Abstract

ABSTRACTS

Molecular Mechanism of Pathogenesis by Human T-cell Leukemia Virus Type I

Masao Matsuoka

Human T-cell leukemia virus type I (HTLV-I) is a causal agent of adult T-cell leukemia (ATL). HTLV-I belongs to complex retrovirus, which encodes several regulatory and accessory genes. Among these genes, the tax gene is considered to play a central role in the leukemogenesis. However, Tax expression is frequently inactivated in ATL cells. We observed that 5'long terminal repeat (LTR) was frequently methylated or deleted in ATL cells while 3'LTR was intact in all ATL cells, suggesting an importance of 3'LTR in leukemogenesis. The HTLV-I bZIP factor (HBZ) gene is encoded by the minus strand of provirus, which is transcribed from 3'LTR. Detailed analyses of defective provirus without 5'LTR demonstrated that only HBZ gene and its expression were intact in ATL cells. Functional analyses of HBZ gene on proliferation of T-lymphocytes reveal that HBZ RNA promotes proliferation of T-lymphocytes. DNA micorarray analyses showed that HBZ RNA and protein differentially influence transcriptions of cellular genes. E2F1 and its target genes were upregulated by HBZ RNA whereas other genes were upregulated by HBZ protein. In the transgenic mice that expressing HBZ gene under control of CD4 specific promoter, the number of CD4+ cells increased. After a long latent period, a part of transgenic mice developed T-cell lymphomas. This finding showed oncogenic potential of HBZ gene. Moreover, CD4+ T-cells of the HBZ transgenic mice infiltrate into skin and lung, indicating that HBZ gene is associated with infiltrative phenotype of HTLV-I infected cells. This phenotype is likely associated with pathogenesis of HTLV-I associated diseases, such as HTLV-I associated myelopathy or uveitis. These data demonstrated the HBZ should be implicated in pathogenesis of not only ATL but also HTLV-I associated dieases.

Pathogenic Roles of Hepatitis C Virus Replication

Kunitada Shimotohno

Hepatitis C virus (HCV) is a major causative agent to develop chronic hepatitis, cirrhosis and hepatocellular carcinoma. Patients infected with HCV also frequently develop diseases such as diabetes as well as steatosis, which are believed to prognose exacerbation of liver diseases. There are accumulating evidence to show that HCV capsid protein (Core) is involved in the development of these diseases by in vitro experiments as well as by statistical analysis of manifestation of liver diseases. However, role of modulation of insulin signaling and lipid metabolism on HCV replication remains elusive. Analyzing HCV replication using a cultured cell line, we found the importance of lipid synthesis for generation of HCV replication complex, an organelle responsible for HCV RNA synthesis, as well as for efficient production of infectious virus particles.
The HCV replication complex surrounded by membrane bilayer is generated upon HCV infection. Synthesis of HCV RNA, both positive and negative stranded, is conducted within this complex and this function of the complex is abolished by disruption of membrane. Further, statin, an inhibitor of lipid synthesis, inhibits the activity of HCV replication, suggesting importance of de novo synthesis of lipid for generation of the replication complex. Another evidence to show the importance of lipid metabolism came from observation of substantial increase of lipid droplet in HCV replicating cells. The lipid droplet and its surrounding area constitute the environment from where infectious HCV particles are produced. HCV Core associates with the surface membrane of the lipid droplet and, then, recruits other HCV proteins around the lipid droplet to constitute HCV replication complex, which is required to produce infectious virus particles. It has been shown that HCV Core trans-activates nuclear hormone receptors such as RAR and RXR. These hormone receptors activate genes involving in lipid metabolism. Taking all these observations together, lipid synthesis activated by HCV Core and association of HCV Core with the lipid droplet may be therapeutic targets for prevention of HCV proliferation as well as prevention of disease progression such as steatosis.

Development of Antiviral Therapeutics of HIV Infection: From AZT to Darunavir

Hiroaki Mitsuya

Despite the discovery of the first human pathogenic retrovirus HTLV-1, virtually no attempt was made to explore antiretroviral therapy since it was believed that once target cells were infected by cellular-genome-integrating retrovirus, antiretroviral drugs would do nothing to the progress of the retrovirus-associated diseases. The first three dideoxynucleoside reverse transcriptase inhibitors (zidovudine or AZT, didanosine, and zalcitabine), developed in 1985, made changes. After these first drugs proved to be efficacious in patients with HIV infection, a number of antiviral agents were added to the armamentarium of the fight against HIV infection. Combination chemotherapy using such antiretroviral agents or HAART has had a major impact on the morbidity and mortality of patients with HIV infection. However, we have faced multiple major problems, which represent the challenges different than we faced in the development of the first drugs. They include (i) drug-related toxicities, (ii) emergence of drug-resistant HIV variants, (iii) only partial restoration of immunologic functions, (iv) paradoxical flame-up of inflammation, and (v) increased cost of antiviral therapy. Nevertheless, extensive knowledge of molecular, biochemical, and structural interactions of antiretroviral agents and targeted viral components has been acquired. We are obviously at a new forefront in the therapy of HIV infection.
One new area in the development of antiretroviral agents is predictive modeling, which maximizes our chances of success. I will discuss an approach of combining site-directed mutagenesis-based data and molecular modeling, which represents a novel strategy for gaining structural insights for thte design of novel drugs. One of recently FDA-approved protease inhibitors, darunavir that we recently developed, was designed based on such structural approach. We most recently discovered that darunavir and a group of newly designed and synthesized agents block the dimerization process of HIV protease, an essential step in the replication cycle of HIV. Further improved approaches to explore new treatment modalities should be continued in the hope that with new and more potent antiviral agents, we will certainly be able to control HIV diseases more efficiently and effectively.

Trend in Oncology: Targeting the Kinome

Motowo Nakajima and Doriano Fabbro

Protein kinases are key elements of protein phosphorylation-based intracellular signaling networks. Recent phosphoproteomic analyses have revealed that a very large proportion of intracellular proteins is phosphorylated, and that a significant fraction of all protein phosphorylations can modify cellular responses to external stimuli. Thus there has been increasing interest in the number of protein kinases constituting intracellular signaling networks as disease therapeutic targets.
In fact mutations in protein kinase genes are increasingly found to be causal in human diseases. Out of 518 protein kinase genes more than 150 kinase genes have been implicated in various diseases, and approximately 120 tyrosine and serine kinases have been associated with cancers. There have been more than 40 clinical targets for cancers to date since 2001. The mechanisms of protein kinase involvement in cause of cancer are activating or inactivating mutations, and overexpression or underexpression. Such examples are genomic rearrangements of Bcr-Abl in chronic myelogenous leukaemia (CML), mutations in c-Kit in gastrointestinal stromal tumor (GIST), mutations in B-Raf in melanoma, enforced dimerization by overexpression of EGFRs, PDGFR, IGF-IR, and c-Kit in a variety of cancers, and ectopic expression of VEGFs/VEGFRs, Ang/Tie2, and EphB/ephrins in angiogenic cancers. Genomic analyses of protein kinase exons and regulatory regions for mutations in cancer have yielded many additional cancer-causing protein kinase gene candidates. These findings have led to intensive efforts to develop specific protein kinase inhibitors as cancer therapeutics, and major efforts are underway both in pharmaceutical industry and academia.
Eleven small molecular compounds and 4 monoclonal antibodies for kinase-mediated signal transduction inhibition have been registered for therapeutics worldwide. Seven tyrosine kinase inhibitors (TKIs) (Gleevec, Iressa, Tarceva, Sutent, Sprycel, Tykerb, Torisel) and one serine/threonine/tyrosine kinase inhibitors (Nexavar) are approved for clinical use in cancer therapy in the US. Currently 180 kinase inhibitors are tested in 300 clinical trials for 40 cancer diseases, both singly and in combination with approved chemotherapy drugs. We can predict that increasing numbers of protein kinase-targeted drugs will become available for treatment of cancers and other diseases in the next decade.

Molecular Mechanism of Anti-Cancer Therapies

Hideyuki Saya

Mitotic catastrophe is an important mechanism for the induction of cell death in cancer cells by antineoplastic agents. We characterized the dynamics and regulation of mitotic catastrophe induced by anti-microtubules agents such as paclitaxel by using time-lapse microscopy. Cells entering mitosis arrested at metaphase without chromosome segregation and subsequently collapsed. In the metaphase-arrested cells, mitotic checkpoint was activated. Furthermore, inhibition of mitotic checkpoint function as a result of depletion of BubR1 or Mad2 by RNAi led cells to escape from mitotic catastrophe and to undergo mitotic slippage. Our data suggest that dysfunction of the mitotic checkpoint in cancer may confer resistance to anti-microtubules agents.
To identify the critical signal pathways to regulate the paclitaxel-induced mitotic catastrophe, activation of various kinases were examined. Among diverse intracellular kinases, p38 MAP kinase was significantly and specifically activated when cells underwent mitotic death. The activation of p38 requires the long-term metaphase arrest and is triggered as cell goes from metaphase to anaphase. Based on these data, we would like to propose the new resistance mechanisms against chemotherapeutic agents in the symposium.

Alterations of the EGFR and Related Genes as a Biomarker of EGFR-TKI Treatment in Lung Cancer

Tetsuya Mitsudomi

Subsets of patients with non-small cell lung cancer (NSCLC) respond remarkably well to small molecule tyrosine kinase inhibitors (TKI) specific for epidermal growth factor receptor (EGFR) such as gefitinib or erlotinib. Those patients are typically of East Asian ethnicity, female gender, no history of smoking or those with adenocarcinoma. In 2004, it was found that NSCLC occurring in patients with above-mentioned characteristics frequently harbors activating mutations of the EGFR gene.
In general, about 80% of NSCLCs with EGFR mutations respond to EGFR-TKIs, whereas some 10% of tumors without the mutations do so. Several investigators claim that EGFR gene copy number is more predictive of response or survival, although EGFR mutations were far better than copy number at least in our cohort. Tumors with mutations of the HER2 gene (3%) and the KRAS gene (14%) do not respond to EGFR-TKI. Of interest, these three mutations had a strict mutual exclusionary relationship.
It is almost inevitable for patients to show disease progression after presenting with an initial marked response to EGFR-TKIs. Secondary mutation occurring in exon 20 of the EGFR gene (T790M) has been associated with the acquired resistance to EGFR-TKI. We were able to confirm that 7/14 patients with acquired resistance after initial dramatic response had T790M, but that no novel mutations were found. New classes of TKIs that are able to overcome T790M are currently being developed. Recently, Engelman et al. reported that amplification of the MET gene is another mechanism of acquired resistance of EGFR-TKI through activation of HER3 pathway. It is known that a small fraction of NSCLCs has T790M or MET amplification even before EGFR-TKI therapy. Obviously, these NSCLC would require another treatment strategy.
Since all but one phase III trials have failed to show a survival advantage of the treatment arm involving EGFR-TKIs, it appears important to select patients by some of biomarkers. Through these efforts, it would be possible to individualize EGFR-TKI treatment for patients suffering from lung cancer.

Imatinib (Gleevec^®)as a Paradigm of Targeted Cancer Therapies

Brian Druker

Imatinib (Gleevec^®) exemplifies the successful development of a rationally designed, molecularly targeted therapy for the treatment of a specific cancer. Imatinib is an inhibitor of the ABL, platelet-derived growth factor receptor, and KIT tyrosine kinases. Given the pathogenetic role of the BCR-ABL tyrosine kinase in chronic myeloid leukemia (CML), this was the first disease selected for clinical trials with imatinib. In the CML clinical trials, patients in all phases of the disease, chronic, accelerated, and blast crisis, responded to imatinib and experienced minimal toxicity. Responses in patients with newly diagnosed chronic phase disease have been durable with a 5-year overall survival of 90%. Relapses in patients with accelerated phase and blast crisis are much more frequent. The most common mechanism of relapse is mutations in the ABL kinase domain that are imatinib resistant. The structural basis for imatinib resistance has been characterized and there are now two new ABL inhibitors for imatinib resistant patients. Clinical trials with imatinib have been expanded to other indications and there are now examples of malignancies driven by each of the targets of imatinib where remarkable results have been seen. The rationale for the use of imatinib in these various diseases and the clinical trial results will be reviewed. Many new agents targeting molecular defects in other cancers are being developed and the translation of the imatinib paradigm to other malignancies will be explored.