Identification of Causative Gene Mutations of Neuroblastoma
New Target Found for Development of Agents to Overcome Intractable Childhood Cancer

[ Background ]

Neuroblastoma is one of the most intractable cancers in childhood. It is thought to arise from nerve precursor cells, and affects one in 7,000 children in the United States. Some neuroblastomas naturally cure without therapies, but many cases of advanced neuroblastoma, accounting for ∼30% of the cases, are resistant to any therapeutic modalities, and only less than 40% of cases can achieve cure even with intensive chemotherapies combined with hematopoietic stem cell transplantation. Compromised quality of life due to the use of large amount of anti-tumor drugs is also a serious problem in long-term survivors. On the other hand, growing lines of evidence now suggest that those drugs that target a key molecule for cancer development could dramatically improve the outcome of cancer treatment. For examples, some lung cancers have excessive activity of epidermal growth factor receptor (EGFR) as a result of the abnormality that occurs in their EGFR gene, and a drug, called Gefitinib, that strongly inhibits the function of EGFR, is shown to be highly effective to those cancers having abnormality of the EGFR gene. Another drug, called Imatinib Mesylate, which was developed to target the BCR/ABL kinase, the causative gene for chronic myeloid leukemia, shows dramatic effects in the treatment of CML. Now effectiveness of these types of new drugs, called molecular targeted-drugs, has been repeatedly demonstrated in the treatment of breast cancer, in addition to lung cancer and CML. Unfortunately, however, no promising molecular targets have been known that can be used to develop neuroblastoma, and thus it is very important to identify the plausible molecule to be targeted for neuroblastoma.

[ Contents and results ]

The project team developed software technology that enabled accurate determination of copy number of cancer genome using high-density SNP arrays, called CNAG ( http://www.genome.umin.jp ), which is now widely used for the analysis of cancer genome. Using this technology, they conducted a large scale cancer genome studies, in collaboration with the Cancer Genomics Project at the University of Tokyo Hospital (Dr. Yuji Taketani, Director), promoted by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and also with the Department of Pediatrics, Graduate School of Medicine, University of Tokyo (Dr. Takashi Igarashi, Prof).
Using the above technology of highly accurate analysis of gene copy numbers, the project team analyzed genomic DNA from a total of 239 neuroblastoma samples in detail and found a common region showing increased genome copy numbers in many neuroblastoma samples (Fig. 1). Because ALK kinase was the only gene included in this region, it was postulated that ALK is a target gene in neuroblastoma, and that excessive activity of ALK due to increased copy number of ALK is related to the development of neuroblastoma. Through the analysis of nucleotide sequence of ALK gene in neuroblastoma samples obtained from the patients, the project team revealed that:
1. ALK gene was mutated in about 6% of neuroblastoma patients (Fig. 2),
2. Most of the mutations were found in advanced neuroblastoma, and
3. Preferentially involved functionally important portions of ALK kinase (Fig. 3).
Moreover, they experimentally demonstrated that:
4. In fact, kinase activity was increased in these mutated ALK (Fig. 4),
5. Mutant ALK gene transformed normal cells to be able to generate tumor in mice, while normal ALK gene did not (Fig.5), and
6. Inhibition of expression of abnormal ALK kinase in neuroblastoma cells lead to their growth suppression (Fig. 6).
From these findings, it becomes clear that ALK gene is a causative gene in neuroblastoma, whose mutation or other abnormality leads to excessive activity of ALK kinase, giving rise to the development of neuroblastoma.

Figure 1. Detection of ALK gene amplification in neuroblastomas

By analyzing DNA obtained from neuroblastoma samples by SNP arrays, a common region of 845,000 bp in length was identified, which underwent recurrent gain of genetic materials (DNA). ALK was the only gene that was included in this region, indicating that ALK is one of the causative genes that are relevant to the development of neuroblastoma. Each red points in the figure shows the measured number of gene copies and the upward shifts of red points indicate gains of gene copy numbers. Note that to the left of the ALK gene, the locus for the MYCN gene exists, which is the first oncogene found in neuroblastoma long ago.

Figure 2. Structure of ALK kinase and distribution of mutations found in neuroblastoma

ALK kinase is a receptor tyrosine kinase localized to cell membrane through its transmembrane (TM) region. The portion to the right of the TM lies within the cell and contains a critical region, called kinase domain, which is responsible for the enzymatic activity of the kinase. Each triangle on the top shows the position of an individual mutation. It shows two hotspots for ALK mutations, where the numbers in the bottom indicate the number of amino-acid from the first amino-acid of the kinase.

Figure 3. Positions of mutations in kinase domain in predicted three-dimensional structure of ALK kinase

The amino-acids frequently involved in neuroblastoma (F1174 and R1275 mutations) take approximate positions to each other. This portion is known to be important for the activity of the ISR family kinases, including ALK.

Figure 4. Increased kinase activity of mutant ALK kinases

This shows kinase activities of two mutant ALKs found in neuroblastoma, as well as that in normal ALK, which were measured after each kinase was expressed in cells. Mutant kinases show increased kinase activity compared with normal ALK.

Figure 5. Evaluation of tumorigenicity of mutant ALK

We introduced normal and mutant ALK kinase in NIH3T3 cells and subcutaneously inoculated them into mice. While cells introduced with normal ALK did not show tumor formation, those introduced with mutant ALK produce tumors in mice, indicating these abnormal kinases have potential to make cell cancerous.

Figure 6. Suppression of cell growth in neuroblastoma cells by inhibition of mutant ALK expression

Expression of ALK can be inhibited by using specific RNA, called siRNA. We inhibited the expression of mutant kinase, and examined its effects on growth of neuroblastoma cells. While untreated cells or cells introduced with non-specific siRNA did not show growth suppression, the cell growth is inhibited by using ALK-specific siRNA in neuroblastoma cells having an ALK mutation, indicating cell proliferation of neuroblastoma depends on the expression of abnormal ALKs.

[ Future potential ]

Through the current study, we revealed that ALK mutations play an important role in the development of advanced neuroblastoma. Given that AKL mutations preferentially found in advanced neuroblastoma cases, the most important implication of our findings is that the development of an ALK inhibitor could dramatically improve the outcome of those children suffering from intractable neuroblastoma. Since inhibitors of EGFR and ABL kinase lead to dramatic success in the improvement of clinical outcome of lung cancer and CML, respectively, an ALK inhibitor could be expected to be a promising agent in the treatment of advanced neuroblastoma. In addition, according to the observation that ALK-deficient mice grow normally, such an inhibitor would not cause serious adverse reactions in children. In conclusion, our findings may contribute to the improvement of the treatment for advanced neuroblastoma in the near future.

[ Publications ]

"Oncogenic mutations of ALK kinase in neuroblastoma"
doi: 10.1038/nature07399

[ Technical terms ]

SNP array

SNP (single nucleotide polymorphism) is a type of DNA polymorphisms, defined by a single nucleotide change. It is the most common form of polymorphisms and more than 10 million common SNPs (e.g. found in >1% of the human population) are thought to exist within the human genome. Recent achievements in the International HapMap Project and a number of large-scale population studies clearly demonstrated its usefulness in exploring the genetic basis of common diseases, including cancers.
The core technology used to conduct these studies was microarray-based genotyping systems, SNP arrays, which enable genotyping of more than one million of SNPs per individual at a time. On a SNP array, hundreds of millions of molecular probes to detect SNPs are synthesized in a small chip, each of which can detect signals from different SNP-containing DNA fragments to be used for determination of SNP types. On the other hand, the same technology can be used to measure copy numbers alterations in cancer genomes. The project team developed a series of software technology, CNAG, to accurately measure SNP-specific signals from the samples. By analyzing copy number changes in cancer genomes using this technology, we can identify genetic targets of cancers.

Tyrosine kinase

Protein consists of a sequence of amino-acids. Tyrosine kinases are those enzymes that specifically phosphorylate the amino acid named, tyrosine, among the sequence. Among other kinases are serine/threonine kinases, which phosphorylate serine or threonine, rather than tyrosine.

Epidermal growth factor receptor (EGFR)

The epidermal growth factor (EGF) is a protein that binds to its receptor, EGF receptor (EGFR) expressed on the cell surface of a number of epithelial tissues, including skin and gastrointestinal mucosa, and promotes the proliferation of the latter cells. The function of EGFR is mediated by its tyrosine kinase activity, which depends on EGF and is induced when EGF binds to the receptor. Some lung cancers have a mutated EGFR, which is constitutively activated regardless of EGF binding, leading to cancer development.

Kinase inhibitor, Gefitinib and Imatinib Mesylate

Gefitinib was an EGFR-specific tyrosine kinase developed by AstraZeneca (IressaR). It specifically inhibits the tyrosine kinase activity of EGFR and has high efficacy to EGFR-mutated lung cancer cases. Imatinib Mesylate is another tyrosine kinase inhibitor, whose action is specific to ABL kinase as well as c-kit kinase and PDGFRs. It is highly effective to those cancers that depend on abnormal activity of these kinases for their growth, including CML (BCR/ABL kinase), Gastrointestinal stromal tumor (c-kit), and other rare types of leukemia.

Chronic myeloid leukemia

A kind of leukemia caused by abnormal activity of BCR/ABL kinase, which is generated by a chromosomal translocation between chromosome 9 and 22. On this translocation, ABLgene on chromosome 9 and BCR gene on chromosome 22 are fused to result in the generation of BCR/ABL fusion protein, and the aberrant tyrosine kinase activity of the latter protein is responsible for the development of CML.

ALK gene

ALK gene was first identified as an abnormal tyrosine kinase gene in a rare type of malignant lymphoma, called anaplastic large cell lymphoma, hence, ALK (anaplastic large cell lymphoma kinase). In this lymphoma, it is fused with another gene, named NPM, to generate NPM-ALK fusion protein. ALK is a kind of receptor tyrosine kinases, like EGFR. The tyrosine kinase activity of ALK is thought to be induced on its ligand, although the physiological ligand of ALK is still unknown. It is also fused to a variety of partner gene and more recently shown to participate in the pathogenesis of some lung cancers, where it is fused with EML4 to generate EML4-ALK. In cancers having ALK-containing fusion genes, the increased tyrosine activity is implicated in carcinogenesis.

PRESS CONTACTS

Seishi Ogawa, MD., Ph.D.,
Associate Professor, Cancer Genomics Project, The University of Tokyo Hospital
Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
TEL: 03-3815-5411 ext 30673
E-mail:

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