Non-solid oncogenes in solid tumors: EML4-ALK fusion genes in lung cancer

Abstract

It is generally accepted that recurrent chromosome translocations play a major role in the molecular pathogenesis of hematological malignancies but not of solid tumors. However, chromosome translocations involving the e26 transformation-specific sequence transcription factor loci have been demonstrated recently in many prostate cancer cases. Furthermore, through a functional screening with retroviral cDNA expression libraries, we have discovered the fusion-type protein tyrosine kinase echinoderm microtubule-associated protein like-4 (EML4)-anaplastic lymphoma kinase (ALK) in non-small cell lung cancer (NSCLC) specimens. A recurrent chromosome translocation, inv(2)(p21p23), in NSCLC generates fused mRNA encoding the amino-terminal half of EML4 ligated to the intracellular region of the receptor-type protein tyrosine kinase ALK. EML4-ALK oligomerizes constitutively in cells through the coiled coil domain within the EML4 region, and becomes activated to exert a marked oncogenicity both in vitro and in vivo. Break and fusion points within the EML4 locus may diverge in NSCLC cells to generate various isoforms of EML4-ALK, which may constitute approximately 5% of NSCLC cases, at least in the Asian ethnic group. In the present review I summarize how detection of EML4-ALK cDNA may become a sensitive diagnostic means for NSCLC cases that are positive for the fusion gene, and discuss whether suppression of ALK enzymatic activity could be an effective treatment strategy against this intractable disorder.

Figure 1

Development of retroviral cDNA expression libraries for oncogene screening. (a) Although oncogenes controlled by a constitutive promoter or enhancer are expressed when introduced into fibroblasts, those controlled by a tissue‐specific promoter‐enhancer (e.g. specific to the hematopoietic system) are not transcribed in fibroblasts and will therefore not be detected by such functional screening. (b) To overcome this limitation, we synthesized cDNA from small quantities of clinical specimens, inserted them into a retroviral plasmid, and generated recombinant retroviral libraries. Theoretically, any type of dividing cell can be infected with such libraries, with the incorporated cDNA being expressed at a high level in the recipient cells under the control of the viral long terminal repeat (LTR).

Figure 2

Anaplastic lymphoma kinase (ALK) fusion proteins. Chromosome rearrangements result in the generation of fusion genes that encode an oligomerization domain (including the coiled coil domain) of nucleophosmin (NPM), tropomyosin (TPM3) or echinoderm microtubule‐associated protein like‐4 (EML4) fused to the intracellular region of the receptor‐type protein tyrosine kinase ALK. WD, WD‐repeat domain.

Figure 3

Diversity in the fusion points between echinoderm microtubule‐associated protein like‐4 (EML4) and anaplastic lymphoma kinase (ALK). (a) EML4 and ALK map in opposite orientations to the short arm of human chromosome 2 (shown in the genome browser of the University of California, Santa Cruz; http://genome.ucsc.edu/cgi‐bin/hggateway). Intron 13 of EML4 is ligated to intron 19 of ALK through a chromosome rearrangement, inv(2)(p21p23), generating the EML4–ALK (variant 1) fusion gene. Primers flanking the fusion point can be used for the molecular detection of EML4–ALK‐positive tumors by polymerase chain reaction. Arrows indicate the direction of transcription. (b) Exon boundaries of EML4 for possible in‐frame fusion to exon 20 of ALK are shown as vertical bars together with the exon numbers at the corresponding positions in the EML4 protein. Reverse tramscription–polymerase chain reaction screening has identified variants (V) 1, 2, 3, and 5 of EML4–ALK, in which exons 13, 20, 6, and 2, respectively, of EML4 cDNA are fused to exon 20 (e20) of ALK cDNA. Unexpectedly, another in‐frame fusion was identified in variant 4 cDNA, in which exon 14 of EML4 was fused via an 11‐bp sequence of unknown origin to the nucleotide at position 50 of ALK exon 20. CC, coiled coil domain; HELP, hydrophobic EMAL‐like protein domain; WD, WD‐repeat domain.

Figure 4

Activation mechanisms for anaplastic lymphoma kinase (ALK) and echinoderm microtubule‐associated protein like‐4 (EML4)–ALK. Wild‐type ALK is thought to undergo transient homodimerization in response to binding of a specific ligand, resulting in its activation and mitogenic signal transduction. In contrast, EML4–ALK is constitutively oligomerized via the coiled coil domain (CC) of EML4, resulting in persistent mitogenic signaling that eventually leads to malignant transformation.

Figure 5

Suppression of the growth of an echinoderm microtubule‐associated protein like‐4 (EML4)–anaplastic lymphoma kinase (ALK)‐positive cell line with an ALK inhibitor. The non‐small cell lung cancer cell line NCI‐H2228, which is positive for EML4–ALK (variant 3), was maintained in spheroid culture for 2 days and was photographed after an additional 5 days of incubation in the absence (upper panel) or presence (lower panel) of 5 nmol/L 2,4‐pyrimidinediamine.^{( 14 )} Scale bars = 4 mm.