Anaplastic Lymphoma Kinase Is Required for Neurogenesis in the Developing Central Nervous System of Zebrafish

10.1371/journal.pone.0063757PLoS ONE85

Anaplastic Lymphoma Kinase as a Therapeutic Target in Anaplastic Large Cell Lymphoma, Non-Small Cell Lung Cancer and Neuroblastoma

Anaplastic lymphoma kinase (ALK), a receptor tyrosine kinase in the insulin receptor superfamily, was originally identified as the oncogenic NPM (nucleophosmin)-ALK fusion protein due to a t (2;5) chromosomal translocation in anaplastic large cell lymphomas. Many other chromosomal rearrangements or gene mutations/amplification leading to enhanced ALK activity have subsequently been identified and characterized in a number of human cancer types. The recent reports of EML4 (echinoderm microtubule-associated protein- like 4)-ALK oncogenic proteins in non-small cell lung cancer (NSCLC) and the identification of ALK activating point mutations and gene amplification in neuroblastoma have indicated ALK as a potential major therapeutic target for human cancers. In this review, the role of oncogenic ALK in development of various human cancers is summarized and the efforts and progress of developing small molecule ALK inhibitors as potential cancer therapeutics are updated. Several small molecule ALK inhibitors from distinctive chemical scaffolds in either clinical or preclinical development stage are highlighted and profiled. The challenges and future directions of developing small molecule ALK inhibitors as cancer therapeutics are discussed. </jats:sec

NPM-ALK Mutants in Kinase Domain Exhibit Altered Kinase Activity and Various Sensitivity to ALK Inhibitors.

Abstract Abnormal expression of constitutively active anaplastic lymphoma kinase chimeric proteins (NPM-ALK) in the pathogenesis of anaplastic large-cell lymphoma (ALCL) is well defined. Recently, small molecule inhibitors have been reported that provide solid proof-of-concept validation that inhibition of ALK is sufficient to attenuate the growth and proliferation of ALK+ALCL cells. Although robust clinical response of ALCL patients to the ALK inhibitors is expected, some of those patients are also anticipated to develop resistance to an ALK inhibitor, most likely associated with single point mutations in the kinase domain of ALK. In this study, the nonsense mutants of NPM-ALK on the phosphate anchor region and the gatekeeper region were generated by site-directed mutagenesis and their kinase activity was measured in cells. NPM-ALK/L182M, L182V and L256M mutants displayed comparable or higher kinase activity in cells relative to NPM-ALK wt, and were able to render BaF3 cells into growth factor-independent growth, while NPM-ALK/L182R, L256R, L256V, L256P and L256Q displayed much weaker or little kinase activity in cells and failed to transform BaF3 cells. While NPM-ALK/L182M and L182V displayed comparable sensitivity to a fused pyrrolocarbazole (FP)-derived ALK inhibitor as NPM-ALK wt, they were &amp;gt;30-fold less sensitive to a diaminopyrimidine (DAP)-derived ALK inhibitor. On the other hand, NPM-ALK/L256M displayed &amp;gt;30-fold less sensitivity to both the FP-derived and the DAP-derived ALK inhibitors. Consistent with NPM-ALK autophosphorylation inhibition, the FP-ALK inhibitor induced growth inhibition and cytotoxicity of BaF3/NPM-ALK/L182M and L182V cells but not L256M cells, and the DAP ALK inhibitor failed to induce growth inhibition and cytotoxicity of all three BaF3/NPM-ALK mutant cell lines in culture. In the absence of an ALK inhibitor, BaF3 cells harboring NPM-ALK mutants did not display growth advantage in culture with 10% serum and in mice over the NPM-ALK wt cells. However, the BaF3/NPM-ALK mutants displayed significant growth advantage in culture with low serum (2%) over the BaF3/NPM-ALKwt cells, suggesting the BAF3/NPM-ALK mutant cells may proliferate better in the stressed conditions. Analyses of binding of ALK inhibitors to ALK wt and mutants in ALK homology models are in progress and the results will be discussed.</jats:p

Structural Analysis of the MAP Kinase ERK2 and Studies of MAP Kinase Regulatory Pathways

This chapter primarily highlights structure–function studies performed collaboratively between the Goldsmith and Cobb laboratories at the University of Texas Southwestern Medical Center in Dallas and includes experiments examining the relationship of MEKKl to the MAP kinase pathway and potential feedback mechanisms in the pathway. Mechanisms regulating the MAP kinase pathway are complicated and inactidvation methods plentiful. Receptor tyrosine kinases regulate the pathway through Ras; heterotrimeric G protein-coupled receptors also use Ras to activate the pathway, although there may also be Ras-independent mechanisms. The MAP kinase pathway is stimulated by numerous hormones, growth factors, and oncogene products including Ras and contributes to their spectrum of actions. The MAP kinases, however, are pleiotropic, phosphorylating many substrates throughout the cell. This pathway has been repeated several times in yeast and mammalian cells, although mechanisms regulating the similar but parallel cascades are sketchier. There are three low-activity forms of each enzyme, the unphosphorylated protein and the two singly phosphorylated forms, that contain phosphate on only tyrosine or only threonine. These two singly phosphorylated ERKs have little more protein kinase activity than unphosphorylated proteins. Because ERKl and ERK2 can autophosphorylate on tyrosine, the form containing only threonine phosphate may, in as yet undefined circumstances, be able to reactivate itself through autophosphorylation. The three-dimensional structure of ERK2 contains the two-domain organization found in all protein kinases whose structures have been determined thus far. The smaller N-terminal domain provides many contacts for ATP, and the larger C-terminal domain contains the major determinants for protein substrate interactions. It is very important to identify the unique features, if any, of these enzyme pairs. It is interesting that phosphorylation by MAP kinase had so little effect on MEK activity, because phosphorylation of MEKl by cdc2 at a site (S286) close to the MAP kinase site (T292) in the C-terminal insert greatly inhibits MEKl activity

Map Kinases Erk1 And Erk2: Pleiotropic Enzymes In A Ubiquitous Signaling Network

MAP kinase is used to refer to at least two distinct proteins, known as the extracellular signal-regulated protein kinases ERK 1 and ERK2. The discussion of proteins that are regulated by ERKs points to the pleiotropic nature of signaling pathways regulated by this family of protein kinases. Relatively, few data are yet in hand to prove that any of these proteins are substrates for these enzymes in vivo. The impact of these enzymes on cell function can be deduced from the recent experiments using mutated enzymes. The ERKl phosphorylation site and lysine mutants have proved useful dominant inhibitors. In Jurkat cells, the mutants inhibit induction of the cytokine IL-2. It was found that ERKl and ERK2 mutants block the ability of ras, serum, and phorbol ester to induce transcription from a TPA response element. ERK2 mutants prevent proliferation caused by activated Raf EGF or small tau antigen. The ubiquitous MAP kinases are activated by a remarkable variety of hormones in differentiated cells and growth factors in dividing cells. Their activation has been linked to the transition from G0 to G1 in the cell cycle and to the induction of differentiated phenotypes. These enzymes are essential components of a universal protein kinase cascade implicated in the control of many cellular processes