Tetsuzo Tauchi

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.

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.

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.