. Our findings raised the possibility that patients with NSCLC with EGFR mutations who express high levels of HGF may benefit from onartuzumab and erlotinib combination therapy, and that HGF can be a novel biomarker for selecting such patients. Mol Cancer Ther; 14(2); 533–41. ©2014 AACR.

Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor-D

Lymphatic spread is an important clinical determinant in the prognosis of many human cancers. The lymphangiogenic factor vascular endothelial growth factor‐D (VEGF‐D) is implicated in the promotion of lymphatic metastasis through the development of lymphatic vessels in some human cancers. In this study, we developed an anti‐VEGF‐D monoclonal antibody, cVE199, and investigated its in vitro properties, in vivo effects against tumors and possible target indications to evaluate its potential as a therapeutic antibody. The cVE199 molecule was revealed to have a specific binding reactivity against human VEGF‐D, as well as a specific inhibitory activity against the binding of human VEGF‐D to VEGFR‐3. In addition, cVE199 was found to inhibit the biological activity of VEGF‐D against lymphatic cells in vitro. Because we determined that a neuroblastoma cell line, SK‐N‐DZ, abundantly expressed VEGF‐D, an in vivo efficacy study was performed using a xenograft model of SK‐N‐DZ. We found that cVE199 significantly decreased lymphatic metastasis of SK‐N‐DZ as well as lymphangiogenesis in primary lesions. Finally, we investigated VEGF‐D expression in human neuroblastoma, finding that the molecule was expressed in 11 of 29 human neuroblastoma specimens (37.9%). In conclusion, we found that a novel anti‐VEGF‐D monoclonal antibody, cVE199, with specific reactivity against human VEGF‐D, prevents lymphatic metastasis of neuroblastoma through the inhibition of lymphangiogenesis in an animal model. In addition, our results show that VEGF‐D is expressed in some cases of human neuroblastomas, which suggests that cVE199 is a potential anti‐metastasis therapeutic antibody in neuroblastoma treatment.

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 2728: Onartuzumab (MetMAb) restores sensitivity to erlotinib in EGFR mutant NSCLC cells expressing HGF

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Onartuzumab (MetMAb) is a humanized monovalent monoclonal antibody that blocks binding of HGF to Met. The combination of onartuzumab + erlotinib is currently being evaluated in a Phase III clinical trial for 2nd/3rd line NSCLC (1). EGFR-activating mutations, including deletions of exon 19 and L858R point mutations, are associated with better response to erlotinib. However, EGFR mutant NSCLC tumors with high HGF expression are less responsive to EGFR small molecule inhibitors (2) (3). Since onartuzumab blocks HGF-induced Met activation, the addition of onartuzumab to erlotinib may be more beneficial than erlotinib alone in such patients. To test this hypothesis preclinically, we engineered the human NSCLC cell line PC9 (exon 19 deletion of EGFR and Met positive) to express hHGF endogenously, and evaluated combination effects of onartuzumab + erlotinib in vitro and in vivo. Methods: PC-9 cells were transfected with hHGF expression plasmids, and a stable clone was selected. For in vitro analyses, parental PC-9 cells and PC-9/hHGF cells were treated with various concentrations of erlotinib combined with 30μg/mL of onartuzumab, and cytotoxicity was examined. Additionally, the phosphorylation status of Met, EGFR, ERK and AKT was examined by western blot analysis of whole cell lysates. For in vivo analyses, PC-9/hHGF cells were inoculated subcutaneously into nude mice. After tumors reached approximately 200 mm3 in volume, onartuzumab (30 mg/kg, IP, Q3W) was administered alone or in combination with erlotinib (50 mg/kg, PO, daily) and tumor volume was measured over time. Results: One stable clone with high expression of hHGF was selected by hHGF ELISA and western blotting. This PC-9/hHGF clone was less sensitive to erlotinib compared to the parental PC-9 in vitro. Adding onartuzumab to erlotinib suppressed proliferation of these PC9/hHGF cells. pERK and pAKT levels were significantly reduced when PC-9/hHGF cells were treated with onartuzumab + erlotinib. In PC-9/hHGF xenograft tumors, onartuzumab or erlotinib alone had modest effects on tumor growth. However, combining onartuzumab and erlotinib dramatically suppressed tumor growth. Conclusions: Overexpression of hHGF decreases response of PC9 cells to erlotinib. Onartuzumab + erlotinib was more efficacious than erlotinib alone on PC9/hHGF cells in vitro and in vivo. These data support the hypothesis that addition of onartuzumab to erlotinib may enhance efficacy of erlotinib in EGFR mutant NSCLC tumors. References: (1) [NCT01456325][1] at www.clinicaltrials.gov (2) S. Yano et al. Cancer Res 68 (2008) (3) A. B. Turke et al. Cancer Cell 17 (2010) 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 2728. doi:1538-7445.AM2012-2728 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01456325&atom=%2Fcanres%2F72%2F8_Supplement%2F2728.atom

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 123: Mechanism of oncogenic signal activation by the novel fusion kinase FGFR3-BAIAP2L1

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Recent cancer genome profiling studies have identified many novel genetic alterations, including rearrangements of genes encoding fibroblast growth factor receptor (FGFR) family members. However, most fusion genes are not functionally well characterized, and the oncogenicity of some fusions as well as their potential sensitivity to targeted therapy are still unclear. In a previous study, we investigated a recently discovered gene fusion between FGFR3 and BAI1-associated protein 2-like 1 (BAIAP2L1). The FGFR3-BAIAP2L1 fusion gene was identified in 4 bladder cancer patients and 2 lung cancer patients via screens involving PCR and a break-apart fluorescence in situ hybridization assay. The functional analysis of FGFR3-BAIAP2L1 transfectant in Rat-2 fibroblast cells (Rat-2\_F3-B) indicated that FGFR3-BAIAP2L1 forms a dimer via the Bin-Amphiphysin-Rvs (BAR) BAIAP2L1 dimerization domain that constitutively activates its FGFR3 kinase activity. CH5183284/Debio 1347*, a selective FGFR inhibitor, effectively inhibits growth of Rat-2\_F3-B both in vitro and in vivo, indicating that the FGFR3 kinase activity is critical for tumorigenic activity of this fusion. These results indicate a potential application of FGFR inhibitors to treat FGFR3-BAIAP2L1fusion gene positive patients. In this study, we further elucidate the mechanism of tumorigenic potential of FGFR3-BAIAP2L1A by a comprehensive gene expression analysis of 4 cell lines (Rat-2\_mock, Rat-2\_FGFR3, Rat-2\_F3-B, and Rat-2\_BAIAP2L1) using RNA sequencing. We identified 143 up-regulated and 67 down-regulated genes specifically engaged by FGFR3-BAIAP2L1. Our gene signature analysis with this gene set revealed that FGFR3-BAIAP2L1 activates growth signals, such as the mitogen-activated protein kinase pathway, and inhibits tumor-suppressive signals, such as the p53, RB1, and CDKN2A pathways. Analysis by Western blot in xenograft tissues confirmed the activation and inactivation status of those pathways. These data suggest that a concurrent regulation of an oncogenic pathway and a tumor-suppressive pathway results in the tumorigenic potential of FGFR3-BAIAP2L1. *CH5183284/Debio 1347 was discovered by Chugai Pharmaceutical Co., Ltd. and is being developed by Debiopharm International S.A. under an exclusive worldwide license. Citation Format: Yoshito Nakanishi, Nukinori Akiyama, Toshiyuki Tsukaguchi, Toshihiko Fujii, Yasuko Satoh, Hideaki Mizuno, Nobuya Ishii, Masahiro Aoki. Mechanism of oncogenic signal activation by the novel fusion kinase FGFR3-BAIAP2L1. [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 123. doi:10.1158/1538-7445.AM2015-123

Abstract 5252: Inhibition of FGFR3-BAIAP2L1 fusion kinase oncogenic potential by CH5183284/Debio 1347, a compound that inhibits FGFR3 kinase activity constitutively activated by BAIAP2L1 BAR domain dimerization

Advances in next-generation sequencing technologies have made possible to identify more efficiently novel fusion proteins in cancer. Among them, fusion kinases are well known as potent oncogenes and promising therapeutic targets for cancer patients. Recently, several FGFR fusion genes, such as FGFR1-TACC1, FGFR2-CCDC6, FGFR3-TACC3, and FGFR3-BAIAP2L1, have been identified in GBM, bladder cancer, and breast cancer as well as other tumor types. In this study, we focused on the FGFR3-BAIAP2L1 (F3-B) gene fusion and investigated its prevalence in clinical samples, tumorigenic activity, mechanism of constitutive activation, and sensitivity to CH5183284/ Debio 1347. We screened cancer tissue panels by RT-PCR for F3-B mRNA and identified F3-B positive specimens in bladder cancer (4.3%: 2/47) and lung cancer (1.2%: 1/83). All these F3-B fusions were confirmed by cDNA sequencing. To expand this study further, we established an FGFR3 break apart FISH assay. In a larger bladder cancer panel, we found 2 additional positive cases (2.2%: 2/89). To investigate the role of F3-B in tumors, we established Rat-2 transfectants with full length F3-B and assessed tumorigenic activity of these cells in in vitro and in vivo settings. Rat-2/F3-B cells acquired sphere forming activity even without FGF1 in vitro, and efficiently formed tumors when subcutaneously inoculated the cells into nude mice. Consistent with these observations, FGFR3 or BAIAP2L1siRNA blocked the proliferation of F3-B positive SW780 cells. Interestingly, the selective FGFR inhibitor, CH5183284/Debio 1347, effectively inhibited the in vivo tumor growth of Rat-2/F3-B and SW780 cells, indicating that F3-B oncogenic activity is depending on FGFR kinase activity. To confirm this, we established Rat-2/F3-B-ΔBAR, which lacks dimerization domain of BAIAP2L1 (BAR domain: Bin-Amphiphysin-Rvs). Phosphorylation of FGFR3 in Rat-2/F3-B-ΔBAR cells was lowered as compared to that in Rat-2/F3-B cells. Furthermore, Rat-2/F3-B-ΔBAR cells exhibited lower spheroid formation activity and slower tumor growth compared with Rat-2/F3-B cells. Taking this information together, the constitutive dimerization though BAR domain is essential for F3-B fusion to exert its potent oncogene activity in tumors. These findings underline the oncogenic potential of FGFR3-BAIAP2L1 gene fusion and warrant further clinical evaluation of the therapeutic potential of CH5183284/Debio 1347 in patients harboring this genetic alteration. Citation Format: Nukinori Akiyama, Yoshito Nakanishi, Toshiyuki Tsukaguchi, Yasuko Satoh, Yasue Nagata, Tsutomu Takahashi, Yukari Nishito, Motoki Ashihara, Nobuya Ishii, Masahiro Aoki. Inhibition of FGFR3-BAIAP2L1 fusion kinase oncogenic potential by CH5183284/Debio 1347, a compound that inhibits FGFR3 kinase activity constitutively activated by BAIAP2L1 BAR domain dimerization. [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 5252. doi:10.1158/1538-7445.AM2014-5252

Abstract LB-327: Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor-D.

Lymphatic spread is an important clinical determinant in the prognosis of many human cancers. The lymphangiogenic factor called vascular endothelial growth factor-D (VEGF-D) is implicated in the promotion of lymphatic metastasis through the development of lymphatic vessels in some human cancers. In this study, we developed an anti-VEGF-D monoclonal antibody, cVE199, and investigated its in vitro properties, in vivo effects against tumors, and possible target indications to evaluate its potential as a therapeutic antibody. The cVE199 molecule was revealed to have a specific binding reactivity against human VEGF-D, as well as a specific inhibitory activity against the binding of human VEGF-D to VEGFR-3. In addition, cVE199 was found to inhibit the biological activity of VEGF-D against lymphatic cells in vitro. Because we determined that a neuroblastoma cell line, SK-N-DZ, abundantly expressed VEGF-D, an in vivo efficacy study was performed using a xenograft model of SK-N-DZ. We found that cVE199 significantly decreased lymphatic metastasis of SK-N-DZ as well as lymphangiogenesis in primary lesions. Finally, we investigated VEGF-D expression in human neuroblastoma, finding that the molecule was expressed in 11 of 29 human neuroblastoma specimens (37.9%). In conclusion, we found that a novel anti-VEGF-D monoclonal antibody, cVE199, with specific reactivity against human VEGF-D prevents lymphatic metastasis of neuroblastoma through the inhibition of lymphangiogenesis in an animal model. In addition, our results show that VEGF-D is expressed in some cases of human neuroblastomas, which suggests that cVE199 is a potential anti-metastasis therapeutic antibody in neuroblastoma treatment. Citation Format: Kenji Kashima, Miho Watanabe, Yasuko Satoh, Junichi Hata, Nobuya Ishii, Yuko Aoki. Inhibition of lymphatic metastasis in neuroblastoma by a novel neutralizing antibody to vascular endothelial growth factor-D. [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 LB-327. doi:10.1158/1538-7445.AM2013-LB-327