Hiroshi Takemori

A novel compound ARN-3236 inhibits salt-inducible kinase 2 and sensitizes ovarian cancer cell lines and xenografts to paclitaxel

Purpose: Salt-inducible kinase 2 (SIK2) is a centrosome kinase required for mitotic spindle formation and a potential target for ovarian cancer therapy. Here, we examine the effects of a novel small-molecule SIK2 inhibitor, ARN-3236, on sensitivity to paclitaxel in ovarian cancer. Experimental Design: SIK2 expression was determined in ovarian cancer tissue samples and cell lines. ARN-3236 was tested for its efficiency to inhibit growth and enhance paclitaxel sensitivity in cultures and xenografts of ovarian cancer cell lines. SIK2 siRNA and ARN-3236 were compared for their ability to produce nuclear–centrosome dissociation, inhibit centrosome splitting, block mitotic progression, induce tetraploidy, trigger apoptotic cell death, and reduce AKT/survivin signaling. Results: SIK2 is overexpressed in approximately 30% of high-grade serous ovarian cancers. ARN-3236 inhibited the growth of 10 ovarian cancer cell lines at an IC50 of 0.8 to 2.6 μmol/L, where the IC50 of ARN-3236 was inversely correlated with endogenous SIK2 expression (Pearson r = −0.642, P = 0.03). ARN-3236 enhanced sensitivity to paclitaxel in 8 of 10 cell lines, as well as in SKOv3ip (P = 0.028) and OVCAR8 xenografts. In at least three cell lines, a synergistic interaction was observed. ARN-3236 uncoupled the centrosome from the nucleus in interphase, blocked centrosome separation in mitosis, caused prometaphase arrest, and induced apoptotic cell death and tetraploidy. ARN-3236 also inhibited AKT phosphorylation and attenuated survivin expression. Conclusions: ARN-3236 is the first orally available inhibitor of SIK2 to be evaluated against ovarian cancer in preclinical models and shows promise in inhibiting ovarian cancer growth and enhancing paclitaxel chemosensitivity. Clin Cancer Res; 23(8); 1945–54. ©2016 AACR.

Role of salt inducible kinase 1 in high glucose-induced lipid accumulation in HepG2 cells and metformin intervention

Aims: To investigate the roles of salt inducible kinase (SIK1) in high glucose‐induced triglyceride accumulation in human hepatoma HepG2 cells as well as in the molecular mechanism by which metformin, a drug to treat diabetes, suppresses high glucose‐induced lipogenesis. Main methods: A cell model for high glucose‐induced hepatic steatosis was prepared by exposing HepG2 cells to high glucose (25 mmol) in the absence or presence of metformin (0.5 mmol). Intracellular triglycerides were visualized by Oil Red O and measured using a triglyceride assay kit. Cell viability was evaluated using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide method. SIK1 overexpression in HepG2 cells was achieved by transient transfection, and the mRNA and protein levels of SIK1 and lipogenic factors were measured using a reverse transcription‐polymerase chain reaction and western blotting, respectively. Key findings: Lipid accumulation in HepG2 cells was obvious after treatment with high glucose for 24 h. In response to high glucose, SIK1 expression was negatively correlated with that of lipogenic factors and lipid accumulation in HepG2 cells. We observed that overexpression of SIK1, or treatment with metformin, suppressed lipogenesis, even in high glucose conditions. Furthermore, treatment with metformin upregulated SIK1 mRNA and protein levels, as well as the active form of SIK1. Significance: SIK1 plays a vital role in high glucose‐induced lipid accumulation, and metformin suppresses lipogenesis via the induction and activation of SIK1.

Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+/K+‐ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate‐mediated excitotoxicity. Under a pyruvate‐restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 μM. The OCR monitoring also revealed the contribution of the N‐methyl‐D‐aspartate receptor and Na+/K+‐ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte‐dependent protection of neurons from glutamate‐mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.

Carnosol suppresses IL-6 production in mouse lungs injured by ischemia-reperfusion operation and in RAW264.7 macrophages treated with lipopolysaccharide

Carnosol is a naturally occurring herbal compound, known for its antioxidative properties. We previously found that carnosol protected mouse lungs from ischemia-reperfusion injury in ex vivo cultures. To elucidate the molecular mechanisms underpinning carnosol-mediated lung protection, we analyzed modes of interleukin-6 (IL-6) gene expression, which is associated with lung ischemia-reperfusion injury. Microarray analysis of mouse lungs suggested that IL-6 mRNA levels were elevated in the mouse lungs subjected to clamp-reperfusion, which was associated with elevated levels of other inflammatory modulators, such as activating transcription factor 3 (ATF3). Carnosol pretreatment lowered the IL-6 protein levels in mouse lung homogenates prepared after the clamp-reperfusion. On the other hand, the ATF3 gene expression was negatively correlated with that of IL-6 in RAW264.7 cells. IL-6 mRNA levels and gene promoter activities were suppressed by carnosol in RAW264.7 cells, but rescued by ATF3 knockdown. When RAW264.7 cells were subjected to hypoxia-reoxygenation, carnosol treatment lowered oxygen consumption after reoxygenation, which was coupled with a correlation with a transient production of mitochondrial reactive oxygen species and following ATF3 gene expression. These results suggest that carnosol treatment could be a new strategy for protecting lungs from ischemia-reperfusion injury by modulating the ATF3-IL-6 axis.

Abstract LB-296: Discovery of ARN-3261 as a potent, selective, orally available SIK2 inhibitor for treating ovarian, endometrial, primary peritoneal, fallopian tube, and triple negative breast cancers

ARN-3261 is an orally bioavailable small molecule inhibitor of the Salt Inducible Kinase 2 (SIK2, 11 nM) and SIK3 (19 nM). Three isoforms of SIK (SIKs) proteins have been reported: SIK1 (SNF1LK), SIK2 (QIK), and SIK3 (QSK). They are the Ser/Thr centrosome kinase family members required for bipolar mitotic spindle formation. The overexpression of SIK2 kinase in 30% of ovarian cancer specimens allows a novel, clinically important new method of treating ovarian cancer by blocking SIK2 kinase activity. In addition to a role in ovarian cancer, SIK2 and SIK3 are prevalent in several other tumor types, including breast, prostate diffuse large B-cell lymphoma, and melanoma cancers. Inhibition of SIK2 has been reported to cause centrosome splitting in interphase, while SIK2 depletion blocked centrosome separation in mitosis and sensitized ovarian cancers to paclitaxel in culture and in vivo Xenograft models. Depletion of SIK2 also delayed G1/S transition and reduced AKT phosphorylation. Higher levels of expression of SIK2 have been shown to be highly correlated with poor survival in patients with high-grade serous ovarian cancers. Using the homology structure of SIK2, fragment-based lead optimization strategies, and screening and structure-activity relationship efforts, we have discovered ARN-3261, a first-in-class novel, selective inhibitor of SIK2 that could prove useful in treating ovarian, endometrial, primary peritoneal, fallopian tube, and triple negative breast cancers. ARN-3261 specifically inhibited SIK2-expressed SKOv3 cells with an IC50 of 92 nM. ARN3261 was effective against ovarian, breast cancer cell lines alone and in combination with Paclitaxel and Cisplatin. ARN-3261 also inhibited ovarian tumor growth significantly at 70% in SKOv3 human ovarian cancer Xenografts in Ncr nu/nu mice in a dose dependent manner at 20, 40, 60, and 100 mg/kg orally. Moreover, ARN-3261 has exhibited excellent in vivo pharmacokinetic, pharmacodynamics, and correlative PK/PD and ADME characteristics. Preliminary in vitro and in vivo tumor up-take studies suggest that ARN-3261 blocks centrosome separation by inhibiting SIK2, thereby enhancing the sensitivity of Paclitaxel. Encouraged by these results, we initiated the IND enabling GLP-Toxicology and safety studies to bring ARN-3261 to the clinic for First-In-Human (FIH) Phase 1 trials. The non-clinical pharmacology data along with Phase 1 POC clinical trial plans will be presented. Citation Format: Hariprasad Vankayalapati, Venkatakrishnareddy Yerramreddy, Jinhua Zhou, Jeffrey A. Handler, Rajendra P. Appalaneni, Ramamohan R. Kancherla, Roy J. Wu, Hiroshi Takemori, Angelique Whitehurst, Amir Anthony Jazaeri, Robert L. Coleman, Zhen Lu, Robert C. Bast. Discovery of ARN-3261 as a potent, selective, orally available SIK2 inhibitor for treating ovarian, endometrial, primary peritoneal, fallopian tube, and triple negative breast cancers [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 LB-296. doi:10.1158/1538-7445.AM2017-LB-296