Different ALK fusion proteins have been identified as responsible of various types of cancer, and their constitutive dimerization and autophosphorylation leads to cellular transforming through a complex signaling network favoring cell cycle progression, survival, cytoskeletal rearrangement and cell migration.
Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumors in nude mice (Manabu Soda et al. 2007). Moreover, the treatment with a small molecule ALK inhibitor led to tumor regression in vivo, demonstrating that the oncogenic ALK signaling is a necessary condition for neoplastic phenotype and that its abrogation could represent a novel strategy for the treatment of NSCLC.
Fusion protein EML4 ALK
The inversion within chromosome 2p leads to formation of a fusion product of 4 kb. Nucleotide sequencing of this genomic fragment revealed that intron 13 of EML4 is distrupted at a point 3.6 Kb downstream of exon 13 and is inverted to connect to a position 297 bp upstream of exon 21 of ALK, yielding EML4-ALK product. The derived protein consists of the amino-terminal portion of echinoderm microtubule-associated protein-like 4 (EML4) and the intracellular region of the anaplastic lymphoma kinase (ALK). EML4 belongs to the family of echinoderm microtubule associated protein-like proteins and is composed of:
- an N-terminal basic region
- a hydrophobic domain (HELP) for the association to microtubules
- the WD repeats that is necessary for protein-interactions.
In the predicted fusion protein, the N-terminal half of EML4 encompassing the basic region, the HELP domain and portion of the WD-repeat region, is fused to the intracellular juxtamembrane region of ALK. The coiled-coil domain within this portion of EML4 mediates the constitutive dimerization and activation of EML4-ALK, which is responsible for the generation of transformed cell foci in culture and the formation by these cells of tumors in nude mice.
EML4-ALK possesses transforming activity that is dependent on its catalytic activity. In fact, protein fusion in which ATP-binding site of the kinase domain is mutated, is not able to form tumors. All domains of EML4 seem to contribute to the oncogenic potential of EML4-ALK, with the basic domain being the most important because it has an important role in dimerization of EML4-ALK.
ALK active chimera are constitutively phosphorylated and EML4-ALK proteins are associated to phosphorylation of STAT3, SHC, Erk1/2 and Shp2.
EML4-ALK as a potential therapeutic target
Several small compounds have recently been shown to inhibit the kinase activity of ALK and to suppress the growth of cells expressing NPM-ALK.
in vitro treatment: To investigate whether use of such ALK inhibitors might be an effective treatment for EML4-ALK NSCLC, Manabu Soda et al. have addicted a chemical inhibitor (WHI-P154) of ALK to the culture medium affected. At a concentration of 10 microM, WHI-P154 rapidly induced the death of these cells. Consistent with these observations, immunoblot analysis revealed that WHI-P154 inhibited the tyrosine phosphorylation of EML4-ALK (Tyr 1604 of wild-type ALK) in a concentration-dependent manner, and that ALK inibitionwas associated to a significant decrease in phosphorylation of several ALK downstream targets, including STAT3, ERK1, SRC, Shp2 and AKT, all known to play a pathogenetic role in other cellular contexts.
in vivo treatment: Manabu Soda et al.(2008) have generated independent mouse lines in which EML4-ALK expression is driven by the SPC promoter (surfactant protein C gene is specifically expressed in alveolar epithelial cells); thus they have generated transgenic lines that express transgenes specifically in lung epithelial cells. Such mice develop hundreds of adenocarcinoma nodules in both lungs within only a few weeks after birth. Immunohistochemical analysis with antibodies to ALK showed a diffuse cytoplasmic staining with granular accentuations in the neoplastic cells, in accord to analysis obtenu by human lung cancer biopsy.another inhibitor, a 2,4-pyrimidinediamine derivative has a median inhibitory concentration for ALK of 10 nM and a high specificity to ALK. M.Soda et al. have examined whether peroral treatment with this compound (10 mg per kg of body weight per day) might inhibit the growth or induce the death of the adenocarcinoma cells in the transgenic mice. It was found that with the ALK inhibitor treatment, a large tumor in the lower lobe of the right lung of mice was reduced to 30% of its original size after only 11 days of the drug treatment and was almost undetectable after treatment for 25 days.
Thus, EML4-ALK activation through multiple pathway promotes tumor cell growth and provides pro-survival signals, whose ablation results in cell cycle arrest and/or apoptosis and tumor regression, in vitro and in vivo.
Given the rapid development of NSCLC induced by EML4- ALK, the tumor cells are likely dependent for growth on the tyrosine kinase activity of the fusion protein. Such ‘‘oncogene
addiction’’ provides a potential target for the development of treatment strategies.
It is likely that expression of EML4-ALK (and probably other accompanying genetic changes) alone is not sufficient to render the cancer cells metastatic. It remains to be determined whether additional abnormalities in other oncogenes or tumor suppressor genes, such as KRAS or LKB1, may lead to the generation of metastatic tumors in EML4-ALK transgenic mice.
The pathogenetic role of ALK fusions or the deregulated expression of wt ALK in solid tumors remains unclear and the tumorigenic contribution of ALK fusions remains to be proved in lung cancer.
- A mouse model for EML4-ALK-positive lung cancer
- Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer
- Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer