Cloning, Expression And Inhibitor Screening Of Anaplastic Lymphomas Kinase (ALK)

Protein phosphorylation is controlled by the coordinate actions of protein kinases and phosphatases and has a crucial role in cell proliferation, differentiation, and transformation. Protein tyrosine kinases are central regulators of signaling pathways. Perturbation of PTK signaling by mutations and other genetic alterations such as chromosomal translocation, interstitial deletion, internal tandem duplication, and amino acid substitution results in deregulated kinase activity and malignant transformation.Tyrosine kinases are involved in the pathogenesis of most cancers. However, few tyrosine kinases have been shown to have a well-defined pathogenetic role in lymphomas. The anaplastic lymphoma kinase (ALK) is the oncogene of most anaplastic large cell lymphomas (ALCL), driving transformation through sophisticated signaling mechanisms.It is generally agreed that ALCL is a distinct subset of T-cell non-Hodgkin lymphomas (NHL) for which a normal cellular counterpart has not yet been established. In the late 1980s, the t(2;5)(p23;q35) translocation was described by several groups as the most frequent event in ALCL and in 1994 the product of this translocation was identified as the receptor tyrosine kinase (RTK) ALK fused to nucleophosmin (NPM). Many groups have since discovered additional translocations in which ALK is fused to other partners.All the alk chromosomal aberrations lead to the expression and constitutive activation of ALK. This transmembrane receptor tyrosine kinase belongs to the insulin receptor superfamily. ALK expression in humans is normally limited to cells of neural origin. The expression of ALK in hematologic neoplasms is largely limited to ALCL tumors of T-cell or null-cell immunophenotype, in which 40% to 60% of these tumors express ALK. In 80% of these cases, ALK expression results from t(2;5)(p23;q35).Despite significant progress in delineating the molecular mechanisms of ALK+ ALCL, the therapeutic approaches to this lymphoma have not changed significantly. The current treatment is based on doxorubicin-containing combination chemotherapy which has severe side effects. This treatment induces complete remission in up to 95% of the patients, but relapse and resistance occur in more than 40% of the cases. The exact cause of the high rate of relapse is not known. The fact that NPM-ALK plays a central role in the development and pathogenesis of ALK+ ALCL tumors makes this chimeric protein a legitimate therapeutic target in this disease. Because of its repeatedly documented oncogenic potential and its expression in this type of malignant lymphoma, specific or selective targeting of NPM-ALK will probably be associated with less toxic effects, as compared with targeting other signaling partners that might be involved in maintaining the biologic functions of the nonneoplastic cells.This study was designed to express the intracellular segment of ALK which contain the tyrosine kinase domain in the baculovirus expression system and further to identify inhibitor of the enzyme. First, a DNA fragment that encodes the intracellular domain of ALK was amplified from the human brain cDNA library using a nested PCR method. The DNA fragment was then inserted into the pBluescript II KS cloning vector. Recombinant plasmids carrying the inserts were identified through blue white screening. After verification by DNA sequencing analysis, the ALK DNA fragment was cut out by using appropriate restrictive endonuclearases and subcloned into pBlueBacHis2B, a transfer vector of the baculovirus expression system. The recombinant transfer plasmid was then co-transfected with the linearized baculovirus genomic DNA construct into Sf9 insect cells to produce recombinant baculovirus expressing the intracellular domain of ALK. The recombinant viruses were further purified and amplified for expression of the ALK enzyme. For screening inhibitors of ALK tyrosine kinase domain, a protein kinase inhibitor library containing 80 potential anti-cancer drugs were used. First, ALK tyrosine kinase assays were performed in the presence of 25μM varied protein kinase inhibitors. Based on this initial screening, 23 compounds were selected for further analysis at a concentration of 1μM. Among them, 2 inhibitors were found to be most effective and their IC50 values were eventually determined. The data indicated that C7 and H11, two most potent inhibitors, displayed IC50 values of around 200nM and 100nM respectively.Tyrosine kinases have become highly attractive targets for development of therapeutic drugs to treat cancers. Naturally, potent inhibitors of ALK are potential drugs for treatment of anaplastic large cell lymphomas. In addition, since mutations of ALK have also been found in subsets of other cancers including lung and breast carcinomas, finding inhibitors of ALK should have general implications for finding a cure for cancer.