Toshiyasu Shimomura

MK-5108, a Highly Selective Aurora-A Kinase Inhibitor, Shows Antitumor Activity Alone and in Combination with Docetaxel

Aurora-A kinase is a one of the key regulators during mitosis progression. Aurora-A kinase is a potential target for anticancer therapies because overexpression of Aurora-A, which is frequently observed in some human cancers, results in aberrant mitosis leading to chromosomal instability and possibly tumorigenesis. MK-5108 is a novel small molecule with potent inhibitory activity against Aurora-A kinase. Although most of the Aurora-kinase inhibitors target both Aurora-A and Aurora-B, MK-5108 specifically inhibited Aurora-A kinase in a panel of protein kinase assays. Inhibition of Aurora-A by MK-5108 in cultured cells induced cell cycle arrest at the G2-M phase in flow cytometry analysis. The effect was confirmed by the accumulation of cells with expression of phosphorylated Histone H3 and inhibition of Aurora-A autophosphorylation by immunostaining assays. MK-5108 also induced phosphorylated Histone H3 in skin and xenograft tumor tissues in a nude rat xenograft model. MK-5108 inhibited growth of human tumor cell lines in culture and in different xenograft models. Furthermore, the combination of MK-5108 and docetaxel showed enhanced antitumor activities compared with control and docetaxel alone–treated animals without exacerbating the adverse effects of docetaxel. MK-5108 is currently tested in clinical trials and offers a new therapeutic approach to combat human cancers as a single agent or in combination with existing taxane therapies. Mol Cancer Ther; 9(1); 157–66

Expression levels of p18INK4C modify the cellular efficacy of cyclin-dependent kinase inhibitors via regulation of Mcl-1 expression in tumor cell lines.

Because cyclin-dependent kinases (CDK) play a pivotal role in cancer progression, the development of CDK inhibitors has attracted attention in antitumor therapy. However, despite significant preclinical and clinical developments, CDK inhibition biomarkers for predicting efficacy against certain cancers in individual patients have not been identified. Here, we characterized a macrocyclic quinoxalin-2-one CDK inhibitor, compound A, and identified a gene biomarker for predicting its efficacy. Compound A showed 100-fold selectivity for CDK family proteins over other kinases and inhibited both E2F transcriptional activity and RNA polymerase II phosphorylation. Compound A treatment resulted in decreased proliferation in various tumor cell lines; however, the apoptosis induction rate differed significantly among the cell lines examined, which was consistent with roscovitine. By comparing the mRNA expression profiles of sensitive and resistant cell lines, we found that expression levels of an endogenous CDK inhibitor, p18INK4C, showed a strong negative correlation to the sensitivity. In fact, p18 status was correlated with the response to CDK inhibitor in an independent data set of multiple myeloma cell lines and silencing p18 expression increased the susceptibility of resistant cells to CDK inhibitors. The analysis of molecular mechanisms revealed that cells with lowered p18 had aberrant CDK6 and E2F activities, which resulted in a transcriptional down-regulation of Mcl-1, a key molecule associated with flavopiridol-induced apoptosis, thereby leading to susceptibility to therapeutic intervention with CDK inhibitors. These results identified a molecular basis for CDK inhibitors to exert an antitumor effect in p18-deficient cancers and support the clinical use of CDK inhibitors. [Mol Cancer Ther 2009;8(6):1460–72]

Biological characterization of 2-aminothiazole-derived Cdk4/6 selective inhibitor in vitro and in vivo

Abnormalities in the p16INK4a/ cyclin-dependent kinase (Cdk)4, 6/ Retinoblastoma (Rb) pathway frequently occur in various human cancers. Thus, Cdk4/6 is an attractive target for cancer therapy. Here we report the biological characterization of a 2-aminothiazole-derived Cdk4/6 selective inhibitor, named Compound A in vitro and in vivo. Compound A potently inhibits Cdk4 and Cdk6 with high selectivity (more than 57-fold) against other Cdks and 45 serine/threonine and tyrosine kinases. Compound A inhibits Rb protein (pRb) phosphorylation at Ser780, inhibits E2F-dependent transcription, and induces cell-cycle arrest at G1 in the T98G human glioma cell line. Among 82 human cells derived from various tissues, cell lines derived from hematological cancers (leukemia/lymphoma) tended to be more sensitive to Compound A in cell proliferation assay. Rb-negative cells tended to be insensitive to Compound A, as we had expected. In a nude rat xenograft model, Compound A inhibited pRb phosphorylation and bromodeoxyuridine (BrdU) incorporation in Eol-1 xenograft tumor at plasma concentration of 510 nM. Interestingly Compound A only moderately inhibited those pharmacodynamic and cell cycle parameters of normal crypt cells in small intestine even at 5 times higher plasma concentration. In F344 rats, Compound A did not cause immunosuppression even at 17 times higher plasma conc. These results suggest that Cdk4/6 selective inhibitors only moderately affects on the cell cycle of normal proliferating tissues and has a safer profile than pan-Cdk inhibitor in vivo.

Abstract C177: TAS-117, a highly selective non-ATP competitive inhibitor of AKT demonstrated antitumor activity in combination with chemotherapeutic agents and molecular targeted drugs.

Background: TAS-117 is a potent and highly selective non-ATP competitive small molecule inhibitor of AKT. AKT controls cellular proliferation and resistance to apoptosis, and is known to be activated in patients with relapsed or refractory tumors. Thus the combination of TAS-117 and cytotoxic drugs could be a promising approach to treat cancers. In addition, recent preclinical and clinical studies suggest that the vertical inhibition of key cancer signaling pathways may be an effective way to minimize crosstalk signaling and compensatory feedback activation. Therefore, we evaluated these combination approaches using TAS-117. Materials and Methods: A PTEN negative human ovarian cancer cell line A2780 and HER2 overexpressing human gastric cancer cell lines NCI-N87 and 4-1ST were used. In vitro combination studies were conducted by using CellTiter Glo™ assays. For in vivo studies, cells were subcutaneously transplanted into the side flank of nude mice. TAS-117, S-1, lapatinib, and everolimus were given by daily oral dosing. Carboplatin and irinotecan were given by weekly i.v. bolus injection. Trastuzumab was given by weekly i.p. bolus injection. Tumor volumes and body weights were measured twice a week. Apoptosis induction and pathway inhibition were determined by immunoblotting. Results: The in vitro combination studies revealed that TAS-117 enhanced the cytotoxicity of cisplatin, SN38, and fluorouracil in A2780 cells. In vivo, TAS-117 significantly improved the antitumor effect of carboplatin or irinotecan in an A2780 xenograft model. TAS-117 also enhanced the antitumor effect of S-1 in a 4-1ST xenograft model. With downstream, mTOR inhibitor everolimus, TAS-117 exhibited synergistic growth inhibition in NCI-N87 cells. Interestingly, cleavage of PARP was induced by the combination of TAS-117 and everolimus but not by either single agent alone. Enhanced antitumor activity in combination with TAS-117 and everolimus was confirmed in the NCI-N87 xenograft model. With an upstream, HER2 inhibitor lapatinib, TAS-117 also exhibited synergistic growth inhibition in vitro and enhanced antitumor activity in vivo in the NCI-N87 model. The combinatorial effect of TAS-117 and HER2 blockade was further evaluated using trastuzumab. TAS-117 strongly enhanced the antitumor effect of trastuzumab in a 4-1ST xenograft model. Conclusion: TAS-117 was effective in combination with chemotherapeutic agents including paclitaxel, carboplatin, irinotecan and S-1. Vertical pathway inhibition by TAS-117 with trastuzumab, lapatinib and everolimus were also effective. Our studies provide a strong rationale for clinical evaluation of our highly selective AKT inhibitor TAS-117 in combination with chemotherapeutic agents and molecular targeted drugs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C177. Citation Format: Koji Ichikawa, Tetsuya Abe, Hideki Nagase, Hitoshi Saito, Ryouto Fujita, Megumu Okada, Kazuhiko Yonekura, Toshiyasu Shimomura, Teruhiro Utsugi. TAS-117, a highly selective non-ATP competitive inhibitor of AKT demonstrated antitumor activity in combination with chemotherapeutic agents and molecular targeted drugs. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C177.

Abstract C178: TAS-117, a highly potent and selective non-ATP competitive inhibitor of AKT, demonstrates a toxicity profile with manageable hyperglycemia in rats.

Background: TAS-117 is a small molecule which inhibits the PI3K-AKT-mTOR signaling pathway, a critical regulator of cell survival, by stimulating cell proliferation and inhibiting apoptosis. One of the most notable AKT-related adverse events is hyperglycemia which has been a dose limiting toxicity (DLT) in some clinical trials. We elucidate the time-dependent changes of hyperglycemia induced by TAS-117 and try an approach to manage the hyperglycemia in the present study. Materials and Methods: TAS-117 was orally administered to male rats repeatedly for 2 weeks (QD) to evaluate the toxicities of TAS-117. Blood glucose and insulin were measured continuously for 24 hours on the first and last day of dosing. Water intake recording and urinalysis were also performed to monitor for diabetes-related findings. Then, we treated the rats with varying anti-diabetic agents, gliclazide, mitiglinide calcium hydrate, pioglitazone hydrochloride, and metformin hydrochloride, in order to ameliorate the observed diabetes-like effects. Results: In continuous monitoring of blood glucose levels, TAS-117 induced transient elevation of blood glucose and insulin following daily oral administration. The peak level of blood glucose was observed between 4 and/or 8 hours after dosing. TAS-117-induced hyperglycemia reverted to baseline by 24 hours after dosing. Blood insulin levels correlatively increased and decreased with changes in blood glucose levels. Additionally, TAS-117 induced diabetes-like changes including body-weight-gain suppression, increases in water intake, urine volume, and urine glucose level were observed. These changes were deemed to be related to the hyperglycemia caused by TAS-117. However, the hyperglycemic effects of TAS-117 were most ameliorated by one of the anti-diabetic agents given, Metformin hydrochloride, an inhibitor of gluconeogenesis. The peak in blood glucose levels in Metformin medicated rats was suppressed up to 31% as compared with that in TAS-117 alone treated rats. Conclusion: TAS-117’s main toxic effects in rats were diabetes-like, including hyperglycemia and blood insulin elevation due to TAS-117’s dysregulation of carbohydrate metabolism. However, these adverse effects were transient and reversible and were controlled by a particular class of anti-diabetic agent. Therefore, TAS-117 has the potential to be administered on a daily schedule without inducing clinically significant hyperglycemia. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C178. Citation Format: Kenji Moriyama, Masahiro Yamaguchi, Shigeo Sugimoto, Hirofumi Matsumura, Fumiko Ninomiya, Kazuhiko Besshi, Fumio Morita, Taiji Hayashi, Ryouto Fujita, Hiroto Fukushima, Toshiyasu Shimomura, Kazuhiko Yonekura, Teruhiro Utsugi. TAS-117, a highly potent and selective non-ATP competitive inhibitor of AKT, demonstrates a toxicity profile with manageable hyperglycemia in rats. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C178.