Masaki Kiga

Antitumor effects of novel highly hydrophilic and non-ATP-competitive MEK1/2 inhibitor, SMK-17

The mitogen-activated protein kinase (MAPK) signal pathway plays a central role in regulating tumor cell proliferation, survival, and differentiation. The components of this pathway, Ras/Raf/MEK/ERK, are frequently activated in human cancers. Targeting this pathway is considered to be a promising anticancer strategy. In particular, MEK is an attractive drug target because of its high selectivity to ERK. We can expect potent growth inhibitory and proapoptotic effects by inhibiting MEK. Here, we report derivatives of N-[2-(2-chloro-4-iodo-phenylamino)-3,4-difluorophenyl]-methanesulfonamide as novel MEK1/2 inhibitors. Among these compounds, we found SMK-17 to be a potent MEK1/2 inhibitor with high aqueous solubility. The in-silico docking study suggested that SMK-17 is bound to an allosteric pocket of MEK1. The kinetic study and the kinase profiler analysis confirmed the allosteric nature of SMK-17. SMK-17 inhibited MEK1 kinase activity in a non-ATP-competitive manner and it was highly selective to MEK1 and 2. SMK-17 inhibited the growth of tumor cell lines in vitro. Especially, it seemed that cell lines harboring highly phosphorylated MEK1/2 and ERK1/2 were highly sensitive to SMK-17. Moreover, unlike previously reported MEK inhibitors, PD184352 or U0126, SMK-17 did not inhibit the phosphorylation of ERK5. In vivo, SMK-17 exhibited potent antitumor activity in animal models on oral administration. SMK-17 selectively blocked the MAPK pathway signaling without affecting other signal pathways, which resulted in significant antitumor efficacy without notable side effects. These findings suggest that SMK-17, an exquisitely selective, orally available MEK1/2 inhibitor, is a useful chemical biology tool for characterizing the function of MEK/MAPK signaling both in vitro and in vivo.

Mitochondrial uncoupler exerts a synthetic lethal effect against β-catenin mutant tumor cells

The wingless/int‐1 (Wnt) signal transduction pathway plays a central role in cell proliferation, survival, differentiation and apoptosis. When β‐catenin: a component of the Wnt pathway, is mutated into an active form, cell growth signaling is hyperactive and drives oncogenesis. As β‐catenin is mutated in a wide variety of tumors, including up to 10% of all sporadic colon carcinomas and 20% of hepatocellular carcinomas, it has been considered a promising target for therapeutic interventions. Therefore, we screened an in‐house natural product library for compounds that exhibited synthetic lethality towards β‐catenin mutations and isolated nonactin, an antibiotic mitochondrial uncoupler, as a hit compound. Nonactin, as well as other mitochondrial uncouplers, induced apoptosis selectively in β‐catenin mutated tumor cells. Significant tumor regression was observed in the β‐catenin mutant HCT 116 xenograft model, but not in the β‐catenin wild type A375 xenograft model, in response to daily administration of nonactin in vivo. Furthermore, we found that expression of an active mutant form of β‐catenin induced a decrease in the glycolysis rate. Taken together, our results demonstrate that tumor cells with mutated β‐catenin depend on mitochondrial oxidative phosphorylation for survival. Therefore, they undergo apoptosis in response to mitochondrial dysfunction following the addition of mitochondrial uncouplers, such as nonactin. These results suggest that targeting mitochondria is a potential chemotherapeutic strategy for tumor cells that harbor β‐catenin mutations.

SMK-17, a MEK1/2-specific inhibitor, selectively induces apoptosis in β-catenin-mutated tumors

Although clinical studies have evaluated several MEK1/2 inhibitors, it is unlikely that MEK1/2 inhibitors will be studied clinically. BRAF mutations have been proposed as a responder marker of MEK1/2 inhibitors in a preclinical study. However, current clinical approaches focusing on BRAF mutations have shown only moderate sensitivity of MEK1/2 inhibitors. This has led to insufficient support for their promoted clinical adoption. Further characterization of tumors sensitive to MEK inhibitors holds great promise for optimizing drug therapy for patients with these tumors. Here, we report that β-catenin mutations accelerate apoptosis induced by MEK1/2 inhibitor. SMK-17, a selective MEK1/2 inhibitor, induced apoptosis in tumor cell lines harboring β-catenin mutations at its effective concentration. To confirm that β-catenin mutations and mutant β-catenin-mediated TCF7L2 (also known as TCF4) transcriptional activity is a predictive marker of MEK inhibitors, we evaluated the effects of dominant-negative TCF7L2 and of active, mutated β-catenin on apoptosis induced by MEK inhibitor. Indeed, dominant-negative TCF7L2 reduced apoptosis induced by MEK inhibitor, whereas active, mutated β-catenin accelerated it. Our findings show that β-catenin mutations are an important responder biomarker for MEK1/2 inhibitors.

Abstract B68: Screening for the compound that induces cell death selectively in β-catenin mutant tumor cells

The Wnt signal transduction pathway plays a central role for the cell proliferation, differentiation and apoptosis. β-catenin, a component of Wnt pathway, translocates to nucleus and forms an active complex with TCF4, leading to activate cell growth signaling, and this activity is tightly regulated by the “destruction complex” consisting of Axin, APC, GSK3β and CK1α. However, when β-catenin is actively mutated, this cell growth signaling would be hyperactive and drive oncogenesis. As β-catenin is mutated in up to 10% of all sporadic colon carcinomas resulting from point mutations or in-frame deletions of serine and threonine residues phosphorylated by GSK3β, mutated β-catenin has been considered as promising targets for therapeutic intervention. Therefore, we screened the compound that induced cell death selectively in tumor cell lines harboring β-catenin mutation from an in-house natural product library, and finally we isolated and found that nonactin, a well-known antibiotics, exhibited this activity. To confirm its apoptosis-inducing ability selectivity in β-catenin mutated cell lines, we examined the effect of nonactin on cell viability in 15 human tumor cell lines. Nonactin potently induced PARP-cleavage in 5 cell lines harboring β-catenin mutation (A427, HCT116, LS-174T, SK-MEL-1, SW48 cells), whereas the others, which do not harbor a mutation of β-catenin activation, were resistant to nonactin. In addition, overexpression of actively mutated β-catenin accelerated apoptosis induced by nonactin in the nonactin-resistant A375 cells, and β-catenin knockdown by siRNA reduced nonactin-induced apoptosis in HCT116 cells. Furthermore, nonactin induced tumor regression only in β-catenin mutated HCT116 xenograft mice. Taken together, these findings suggested that the β-catenin mutated tumor cell lines are highly sensitive to nonactin, and it would be potential drug-seed for tumor cells harboring β-catenin mutation. Citation Format: Yuki Shikata, Masaki Kiga, Etsu Tashiro, Masaya Imoto. Screening for the compound that induces cell death selectively in β-catenin mutant tumor cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B68.

Abstract B184: In vitro and in vivo profiling of novel mTORC1/2 inhibitor DS-3078a.

DS[[Unable to Display Character: ‑]]3078a is a potent, selective small molecule inhibitor of the catalytic site of the mammalian target of rapamycin (mTOR). Our previous study showed DS-3078a to inhibit mTOR kinase activity in a concentration-dependent manner with an IC50 (concentration resulting in 50% of maximum inhibition) of approximately single digit nano molar. Here the antitumor potency of DS-3078a against various cancer cell lines in vitro and in vivo is shown. DS-3078a was applied to a 230 cell line panel assay to evaluate cell growth inhibition and apoptosis induction activity. In this study, DS-3078a was found to be a highly cytostatic compound with strong cell growth inhibiting activity. DS-3078a exhibited GI50s lower than 500 nM against over 200 cell lines and a 5-fold and higher caspase activation against only 36 cell lines. Among 12 in vivo xenograft models, 9 of 12 xenograft models demonstrated greater than a 50% tumor growth inhibition response at half of MTD dosing. There are several well-known selective mTOR kinase inhibitors including AZD8055 and OSI-027. When comparing the cellular response among these compounds, DS-3078a was found to have a weaker induction of the sub G1 population against AN3 CA human endometrial carcinoma cell line than the other two compounds, despite its similar kinase inhibiting profile against them. In a previous study, an increase of the phospho Akt(T308) by DS-3078a treatment in A498 human renal cell carcinoma, which was a sign of negative feedback loop disruption by inhibition of mTORC1 was demonstrated. In this model, AZD8055 and OSI-027 did not increase phospho Akt(T308), which implies inhibition of the PI-3K signal at higher concentrations. To understand the impact of the cytostatic characteristics of DS-3078a, in vivo mTOR signal inhibition at MTD using tumor bearing mouse was measured and compared to the signal inhibition pattern of AZD8055 and the PI-3K/mTOR dual inhibitor DS-7423. DS-3078a fully inhibited mTORC1 and mTORC2 downstream signals for 24 hr, while signal inhibition by DS-7423 was reduced by 18 hr. AZD8055 showed a similar pattern to DS-7423 at MTD dosing. This study highlights the unique characteristics of DS-3078a which may offer a unique differentiation of response and toxic profile compared to other mTOR inhibiting compounds in the clinical setting. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B184. Citation Format: Kenichi Wakita, Masaki Kiga, Noriko Togashi. In vitro and in vivo profiling of novel mTORC1/2 inhibitor DS-3078a. [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 B184.

Abstract B133: Antitumor effects of novel high-hydrophilic and non-ATP-competitive MEK1/2 inhibitor SMK-17.

The mitogen-activated protein kinase (MAPK) signal pathway plays a central role in regulating tumor cell proliferation, survival, and differentiation. The components of this pathway, Ras/Raf/MEK/ERK, are frequently activated in human cancers. Targeting this pathway is considered to be a promising anticancer strategy. In particular, MEK is an attractive drug target due to its high selectivity to ERK. We can expect potent growth inhibitory and proapoptotic effects by inhibiting MEK. We synthesized derivatives of N-[2-(2-Chloro-4-iodo-phenylamino)-3,4-difluorophenyl] -methanesulfonamide as novel MEK1/2 inhibitors. Among these compounds, we found SMK-17 to be a potent MEK1/2 inhibitor with high aqueous solubility. The in silico docking study suggested that SMK-17 be bound to an allosteric pocket of MEK1. The kinetics study and kinase profiler analysis confirmed the allosteric character of SMK-17. SMK-17 inhibited MEK1 kinase activity by a non-ATP-competitive manner and it was highly selective to MEK1 and 2. SMK-17 inhibited the growth of tumor cell lines in vitro. Especially, it seemed that cell lines harboring highly phosphorylated MEK1/2 and ERK1/2 were highly sensitive to SMK-17. Moreover, unlike previously reported MEK inhibitors, PD184352 or U0126, SMK-17 did not inhibit phosphorylation of ERK5. In vivo, SMK-17 exhibited potent antitumor activity in animal models by oral administration. SMK-17 selectively blocked the MAPK pathway signaling without affecting other signal pathways, which resulted in significant antitumor efficacy without notable side effects. In conclusion, we found very potent MEK inhibitor SMK-17 from our structure-activity correlation study of new derivative series of diphenyl amine sulfonamide. Kinase profiler and kinetics study revealed that SMK-17 is a non-ATP-competitive and highly selective MEK1/2 inhibitor. Moreover, SMK-17 exhibited potent antitumor activity in animal models by oral administration. SMK-17 selectively blocked the MAPK pathway signaling without affecting other signal pathways both in vitro and in vivo. These findings suggest that SMK-17 is a useful chemical biology tool for characterizing the function of MEK/MAPK signaling. Furthermore, we are now investigating the combination effects of SMK-17 with several drugs, and studying about prediction marker of this compound. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B133.