Masao Iwata

Antitumor activities of the targeted multi-tyrosine kinase inhibitor lenvatinib (E7080) against RET gene fusion-driven tumor models

RET gene fusions are recurrent oncogenes identified in thyroid and lung carcinomas. Lenvatinib is a multi-tyrosine kinase inhibitor currently under evaluation in several clinical trials. Here we evaluated lenvatinib in RET gene fusion-driven preclinical models. In cellular assays, lenvatinib inhibited auto-phosphorylation of KIF5B-RET, CCDC6-RET, and NcoA4-RET. Lenvatinib suppressed the growth of CCDC6-RET human thyroid and lung cancer cell lines, and as well, suppressed anchorage-independent growth and tumorigenicity of RET gene fusion-transformed NIH3T3 cells. These results demonstrate that lenvatinib can exert antitumor activity against RET gene fusion-driven tumor models by inhibiting oncogenic RET gene fusion signaling.

Biological validation that SF3b is a target of the antitumor macrolide pladienolide

Pladienolide is a naturally occurring macrolide that binds to the SF3b complex to inhibit mRNA splicing. It has not been fully validated whether the splicing impairment is a relevant mechanism for the potent antitumor activity of pladienolide. We established pladienolide‐resistant clones from WiDr and DLD1 colorectal cancer cells that were insensitive to the inhibitory action of pladienolide on cell proliferation and splicing. An mRNA‐Seq differential analysis revealed that these two cell lines have an identical mutation at Arg1074 in the gene for SF3B1, which encodes a subunit of the SF3b complex. Reverse expression of the mutant protein transferred pladienolide resistance to WiDr cells. Furthermore, immunoprecipitation analysis using a radiolabeled probe showed that the mutation impaired the binding affinity of paldienolide to its target. These results clearly demonstrate that pladienolide exerts its potent activity by targeting SF3b and also suggest that inhibition of SF3b is a promising drug target for anticancer therapy.

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.

Novel ATP-Competitive MEK Inhibitor E6201 Is Effective against Vemurafenib-Resistant Melanoma Harboring the MEK1-C121S Mutation in a Preclinical Model

Many clinical cases of acquired resistance to the BRAF inhibitor vemurafenib have recently been reported. One of the causes of this acquired resistance is the BRAF downstream kinase point mutation MEK1-C121S. This mutation confers resistance to not only vemurafenib, but also to the allosteric MEK inhibitor selumetinib (AZD6244). Here, we investigated the pharmacologic activities and effectiveness of the novel MEK inhibitor E6201 against BRAF (v-raf murine sarcoma viral oncogene homolog B1)-V600E mutant melanoma harboring the MEK1-C121S mutation. A cell-free assay confirmed that E6201 is an ATP-competitive MEK inhibitor, meaning it has a different binding mode with MEK compared with allosteric MEK inhibitors. E6201 is more effective against BRAF-V600E mutant melanoma compared with BRAF wild-type melanoma based on MEK inhibition. We found that the acquired MEK1-C121S mutation in BRAF-V600E mutant melanoma conferred resistance to both vemurafenib and selumetinib but not E6201. The effectiveness of E6201 in this preclinical study is a result of its binding with MEK1 far from the C121S point mutation so the mutation is unable to influence the MAPK pathway inhibitory activity. These results support further clinical investigation of E6201. Mol Cancer Ther; 13(4); 823–32. ©2014 AACR.

High antitumor activity of pladienolide B and its derivative in gastric cancer

The antitumor activity of pladienolide B, a novel splicing inhibitor, against gastric cancer is totally unknown and no predictive biomarker of pladienolide B efficacy has been reported. We investigated the antitumor activity of pladienolide B and its derivative on gastric cancer cell lines and primary cultured cancer cells from carcinomatous ascites of gastric cancer patients. The effect of pladienolide B and its derivative on six gastric cancer cell lines was investigated using a MTT assay and the mean IC50 values determined to be 1.6 ± 1.2 (range, 0.6–4.0) and 1.2 ± 1.1 (range, 0.4–3.4) nM, respectively, suggesting strong antitumor activity against gastric cancer. The mean IC50 value of pladienolide B derivative against primary cultured cells from 12 gastric cancer patients was 4.9 ± 4.7 nM, indicative of high antitumor activity. When 18 SCID mice xenografted with primary cultured cells from three patients were administered the pladienolide B derivative intraperitoneally, all tumors completely disappeared within 2 weeks after treatment. Histological examination revealed a pathological complete response for all tumors. In the xenograft tumors after treatment with pladienolide B derivative, immature mRNA were detected and apoptotic cells were observed. When the expressions of cell‐cycle proteins p16 and cyclin E in biopsied gastric cancer specimens were examined using immunohisctochemistry, positivities for p16 and cyclin E were significantly and marginally higher, respectively, in the low‐IC50 group compared with the high‐IC50 group, suggesting the possibility that they might be useful as predictive biomarkers for pladienolide B. In conclusion, pladienolide B was very active against gastric cancer via a mechanism involving splicing impairment and apoptosis induction.

Radiolabeled liposome imaging determines an indication for liposomal anticancer agent in ovarian cancer mouse xenograft models

Liposomal anticancer agents can effectively deliver drugs to tumor lesions, but their therapeutic effects are enhanced in only limited number of patients. Appropriate biomarkers to identify responder patients to these liposomal agents will improve their treatment efficacies. We carried out pharmacological and histopathological analyses of mouse xenograft models bearing human ovarian cancers (Caov‐3, SK‐OV‐3, KURAMOCHI, and TOV‐112D) to correlate the therapeutic effects of doxorubicin‐encapsulated liposome (Doxil®) and histological characteristics linked to the enhanced permeability and retention effect. We next generated 111In‐encapsulated liposomes to examine their capacities to determine indications for Doxil® treatment by single‐photon emission computed tomography (SPECT)/CT imaging. Antitumor activities of Doxil® were drastically enhanced in Caov‐3, moderately in SK‐OV‐3, and minimally in KURAMOCHI and TOV‐112D when compared to doxorubicin. Microvessel density and vascular perfusion were high in Caov‐3 and SK‐OV‐3, indicating a close relation with the enhanced antitumor effects. Next, 111In‐encapsulated liposomes were given i.v. to the animals. Their tumor accumulation and area under the curve values over 72 h were high in Caov‐3, relatively high in SK‐OV‐3, and low in two other tumors. Importantly, as both Doxil® effects and liposomal accumulation varied in the SK‐OV‐3 group, we individually obtained SPECT/CT images of SK‐OV‐3‐bearing mouse (n = 11) before Doxil® treatment. Clear correlation between liposomal tumor accumulation and effects of Doxil® was confirmed (R2 = 0.73). Taken together, our experiments definitely verified that enhanced therapeutic effects through liposomal formulations of anticancer agents depend on tumor accumulation of liposomes. Tumor accumulation of the radiolabeled liposomes evaluated by SPECT/CT imaging is applicable to appropriately determine indications for liposomal antitumor agents.

E7090, a Novel Selective Inhibitor of Fibroblast Growth Factor Receptors, Displays Potent Antitumor Activity and Prolongs Survival in Preclinical Models

The FGFR signaling pathway has a crucial role in proliferation, survival, and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR genetic abnormalities, such as gene fusion, mutation, and amplification, have been implicated in several types of cancer. Therefore, FGFRs are considered potential targets for cancer therapy. E7090 is an orally available and selective inhibitor of the tyrosine kinase activities of FGFR1, -2, and -3. In kinetic analyses of the interaction between E7090 and FGFR1 tyrosine kinase, E7090 associated more rapidly with FGFR1 than did the type II FGFR1 inhibitor ponatinib, and E7090 dissociated more slowly from FGFR1, with a relatively longer residence time, than did the type I FGFR1 inhibitor AZD4547, suggesting that its kinetics are more similar to the type V inhibitors, such as lenvatinib. E7090 showed selective antiproliferative activity against cancer cell lines harboring FGFR genetic abnormalities and decreased tumor size in a mouse xenograft model using cell lines with dysregulated FGFR. Furthermore, E7090 administration significantly prolonged the survival of mice with metastasized tumors in the lung. Our results suggest that E7090 is a promising candidate as a therapeutic agent for the treatment of tumors harboring FGFR genetic abnormalities. It is currently being investigated in a phase I clinical trial. Mol Cancer Ther; 15(11); 2630–9. ©2016 AACR.

Abstract 5177: E7386 : First-in-class orally active CBP/beta-catenin modulator as an anticancer agent

Carcinogenesis is often accelerated by the aberrant activation of components molecules of Wnt signaling pathway, especially, APC and beta-catenin are frequently reported to be mutated in various cancers. Therefore, Wnt signal pathway is thought to be one of the attractive therapeutic targets. PRI-724 generated by PRISM Pharma is a small molecule inhibitor of beta-catenin and its transcriptional coactivator CREB binding protein (CBP) thereby specific modulating Wnt/beta-catenin signaling pathway by intravenous continuous infusion. Here we firstly generated orally active small molecular inhibitor, E7386. E7386 disrupted the interaction between beta-catenin and CBP in co-immunoprecipitation assay. E7386 inhibited canonical Wnt signaling pathway /TCF reporter gene activity in LiCl-stimulated HEK-293 and MDA-MB-231 in a dose dependent manner with IC50 values of 55 nmol/L and 73 nmol/L, respectively. E7386 modulated the expression of Wnt signaling pathway related genes including AXIN2 and other genes, which were down-regulated by artificial knockdown of beta-catenin. These results indicate that E7386 controls the expression of Wnt target genes through modulation of beta-catenin/CBP interaction. Next we investigated anti-polyposis effect in ApcMin/+ mice as an in vivo proof of mechanism model. ApcMin/+ mice develops polyps in the intestinal tract caused by the aberrant activation of Wnt/beta-catenin signaling pathway. Oral administration of E7386 significantly suppressed the number of polyposis in a dose dependent manner at the dose range from 8.5 to 50 mg/kg. In addition, E7386 significantly changed the expressions of Wnt related genes in whisker follicle of ApcMin/+mice model. Finally, we investigated anti-tumor activity of E7386 in vitro tumor proliferation panel against 28 human tumor cell lines. E7386 showed relatively potent anti-proliferative activity against cancer cell lines harboring exclusively mutated Wnt signaling pathway molecules such as APC or beta-catenin. E7386 also showed significant antitumor activity in a dose dependent manner on human tumor cell line xenograft harboring APC mutation. Taken together, E7386 is a first in class orally active CBP/beta-catenin modulator and showed potent inhibitory activity against aberrant activation of Wnt/beta-catenin signaling pathway. Citation Format: Kazuhiko Yamada, Yusaku Hori, Atsumi Yamaguchi, Masahiro Matsuki, Shuntaro Tsukamoto, Akira Yokoi, Taro Semba, Yoichi Ozawa, Satoshi Inoue, Yuji Yamamoto, Kentaro Iso, Kazutaka Nakamoto, Hitoshi Harada, Naoki Yoneda, Atsushi Takemura, Masayuki Matsukura,, Kenji Kubara, Takenao Odagami, Masao Iwata, Akihiko Tsuruoka, Toshimitsu Uenaka, Junji Matsui, Tomohiro Matsushima, Kenich Nomoto, Hiroyuki Kouji, Takashi Owa. E7386 : First-in-class orally active CBP/beta-catenin modulator as an anticancer agent [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 5177. doi:10.1158/1538-7445.AM2017-5177

Abstract 3778: In vitro and in vivo characterization of E7090, a novel and selective FGFR inhibitor, for the treatment of endometrial cancer

Genetic abnormalities (gene fusion, mutation and amplification) of Fibroblast Growth Factor Receptor (FGFR) family members are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, tumor angiogenesis, and drug resistance. FGFR2 is mutated in about 10% of endometrial cancer patients and is associated with a higher risk of recurrence. E7090, an oral available FGFR1, 2, and 3 inhibitor that the chemical-structure and basic kinase inhibitory activity of this compound have been presented at AACR2015, is currently under a first-in-human study (NCT02275910) in Japan. In this presentation, the efficacy of E7090 against human endometrial cancer was examined. E7090 exhibited selective potent antiproliferative activity against 3 human endometrial cancer cell lines harboring FGFR2 mutation with IC 50 values of approximately 10 nM among 8 endometrial cancer cell lines. These cell lines harbor S252W mutation (extracellular domain mutation; MFE280) or N549K mutation (kinase domain mutation; AN3CA and MFE296), that are the most common mutations of FGFR2 in endometrial cancer. E7090 also showed antiproliferative activity against Ba/F3 cells expressing mutated type of FGFR2, confirming the inhibitory activity of E7090 against mutated FGFR2. The antiproliferative activity of E7090 against these cell lines was based on inhibition of phosphorylated FRS2α and ERK1/2 as downstream molecules of FGF/FGFR signaling. Especially, the antiproliferative activities of E7090 against AN3CA and MFE296 harboring N550K mutation were more potent compared to these of other FGFR specific inhibitors. In addition, oral administration of E7090 resulted in significant tumor growth inhibition in MFE280, AN3CA and MFE296 xenograft models in mice. Especially, E7090 showed tumor regression in AN3CA xenograft model with a delta T/C value of -66% at a dose of 50 mg/kg. These results indicated that E7090 showed potent inhibitory activity against mutated type of FGFR2 both in vitro and in vivo, and suggest that E7090 may provide therapeutic opportunities for endometrial cancer harboring FGFR2 mutations. Citation Format: Saori Watanabe Miyano, Yuji Yamamoto, Kotaro Kodama, Naoko Hata Sugi, Yukinori Minoshima, Junji Matsui, Masao Iwata. In vitro and in vivo characterization of E7090, a novel and selective FGFR inhibitor, for the treatment of endometrial cancer. [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 3778.

Abstract 770: E7090: A potent and selective FGFR inhibitor with activity in multiple FGFR-driven cancer models with distinct mechanisms of activation

The fibroblast growth factor (FGF) signaling pathway comprises 18 ligands and 4 FGF receptor subtypes, FGFR1, 2, 3 and 4, which are known as receptor-type tyrosine kinases corresponding to those ligands. Upon ligand binding, FGFRs activate an array of downstream signaling pathways, such as the mitogen activated protein kinase (MAPK) and the phosphoinositide-3-kinase (PI3K)/Akt pathways. Genetic abnormalities (gene fusion, mutation and amplification) of FGFRs are known to cause constitutive activation of their signaling pathways, which play an important role in proliferation, survival and migration of cancer cells, tumor angiogenesis, drug resistance, etc. These abnormalities have also been reported to be involved in different cancer types including lung, breast, endometrial, gastric, and bladder cancer so far. Therefore, FGFRs are considered as one of potential targets for cancer therapy. E7090 is an orally available, selective and potent inhibitor of FGFR1, 2 and 3 tyrosine kinase activities. E7090 displayed inhibition of FGFR1, 2, and 3 kinase activities with the IC50 values of approximately 1 nmol/L. E7090 also inhibited the growth of SNU-16, human gastric cancer cell line harboring FGFR2 amplification with an IC50 value of 3 nmol/L. This activity was about 60-fold stronger than that against VEGF-stimulated HUVEC growth. Kinase profiling assay consisting of 93 kinases including non-receptor and serine/threonine kinases, also demonstrated that E7090 inhibited limited kinases including FGFR-1, -2 and -3. In addition, E7090 inhibited proliferation of human cancer cell lines harboring various types of FGFRs gene abnormalities such as amplification, mutation, or translocation in vitro, which are confirmed by the inhibition of FGFR signaling. E7090 also showed significant antitumor activities on various human xenografts harboring FGFRs gene abnormalities in a dose dependent manner and demonstrated tumor shrinkage at several doses in some models. Furthermore, pharmacodynamics analysis revealed that E7090 inhibited phosphorylation of FGFRs in SNU-16 xenograft tumors in a dose-dependent manner. Overall, the in vitro and in vivo studies confirm that E7090 is a potent and selective FGFRs inhibitor, showing promising antitumor activities with wider therapeutic windows in preclinical cancer models harboring FGFRs gene abnormalities. E7090 has been advanced through preclinical development and we disclose here the first details of its preclinical profile. Citation Format: Saori Watanabe Miyano, Yuji Yamamoto, Kotaro Kodama, Setsuo Funasaka, Satoshi Nagao, Naoko Hata Sugi, Hiroko Kuramochi, Katsuyuki Ishikawa, Kiyoshi Okamoto, Yukinori Minoshima, Takayuki Nakagawa, Yusuke Nakatani, Yuki Karoji, Isao Ohashi, Yoshinobu Yamane, Keigo Tanaka, Toshimi Okada, Tomohiro Matsushima, Junji Matsui, Masao Iwata, Akihiko Tsuruoka, Toshimitsu Uenaka. E7090: A potent and selective FGFR inhibitor with activity in multiple FGFR-driven cancer models with distinct mechanisms of activation. [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 770. doi:10.1158/1538-7445.AM2015-770