Anaplastic lymphoma kinase (ALK): Structure, oncogenic activation, and pharmacological inhibition

Abstract

Anaplastic lymphoma kinase was first described in 1994 as the NPM-ALK fusion protein that is expressed in the majority of anaplastic large-cell lymphomas. ALK is a receptor protein-tyrosine kinase that was more fully characterized in 1997. Physiological ALK participates in embryonic nervous system development, but its expression decreases after birth. ALK is a member of the insulin receptor superfamily and is most closely related to leukocyte tyrosine kinase (Ltk), which is a receptor protein-tyrosine kinase. Twenty different ALK-fusion proteins have been described that result from various chromosomal rearrangements, and they have been implicated in the pathogenesis of several diseases including anaplastic large-cell lymphoma, diffuse large B-cell lymphoma, and inflammatory myofibroblastic tumors. The EML4-ALK fusion protein and four other ALK-fusion proteins play a fundamental role in the development in about 5% of non-small cell lung cancers. The formation of dimers by the amino-terminal portion of the ALK fusion proteins results in the activation of the ALK protein kinase domain that plays a key role in the tumorigenic process. Downstream signaling from ALK fusion proteins involves the Ras/Raf/MEK/ERK1/2 cell proliferation module and the JAK/STAT cell survival pathway. Furthermore, nearly two dozen ALK activating mutations participate in the pathogenesis of childhood neuroblastomas along with ALK overexpression. The occurrence of oncogenic ALK, particularly in non-small cell lung cancer, has generated considerable interest and effort in developing ALK inhibitors. Currently, crizotinib has been approved by the US Food and Drug Administration for the treatment of ALK-positive non-small cell lung cancer along with an approved fluorescence in situ hybridization kit used for the diagnosis of the disease. The emergence of crizotinib drug resistance with a median occurrence at approximately 10 months after the initiation of therapy has stimulated the development of second-generation drugs for the treatment of non-small cell lung cancer and other disorders. About 28% of the cases of crizotinib resistance are related to nearly a dozen different mutations of ALK in the EML4-ALK fusion protein; the other cases of resistance are related to the upregulation of alternative signaling pathways or to undefined mechanisms. It is remarkable that the EML4-ALK fusion protein was discovered in 2007 and crizotinib was approved for the treatment of ALK-positive non-small cell lung cancer in 2011, which is a remarkably short timeframe in the overall scheme of drug discovery.

Introduction

The human protein kinase family consists of 518 genes thereby making it one of the largest gene families [1]. These enzymes catalyze the following reaction:

MgATP⁻¹ + proteinOH → proteinOPO₃²⁻ + MgADP + H⁺

Based upon the nature of the phosphorylated OH group, these proteins are classified as protein-serine/threonine kinases (385 members), protein-tyrosine kinases (90 members), and tyrosine-kinase like proteins (43 members). Moreover, there are 106 protein kinase pseudogenes. Of the 90 protein-tyrosine kinases, 58 are receptor and 32 are non-receptor kinases. A small group of dual-specificity kinases including MEK1 and MEK2 catalyze the phosphorylation of both tyrosine and threonine in target proteins; dual-specificity kinases possess molecular features that place them within the protein-serine/threonine kinase family. Protein phosphorylation is the most widespread class of post-translational modification used in signal transduction. Families of protein phosphatases catalyze the dephosphorylation of proteins thus making phosphorylation-dephosphorylation an overall reversible process [2].

Protein kinases play a predominant regulatory role in nearly every aspect of cell biology [1]. They regulate apoptosis, cell cycle progression, cytoskeletal rearrangement, differentiation, development, the immune response, nervous system function, and transcription. Moreover, dysregulation of protein kinases occurs in a variety of diseases including cancer, diabetes, and autoimmune, cardiovascular, inflammatory, and nervous disorders. Considerable effort has been expended to determine the physiological and pathological functions of protein-kinase signal transduction pathways during the past 30 years.

Besides their overall importance in signal transduction, protein kinases represent attractive drug targets. See Ref. [3] for a classification of six types of small molecule protein kinase inhibitors and Supplementary Table 1 for a list of 18 US Food and Drug Administration approved protein kinase inhibitors and Supplementary Fig. 1 for their structures. Supplementary Table 1 includes four drugs that were approved in 2012 including (a) axitinib, which is used in the treatment of renal cell cancer, (b) bosutinib, which is used in the treatment of Philadelphia chromosome positive chronic myelogenous leukemia, (c) regorafenib, which is used in the treatment of colorectal cancer, and (d) tofacitinib (pronounced toe” fa sye’ ti nib), which is used in the treatment of rheumatoid arthritis. Nine of the agents listed in the Supplementary table are multi-kinase inhibitors, and future studies will address the importance and generality of the therapeutic effectiveness of such drugs.

. US Food and Drug Administration approved small molecule protein kinase inhibitors.^a,b

One characteristic property of protein kinases is that they are stringently regulated, and the mechanisms for the interconversion of dormant and active enzymes are often intricate. Taylor et al. refer to this interconversion as switching [4]. Furthermore, protein kinases have not evolved to catalyze the phosphorylation of thousands of molecules per minute like a general metabolic enzyme such as hexokinase (k_cat = 6000 min⁻¹) [5]. When a receptor protein-tyrosine kinase is activated by its ligand, the chief phosphorylated product is the receptor itself by the process of autophosphorylation as described in Section 4.1. Thus, classical steady-state enzyme kinetics, which is based on the premise that the substrate concentration greatly exceeds that of the enzyme, fails to apply to the physiological function of protein kinases when the concentrations of the enzyme and substrate are similar. For example, Fujioka et al. measured the content of MEK and ERK in HeLa cells and found that the concentrations of these protein kinases were 1.4 μM and 0.96 μM, respectively [6]. ERK is the only known substrate of MEK, and in this case the substrate concentration is less than that of the enzyme.

Taylor et al. argue that pre-steady-state kinetics for the phosphoryl transfer is important in the analysis of protein kinase function [4]. Grant and Adams found that the pre-steady-state phosphoryl transfer rate of PKA is significantly faster than the steady-state k_cat (30,000 versus 1200 min⁻¹) [7]. The rapid pre-steady-state rate of enzymes, which lasts about 20 ms for PKA, is called a burst. Such kinetics are consistent with the rapid and transient reactions that have been optimized for the specific and local phosphorylation of proteins involved in a signaling pathway. Taylor et al. suggest that pre-steady-state kinetics should be used routinely in the assessment of protein kinase signaling mechanisms [4].