ALK receptor activation, ligands and therapeutic targeting in glioblastoma and in other cancers

Abstract

The intracellular anaplastic lymphoma kinase (ALK) fragment shows striking homology with members of the insulin receptor family and was initially identified as an oncogenic fusion protein resulting from a translocation in lymphoma and more recently in a range of cancers. The full-length ALK transmembrane receptor of ~220 kDa was identified based on this initial work. This tyrosine kinase receptor and its ligands, the growth factors pleiotrophin (PTN) and midkine (MK) are highly expressed during development of the nervous system and other organs. Each of these genes has been implicated in malignant progression of different tumor types and shown to alter phenotypes as well as signal transduction in cultured normal and tumor cells. Beyond its role in cancer, the ALK receptor pathway is thought to contribute to nervous system development, function, and repair, as well as metabolic homeostasis and the maintenance of tissue regeneration. ALK receptor activity in cancer can be up-regulated by amplification, overexpression, ligand binding, mutations in the intracellular domain of the receptor and by activity of the receptor tyrosine phosphatase PTPRz. Here we discuss the evidence for ligand control of ALK activity as well as the potential prognostic and therapeutic implications from gene expression and functional studies. An analysis of 18 published gene expression data sets from different cancers shows that overexpression of ALK, its smaller homolog LTK (leukocyte tyrosine kinase) and the ligands PTN and MK in cancer tissues from patients correlate significantly with worse course and outcome of the disease. This observation together with preclinical functional studies suggests that this pathway could be a valid therapeutic target for which complementary targeting strategies with small molecule kinase inhibitors as well as antibodies to ligands or the receptors may be used.

Keywords: anaplastic lymphoma kinase; growth factor; midkine; pleiotrophin; signal transduction.

FIGURE 1

Anaplastic lymphoma kinase (ALK) receptor domains, pathways, inhibitors, and homologies. Amino acid positions flanking different domains in ALK are shown. Small molecule kinase inhibitors and antibodies are indicated as is the ALK translocation breakpoint. The amino acid sequence overlap and homology of the ECD of LTK is shown. Also, the homology with the closest related kinases is indicated for the kinase domain. Mutations discussed in the text and also in (Wellstein and Toretsky, 2011). The LBD was identified by phage display of a brain cDNA library against immobilized PTN (Stoica et al., 2001). The LBD is within an MAM domain that is frequently found in the ECDs of a diverse family of transmembrane proteins (Prosite data base PDOC 00604). SigP, signal peptide; LBD, ligand binding domain; ECD, extracellular domain; ICD, intracellular domain; TM, transmembrane domain; PTN, pleiotrophin; MK, midkine; LTK, leukocyte tyrosine kinase; IGF1R, insulin-like growth factor 1 receptor; InsR, insulin receptor.

FIGURE 2

Binding of PTN and MK to the ALK receptor in intact cells. (A) Competition for ALK binding of ³⁵S-PTN in 32D/ALK cells. Competitors were: anti-PTN antibody, anti-ECD antibody, ECD protein, unlabeled PTN. (B) Titration of equilibrium binding of ³⁵S-PTN to 32D/ALK and to 32D/control cells. A Scatchard analysis of specific binding is shown in the inset. Modified from Stoica et al. (2001). (C) Competition of PTN for ALK receptor binding of ³⁵S-MK. Binding of ³⁵S-MK to 32D/ALK (filled symbols) and 32D/control cells (open symbols) was competed by different concentrations of PTN. Modified from Stoica et al. (2002).

FIGURE 3

Cross-talk between stromal and cancer cells via the PTN/MK–ALK pathway. PTN and MK are heparin-binding proteins released from cancer or stromal cells. They can bind at nanomolar affinity to glycosaminoglycans (GAGs) such as the heparan sulfate side chains of proteoglycan (HSPG) as well as chondroitin sulfate (CS; Deepa et al., 2002). CS is proposed as a co-receptor for MK (Muramatsu, 2010) as are other GAGs (Li et al., 2010). Also N-syndecan could function as such a co-receptor (Raulo et al., 1994). Both ligands and receptor can be found up-regulated in the stroma of cancer as well as in cancer epithelia (see text).

FIGURE 4

Expression of PTN or ALK in brain tumors in patients with different survival. Data from the Oncomine data base showing the expression levels of PTN (Phillips et al., 2006) or of ALK (Shai et al., 2003) in brain tumor samples. Tumor expression data were separated into those from patients alive or dead at 5 years (PTN) or 3 years (ALK) respectively. The data are presented as log values after normalization to the respective gene expression array and are shown as provided by Oncomine. Further studies are provided in Tables 1 and 2.

FIGURE 5

Effect of anti-ALK antibody on U87 GBM cell invasion of an endothelial cell monolayer. Endothelial cell monolayers were formed on electrodes and the intactness of the monolayer was monitored by electrical impedance sensing. Upon addition of the U87 cells the monolayer is disrupted and this is reflected in real-time as a decrease in electrical resistance of the monolayer. Inclusion of an anti-ALK antibody prevents this disruption (Stylianou et al., 2009).