Developing smart drugs

Apr 30 2004

Dana-Farber Cancer Institute Researchers, sensing an opening in the cancer battle, are mounting a quick thrust to flush out suspected molecular cancer triggers in tumor cells. Drug companies can then select specific compounds that block these triggers, turning off the cells' stimulus to grow, but leaving normal cells unaffected.

The scientists are scanning the DNA from human tumor samples, sifting through their genetic blueprints for genes that produce abnormal growth-stimulating proteins known as tyrosine kinases. These mutant proteins behave like stuck "on" switches, causing unruly cell growth. The beauty of the plan, the investigators say, is that pharmaceutical companies have already developed hundreds of different drugs that inhibit tyrosine kinases, so they can quickly move these agents into clinical trials in humans. The approach could shave years off the customarily long delay between finding a vulnerable target in cancer cells and then testing new drugs to attack it.

Kinases are enzymes in cells that regulate their behavior, including when they should grow and when they should rest. Damaged kinases have been found in many types of cancer, where they spur the cells into runaway malignant growth. Many of those kinases belong to the group called tyrosine kinases. Of the 500 kinases in the human body, 94 of them are of the tyrosine type.

The new campaign was inspired by Dana-Farber research on tyrosine kinases in the 1980s and '90s that paved the way for the highly successful cancer drug Gleevec. It was among the first agents to take aim at specific mutant tyrosine kinases as a cancer therapy. Gleevec has stopped or shrunk tumors in many patients with chronic myeloid leukemia (CML) and gastrointestinal stromal tumor (GIST), a disease that had been virtually untreatable.

But these are two relatively rare cancers. Now the scientists want to open a wider front against more common forms. They have begun culling DNA from human tumors, hoping to process about 500 samples within a year in search of overactive tyrosine kinase proteins. They're hunting them by sequencing their blueprints -- the DNA in the genes that produce the proteins.

As they discover new mutated kinases, the scientists will disseminate the findings so that pharmaceutical companies can match them with inhibitors already on the shelf. Development of these drugs has sped forward in recent years after it became evident that blocking kinase-stimulated cell growth was a promising new approach for combating cancer.

The investigators say the stars are aligned to press this search, named the Kinase Project. Todd Golub, MD, who is affiliated with both institutions, notes that Dana-Farber researchers had the foresight several years ago to begin freezing patient tumor samples and "waiting for the day they had technology in hand to make these kinds of discoveries."

"It took 20 years from the time mutant kinases were discovered to the approval of Gleevec," says Dana-Farber's Thomas Roberts, PhD, who helped lay the groundwork for thwarting kinases, particularly in blood cancers. He did so with DFCI colleagues Charles Stiles, PhD, James Griffin, MD, and Brian Druker, MD, who's now at the Oregon Health Sciences Center. "If we find an entirely novel mutant kinase today," Roberts adds, "it would take two years or less" to begin clinical trials of an inhibitor (drug) to block it.

Gleevec, formerly known as STI-571, had been developed by Ciba-Geigy Corp. in Switzerland as one of many kinase inhibitors. The company, now called Novartis after a merger, was persuaded to try it against the uncommon and hard-to-treat cancer CML, caused by a chromosome abnormality that triggers a runaway kinase signal.

Initially approved only for CML, Gleevec rapidly gained federal approval for use against GIST in early 2002, thanks in part to research and advocacy by Dana-Farber's George Demetri, MD. It is now viewed as the wave of the future in cancer drugs.

Unlike standard chemotherapy drugs, kinase inhibitors are so specific that they spare normal cells and tissues. As a result, their side effects are much milder, and they can be taken orally rather than needing to be injected into veins.

In a more recent discovery, Dana-Farber investigator Scott Armstrong, MD, and colleagues found a mutant kinase called FLT-3 in cells of a particularly aggressive type of infant leukemia. With this distinctive abnormal kinase, the disease warranted a new, specific name: mixed lineage leukemia. As with the case of CML and Gleevec, inhibitors already existed -- four of them, in fact -- for this kinase. Today, one inhibitor-based treatment is already in clinical trials, showing activity against a type of adult leukemia known as acute myelogenous leukemia. It is about to be tried in infants.

The kinase hunt can be traced to a conversation between Sellers and Meyerson while flying to Montreux, Switzerland, in 2001 for a Novartis conference. "We were talking about the fact that if you really want to understand cancer, you need to understand the complete pattern of genetic changes in cancer cells that accounts for their malevolent behavior," recalls Meyerson, a molecular and cellular oncology researcher whose chief interest is lung cancer.

"Bill said, 'Let's start with the [mutant] kinases, because there are already drugs that can inhibit them,'" Meyerson says. He agreed. "The biggest impact we could have on cancer would be to find mutations that could immediately be translated into therapies."

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.

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.

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."

Kinases are proteins of a special category called enzymes, which spark chemical reactions throughout the body to make it grow and function. Many kinases act as on-off switches inside cells, controlling the continuous flow of chemical signals that tell the cell what to do.

A kinase sends a "go" message by adding a chemical unit called a phosphate to another protein, changing its shape or altering its function. The Kinase Project now under way at Dana-Farber is focusing on tyrosine kinases, so named because they add the phosphate to tyrosine, an amino acid component of the protein.

Many kinases, largely the tyrosine kinases, have been implicated in cancer. These on-off proteins may get stuck in the "on" position, continuously sending signals for the cell to grow and divide, and leading to uncontrolled proliferation of cells and tumor formation. A "stuck" signal switch is often caused by a harmful change, or mutation, in the gene that makes the kinase.

In the last decade or so, drug companies have developed many hundreds of compounds that fit into a specific kinase like a key in a lock. These kinase inhibitors turn off the switch, blocking the signals that are stimulating the cancer. In some cases, this halts or shrinks the tumor.