Maki Hasegawa

TAK-960, a Novel, Orally Available, Selective Inhibitor of Polo-Like Kinase 1, Shows Broad-spectrum Preclinical Antitumor Activity in Multiple Dosing Regimens

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.

Regulation of angiogenesis in the bone marrow of myelodysplastic syndromes transforming to overt leukaemia

To investigate the regulatory mechanisms of angiogenesis in the development of myelodysplastic syndromes (MDS) and its progression to overt leukaemia (OL), bone marrow samples from control, paired samples from MDS patients before and after transformation to OL (MDS → OL) and de novo acute myeloid leukaemia (AML) were analysed. Immunohistochemical staining showed a significant increase of bone marrow microvascular density (MVD) in MDS and de novo AML compared with controls. Surprisingly, in MDS, MVD significantly decreased upon transformation to OL, which was also significantly lower than the MVD of de novo AML. This evidence was strengthened by the pattern of angiogenic mediator gene expression, confirming the importance of various angiogenic mediators including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumour necrosis factor α (TNFα), hepatocyte growth factor (HGF) and the angiopoietin family of mediators (Ang‐1 and Ang‐2) as well as the receptors for angiogenic mediators, such as VEGF receptor 2 (VEGFR2) and the tyrosine kinase receptor, TIE2. By contrast, the anti‐angiogenic mediator, transforming growth factor‐β (TGFβ) exhibited significantly higher expression in the bone marrow of MDS → OL, indicating the importance of this cytokine as the suppressive factor of angiogenesis in MDS. These findings indicate that the bone marrow microenvironment in MDS → OL and de novo AML differs remarkably, suggesting the different efficacy of anti‐angiogenic therapy between de novo AML and leukaemia secondary to MDS.

Friend Leukemia Virus Infection Enhances DNA Damage-Induced Apoptosis of Hematopoietic Cells, Causing Lethal Anemia in C3H Hosts

ABSTRACT Exposure of hematopoietic progenitors to gamma irradiation induces p53-dependent apoptosis. However, host responses to DNA damage are not uniform and can be modified by various factors. Here, we report that a split low-dose total-body irradiation (TBI) (1.5 Gy twice) to the host causes prominent apoptosis in bone marrow cells of Friend leukemia virus (FLV)-infected C3H mice but not in those of FLV-infected DBA mice. In C3H mice, the apoptosis occurs rapidly and progressively in erythroid cells, leading to lethal host anemia, although treatment with FLV alone or TBI alone induced minimal apoptosis in bone marrow cells. A marked accumulation of P53 protein was demonstrated in bone marrow cells from FLV-infected C3H mice 12 h after treatment with TBI. Although a similar accumulation of P53 was also observed in bone marrow cells from FLV-infected DBA mice treated with TBI, the amount appeared to be parallel to that of mice treated with TBI alone and was much lower than that of FLV- plus TBI-treated C3H mice. To determine the association of p53 with the prominent enhancement of apoptosis in FLV- plus TBI-treated C3H mice, p53 knockout mice of the C3H background (C3H p53−/− ) were infected with FLV and treated with TBI. As expected, p53 knockout mice exhibited a very low frequency of apoptosis in the bone marrow after treatment with FLV plus TBI. Further, C3H p53−/− → C3H p53+/+ bone marrow chimeric mice treated with FLV plus TBI survived even longer than the chimeras treated with FLV alone. These findings indicate that infection with FLV strongly enhances radiation-induced apoptotic cell death of hematopoietic cells in host animals and that the apoptosis occurs through a p53-associated signaling pathway, although the response was not uniform in different host strains.

In vivo distribution of receptor for ecotropic murine leukemia virus and binding of envelope protein of Friend Murine leukemia virus

Summary. Ecotropic infection by Murine leukemia virus (MuLV) infection is initiated by the interaction between the receptor-binding domain of the viral surface glycoprotein (SU) and the cell-surface receptor, mCAT-1. To study the in vivo localization of viral binding site in mice, green fluorescence protein (GFP)-tagged Friend SU (F-SU/GFP) was incubated with tissue sections. Lymphohematopoietic organs and a part of the glandular tissues of C3H as well as C57BL/6 mice revealed positive signals for F-SU/GFP binding on the cell surface. In contrast, C4W mice, which is a partial congenic mouse strain carrying the Fv-4r gene on a BALB/c genetic background, exhibited negative signals in most of the organs except for a very weak binding in the pancreas. The expression of mCAT-1 mRNA determined by reverse transcriptase (RT)-polymerase chain reaction (PCR) revealed a similar distribution in C3H, C57BL/6 and C4W mice. Most of the organs including lymphohematopoietic organs and glandular organs revealed significant expression of mRNA for mCAT-1 gene, while the liver, heart and muscle did not. The results from binding assay were consistent with the fact that Friend MuLV-induced pathogenesis was usually associated with lymphohematopoietic systems, although mRNA expression for mCAT-1 was rather ubiquitous. The discrepancy between F-SU/GFP binding and mRNA expression for mCAT-1 in lymphohematopoietic organs of C4W mice would support the receptor interference effect by the Fv-4r gene causing the resistance of C4W mouse to Friend MuLV infection.

A novel role for acinus and MCM2 as host-specific signaling enhancers of DNA-damage-induced apoptosis in association with viral protein gp70

The interaction of viral proteins with host-cellular proteins elicits the activation of numerous cellular signal transduction pathways possibly leading to the viral pathogenesis. We previously demonstrated that infection with Friend leukemia virus (FLV) radiosensitizes murine hematopoietic cells via a p53-dependent apoptotic pathway in C3H hosts. Here, we show that the transduction of the env-gene (gp70) of Friend murine leukemia virus (F-MuLV) sensitized C3H-derived myeloid leukemia cells to DNA-damage (ionizing radiation as well as doxorubicin)-induced apoptosis through the activation of DNA-dependent protein kinase (DNA-PK) and P53. Knockdown of DNA-PK by siRNA inhibited the radiosensitization induced by gp70. In association with gp70 and DNA-PK, the acinus and MCM2 proteins were host-specifically overexpressed in C3H-derived cells. Taken together, these data suggested that gp70 enhances cellular DNA-damage-induced signaling in association with host-specific cellular proteins including acinus and MCM2 resulting in the activation of DNA-PK to phosphorylate P53. This in vitro study clearly indicates that the enhancement of DNA-damage-induced apoptosis by gp70 is not caused by the bone marrow environment of the host but is introduced by modified signaling in hematopoietic cells. The mechanisms involved in the ability of a viral protein to regulate cellular gene expression could provide invaluable insight into the manipulation of cellular pro-apoptotic signaling and the development of novel therapeutic strategies.

Significant increase in the expression of matrix metalloproteinase 7 in primary CNS lymphoma

The aim of this study is to compare the expression pattern of matrix metalloproteinases (MMP) in primary CNS lymphoma (PCNSL) with that of nodal lymphoma (NL). PCNSL possesses a characteristic pattern of brain infiltration, specifically, perivascular aggregation and destruction of the surrounding connective tissue. Hypotheses have been proposed that this infiltration pattern indicates the infiltration of lympoma cells from outside the CNS. However, the mechanism of this infiltration pattern has not been clearly explained. Here we performed quantitative analysis of mRNA expression of MMP1, 2, 3, 7 and 9 in lymphoma cells from 10 cases of PCNSL, all of which were diagnosed as diffuse large B‐cell lymphoma (DLBCL), and 14 cases of nodal DLBCL. Immunohistochemistry was also performed for phenotyping of lymphoma cells and for examining the localization of MMPs. The expression level of MMP7 in PCNSL (median ratio to normal lymophocytes, 45.6) was significantly higher than the level in NL (median, 7.55). By contrast, the expression levels of MMP2 (median, 31.7) and MMP9 (median, 1.30) in PCNSL were significantly lower than those in NL (median, 120 and 16.5, respectively). The expression levels of MMP1 and MMP3 were very low and not different between PCNSL and NL (almost comparable to those in normal lymphocytes). Immunohistochemical phenotyping revealed that the frequency of non‐germinal center type DLBCL was significantly higher in PCNSL than in NL. These results, notably the significantly increased expression of MMP7 in PCNSL, suggest the involvement of this MMP in the characteristic infiltration pattern of PCNSL. They also support the existing hypothesis that PCNSL and NL are of different origins.

Cytological basis for enhancement of radiation-induced mortality by Friend leukaemia virus infection

Purpose: To analyse the cytological basis for enhancement of radiation‐induced mortality by Friend leukaemia virus infection. Materials and methods: Cellularity in haematopoietic tissues of C3H mice infected with FLV and/or whole‐body irradiation was examined. Results: When mice were treated with a sublethal dose (3 Gy) of irradiation at 1 week after virus infection, most manifested a severe loss of cellularity in the spleen, bone marrow and peripheral blood 2 weeks after irradiation. More than 90% of the mice died within 1 month post‐irradiation. However, this deleterious effect of virus infection on the survival of irradiated mice was observed only when they were irradiated at around 1 week after virus inoculation. Strain differences in the sensitivity to this effect were observed among virus‐sensitive strains of mice. Conclusions: The results indicate that Friend leukaemia virus infection can cause enhancement of radiation sensitivity of haematopoietic cells in host animals in a restricted manner in terms of genetic background and the interval between infection and irradiation.

Studies of CDK 8/19 inhibitors: Discovery of novel and selective CDK8/19 dual inhibitors and elimination of their CYP3A4 time-dependent inhibition potential

In this article, synthetic studies around a pyridylacrylamide-based hit compound (1), utilizing structure-based drug design guided by CDK8 docking models, is discussed. Modification of the pendant 4-fluorophenyl group to various heteroaromatic rings was conducted aiming an interaction with the proximal amino acids, and then replacement of the morpholine ring was targeted for decreasing potential of time-dependent CYP3A4 inhibition. These efforts led to the compound 4k, with enhanced CDK8 inhibitory activity and no apparent potential for time-dependent CYP3A4 inhibition (CDK8 IC50: 2.5nM; CYP3A4 TDI: 99% compound remaining). Compound 4k was found to possess a highly selective kinase inhibition profile, and also showed favorable pharmacokinetic profile. Oral administration of 4k (15mg/kg, bid. for 2weeks) suppressed tumor growth (T/C 29%) in an RPMI8226 mouse xenograft model.