Seeking likely suspects
The DNA blueprint that governs how the body is constructed and operates is encoded in different combinations of four types of chemical units, or nucleotides, that make up genes. The order of the nucleotides (specified as A, G, T, and C) is called the sequence of the gene, and it spells out a unique message that may be hundreds or thousands of nucleotides in length. The genetic alterations in cancer cells can be likened to typographical errors, or big chunks of the sequence that are scrambled, deleted, or misplaced.
Dana-Farber scientists are extracting the DNA from tumor samples, checking for quality, isolating the kinase gene segments of interest, and preparing them to be deciphered by robots.
Thanks to the just-completed Human Genome Project, the normal, healthy sequences of most of the estimated 30,000 genes in people are available in a database. One way of highlighting abnormal genes linked to cancer is to compare the normal sequence with that of genes taken from tumors. Rather than comb the entire 3 billion letters of the human DNA script, though, scientists are focusing on likely suspects. So, to initially harvest the low-hanging fruit, Meyerson and Sellers proposed sequencing types of genes that have already been implicated in many kinds of cancer — the so-called tyrosine kinases.
Roberts notes that members of another large kinase group, known as serine-threonine kinases, are also under growing scrutiny as important cancer triggers.
While the actual sequencing of DNA will be carried out at the Whitehead Institute/MIT Center for Genome Research, Dana-Farber scientists are extracting it from tumor samples, checking for quality, isolating the kinase gene segments of interest, and preparing them to be deciphered by the Whitehead robots.
Dana-Farber colleagues Matthew Meyerson, MD, PhD, (left) and William Sellers, MD, launched the project to find mutant kinases in cancer cells and match them with inhibitor drugs.
Much of this work is performed in Sellers' laboratory by postdoctoral fellow Guillermo Paez, PhD, whose computer screen saver is a colorful molecular diagram of a tyrosine kinase called the platelet-derived growth factor receptor. Paez works with tumor specimens, frozen in liquid nitrogen, that come from the DFCI tissue bank or from collaborators at other facilities.
Paez extracts the DNA and singles out the particular blocks of DNA code, called exons, where kinase mutations are likely to be found. The most exacting step is designing "primers," short DNA sequences that he attaches to each exon so it can be copied many times over for sequencing. "We have to design a specific primer for each exon of each kinase," says Paez.
Already, the Sellers lab has made and tested more than 1,700 sets of primers for attachment to the exons of 94 different tyrosine kinase genes in each tumor sample. For every test run, Paez and his co-workers combine the primers with 94 samples of DNA from one human tumor — a separate sample for each kinase gene that will be scanned for mutations. At the Whitehead, robots read the genes' sequences in search of "typos." Sellers estimates 1.7 million "reads" will be needed to search for mutant tyrosine kinase genes in hundreds of different tumors.
One challenge is that the Kinase Project hasn't yet been fully funded. The investigators have been leveraging existing grants to get started, but they estimate they'll need at least $6 million to see it through. Institute staff are seeking funding sources for this effort, deemed a strategic priority for Dana-Farber.
"One way or another, we're going to accomplish this," states DFCI President Edward J. Benz Jr., MD. "The project is one of our most important contributions to waging the war on cancer."
