Shuntarou Tsuchiya

Antitumor Activity of the Selective Pan-RAF Inhibitor TAK-632 in BRAF Inhibitor-Resistant Melanoma

The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells. Somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, the BRAF inhibitors vemurafenib and dabrafenib have emerged as promising agents for the treatment of melanoma patients with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild-type BRAF (including NRAS-mutant melanoma) are clinical concerns. Here, we demonstrate that the selective pan-RAF inhibitor TAK-632 suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. Our analysis using RNAi and TAK-632 in preclinical models reveals that the MAPK pathway of NRAS-mutated melanoma cells is highly dependent on RAF. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. As a result, TAK-632 demonstrates potent antiproliferative effects both on NRAS-mutated melanoma cells and BRAF-mutated melanoma cells with acquired resistance to BRAF inhibitors through NRAS mutation or BRAF truncation. Furthermore, we demonstrate that the combination of TAK-632 and the MAPK kinase (MEK) inhibitor TAK-733 exhibits synergistic antiproliferative effects on these cells. Our findings characterize the unique features of TAK-632 as a pan-RAF inhibitor and provide rationale for its further investigation in NRAS-mutated melanoma and a subset of BRAF-mutated melanomas refractory to BRAF inhibitors.

Anti-angiogenic and anti-tumor effects of TAK-593, a potent and selective inhibitor of vascular endothelial growth factor and platelet-derived growth factor receptor tyrosine kinase.

We recently reported that TAK‐593, a novel imidazo[1,2‐b]pyridazine derivative, is a highly potent and selective inhibitor of the vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) receptor tyrosine kinase families. Moreover, TAK‐593 exhibits a uniquely long‐acting inhibitory profile towards VEGF receptor 2 (VEGFR2) and PDGF receptor β (PDGFRβ). In this study, we demonstrated that TAK‐593 potently inhibits VEGF‐ and PDGF‐stimulated cellular phosphorylation and proliferation of human umbilical vein endothelial cells and human coronary artery smooth muscle cells. TAK‐593 also potently inhibits VEGF‐induced tube formation of endothelial cells co‐cultured with fibroblasts. Oral administration of TAK‐593 exhibited strong anti‐tumor effects against various human cancer xenografts along with good tolerability despite a low level of plasma exposure. Even after the blood and tissue concentrations of TAK‐593 decreased below the detectable limit, a pharmacodynamic marker (phospho VEGFR2) was almost completely suppressed, indicating that its long duration of enzyme inhibition might contribute to the potent activity of TAK‐593. Immunohistochemical staining indicated that TAK‐593 showed anti‐proliferative and pro‐apoptotic effects on tumors along with a decrease of vessel density and inhibition of pericyte recruitment to microvessels in vivo. Furthermore, dynamic contrast‐enhanced magnetic resonance imaging revealed that TAK‐593 reduced tumor vessel permeability prior to the onset of anti‐tumor activity. In conclusion, TAK‐593 is an extremely potent VEGFR/PDGFR kinase inhibitor whose potent anti‐angiogenic activity suggests therapeutic potential for the treatment of solid tumors.

Pharmacological evaluation of pioglitazone and candesartan cilexetil in a novel mouse model of non-alcoholic steatohepatitis, modified choline-deficient, amino acid-defined diet fed low-density lipoprotein receptor knockout mice

Low‐density lipoprotein receptor knockout (LDLR‐KO) mice fed a modified choline‐deficient and amino acid‐defined (mCDAA) diet show non‐alcoholic steatohepatitis (NASH)‐like pathophysiology. In order to pharmacologically benchmark this model, effects of pioglitazone (a thiazolidinedione) and candesartan cilexetil (an angiotensin II type 1 receptor blocker) on steatosis and liver fibrosis were examined.

Non-alcoholic steatohepatitis-associated hepatic fibrosis and hepatocellular carcinoma in a combined mouse model of genetic modification and dietary challenge

Experimental models of non‐alcoholic steatohepatitis (NASH) are still required for understanding the pathophysiology of this disease. This study aimed to examine whether disease progression is accelerated by combining dyslipidemic genetic modification and dietary challenges and develop NASH‐associated hepatic fibrosis, cirrhosis, and carcinoma in a short period.

Abstract 2518: TAK-733 is a Potent and Selective Inhibitor of Mammalian MEK Kinases Capable of Inducing Apoptosis in BRAF Driven Cells Both in vitro and in vivo

Mutations in BRAF a serine/threonine protein kinase are frequently observed in human cancers. A valine to glutamic acid mutation at amino acid position 600 accounts for nearly 80% of all the activating mutations found for this kinase. This mutation (V600E) forces BRAF to become constitutively active and phosphorylate its substrates regardless of upstream signaling. Numerous cancers in tissues such as skin, colon, thyroid, and ovary have been attributed to uncontrolled proliferation driven by this mutation. TAK-733 represents a novel and distinct chemotype which is capable of allosteric inhibition of the BRAF substrates MEK-1 and MEK-2 at low nanomolar concentrations. Viability assays in established human cancer cell lines which bear the BRAF V600E mutation demonstrate this compound was also capable of lowering NADH levels with single digit nanomolar EC50s. Loss of NADH production can be attributed to the induction of programmed cell death pathways involving Caspases 3 and 7. Induction of these apoptosis markers was confirmed in vitro by a Caspase-GLO assay. TAK-733 induces apoptosis more potently in COLO 205 (BRAF V600E) human colo-rectal carcinoma cells when compared to other MEK inhibitors. Pharmacodynamic analysis of COLO 205 tumor bearing mice confirmed TAK-733, unlike other MEK inhibitors tested, is capable of inducing apoptosis 36 hours after a single oral administration. To determine whether the anti-tumor effect of TAK-733 is associated with apoptosis, A375 (BRAF V600E) human melanoma tumors were harvested at sacrifice and protein extracted to determine cleavage products of PARP. A single oral treatment with 1, 10, and 30 mg/kg TAK-733 increased protein expression of cleaved PARP in a dose and time dependent manner. Elevated cleaved PARP protein expression was detected as early as 0.5-hours in tumors following 30 mg/kg dose and detectable at all groups by 4-hours. The increase cleaved PARP protein levels persisted for at least 24-hours and returned to control expression levels by 48-hours. Daily oral administration of 1, 10, and 30 mg/kg/day TAK-733 for 14 days potently inhibited tumor growth resulting in tumor growth delay of 3.4, 10.8, and 26.6 days in A375 cell implanted mice. A 60% partial regression response rate (tumor volume reduced by more than 50% of initial volume) was observed in mice that received 30 mg/kg/day TAK-733. These data suggest a possible explanation as to why TAK-733 is capable of inducing regressions in COLO 205 and A375 tumor growth inhibition studies, and may represent a differentiation point from other MEK inhibitors currently in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2518.

Abstract 2516: Antitumor effects of TAK-733, a novel MEK1/2 inhibitor, against mesothelioma cells

Malignant mesothelioma (MM) is a highly aggressive tumor characterized by local invasion of the pleura and metastasis, and is regarded as an almost incurable cancer with increasing incidence worldwide. Various cytokines and growth factors are reported to play important roles in the physiology of MM, possibly through MEK/ERK cascade. Thus, antitumor effects of TAK-733, a highly potent, selective, and non-ATP competitive inhibitor of mitogen-activated protein kinase kinase 1/2 (MEK1/2), were investigated against MM cell lines, NCI-H226, MSTO-211H, NCI-H2052, NCI-H2452, and NCI-H28 harboring wild-type KRAS and BRAF and display moderate levels of basal ERK phosphorylation. TAK-733 completely inhibited the ERK phosphorylation of 5 mesothelioma cell lines at 1-10 nM. TAK-733 also inhibited the proliferation of these cell lines in a dose dependent manner (IC 50 = 4.9-240 nM). Treatment with TAK-733 clearly induced G1 cell cycle arrest with cyclin D1 down regulation and without up regulation of cleaved PARP in NCI-H226 and MSTO-211H cells. Once daily, oral administration of 1, 3, and 10 mg/kg TAK-733 potently inhibited the tumor growth of MSTO-211H xenograft in nude mice with T/C values of 31, 8, and −4% (T/C: treated per control on tumor volume growth over treatment period), respectively, with good tolerability. TAK-733 also showed potent antitumor activities against NCI-H226 and NCI-H2052 xenografts. Additionally, in an intrapleural MSTO-211H xenograft nude mouse model, daily oral administration of 3 and 10 mg/kg TAK-733 for 14 days significantly increased survival. These results demonstrate that TAK-733 is useful for the treatment of malignant mesothelioma with an activate MEK-ERK pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2516.

Combination effects of alogliptin and pioglitazone on steatosis and hepatic fibrosis formation in a mouse model of non-alcoholic steatohepatitis

This study aimed to evaluate the effects of combination therapy with a dipeptidyl peptidase-4 inhibitor, alogliptin, and a peroxisome proliferator-activated receptor-γ agonist, pioglitazone, in a preclinical model of nonalcoholic steatohepatitis using low-density lipoprotein receptor-knockout mice fed a modified choline-deficient l-amino acid-defined diet. Monotherapy with either alogliptin (10-200 mg/kg) or pioglitazone (6-20 mg/kg) significantly decreased hepatic triglyceride content and fibrosis. The concomitant treatment of alogliptin (30 mg/kg), pioglitazone (20 mg/kg) also decreased hepatic triglyceride and hepatic collagen-I mRNA at greater extent compared to monotherapy. Hepatic expression of CD11b mRNA and monocyte chemoattractant protein-1 were also reduced by the concomitant treatment. These results suggest that via an anti-inflammatory potential in addition to anti-metabolic effects, the combination therapy of alogliptin and pioglitazone may provide therapeutic benefits to type 2 diabetes patients with nonalcoholic steatohepatitis, which will be proven in controlled clinical trials.

Abstract 4247: Characterization of the selective pan-RAF inhibitor TAK-632 with antitumor activity in BRAF inhibitor-resistant melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Melanoma is one of the deadliest and most aggressive forms of skin cancer, arising from the malignant transformation of pigment-producing cells, melanocytes. The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells and somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, it has been reported that the BRAF inhibitors vemurafenib and dabrafenib showed high response rates and improved overall survival in melanomas with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild-type BRAF (including NRAS mutant melanoma) are clinical concerns. In this preclinical study, we describe the biological characterization of TAK-632 as a potent and selective pan-RAF inhibitor that suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. We used both genetic and chemical approaches to investigate the dependence of NRAS-mutated melanoma and BRAF inhibitor-resistant BRAF mutant melanoma cells on RAF. Our analysis reveals that the MAPK pathway and proliferation of these cells is highly dependent on RAF. Such dependence was not observed in several RAS/RAF-wild type and KRAS-mutated cells. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. Furthermore, we demonstrate that the combination of TAK-632 and various MAPK kinase (MEK) inhibitors exhibits synergistic anti-proliferative effects on these cells. Our findings indicate that TAK-632 has favorable characteristics in terms of suppressing the proliferation of NRAS mutant melanoma and BRAF inhibitor-resistant BRAF mutant melanoma cells. Citation Format: Akito Nakamura, Takeo Arita, Shuntarou Tsuchiya, Jill Donelan, Jouhara Chouitar, Elizabeth Carideo, Katherine Galvin, Masanori Okaniwa, Tomoyasu Ishikawa, Sei Yoshida. Characterization of the selective pan-RAF inhibitor TAK-632 with antitumor activity in BRAF inhibitor-resistant melanoma. [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 4247. doi:10.1158/1538-7445.AM2014-4247

Abstract 2520: TAK-733, a novel MEK1/2 inhibitor, inhibits tumor angiogenesis without affecting vascular permeability

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC TAK-733 is a highly potent, selective, and non-ATP competitive inhibitor of mitogen-activated protein kinase kinase 1/2 (MEK1/2). In preclinical studies, TAK-733 exhibits potent antitumor activity against a variety of human tumor xenografts, but does not affect vascular permeability. The effects of TAK-733 on tumor angiogenesis were investigated in this preclinical study. TAK-733 completely inhibited the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)-induced ERK phosphorylation in human umbilical vein endothelial cells (HUVECs) at 100 nM. TAK-733 also inhibited VEGF- and FGF-induced HUVEC proliferation (IC50 = 1.3 and 18 nM, respectively) and HUVEC tube formation cocultured with fibroblasts (IC50 = 0.49 and 0.34 nM, respectively). The anti-angiogenic effect of TAK-733 in vivo was evaluated in a Matrigel plug assay following daily oral administration of TAK-733 at 3 and 10 mg/kg for 7 days. TAK-733 significantly inhibited the bFGF-induced hemoglobin accumulation in Matrigel plugs. TAK-733 significantly suppressed micro vessel density, examined by immunostaining with an anti-CD31 (PECAM) antibody, in human lung carcinoma A549 xenografts 4 and 7 days after initial treatment of 3 and 10 mg/kg. This was associated with significant antitumor effects in these xenograft models. These results indicate that potent anti-angiogenic activity of TAK-733 contributes to the anti-tumor activities. Angiogenesis inhibitors that modulate VEGF or VEGFR have all displayed hypertensive toxicity. Lack of nitric oxide production as a result of VEGF inhibition produces vasoconstriction and decreased vascular permeability leading to hypertensive effects (Fukumura et. al, PNAS 98, p2604, 2001). The effects of TAK-733 and VEGFR tyrosine kinase inhibitor on tumor interstitial fluid pressure (IFP) were examined by using wick-in-needle method in A549 xenograft model. TAK-733 did not affect the tumor IFP 6 hours after single administration of 30 mg/kg, while the VEGFR tyrosine kinase inhibitor significantly decreased it at 60 mg/kg. Consistent with the effects on tumor IFP, TAK-733 did show change of the vascular permeability unlike the VEGFR tyrosine kinase inhibitor by means of the Evans blue extravasation technique in A549 xenograft model. Although TAK-733 shows potent antiangiogenic effect, these profiles of TAK-733 on vascular permeability would not be expected to induce the typical toxicity of VEGF inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2520.

Abstract 2523: BRAF is not the only predictor of sensitivity in vitro and in vivo for TAK-733, a selective potent inhibitor of MEK1/2

MEK has emerged as an attractive target for pharmacologic intervention in cancer as the RAS/RAF/MEK/ERK cascade has proven to play a central role in the signaling required for cellular transformation and proliferation. Inhibition of MEK impacts a diverse array of additional cellular events, including differentiation, apoptosis, and angiogenesis. Identification of an orally active inhibitor of MEK which shows significant efficacy as a mono-therapy for the treatment of cancer would be ideal. TAK-733 is a potent non-ATP competitive allosteric inhibitor of MEK1/2 with IC 50 values for the inhibition of MEK1/2 signaling of 2-5 nM at high (400 μM) and low (10 μM) ATP concentrations. TAK-733 was selective for mammalian MEK-1/2 in a panel of 71 enzyme assays. These included phosphodiesterases, 17 kinases (IC 50 values >25 µM), cyclases, and 14 other enzymes (IC 50 values >10 µM). Using an MTS (a tetrazolium salt) viability assay, TAK-733 was found to inhibit proliferation of numerous cancer cell lines expressing BRAF, KRAS, NRAS, PI3K, MEK1, EGFR mutations, cMET amplification, and wild type MAPK signaling with EC 50 values ranging from 2 to 90 nM. A quantitative PK/PD relationship was established in the HT-29 human colorectal carcinoma murine xenograft model. This model showed a range of PD activity (ie, from no effect to maximum effect) which correlated well with tumor and plasma TAK-733 concentrations. The relationship between TAK-733 plasma concentration (PK) and target modulation (PD) was evaluated by modeling percent inhibition of tumor ERK phosphorylation and plasma TAK-733 concentrations at 4 hours postdose, using an inhibitory sigmoidal concentration response model. The plasma EC 50 was calculated to be 45 ng/mL. TAK-733 exhibited broad-spectrum in vivo antitumor activity against a panel of human tumor xenografts with and without mutations in the MAPK pathway. These xenografts were of colon (COLO 205/ BRAF V600E and HT-29/ BRAF V600E/PI3K P449T), NSCLC (A549/ KRAS G12S, PC-9/ EGFRΔE746-A750, NCI-H1975/ EGFR L858R T790M), AML (HL-60/ NRAS), cholangio-carcinoma (EGI-1/ KRAS G12D) and melanoma (A375/ BRAF V600E) origins. A high incidence of tumor regressions was observed in this collection of cancer models. Collectively, preclinical pharmacology studies indicate TAK-733 is a specific inhibitor of MEK with the potential to significantly impair growth of a large number of cancers relying on the MAPK signaling cascade for growth and survival. and provides a strong rationale for study of this agent in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2523.