Hisayuki Yao

Targeting the Wnt/β-catenin signaling pathway in human cancers

Introduction: The Wnt/β-catenin signaling pathway plays a pivotal role in the regulation of cell growth, cell development and the differentiation of normal stem cells. Constitutive activation of the Wnt/β-catenin signaling pathway is found in many human cancers, and is thus an attractive target for anti-cancer therapy. Specific inhibitors of this pathway have been keenly researched and developed. Areas covered: This review discusses the potential of inhibiting the Wnt/β-catenin signaling pathway, as a therapeutic approach for cancer, along with an overview of the development of specific inhibitors. Expert opinion: Cancer stem cells (CSCs) play a significant role in the development and recurrence of several cancers, and Wnt/β-catenin signaling is important for the proliferation of CSCs. Inhibition of Wnt/β-catenin signaling is therefore a promising treatment approach. Progress has been made in the development of screening methods to identify Wnt/β-catenin signaling inhibitors. Biomarker-based screening is an effective and promising method for the identification of compounds of interest.

Identification of IL-7–Producing Cells in Primary and Secondary Lymphoid Organs Using IL-7–GFP Knock-In Mice

IL-7 is a cytokine crucial for development and maintenance of lymphocytes and other hematopoietic cells. However, how IL-7–expressing cells are distributed in lymphoid organs is not well known. To address this question, we established and analyzed IL-7–GFP knock-in mice. Thymic epithelial cells (TECs) expressed high GFP levels in the cortex and medulla, as detected with an anti-GFP Ab. Thymic mesenchymal cells also expressed GFP. Flow cytometry analysis suggested that cortical TECs expressed higher GFP levels than did medullary TECs. In bone marrow, immunohistochemistry indicated high levels of GFP in many VCAM-1+ mesenchymal stromal cells and in some VCAM-1− cells. Additionally, half of the VCAM-1+CD31− stromal cells and some platelet-derived growth factor receptor α+ stromal cells were GFP+, as detected by flow cytometry. Moreover, we detected GFP expression in fibroblastic reticular cells in the T cell zone and cortical ridge of lymph nodes. Remarkably, lymphatic endothelial cells (LECs) expressed GFP at high levels within the lymph node medulla, skin epidermis, and intestinal tissues. Additionally, we detected abundant IL-7 transcripts in isolated LECs, suggesting that LECs produce IL-7, a heretofore unknown finding. Furthermore, GFP is expressed in a subpopulation of intestinal epithelial cells, and that expression was markedly upregulated in a dextran sulfate sodium-induced acute colitis model. Overall, IL-7–GFP knock-in mice serve as a unique and powerful tool to examine the identity and distribution of IL-7–expressing cells in vivo.

Activity of the multitargeted kinase inhibitor, AT9283, in imatinib-resistant BCR-ABL-positive leukemic cells

Despite promising clinical results from imatinib mesylate and second-generation ABL tyrosine kinase inhibitors (TKIs) for most BCR-ABL(+) leukemia, BCR-ABL harboring the mutation of threonine 315 to isoleucine (BCR-ABL/T315I) is not targeted by any of these agents. We describe the in vitro and in vivo effects of AT9283 (1-cyclopropyl-3[5-morpholin-4yl methyl-1H-benzomidazol-2-yl]-urea), a potent inhibitor of several protein kinases, including Aurora A, Aurora B, Janus kinase 2 (JAK2), JAK3, and ABL on diverse imatinib-resistant BCR-ABL(+) cells. AT9283 showed potent antiproliferative activity on cells transformed by wild-type BCR-ABL and BCR-ABL/T315I. AT9283 inhibited proliferation in a panel of BaF3 and human BCR-ABL(+) cell lines both sensitive and resistant to imatinib because of a variety of mechanisms. In BCR-ABL(+) cells, we confirmed inhibition of substrates of both BCR-ABL (signal transducer and activator of transcription-5) and Aurora B (histone H3) at physiologically achievable concentrations. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3/BCR-ABL, human BCR-ABL(+) cell lines, or primary patient samples expressing BCR-ABL/T315I or glutamic acid 255 to lysine, another imatinib-resistant mutation. These data together support further clinical investigation of AT9283 in patients with imatinib- and second-generation ABL TKI-resistant BCR-ABL(+) cells, including T315I.

AV-65, a novel Wnt/β-catenin signal inhibitor, successfully suppresses progression of multiple myeloma in a mouse model.

Multiple myeloma (MM) is a malignant neoplasm of plasma cells. Although new molecular targeting agents against MM have been developed based on the better understanding of the underlying pathogenesis, MM still remains an incurable disease. We previously demonstrated that β-catenin, a downstream effector in the Wnt pathway, is a potential target in MM using RNA interference in an in vivo experimental mouse model. In this study, we have screened a library of more than 100 000 small-molecule chemical compounds for novel Wnt/β-catenin signaling inhibitors using a high-throughput transcriptional screening technology. We identified AV-65, which diminished β-catenin protein levels and T-cell factor transcriptional activity. AV-65 then decreased c-myc, cyclin D1 and survivin expression, resulting in the inhibition of MM cell proliferation through the apoptotic pathway. AV-65 treatment prolonged the survival of MM-bearing mice. These findings indicate that this compound represents a novel and attractive therapeutic agent against MM. This study also illustrates the potential of high-throughput transcriptional screening to identify candidates for anticancer drug discovery.

Galectin-9 ameliorates acute GVH disease through the induction of T-cell apoptosis

Galectins comprise a family of animal lectins that differ in their affinity for β‐galactosides. Galectin‐9 (Gal‐9) is a tandem‐repeat‐type galectin that was recently shown to function as a ligand for T‐cell immunoglobin domain and mucin domain‐3 (Tim‐3) expressed on terminally differentiated CD4+ Th1 cells. Gal‐9 modulates immune reactions, including the induction of apoptosis in Th1 cells. In this study, we investigated the effects of Gal‐9 in murine models of acute GVH disease (aGVHD). First, we demonstrated that recombinant human Gal‐9 inhibit MLR in a dose‐dependent manner, involving both Ca2+ influx and apoptosis in T cells. Next, we revealed that recombinant human Gal‐9 significantly inhibit the progression of aGVHD in murine BM transplantation models. In conclusion, Gal‐9 ameliorates aGVHD, possibly by inducing T‐cell apoptosis, suggesting that gal‐9 may be an attractive candidate for the treatment of aGVHD.

C/EBPβ Is Involved in the Amplification of Early Granulocyte Precursors during Candidemia-Induced “Emergency” Granulopoiesis

Granulopoiesis is tightly regulated to meet host demands during both “steady-state” and “emergency” situations, such as infections. The transcription factor CCAAT/enhancer binding protein β (C/EBPβ) plays critical roles in emergency granulopoiesis, but the precise developmental stages in which C/EBPβ is required are unknown. In this study, a novel flow cytometric method was developed that successfully dissected mouse bone marrow cells undergoing granulopoiesis into five distinct subpopulations (#1–5) according to their levels of c-Kit and Ly-6G expression. After the induction of candidemia, rapid mobilization of mature granulocytes and an increase in early granulocyte precursors accompanied by cell cycle acceleration was followed by a gradual increase in granulocytes originating from the immature populations. Upon infection, C/EBPβ was upregulated at the protein level in all the granulopoietic subpopulations. The rapid increase in immature subpopulations #1 and #2 observed in C/EBPβ knockout mice at 1 d postinfection was attenuated. Candidemia-induced cell cycle acceleration and proliferation of hematopoietic stem/progenitors were also impaired. Taken together, these data suggest that C/EBPβ is involved in the efficient amplification of early granulocyte precursors during candidemia-induced emergency granulopoiesis.

Rakicidin A effectively induces apoptosis in hypoxia adapted Bcr-Abl positive leukemic cells

Treatment with Abl tyrosine kinase inhibitors (TKI) drastically improves the prognosis of chronic myelogenous leukemia (CML) patients. However, quiescent CML cells are insensitive to TKI and can lead to relapse of the disease. Thus, research is needed to elucidate the properties of these quiescent CML cells, including their microenvironment, in order to effectively target them. Hypoxia is known to be a common feature of solid tumors that contributes to therapeutic resistance. Leukemic cells are also able to survive and proliferate in severely hypoxic environments. The hypoxic conditions in the bone marrow (BM) allow leukemic cells that reside there to become insensitive to cell death stimuli. To target leukemic cells in hypoxic conditions, we focused on the hypoxia‐selective cytotoxin, Rakicidin A. A previous report showed that Rakicidin A, a natural product produced by the Micromonospora strain, induced hypoxia‐selective cytotoxicity in solid tumors. Here, we describe Rakicidin A‐induced cell death in hypoxia‐adapted (HA)‐CML cells with stem cell‐like characteristics. Interestingly, apoptosis was induced via caspase‐dependent and ‐independent pathways. In addition, treatment with Rakicidin A in combination with the TKI, imatinib, resulted in synergistic cytotoxicity against HA‐CML cells. In conclusion, Rakicidin A is a promising compound for targeting TKI‐resistant quiescent CML stem cells in the hypoxic BM environment. (Cancer Sci 2011; 102: 591–596)

Growth inhibition of imatinib-resistant CML cells with the T315I mutation and hypoxia-adaptation by AV65--a novel Wnt/β-catenin signaling inhibitor.

We investigated the effect of a novel Wnt/β-catenin signaling inhibitor, AV65 on imatinib mesylate (IM)-sensitive and -resistant human chronic myeloid leukemia (CML) cells in vitro. AV65 inhibited the proliferation of various CML cell lines including T315I mutation-harboring cells. AV65 reduced the expression of β-catenin in CML cells, resulting in the induction of apoptosis. Moreover, AV65 inhibited the proliferation of hypoxia-adapted primitive CML cells that overexpress β-catenin. The combination of AV65 with IM had a synergistic inhibitory effect on the proliferation of CML cells. These findings suggest that AV65 could be a novel therapeutic agent for the treatment of CML.

Osteoclasts are involved in the maintenance of dormant leukemic cells

Osteoclasts (OCs) are specialized cells for the resorption of bone matrix that have also been recently reported to be involved in the mobilization of hematopoietic progenitor cells. When Ba/F3 cells expressing wild-type bcr-abl were co-cultured with osteoblasts (OBs), OCs, and bone slices, their proliferation was significantly suppressed, and the Ki-67 negative population, which is believed to be in G(0) phase, was increased. The results of our in vitro experiments suggest that OCs could be involved in the maintenance of dormant leukemic cells in the bone marrow (BM) microenvironment through the release of soluble factors, one of which could be TGF-beta.

CCAAT/enhancer-binding protein β expressed by bone marrow mesenchymal stromal cells regulates early B-cell lymphopoiesis.

The transcription factor CCAAT/enhancer‐binding protein β (C/EBPβ) regulates the differentiation of a variety of cell types. Here, the role of C/EBPβ expressed by bone marrow mesenchymal stromal cells (BMMSCs) in B‐cell lymphopoiesis was examined. The size of the precursor B‐cell population in bone marrow was reduced in C/EBPβ‐knockout (KO) mice. When bone marrow cells from C/EBPβ‐KO mice were transplanted into lethally irradiated wild‐type (WT) mice, which provide a normal bone marrow microenvironment, the size of the precursor B‐cell population was restored to a level equivalent to that generated by WT bone marrow cells. In coculture experiments, BMMSCs from C/EBPβ‐KO mice did not support the differentiation of WT c‐Kit+ Sca‐1+ Lineage− hematopoietic stem cells (KSL cells) into precursor B cells, whereas BMMSCs from WT mice did. The impaired differentiation of KSL cells correlated with the reduced production of CXCL12/stromal cell‐derived factor‐1 by the cocultured C/EBPβ‐deficient BMMSCs. The ability of C/EBPβ‐deficient BMMSCs to undergo osteogenic and adipogenic differentiation was also defective. The survival of leukemic precursor B cells was poorer when they were cocultured with C/EBPβ‐deficient BMMSCs than when they were cocultured with WT BMMSCs. These results indicate that C/EBPβ expressed by BMMSCs plays a crucial role in early B‐cell lymphopoiesis. Stem Cells 2014;32:730–740