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Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase involved in key processes during mitosis. Human PLK1 has been shown to be overexpressed in various human cancers, and elevated levels of PLK1 have been associated with poor prognosis, making it an attractive target for anticancer therapy. TAK-960 [4-[(9-cyclopentyl-7,7-difluoro-5-methyl-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino]-2-fluoro-5-methoxy-N-(1-methylpiperidin-4-yl) benzamide] is a novel, investigational, orally bioavailable, potent, and selective PLK1 inhibitor that has shown activity in several tumor cell lines, including those that express multidrug-resistant protein 1 (MDR1). Consistent with PLK1 inhibition, TAK-960 treatment caused accumulation of G2–M cells, aberrant polo mitosis morphology, and increased phosphorylation of histone H3 (pHH3) in vitro and in vivo. TAK-960 inhibited proliferation of multiple cancer cell lines, with mean EC50 values ranging from 8.4 to 46.9 nmol/L, but not in nondividing normal cells (EC50 >1,000 nmol/L). The mutation status of TP53 or KRAS and MDR1 expression did not correlate with the potency of TAK-960 in the cell lines tested. In animal models, oral administration of TAK-960 increased pHH3 in a dose-dependent manner and significantly inhibited the growth of HT-29 colorectal cancer xenografts. Treatment with once daily TAK-960 exhibited significant efficacy against multiple tumor xenografts, including an adriamycin/paclitaxel-resistant xenograft model and a disseminated leukemia model. TAK-960 has entered clinical evaluation in patients with advanced cancers. Mol Cancer Ther; 11(3); 700–9. ©2011 AACR.

PDF file - 85K, Relationship between TP53 (A) or KRAS (B) gene status and TAK 960 concentration producing EC50 values. Each data point represents a mean concentration producing 50% efficacy (EC50) value for TAK 960 in an individual cell line.

PDF file - 144K, Comparison of dosing regimens.

PDF file - 61K

PDF file - 130K, Anti-tumor activity of TAK-960 in MV4-11luc disseminated leukemia model.

Colorectal cancer (CRC) is the third leading cause of cancer and cancer-related death. A large fraction of the CRC patients diagnosed with de novo metastatic disease do not benefit from standard of care and experience substantial side effects. Therefore, there is an urgent need for preclinical models that help predict patient response in the clinic. Patient-derived organoids (PDOs or HUB Organoids®) represent a significant breakthrough as preclinical models as they are directly established from patient tissue and faithfully recapitulate patient disease. HUB Organoid Technology can already be applied to preclinical drug screening, however, to provide direct patient benefit the turnaround time between patient diagnosis and PDO-based results must be shortened. In collaboration with Yamaha Motor, the Yamaha CELL HANDLERTM has been validated to automatically pick-and-place with high accuracy a significantly reduced number of organoids per screening plate, compared to standard procedures. In addition, an image-based readout was developed that enables a precise quantification of organoid number ensuring high assay quality. Organoid responses to chemotherapy and targeted agents were validated using this newly automated system, confirming comparable PDO-patient drug sensitivity profiles, and known drug responses based on genetic dependencies. In summary we describe the development of an automated workflow that combines patient-representative HUB Organoids and state-of-the-art robotics by Yamaha CELL HANDLER. This workflow down-scales the number of organoids needed per screening well and allows to efficiently predict patient response, thereby reducing the diagnostic turnaround time and increasing patient benefits. Citation Format: Esmee Koedoot, Inez van Weersch, Gakuro Harada, Masahiko Watanabe, Hamdy Warda, Hideaki Kyan, Yasmine Abouleila, Takahiko Kumagai, Yuichi Hikichi, René Overmeer, Jeanine Roodhart, Kiyotaka Matsuno, Carla Verissimo, Sylvia F. Boj. Patient in the lab: Down-scaling patient-derived organoid screening for diagnostic purposes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 157.

Using structure-based drug design, we identified and optimized a novel series of pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced solid malignancies.

Using structure-based drug design, we identified a novel series of 5,6-dihydroimidazolo[1,5-f]pteridine PLK1 inhibitors. Rational improvements to compounds of this class resulted in single-digit nanomolar enzyme and cellular activity against PLK1, and oral bioavailability. Compound 1 exhibits >7 fold induction of phosphorylated Histone H3 and is efficacious in an in vivo HT-29 tumor xenograft model.

Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase involved in key processes during mitosis. Human PLK1 has been shown to be overexpressed in various human cancers, and elevated levels of PLK1 have been associated with poor prognosis, making it an attractive target for anticancer therapy. TAK-960 [4-[(9-cyclopentyl-7,7-difluoro-5-methyl-6-oxo-6,7,8,9-tetrahydro-5H-pyrimido[4,5-b][1,4]diazepin-2-yl)amino]-2-fluoro-5-methoxy-N-(1-methylpiperidin-4-yl) benzamide] is a novel, investigational, orally bioavailable, potent, and selective PLK1 inhibitor that has shown activity in several tumor cell lines, including those that express multidrug-resistant protein 1 (MDR1). Consistent with PLK1 inhibition, TAK-960 treatment caused accumulation of G2–M cells, aberrant polo mitosis morphology, and increased phosphorylation of histone H3 (pHH3) in vitro and in vivo. TAK-960 inhibited proliferation of multiple cancer cell lines, with mean EC50 values ranging from 8.4 to 46.9 nmol/L, but not in nondividing normal cells (EC50 >1,000 nmol/L). The mutation status of TP53 or KRAS and MDR1 expression did not correlate with the potency of TAK-960 in the cell lines tested. In animal models, oral administration of TAK-960 increased pHH3 in a dose-dependent manner and significantly inhibited the growth of HT-29 colorectal cancer xenografts. Treatment with once daily TAK-960 exhibited significant efficacy against multiple tumor xenografts, including an adriamycin/paclitaxel-resistant xenograft model and a disseminated leukemia model. TAK-960 has entered clinical evaluation in patients with advanced cancers. Mol Cancer Ther; 11(3); 700–9. ©2011 AACR.

Flat adenomas in the colon are associated with a relatively higher potential for malignancy. Distinct genes may be involved in the development of flat adenoma. The aim of this study was to profile gene expression changes in flat adenomas in the colon.A genomewide expression analysis was carried out by using flat adenoma and adjacent normal mucosa in the colon to detect differences in gene expression. Because the right and left colon have different embryonic origins, each sample was classified according to its location, and the gene expression levels between flat adenoma and adjacent normal mucosa were also compared among samples derived from the right or left colon.A total of 180 genes were differentially expressed between flat adenoma and normal mucosa in the colon, including matrix metalloproteinase 7 (MMP7), cadherin 3 (CDH3), S100P, and dual oxidase 2 (DUOX2). In addition, a total of 89 and 49 genes were differentially expressed between flat adenoma and normal mucosa among the samples from the right and left colon, respectively. Subsequent quantitative real-time reverse transcriptase-polymerase chain reaction supported the reliability of the expression analysis. Immunohistochemical analysis confirmed differential CDH3 and MMP7 protein expression.This is the first report characterizing the genes differentially expressed in flat adenomas using a microarray analysis. Considerable differences in the gene expression profiles of flat adenomas also exist between the right and left colon. These data should lead to new insights into the pathogenesis of flat adenomas in the colon as well as to new therapeutic strategies.

In order to elucidate the mechanism of cartilage degradation in osteoarthritis (OA), we established a cell assay system. Under the stimulation of all-trans retinoic acid (ATRA), the human chondrosarcoma cell line HCS-2/8 increased proteoglycan release from inactivated bovine nasal cartilage (BNC) and the results suggested the involvement of membrane-bound metalloproteinase(s). Therefore, we focused on the induction of a disintegrin and metalloproteinase (ADAM) superfamily upon ATRA stimulation. Of all ADAMs tested, only ADAM28 was induced by ATRA in HCS-2/8 cells and also in human primary chondrocytes. We found that transfection of ADAM28 or its alternatively spliced soluble form augmented proteoglycan release in the cell assay; however, a mutant soluble form in which a portion of the disintegrin domain was deleted did not have proteoglycan-releasing activity, implying the importance of the domain for enzyme localization and substrate recognition for cartilage degradation in OA.

Background: Polo-like kinase 1 (PLK1) plays an essential role in mitosis. Human PLK1 has been shown to be overexpressed in various human cancers, and has been associated with poor prognosis. TAK-960 is a highly selective, orally bioavailable PLK1 inhibitor that inhibits proliferation in multiple cancer cell lines; however, some cancer cell lines are insensitive to TAK-960. To investigate mechanisms of sensitivity to TAK-960 treatment and to identify a potential gene signature that might be useful for clinical development, we examined gene expression, mutation status, and copy number information in cancer cell lines with different TAK-960 sensitivities. Methods: A total of 100 cancer cell lines were treated with 15–5,000 nmol/L of TAK-960 at ONCOTEST GmbH (Freiburg, Germany) using a clonogenic assay. Cell lines with EC50 values 1,000 nmol/L were defined as sensitive and resistant cell lines, respectively. Gene expression data generated using DNA microarray were obtained from ONCOTEST and used to detect sensitive and resistant cell lines which were then analyzed using Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood City, CA) and Molecular Concepts Analysis (Rhodes D, et al Neoplasia 2007). In vivo antitumor activity of TAK-960 in 15 mouse xenograft models was evaluated by the treated/control (T/C) tumor volume ratio after once daily oral administration of TAK-960 10 mg/kg for 2 weeks. Mutation status and copy number information of cell lines used to establish the xenograft models were obtained from the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Results: Analysis of gene expression data from ONCOTEST cancer cell lines revealed that expression of several genes implicated in the cell-to-cell signaling and interaction, hematological system development and function, hepatic system disease, cell death, tumor morphology, and cellular development network including CDKN2A, CD36, TLR4, TNFRSF11A, IGFBP1, TIMP3 and several chemokine genes were different in sensitive and resistant cell lines. In addition, xenograft tumors (A549, MES-SA/D×5, 786-O, Caki-1, and Ma-1) with a deletion or mutation of CDKN2A, which is a tumor suppressor gene encoding the p16 cell cycle regulatory protein, did not respond to TAK-960 compared with CDKN2A wild-type xenograft tumors (BT474, HT-29, MV4–11, PC-3, H1299, and A2780). Conclusions: These results indicate that CDKN2A status correlated with sensitivity and thus suggest that CDKN2A status may be a useful predictive biomarker of TAK-960 antitumor activity in clinical studies. The role of CDKN2A in modulation of sensitivity to TAK-960 is under investigation. 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 A207.

Eph receptors are members of receptor tyrosine kinase family. They are activated by binding of their ligands, ephrins, which regulate numerous physiological responses, including axon guidance, and pattern formation during development. In addition to their important roles during development, several members of the Eph receptor family have been reported to function in carcinogenesis. Recently EphA10 was isolated as the 16th member of the Eph receptor family. EphA10 is expressed in normal testis and binds to ephrin-A. However, biological functions of EphA10 remain unclear. We demonstrate here that EphA10 functions as a dominant-negative receptor for EphA signaling. Our results show that EphA10 is over-expressed in cancer tissues, especially breast and lung cancer tissues as compared with patient-matched adjacent normal tissues. EphA10 binds to ephrin-A2, 3, 4, and 5. Analysis of the amino acid sequence of EphA10 revealed that EphA10 does not have Lys and Asp in the kinase domain, which are necessary for kinase activity nor two Tyr in the juxtamembrane domain, which are necessary for phosphorylation. As expected from the amino acid sequence, no intrinsic tyrosine kinase activity of EphA10 was detected despite its interaction with ephrin-A. To further investigate the mechanism of EphA10 signaling, we tested the role of EphA10 in modulating ephrin-A4 activity. In our experiment, ephrin-A4 suppressed phosphorylation of ERK1/2 in HEK293 cells. EphA10 over-expression in HEK293 cells reduced the effect of ephrin-A4 on ERK1/2. Conversely, reduction of EphA10 expression by antisense oligonucleotides induced apoptosis in the breast cancer cell line ZR-75-1. In addition to its role in shunting ephrin-A response through binding to ephrin-A directly, we found that EphA10 formed a hetero-complex with functional EphA7, suggesting that EphA10 may block EphA signaling by forming non-functional hetero complexes with other functional EphA receptors. Our studies demonstrated that EphA10 has anti-apoptotic function in cancer cells by blocking EphA signaling through its dominant-negative activity. Thus, EphA10 potentially plays an important role in cancer cell survival. 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 3951.