Toshimitsu Uenaka

Selective Inhibition of EZH2 by EPZ-6438 Leads to Potent Antitumor Activity in EZH2-Mutant Non-Hodgkin Lymphoma

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842–54. ©2014 AACR.

E7080, a novel inhibitor that targets multiple kinases, has potent antitumor activities against stem cell factor producing human small cell lung cancer H146, based on angiogenesis inhibition

E7080 is an orally active inhibitor of multiple receptor tyrosine kinases including VEGF, FGF and SCF receptors. In this study, we show the inhibitory activity of E7080 against SCF‐induced angiogenesis in vitro and tumor growth of SCF‐producing human small cell lung carcinoma H146 cells in vivo. E7080 inhibits SCF‐driven tube formation of HUVEC, which express SCF receptor, KIT at the IC50 value of 5.2 nM and it was almost identical for VEGF‐driven one (IC50 = 5.1 nM). To assess the role of SCF/KIT signaling in tumor angiogenesis, we evaluated the effect of imatinib, a selective KIT kinase inhibitor, on tumor growth of H146 cells in nude mice. Imatinib did not show the potent antitumor activity in vitro (IC50 = 2,200 nM), because H146 cells did not express KIT. However, oral administration of imatinib at 160 mg/kg clearly slowed tumor growth of H146 cells in nude mice, accompanied by decreased microvessel density. Oral administration of E7080 inhibited tumor growth of H146 cells at doses of 30 and 100 mg/kg in a dose‐dependent manner and caused tumor regression at 100 mg/kg. While anti‐VEGF antibody also slowed tumor growth, it did not cause tumor regression. These results indicate that KIT signaling has a role in tumor angiogenesis of SCF‐producing H146 cells, and E7080 causes regression of H146 tumors as a result of antiangiogenic activity mediated by inhibition of both KIT and VEGF receptor signaling. E7080 may provide therapeutic benefits in the treatment of SCF‐producing tumors.

Multi-Kinase Inhibitor E7080 Suppresses Lymph Node and Lung Metastases of Human Mammary Breast Tumor MDA-MB-231 via Inhibition of Vascular Endothelial Growth Factor-Receptor (VEGF-R) 2 and VEGF-R3 Kinase

Purpose: Vascular endothelial growth factor (VEGF)-C/VEGF-receptor 3 (VEGF-R3) signal plays a significant role in lymphangiogenesis and tumor metastasis based on its effects on lymphatic vessels. However, little is known about the effect of inhibiting VEGF-R3 on lymphangiogenesis and lymph node metastases using a small-molecule kinase inhibitor. Experimental Design: We evaluated the effect of E7080, a potent inhibitor of both VEGF-R2 and VEGF-R3 kinase, and bevacizumab on lymphangiogenesis and angiogenesis in a mammary fat pad xenograft model of human breast cancer using MDA-MB-231 cells that express excessive amounts of VEGF-C. Lymphangiogenesis was determined by lymphatic vessel density (LVD) and angiogenesis by microvessel density (MVD). Results: In contrast to MDA-MB-435 cells, which expressed a similar amount of VEGF to MDA-MB-231 cells with an undetectable amount of VEGF-C, only MDA-MB-231 exhibited lymphangiogenesis in the primary tumor. E7080 but not bevacizumab significantly decreased LVD within the MDA-MB-231 tumor. E7080 and bevacizumab decreased MVD in both the MDA-MB-231 and MDA-MB-435 models. E7080 significantly suppressed regional lymph nodes and distant lung metastases of MDA-MB-231, whereas bevacizumab significantly inhibited only lung metastases. E7080 also decreased both MVD and LVD within the metastatic nodules at lymph nodes after resection of the primary tumor. Conclusions: Inhibition of VEGF-R3 kinase with E7080 effectively decreased LVD within MDA-MB-231 tumors, which express VEGF-C. Simultaneous inhibition of both VEGF-R2 and VEGF-R3 kinases by E7080 may be a promising new strategy to control regional lymph node and distant lung metastases.

Paracrine Receptor Activation by Microenvironment Triggers Bypass Survival Signals and ALK Inhibitor Resistance in EML4-ALK Lung Cancer Cells

Purpose: Cancer cell microenvironments, including host cells, can critically affect cancer cell behaviors, including drug sensitivity. Although crizotinib, a dual tyrosine kinase inhibitor (TKI) of ALK and Met, shows dramatic effect against EML4-ALK lung cancer cells, these cells can acquire resistance to crizotinib by several mechanisms, including ALK amplification and gatekeeper mutation. We determined whether microenvironmental factors trigger ALK inhibitor resistance in EML4-ALK lung cancer cells. Experimental Design: We tested the effects of ligands produced by endothelial cells and fibroblasts, and the cells themselves, on the susceptibility of EML4-ALK lung cancer cell lines to crizotinib and TAE684, a selective ALK inhibitor active against cells with ALK amplification and gatekeeper mutations, both in vitro and in vivo. Results: EML4-ALK lung cancer cells were highly sensitive to ALK inhibitors. EGF receptor (EGFR) ligands, such as EGF, TGF-α, and HB-EGF, activated EGFR and triggered resistance to crizotinib and TAE684 by transducing bypass survival signaling through Erk1/2 and Akt. Hepatocyte growth factor (HGF) activated Met/Gab1 and triggered resistance to TAE684, but not crizotinib, which inhibits Met. Endothelial cells and fibroblasts, which produce the EGFR ligands and HGF, respectively, decreased the sensitivity of EML4-ALK lung cancer cells to crizotinib and TAE684, respectively. EGFR-TKIs resensitized these cells to crizotinib and Met-TKI to TAE684 even in the presence of EGFR ligands and HGF, respectively. Conclusions: Paracrine receptor activation by ligands from the microenvironment may trigger resistance to ALK inhibitors in EML4-ALK lung cancer cells, suggesting that receptor ligands from microenvironment may be additional targets during treatment with ALK inhibitors. Clin Cancer Res; 18(13); 3592–602. ©2012 AACR.

Combined Therapy with Mutant-Selective EGFR Inhibitor and Met Kinase Inhibitor for Overcoming Erlotinib Resistance in EGFR-Mutant Lung Cancer

Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) erlotinib and gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, Met amplification, and HGF overexpression, thereafter relapsing. Thus, it is urgent to develop novel agents to overcome EGFR-TKI resistance. We have tested the effects of the mutant-selective EGFR-TKI WZ4002 and the mutant-selective Met-TKI E7050 on 3 EGFR mutant lung cancer cell lines resistant to erlotinib by different mechanisms: PC-9/HGF cells with an exon 19 deletion, H1975 with an L858R mutation, and HCC827ER with an exon 19 deletion, with acquired resistance to erlotinib because of HGF gene transfection, gatekeeper T790M mutation, and Met amplification, respectively. WZ4002 inhibited the growth of H1975 cells with a gatekeeper T790M mutation, but did not inhibit the growth of HCC827ER and PC-9/HGF cells. HGF triggered the resistance of H1975 cells to WZ4002, whereas E7050 sensitized HCC827ER, PC-9/HGF, and HGF-treated H1975 cells to WZ4002, inhibiting EGFR and Met phosphorylation and their downstream molecules. Combined treatment potently inhibited the growth of tumors induced in severe-combined immunodeficient mice by H1975, HCC827ER, and PC-9/HGF cells, without any marked adverse events. These therapeutic effects were associated with the inhibition of EGFR and Met phosphorylation in vivo. The combination of a mutant-selective EGFR-TKI and a Met-TKI was effective in suppressing the growth of erlotinib-resistant tumors caused by gatekeeper T790M mutation, Met amplification, and HGF overexpression. Further evaluations in clinical trials are warranted. Mol Cancer Ther; 11(10); 2149–57. ©2012 AACR.

Met Kinase Inhibitor E7050 Reverses Three Different Mechanisms of Hepatocyte Growth Factor–Induced Tyrosine Kinase Inhibitor Resistance in EGFR Mutant Lung Cancer

Purpose: Hepatocyte growth factor (HGF) induces resistance to reversible and irreversible epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR-TKI) in EGFR mutant lung cancer cells by activating Met and the downstream phosphoinositide 3-kinase (PI3K)/Akt pathway. Moreover, continuous exposure to HGF accelerates the emergence of EGFR-TKI–resistant clones. We assayed whether a new Met kinase inhibitor, E7050, which is currently being evaluated in clinical trials, could overcome these three mechanisms of resistance to EGFR-TKIs. Experimental Design: The effects of E7050 on HGF-induced resistance to reversible (gefitinib), irreversible (BIBW2992), and mutant-selective (WZ4002) EGFR-TKIs were determined using the EGFR mutant human lung cancer cell lines PC-9 and HCC827 with an exon 19 deletion and H1975 with an T790M secondary mutation. PC-9 cells were mixed with HGF-producing fibroblasts, MRC-5 cells, and subcutaneously inoculated into severe combined immunodeficient mice, and the therapeutic effects of E7050 plus gefitinib were assayed. Results: E7050 circumvented resistance to all of the reversible, irreversible, and mutant-selective EGFR-TKIs induced by exogenous and/or endogenous HGF in EGFR mutant lung cancer cell lines, by blocking the Met/Gab1/PI3K/Akt pathway in vitro. E7050 also prevented the emergence of gefitinib-resistant HCC827 cells induced by continuous exposure to HGF. In the in vivo model, E7050 plus gefitinib resulted in marked regression of tumor growth associated with inhibition of Akt phosphorylation in cancer cells. Conclusions: A new Met kinase inhibitor, E7050, reverses the three HGF-induced mechanisms of gefitinib resistance, suggesting that E7050 may overcome HGF-induced resistance to gefitinib and next-generation EGFR-TKIs. Clin Cancer Res; 18(6); 1663–71. ©2012 AACR.

E7080, a Multi–Tyrosine Kinase Inhibitor, Suppresses the Progression of Malignant Pleural Mesothelioma with Different Proangiogenic Cytokine Production Profiles

Purpose: Malignant pleural mesothelioma (MPM) is a biologically heterogeneous malignant disease with a poor prognosis. We reported previously that the anti–vascular endothelial growth factor (VEGF) antibody, bevacizumab, effectively inhibited the progression of VEGF-high-producing (but not VEGF-low-producing) MPM cells in orthotopic implantation models, indicating the need for novel therapeutic strategies to improve the poor prognosis of this disease. Therefore, we focused on the multi–tyrosine kinase inhibitor E7080 and assessed its therapeutic efficacy against MPM cells with different proangiogenic cytokine production profiles. Experimental Design: The efficacy of E7080 was assayed in orthotopic implantation of severe combined immunodeficient mouse models with three human MPM cell lines (MSTO-211H, NCI-H290, and Y-MESO-14). Results: With regard to proangiogenic cytokine production profiles, MSTO-211H and Y-MESO-14 cells were MPM cells producing high levels of fibroblast growth factor-2 and VEGF, respectively. NCI-H290 cells produced low levels of fibroblast growth factor-2 and VEGF compared with the other two cell lines. E7080 potently suppressed the phosphorylation of VEGF receptor-2 and FGF receptor 1 and, thus, inhibited proliferation of endothelial cells, but not that of the MPM cell lines, in vitro. Orthotopically inoculated MSTO-211H cells produced only thoracic tumors, whereas NCI-H290 and Y-MESO-14 cells also developed pleural effusions. Treatment with E7080 potently inhibited the progression of these three MPM cell lines and markedly prolonged mouse survival, which was associated with decreased numbers of tumor-associated vessels and proliferating MPM cells in the tumor. Conclusions: These results strongly suggest broad-spectrum activity of E7080 against MPM with different proangiogenic cytokine production profiles in humans. (Clin Cancer Res 2009;15(23):7229–37)

Dual inhibition of Met kinase and angiogenesis to overcome HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

Acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) is a serious problem in the management of EGFR mutant lung cancer. We recently reported that hepatocyte growth factor (HGF) induces resistance to EGFR-TKIs by activating the Met/PI3K pathway. HGF is also known to induce angiogenesis in cooperation with vascular endothelial growth factor (VEGF), which is an important therapeutic target in lung cancer. Therefore, we hypothesized that dual inhibition of HGF and VEGF may be therapeutically useful for controlling HGF-induced EGFR-TKI-resistant lung cancer. We found that a dual Met/VEGF receptor 2 kinase inhibitor, E7050, circumvented HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer cell lines by inhibiting the Met/Gab1/PI3K/Akt pathway in vitro. HGF stimulated VEGF production by activation of the Met/Gab1 signaling pathway in EGFR mutant lung cancer cell lines, and E7050 showed an inhibitory effect. In a xenograft model, tumors produced by HGF-transfected Ma-1 (Ma-1/HGF) cells were more angiogenic than vector control tumors and showed resistance to gefitinib. E7050 alone inhibited angiogenesis and retarded growth of Ma-1/HGF tumors. E7050 combined with gefitinib induced marked regression of tumor growth. Moreover, dual inhibition of HGF and VEGF by neutralizing antibodies combined with gefitinib also markedly regressed tumor growth. These results indicate the therapeutic rationale of dual targeting of HGF-Met and VEGF-VEGF receptor 2 for overcoming HGF-induced EGFR-TKI resistance in EGFR mutant lung cancer.

Antitumour activity of oral E7080, a novel inhibitor of multiple tyrosine kinases, in human sarcoma xenografts.

E7080 is an inhibitor of multiple tyrosine kinases, several of which have pro‐angiogenic properties, including receptors for VEGF, FGF, SCF and PDGF. We undertook our study to evaluate the preclinical activity of E7080 in human sarcomas. The antitumour activity of orally administered E7080 was tested in ten human tumour xenografts representing different sarcoma histotypes. Concomitant changes in microvessel density were assayed by immunohistochemistry to CD31. Immunohistochemistry was also used to assess the expression of kinases that E7080 is known to inhibit. The MTS assay was applied to determine effects on tumour cell viability in vitro. At the Q1D5 × 2 schedule, E7080 (30 mg/kg) was active (T/C<40%) in 7/10 xenografts. The effects were accompanied by marked decrease in microvessel densities. Given at the Q1D5 × 4 schedule, E7080 (30, 10, 3 mg/kg) showed antitumour activity in a dose dependent manner in two different xenografts. E7080 growth inhibition did not correlate with the expression of VEGFR1‐3, PDGFRA, PDGFRB, FGFR1 or KIT on tumour cells but was significantly correlated with expression of VEGFR2 on tumour microvessels. In vitro E7080 did not show potent effects on tumour cell viability in four different sarcoma cell lines, with IC50 values ≥10 μM. In conclusion, E7080 showed broad in vivo antitumour activity in sarcoma, mainly attributable to angiogenesis inhibition. E7080 was also active in xenografts resistant to one or more clinically relevant reference drugs given at MTD (doxorubicin, cisplatin or ifosfamide). The present results encourage further investigation of a potential role of E7080 in sarcoma therapy in the clinic.

E7080 Suppresses Hematogenous Multiple Organ Metastases of Lung Cancer Cells with Nonmutated Epidermal Growth Factor Receptor

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors improve the prognosis of patients with EGFR mutant lung cancer, the prognosis of patients with nonmutant EGFR lung cancer, especially those with metastases, is still extremely poor. We have assessed the therapeutic efficacy of E7080, an orally available inhibitor of multiple tyrosine kinases including VEGF receptor 2 (VEGFR-2) and VEGFR-3, in experimental multiple organ metastasis of lung cancer cell lines without EGFR mutations. E7080 markedly inhibited the in vitro proliferation of VEGF-stimulated microvascular endothelial cells. Intravenous inoculation into natural killer cell–depleted severe combined immunodeficient mice of the small cell lung cancer cell lines H1048 (producing low amounts of VEGF) and SBC-5 (producing intermediate amounts of VEGF) resulted in hematogenous metastases into multiple organs, including the liver, lungs, kidneys, and bones, whereas intravenous inoculation of PC14PE6, a non–small cell lung cancer cell line producing high amounts of VEGF, resulted in lung metastases followed by massive pleural effusion. Daily treatment with E7080 started after the establishment of micrometastases significantly reduced the number of large (>2 mm) metastatic nodules and the amount of pleural effusion, and prolonged mouse survival. Histologically, E7080 treatment reduced the numbers of endothelial and lymph endothelial cells and proliferating tumor cells and increased the number of apoptotic cells in metastatic nodules. These results suggest that E7080 has antiangiogenic and antilymphangiogenic activity and may be of potential therapeutic value in patients with nonmutant EGFR lung cancer and multiple organ metastases. Mol Cancer Ther; 10(7); 1218–28. ©2011 AACR.