Kazuma Ohyashiki

Telomerase inhibition with a novel G-quadruplex-interactive agent, telomestatin: in vitro and in vivo studies in acute leukemia

The telomerase complex is responsible for telomere maintenance and represents a promising neoplasia therapeutic target. Recently, we have demonstrated that treatment with a G-quadruplex-interactive agent, telomestatin reproducibly inhibited telomerase activity in the BCR-ABL-positive leukemic cell lines. In the present study, we investigated the mechanisms of apoptosis induced by telomerase inhibition in acute leukemia. We have found the activation of caspase-3 and poly-(ADP-ribose) polymerase in telomestatin-treated U937 cells (PD20) and dominant-negative DN-hTERT-expressing U937 cells (PD25). Activation of p38 mitogen-activated protein (MAP) kinase and MKK3/6 was also found in telomestatin-treated U937 cells (PD20) and dominant-negative DN-hTERT-expressing U937 cells (PD25); however, activation of JNK and ASK1 was not detected in these cells. To examine the effect of p38 MAP kinase inhibition on growth properties and apoptosis in telomerase-inhibited cells, we cultured DN-hTERT-expressing U937 cells with or without SB203580. Dominant-negative-hTERT-expressing U937 cells stopped proliferation on PD25; however, a significant increase in growth rate was observed in the presence of SB203580. Treatment of SB203580 also reduced the induction of apoptosis in DN-hTERT-expressing U937 cells (PD25). These results suggest that p38 MAP kinase has a critical role for the induction of apoptosis in telomerase-inhibited leukemia cells. Further, we evaluated the effect of telomestatin on the growth of U937 cells in xenograft mouse model. Systemic intraperitoneal administration of telomestatin in U937 xenografts decreased tumor telomerase levels and reduced tumor volumes. Tumor tissue from telomestatin-treated animals exhibited marked apoptosis. None of the mice treated with telomestatin displayed any signs of toxicity. Taken together, these results lay the foundations for a program of drug development to achieve the dual aims of efficacy and selectivity in vivo.

Transcriptional profiling of Epstein-Barr virus (EBV) genes and host cellular genes in nasal NK/T-cell lymphoma and chronic active EBV infection.

Nasal NK/T-cell lymphoma is an aggressive subtype of non-Hodgkin lymphoma (NHL) that is closely associated with Epstein–Barr virus (EBV). The clonal expansion of EBV-infected NK or T cells is also seen in patients with chronic active EBV (CAEBV) infection, suggesting that two diseases might share a partially similar mechanism by which EBV affects host cellular gene expression. To understand the pathogenesis of EBV-associated NK/T-cell lymphoproliferative disorders (LPD) and design new therapies, we employed a novel EBV DNA microarray to compare patterns of EBV expression in six cell lines established from EBV-associated NK/T-cell LPD. We found that expression of BZLF1, which encodes the immediate-early gene product Zta, was expressed in SNK/T cells and the expression levels were preferentially high in cell lines from CAEBV infection. We also analyzsd the gene expression patterns of host cellular genes using a human oligonucleotide DNA microarray. We identified a subset of pathogenically and clinically relevant host cellular genes, including TNFRSF10D, CDK2, HSPCA, IL12A as a common molecular biological properties of EBV-associated NK/T-cell LPD and a subset of genes, such as PDCD4 as a putative contributor for disease progression. This study describes a novel approach from the aspects of viral and host gene expression, which could identify novel therapeutic targets in EBV-associated NK/T-cell LPD.

The Second Generation of BCR-ABL Tyrosine Kinase Inhibitors

Imatinib was developed as the first molecularly targeted therapy to specifically inhibit the BCR-ABL kinase in Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML). Because of the excellent hematologic and cytogenetic responses, imatinib has moved toward first-line treatment for newly diagnosed CML. However, the emergence of resistance to imatinib remains a major problem in the treatment of Ph-positive leukemia. Several mechanisms of imatinib resistance have been identified, including BCR-ABL gene amplification that leads to overexpression of the BCR-ABL protein, point mutations in the BCR-ABL kinase domain that interfere with imatinib binding, and point mutations outside of the kinase domain that allosterically inhibit imatinib binding to BCR-ABL.The need for alternative or additional treatment for imatinib-resistant BCR-ABL-positive leukemia has guided the way to the design of a second generation of targeted therapies, which has resulted mainly in the development of novel small-molecule inhibitors such as AMN107, dasatinib, NS-187, and ON012380. The major goal of these efforts is to create new compounds that are more potent than imatinib and/or more effective against imatinib-resistant BCR-ABL clones. In this review, we discuss the next generation of BCR-ABL kinase inhibitors for overcoming imatinib resistance.

Quantitative relationship between functionally active telomerase and major telomerase components (hTERT and HTR) in acute leukaemia cells

Functionally active telomerase is affected at various steps including transcriptional and post-transcriptional levels of major telomerase components (hTR and human telomerase reverse transcriptase (hTERT)). We therefore developed a rapid and sensitive method to quantify hTERT and its splicing variants as well as the hTR by a Taqman real-time reverse transcriptase–polymerase chain reaction to determine whether their altered expression may contribute to telomere attrition in vivo or not. Fresh leukaemia cells obtained from 38 consecutive patients were used in this study. The enzymatic level of telomerase activity measured by TRAP assay was generally associated with the copy numbers of full-length hTERT+α+β mRNA (P=0.0024), but did not correlate with hTR expression (P=0.6753). In spite of high copy numbers of full-length hTERT mRNA, telomerase activity was low in some cases correlating with low copy numbers of hTR, raising the possibility that alteration of the hTRu2009:u2009hTERT ratio may affect functionally active telomerase activity in vivo. The spliced nonactive hTERT mRNA tends to be lower in patients with high telomerase activity, suggesting that this epiphenomenon may play some role in telomerase regulation. An understanding of the complexities of telomerase gene regulation in biologically heterogeneous leukaemia cells may offer new therapeutic approaches to the treatment of acute leukaemia.

JAK2(V617F) mutational status as determined by semiquantitative sequence-specific primer-single molecule fluorescence detection assay is linked to clinical features in chronic myeloproliferative disorders.

JAK2 V617F mutational status as determined by semiquantitative sequence-specific primer-single molecule fluorescence detection assay is linked to clinical features in chronic myeloproliferative disorders

Induction of heme oxygenase-1 by cobalt protoporphyrin enhances the antitumour effect of bortezomib in adult T-cell leukaemia cells

Adult T-cell leukaemia (ATL) is a lethal neoplasia derived from HTLV-1-infected T lymphocytes frequently exhibiting nuclear factor-κB (NF-κB) activation. Despite the use of various treatment regimens, the prognosis of ATL is poor, and new treatment strategies are urgently needed. We therefore explored the effect and the molecular mechanism of a proteasome inhibitor, bortezomib, in ATL cells. We found bortezomib-induced cell death, and bortezomib suppressed constitutive NF-κB activation via I-κB stabilisation in three ATL cell lines (TaY, MT-2 and MT-4). An oligonucleotide DNA microarray analysis of TaY cells revealed upregulation of genes encoding heat shock proteins (HSPA1A, STIP1, HSPA1B, and HSPCA), genes related to protein folding (CDC37 and ANAPC5), Fas-associated factor 1(FAF1) and an oxidative stress-related gene, heme oxygenase-1(HMOX-1), known to be a target gene of hypoxia-inducible gene-1 alpha (HIF-1 alpha). Cobalt protoporphyrin induced HMOX-1, instead of HIF-1 alpha expression and increased bortezomib-induced apoptosis in the presence of pharmacologically effective doses of bortezomib. In contrast, zinc protoporphyrin downregulated HMOX-1 expression, thereby partially inhibiting bortezomib-induced cell death. This indicates that HMOX-1 may modulate anticancer effects of bortezomib in ATL cells, and could be a molecular target in treating ATL patients.

Identification and functional signature of genes regulated by structurally different ABL kinase inhibitors.

Dasatinib is an ATP-competitive, multi-targeted SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical standpoint, dasatinib is particularly attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant chronic myeloid leukemia patients. The fact because the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on wild-type p210 BCR-ABL-expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16u2009h, and gene expression data were obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib-responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The distinct genes that are selectively modulated by dasatinib are cyclin-dependent kinase 2 (CDK2) and CDK8, which had a maximal reduction of <5-fold in microarray screen. To assess the functional importance of dasatinib regulated genes, we used RNA interference to determine whether reduction of CDK2 and CDK8 affected the growth inhibition. K562 and TF-1BCR-ABL cells, pretreated with CDK2 or CDK8 small interfering RNA, showed additive growth inhibition with imatinib, but not with dasatinib. These findings demonstrate that the additive/synergistic growth inhibition by imatinib and dasatinib may be mediated in part by CDK2 and CDK8.

Imatinib Mesylate in Combination with Other Chemotherapeutic Agents for Chronic Myelogenous Leukemia

Imatinib therapy is an important contribution to the management of patients with chronic myelogenous leukemia (CML). Despite high rates of hematologic and cytogenetic responses to imatinib therapy, the emergence of resistance to imatinib has been recognized as a major problem in the treatment of CML. Experimental and clinical studies suggest that imatinib as a single drug may not be sufficient to eradicate BCR-ABL-positive stem cells. Therefore, whether combinations of imatinib with other agents can increase the length of molecular remission and whether such combinations can prolong survival should be determined by large-scale clinical studies. In this review, we discuss efficacious combinations for future clinical trials. These regimens combine imatinib with conventional chemotherapeutic agents or with inhibitors of other signal transduction molecules that may be preferentially activated in CML cells.