Miki Takeuchi

ABT-737 is a useful component of combinatory chemotherapies for chronic myeloid leukaemias with diverse drug-resistance mechanisms

The effect of ABT‐737, a BH3‐mimicking inhibitor for anti‐apoptotic Bcl‐2 and Bcl‐XL, but not Mcl‐1, against Bcr‐Abl‐positive (Bcr‐Abl+) leukaemic cells was examined. ABT‐737 potently induced apoptosis in Bcr‐Abl+ chronic myeloid leukaemia (CML) cell lines and primary CML samples in vitro and prolonged the survival of mice xenografted with BV173 cells, a CML cell line. Higher expression of anti‐apoptotic Bcl‐2 proteins reduced cell killing by ABT‐737 in each cell line, but there was no correlation between the sensitivities to ABT‐737 and the specific expression patterns of Bcl‐2 family proteins among cell lines. Thus, the cell killing effect of ABT‐737 must be determined not only by the expression patterns of Bcl‐2 family proteins but also by other mechanisms, such as high expression of Bcr‐Abl, or a drug‐efflux pump, in CML cells. ABT‐737 augmented the cell killing effect of imatinib in Bcr‐Abl+ cells with diverse drug‐resistance mechanisms unless leukaemic cells harboured imatinib‐insensitive Abl kinase domain mutations, such as T315I. The combination of homoharringtonine that reduces Mcl‐1 enhanced the killing by ABT‐737 strongly in Bcr‐Abl+ cells even with T315I mutation. These results suggest that ABT‐737 is a useful component of chemotherapies for CML with diverse drug‐resistance mechanisms.

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia.

Bcr-Abl is the cause of Philadelphia-positive (Ph+) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph+ leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-XL, greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph+ leukemic cells.

Administration of PLK-1 small interfering RNA with atelocollagen prevents the growth of liver metastases of lung cancer

Liver metastasis is one of the most important prognostic factors in lung cancer patients. However, current therapies are not sufficient. RNA interference provides us a powerful and promising approach for treating human diseases including cancers. Herein, we investigated the in vitro effects of PLK-1 small interfering RNA (siRNA) on human lung cancer cell lines and the in vivo usage of PLK-1 siRNA with atelocollagen as a drug delivery system in a murine liver metastasis model of lung cancer. PLK-1 was overexpressed in cell lines and in cancerous tissues from lung cancer patients. PLK-1 siRNA treatment inhibited growth and induced apoptosis in a concentration-dependent manner. To verify in vivo efficacy, we confirmed that atelocollagen was a useful drug delivery system in our model of implanted luciferase-labeled A549LUC cells by detecting reduced bioluminescence after an i.v. injection of luciferase GL3 siRNA/atelocollagen. PLK-1 siRNA/atelocollagen was also successfully transfected into cells and inhibited the progression of metastases. This study shows the efficacy of i.v. administration of PLK-1 siRNA/atelocollagen for liver metastases of lung cancer. We believe siRNA therapy will be a powerful and promising strategy against advanced lung cancer. [Mol Cancer Ther 2008;7(9):2904–12]

β-Catenin Small Interfering RNA Successfully Suppressed Progression of Multiple Myeloma in a Mouse Model

Purpose: β-catenin is the downstream effector of the Wnt signaling pathway, and it regulates cell proliferation. β-catenin overexpression correlates positively with prognosis in several types of malignancies. We herein assessed its effects on growth of multiple myeloma cells using a xenograft model. Experimental Design: We first investigated the expression of β-catenin in multiple myeloma cell lines and multiple myeloma cells obtained from patients. Next, we investigated the growth inhibitory effects of β-catenin small interfering RNA on the growth of multiple myeloma cells in vivo. Six-week-old male BALB/c nu/nu mice were inoculated s.c. in the right flank with 5 × 106 RPMI8226 cells, followed by s.c. injections of β-catenin small interfering RNA, scramble small interfering RNA, or PBS/atelocollagen complex twice a week for a total of eight injections. Results: Significantly higher levels of β-catenin expression were observed in multiple myeloma cell lines and in samples from patients with multiple myeloma than those found in mononuclear cells obtained from healthy volunteers. In in vivo experiments, no inhibitory effects were observed following treatment with scramble small interfering RNA or PBS/atelocollagen complexes, whereas treatment with β-catenin small interfering RNA/atelocollagen complex significantly inhibited growth of multiple myeloma tumors (P < 0.05). Conclusions: β-catenin small interfering RNA treatment inhibited the growth of multiple myeloma tumors in a xenograft model. To our knowledge, this is the first report showing that the treatment with β-catenin small interfering RNA produces an inhibitory effects on growth of hematologic malignancies in vivo. Because treatment with β-catenin small interfering RNA inhibited growth of multiple myeloma cells, β-catenin is the attractive novel target for treating multiple myeloma.

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.

Glyoxalase-I is a novel target against Bcr-Abl + leukemic cells acquiring stem-like characteristics in a hypoxic environment

Abl tyrosine kinase inhibitors (TKIs) such as imatinib and dasatinib are ineffective against Bcr-Abl+ leukemic stem cells. Thus, the identification of novel agents that are effective in eradicating quiescent Bcr-Abl+ stem cells is needed to cure leukemias caused by Bcr-Abl+ cells. Human Bcr-Abl+ cells engrafted in the bone marrow of immunodeficient mice survive under severe hypoxia. We generated two hypoxia-adapted (HA)-Bcr-Abl+ sublines by selection in long-term hypoxic cultures (1.0% O2). Interestingly, HA-Bcr-Abl+ cells exhibited stem cell-like characteristics, including more cells in a dormant, increase of side population fraction, higher β-catenin expression, resistance to Abl TKIs, and a higher transplantation efficiency. Compared with the respective parental cells, HA-Bcr-Abl+ cells had higher levels of protein and higher enzyme activity of glyoxalase-I (Glo-I), an enzyme that detoxifies methylglyoxal, a cytotoxic by-product of glycolysis. In contrast to Abl TKIs, Glo-I inhibitors were much more effective in killing HA-Bcr-Abl+ cells both in vitro and in vivo. These findings indicate that Glo-I is a novel molecular target for treatment of Bcr-Abl+ leukemias, and, in particular, Abl TKI-resistant quiescent Bcr-Abl+ leukemic cells that have acquired stem-like characteristics in the process of adapting to a hypoxic environment.

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.