Osamu Tohyama

E7050: A dual c-Met and VEGFR-2 tyrosine kinase inhibitor promotes tumor regression and prolongs survival in mouse xenograft models

c‐Met is the cellular receptor for hepatocyte growth factor (HGF) and is known to be dysregulated in various types of human cancers. Activation of the HGF/c‐Met pathway causes tumor progression, invasion, and metastasis. Vascular endothelial growth factor (VEGF) is also known as a key molecule in tumor progression through the induction of tumor angiogenesis. Because of their key roles in tumor progression, these pathways provide attractive targets for therapeutic intervention. We have generated a novel, orally active, small molecule compound, E7050, which inhibits both c‐Met and vascular endothelial growth factor receptor (VEGFR)‐2. In vitro studies indicate that E7050 potently inhibits phosphorylation of both c‐Met and VEGFR‐2. E7050 also potently represses the growth of both c‐met amplified tumor cells and endothelial cells stimulated with either HGF or VEGF. In vivo studies using E7050 showed inhibition of the phosphorylation of c‐Met and VEGFR‐2 in tumors, and strong inhibition of tumor growth and tumor angiogenesis in xenograft models. Treatment of some tumor lines containing c‐met amplifications with high doses of E7050 (50–200 mg/kg) induced tumor regression and disappearance. In a peritoneal dissemination model, E7050 showed an antitumor effect against peritoneal tumors as well as a significant prolongation of lifespan in treated mice. Our results indicate that E7050 is a potent inhibitor of c‐Met and VEGFR‐2 and has therapeutic potential for the treatment of cancer. (Cancer Sci 2009)

Antitumor Activity of Lenvatinib (E7080): An Angiogenesis Inhibitor That Targets Multiple Receptor Tyrosine Kinases in Preclinical Human Thyroid Cancer Models

Inhibition of tumor angiogenesis by blockading the vascular endothelial growth factor (VEGF) signaling pathway is a promising therapeutic strategy for thyroid cancer. Lenvatinib mesilate (lenvatinib) is a potent inhibitor of VEGF receptors (VEGFR1–3) and other prooncogenic and prooncogenic receptor tyrosine kinases, including fibroblast growth factor receptors (FGFR1–4), platelet derived growth factor receptor α (PDGFRα), KIT, and RET. We examined the antitumor activity of lenvatinib against human thyroid cancer xenograft models in nude mice. Orally administered lenvatinib showed significant antitumor activity in 5 differentiated thyroid cancer (DTC), 5 anaplastic thyroid cancer (ATC), and 1 medullary thyroid cancer (MTC) xenograft models. Lenvatinib also showed antiangiogenesis activity against 5 DTC and 5 ATC xenografts, while lenvatinib showed in vitro antiproliferative activity against only 2 of 11 thyroid cancer cell lines: that is, RO82-W-1 and TT cells. Western blot analysis showed that cultured RO82-W-1 cells overexpressed FGFR1 and that lenvatinib inhibited the phosphorylation of FGFR1 and its downstream effector FRS2. Lenvatinib also inhibited the phosphorylation of RET with the activated mutation C634W in TT cells. These data demonstrate that lenvatinib provides antitumor activity mainly via angiogenesis inhibition but also inhibits FGFR and RET signaling pathway in preclinical human thyroid cancer models.

Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, shows broad antitumor activity in human tumor xenograft models associated with microvessel density and pericyte coverage

BackgroundLenvatinib is an oral inhibitor of multiple receptor tyrosine kinases (RTKs) targeting vascular endothelial growth factor receptor (VEGFR1-3), fibroblast growth factor receptor (FGFR1-4), platelet growth factor receptor α (PDGFR α), RET and KIT. Antiangiogenesis activity of lenvatinib in VEGF- and FGF-driven angiogenesis models in both in vitro and in vivo was determined. Roles of tumor vasculature (microvessel density (MVD) and pericyte coverage) as biomarkers for lenvatinib were also examined in this study.MethodWe evaluated antiangiogenesis activity of lenvatinib against VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. Effects of lenvatinib on in vivo angiogenesis, which was enhanced by overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells, were examined in the mouse dorsal air sac assay. We determined antitumor activity of lenvatinib in a broad panel of human tumor xenograft models to test if vascular score, which consisted of high MVD and low pericyte coverage, was associated with sensitivity to lenvatinib treatment. Vascular score was also analyzed using human tumor specimens with 18 different types of human primary tumors.ResultLenvatinib inhibited VEGF- and FGF-driven proliferation and tube formation of HUVECs in vitro. In vivo angiogenesis induced by overexpressed VEGF (KP-1/VEGF transfectants) or FGF (KP-1/FGF transfectants) was significantly suppressed with oral treatments of lenvatinib. Lenvatinib showed significant antitumor activity in KP-1/VEGF and five 5 of 7 different types of human tumor xenograft models at between 1 to 100 mg/kg. We divided 19 human tumor xenograft models into lenvatinib-sensitive (tumor-shrinkage) and relatively resistant (slow-growth) subgroups based on sensitivity to lenvatinib treatments at 100 mg/kg. IHC analysis showed that vascular score was significantly higher in sensitive subgroup than relatively resistant subgroup (p < 0.0004). Among 18 types of human primary tumors, kidney cancer had the highest MVD, while liver cancer had the lowest pericyte coverage, and cancers in Kidney and Stomach had highest vascular score.ConclusionThese results indicated that Lenvatinib inhibited VEGF- and FGF-driven angiogenesis and showed a broad spectrum of antitumor activity with a wide therapeutic window. MVD and pericyte-coverage of tumor vasculature might be biomarkers and suggest cases that would respond for lenvatinib therapy.

Eribulin mesylate reduces tumor microenvironment abnormality by vascular remodeling in preclinical human breast cancer models

Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B and an inhibitor of microtubule dynamics. Some tubulin‐binding drugs are known to have antivascular (antiangiogenesis or vascular‐disrupting) activities that can target abnormal tumor vessels. Using dynamic contrast‐enhanced MRI analyses, here we show that eribulin induces remodeling of tumor vasculature through a novel antivascular activity in MX‐1 and MDA‐MB‐231 human breast cancer xenograft models. Vascular remodeling associated with improved perfusion was shown by Hoechst 33342 staining and by increased microvessel density together with decreased mean vascular areas and fewer branched vessels in tumor tissues, as determined by immunohistochemical staining for endothelial marker CD31. Quantitative RT‐PCR analysis of normal host cells in the stroma of xenograft tumors showed that eribulin altered the expression of mouse (host) genes in angiogenesis signaling pathways controlling endothelial cell–pericyte interactions, and in the epithelial–mesenchymal transition pathway in the context of the tumor microenvironment. Eribulin also decreased hypoxia‐associated protein expression of mouse (host) vascular endothelial growth factor by ELISA and human CA9 by immunohistochemical analysis. Prior treatment with eribulin enhanced the anti‐tumor activity of capecitabine in the MDA‐MB‐231 xenograft model. These findings suggest that eribulin‐induced remodeling of abnormal tumor vasculature leads to a more functional microenvironment that may reduce the aggressiveness of tumors due to elimination of inner tumor hypoxia. Because abnormal tumor microenvironments enhance both drug resistance and metastasis, the apparent ability of eribulin to reverse these aggressive characteristics may contribute to its clinical benefits.

Multitargeting strategy using lenvatinib and golvatinib: Maximizing anti-angiogenesis activity in a preclinical cancer model

Almost all cancers show intrinsic and/or evasive resistance to vascular endothelial growth factor (VEGF) inhibitors by multiple mechanisms. Serum angiopoietin‐2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. From these clinical observations, the Ang2 and Tie2 (its receptor) axis has been focused on as a promising target. Here, we show a novel strategy to circumvent the resistance by combining multi‐tyrosine kinase inhibitors lenvatinib (VEGF receptor, fibroblast growth factor receptor, and RET inhibitor) and golvatinib (E7050; c‐Met, Tie2, and EphB4 inhibitor). Tie2 identifies a highly pro‐angiogenic macrophage subset, Tie2‐expressing macrophages (TEM). Angi‐Tie2 and EphB4‐EphrinB2 signaling plays critical roles in pericyte‐mediated vessel stabilization. In vitro analyses suggested that golvatinib combined with lenvatinib inhibited pericyte‐mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib and lenvatinib inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable, and no macroscopic change was observed. These preclinical studies suggest that modulation of the tumor microenvironment by a strategic and well‐tolerated combination of multi‐targeting tyrosine kinase inhibitors may sensitize cancer to VEGF inhibitors.

Targeting of tumor growth and angiogenesis underlies the enhanced antitumor activity of lenvatinib in combination with everolimus

The combination of lenvatinib, a multiple receptor tyrosine kinase inhibitor, plus everolimus, a mammalian target of rapamycin (mTOR) inhibitor, significantly improved clinical outcomes versus everolimus monotherapy in a phase II clinical study of metastatic renal cell carcinoma (RCC). We investigated potential mechanisms underlying the antitumor activity of the combination treatment in preclinical RCC models. Lenvatinib plus everolimus showed greater antitumor activity than either monotherapy in three human RCC xenograft mouse models (A‐498, Caki‐1, and Caki‐2). In particular, the combination led to tumor regression in the A‐498 and Caki‐1 models. In the A‐498 model, everolimus showed antiproliferative activity, whereas lenvatinib showed anti‐angiogenic effects. The anti‐angiogenic activity was potentiated by the lenvatinib plus everolimus combination in Caki‐1 xenografts, in which fibroblast growth factor (FGF)‐driven angiogenesis may contribute to tumor growth. The combination showed mostly additive activity in vascular endothelial growth factor (VEGF)‐activated, and synergistic activity against FGF‐activated endothelial cells, in cell proliferation and tube formation assays, as well as strongly suppressed mTOR‐S6K‐S6 signaling. Enhanced antitumor activities of the combination versus each monotherapy were also observed in mice bearing human pancreatic KP‐1 xenografts overexpressing VEGF or FGF. Our results indicated that simultaneous targeting of tumor cell growth and angiogenesis by lenvatinib plus everolimus resulted in enhanced antitumor activity. The enhanced inhibition of both VEGF and FGF signaling pathways by the combination underlies its superior anti‐angiogenic activity in human RCC xenograft models.

Microregional antitumor activity of a small-molecule hypoxia-inducible factor 1 inhibitor.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that play crucial roles in the adaptation of cancer cells to hypoxia. HIF-1α overexpression has been associated with poor prognosis in patients with various types of cancer. Here, we describe ER-400583-00 as a novel HIF-1 inhibitor. ER-400583-00 suppressed the production of HIF-1α protein in response to hypoxia, with a half-maximal inhibitory concentration value of 3.7 nM in human U251 glioma cells. The oral administration of 100 mg/kg ER-400583-00 to mice bearing U251 tumor xenografts resulted in a rapid suppression of HIF-1α that persisted for 24 h. Immunohistochemical analysis revealed that ER-400583-00 suppressed the proliferation of cancer cells most prominently in areas distal to the region of blood perfusion, where HIF-1α-expressing hypoxic cancer cells were located. These hypoxic cancer cells were resistant to radiation therapy. ER-400583-00 showed a synergistic interaction with radiation therapy in terms of antitumor activity. These data suggest that HIF-1 blockade by small compounds may have therapeutic value in cancer, especially in combination with radiation therapy.

Abstract 2980: Maximizing the efficacy of anti-angiogenesis cancer therapy: A multi-targeting strategy by tyrosine kinase inhibitors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Although vascular endothelial growth factor (VEGF) inhibitors provide significant clinical benefit, they often require dose reductions or even withdrawals due to their severe toxicities. Furthermore, almost all cancers show intrinsic and/or evasive resistance to VEGF inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. Response to lenvatinib (E7080; VEGFR1-3 inhibitor) is also reported to correlate with low Ang2 level in differentiated thyroid cancer and endometrial cancer. From these clinical observations, Ang2 and its receptor Tie2 has been focused as promising targets. Here, we demonstrated mechanisms of resistance induced by Ang2 and a novel strategy to circumvent the resistance by combination of multi-tyrosine kinase inhibitors (TKIs), lenvatinib and golvatinib (E7050; c-Met, Tie2, EphB4 inhibitor). Tie2 defines a highly pro-angiogenic macrophage subset, Tie2-expressing macrophage (TEM). Ang-Tie2 and EphB4-EphrinB2 signaling play critical roles in pericyte-mediated vessel stabilization. Ectopic expression of Ang2 in thyroid cancer conferred resistance to lenvatinib and enhanced pericyte-associated endothelial network development and TEM infiltration. In vitro analyses suggested that golvatinib/lenvatinib combination inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib/lenvatinib combination inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable in mice, and no macroscopic change was observed. These results suggest that modulation of tumor microenvironment by strategic and well-tolerated combination of multi-targeting TKIs sensitizes cancer to VEGF inhibitors, which warrants further clinical investigation to determine the clinical benefit of anti-angiogenesis cancer therapy. Citation Format: Youya Nakazawa, Satoshi Kawano, Junji Matsui, Yasuhiro Funahashi, Osamu Tohyama, Hiroki Muto, Takayuki Nakagawa, Tomohiro Matsushima. Maximizing the efficacy of anti-angiogenesis cancer therapy: A multi-targeting strategy by tyrosine kinase inhibitors. [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 2980. doi:10.1158/1538-7445.AM2014-2980

Abstract 3264: Lenvatinib in combination with everolimus demonstrated enhanced antiangiogenesis and antitumor activity in human RCC xenograft models

Lenvatinib mesilate (lenvatinib) is an oral multiple receptor tyrosine kinase (RTK) inhibitor that selectively inhibits the kinase activities of vascular endothelial growth factor (VEGF) receptors VEGFR1 (FLT1), VEGFR2 (KDR), and VEGFR3 (FLT4), in addition to other proangiogenic and oncogenic pathway-related RTKs including fibroblast growth factor (FGF) receptors FGFR1, 2, 3, and 4; the platelet-derived growth factor (PDGF) receptor PDGFRα; KIT; and RET. Lenvatinib showed antitumor activity against various tumor types mainly through its potent inhibition of angiogenesis, and is currently marketed for the treatment of patients with radioactive iodine-refractory differentiated thyroid cancer. Recently, lenvatinib in combination with everolimus has shown longer progression free survival compared to lenvatinib or everolimus alone in renal cell carcinoma in Phase 2 study. The aim of this study is to elucidate the activity of the combination of lenvatinib and everolimus in preclinical human RCC xenograft models. We examined antitumor activity in two human RCC (A-498 and Caki-1) xenograft models orally treated with lenvatinib (10 mg/kg), everolimus (30 mg/kg), and the combination of lenvatinib and everolimus for 1 or 2 weeks. The antitumor proliferation and antiangiogenic effects were evaluated by immunohistochemistry (IHC) using anti Ki67 antibody and anti CD31 antibody, respectively. The induction of apoptosis was detected by TUNEL assay. To analyze the gene expression profile of tumor samples, microarray analysis were also conducted. The antitumor activity of the combination of lenvatinib and everolimus was greater than that of either agent administered alone in A-498 and Caki-1 xenograft models. The combination caused tumor regression and had no remarkable body weight loss. IHC analysis revealed decrease of microvessel density in lenvatinib and combination groups, and also decrease in the proportion of proliferative cells in everolimus treated and combination-treated group in A-498 model. In TUNEL assay, significant induction of apoptosis was observed only in the combination-treatment group. The analysis of gene expression profile in A-498 xenograft tumors also supported these results: lenvatinib alone upregulated hypoxia-related genes and everolimus decreased proliferation-related genes. The combination of these 2 drugs induced blends of the gene expression changes caused by each single treatment. Our results indicate that treatment of lenvatinib in combination with everolimus caused significant antitumor effect by combining the potent antiangiogenic activity of lenvatinib as well as direct antitumor activity of everolimus in A-498 model. These preclinical results provide one of the mechanisms to support the significant clinical benefit observed in RCC with the combination of lenvatinib and everolimus. Citation Format: Yusuke Adachi, Masahiro Matsuki, Atsumi Yamaguchi, Yoichi Ozawa, Kiyochi Okamoto, Kaoru Mitsuhashi, Takayuki Kimura, Taisuke Hoshi, Osamu Tohyama, Kenji Tai, Makoto Ogo, Yasuhiro Funahashi, Junji Matsui. Lenvatinib in combination with everolimus demonstrated enhanced antiangiogenesis and antitumor activity in human RCC xenograft models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3264.

Abstract 3262: Effects of lenvatinib mesilate in combination with everolimus on VEGF and FGF-driven angiogenesis and tumor growth

Lenvatinib mesilate (lenvatinib) is an orally available inhibitor for multiple receptor tyrosine kinases (RTKs) that selectively inhibits the kinase activities of VEGFR1, 2, and 3, in addition to other proangiogenic and oncogenic pathway-related RTKs including FGFR1, 2, 3, and 4; PDGFRα; KIT; and RET. Recently, lenvatinib in combination with everolimus has shown longer progression free survival compared to lenvatinib or everolimus alone in renal cell carcinoma in a Phase 2 study. In this study, we evaluated the effect of lenvatinib in combination with everolimus on VEGF and bFGF-driven angiogenesis to elucidate the mechanism of combination action in preclinical models. Preclinical studies provide a plausible biologic rationale for the significant clinical benefit observed in RCC with the combination of lenvatinib and everolimus. Effects of lenvatinib, everolimus, and its combination on VEGF or bFGF activated intracellular signaling were analyzed in HUVEC by western blotting. Combination effects of lenvatinib and everolimus on VEGF and bFGF-induced proliferation or tube formation of HUVEC were examined using combination indexes (CI). Antitumor activities were tested in the KP-1/VEGF or KP-1/FGF models, where VEGF or FGF-induced tumor angiogenesis and tumor growth were enhanced in nude mice due to overexpressed VEGF or FGF in human pancreatic cancer KP-1 cells. Lenvatinib inhibited the VEGF or bFGF-driven phosphorylation of Erk1/2 (Thr202/Tyr204), S6K (Thr389), and S6K (Thr421/Ser424), and S6 (Ser235/Ser236), indicating the inhibition of both the MAPK pathway and the mTOR-S6K-S6 pathway. Everolimus inhibited the phosphorylation of S6K (Thr389), S6K (Thr421/Ser424), and S6 (Ser235/Ser236), but not Erk1/2. The combination showed greater inhibition for the phosphorylation of S6K (Thr421/Ser424) and S6 (Ser235/Ser236) than each single agent. Inhibitory activity of the combination at several molar ratios was mostly additive for VEGF-driven proliferation (CI: 0.799-1.167) and mostly synergistic for bFGF-driven tube formation (CI: 0.469-0.741). In the KP-1/VEGF or KP-1/FGF xenogtaft models, lenvatinib, everolimus, and the combination (p.o., qd x 14) significantly inhibited tumor growth compared to vehicle. In addition the combination of lenvatinib (7.5 mg/kg) and everolimus (15 mg/kg) showed significantly greater antitumor activity than higher dose of either lenvatinib (10 mg/kg) or everolimus (30 mg/kg) monotherapy. These results demonstrated enhancement of the inhibitory activity against VEGF and FGF-induced angiogenesis by the combination of lenvatinib with everolimus, and the synergistic enhancement against bFGF-induced angiogenesis unlike other VEGFR2 TKIs. The vertical inhibition of angiogenic signaling pathways with lenvatinib (RTK) and everolimus (mTOR) may contribute to enhanced antiangiogenic activity by dual targeting of the mTOR-S6K-S6 pathway. Citation Format: Kaoru Mitsuhashi, Takayuki Kimura, Taisuke Hoshi, Osamu Tohyama, Kenji Tai, Makoto Ogo, Masahiro Matsuki, Atsumi Yamaguchi, Yoichi Ozawa, Yusuke Adachi, Kiyoshi Okamoto, Junji Matsui, Yasuhiro Funahashi. Effects of lenvatinib mesilate in combination with everolimus on VEGF and FGF-driven angiogenesis and tumor growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3262.