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GENETICS Resistance Mutation Found for GleevecHigher Doses May Save Some Patients--Are Multipronged Therapies Next? In the relatively haphazard world of drug discovery, where new medicines are more often discovered by luck than invention, Gleevec has gained iconic status as a model of rational drug design. Yet the truth is, though Gleevec's target--a rogue tyrosine kinase enzyme that results in a chronic form of leukemia--was known for years, its maker, Novartis, was aiming at a different bull's-eye when it hit upon the drug, a tyrosine kinase inhibitor. The Mechanics of Resistance. The Y253F mutation appears to confer resistance by impairing the fit between Abl (dark gray ribbon) and Gleevec (pale gray blob). The residue T315 (red) makes direct contact with the central part of Gleevec, while Y253 in the P-loop folds down from the left side and makes an "induced fit" with the drug. (Image by Brad Brasher) In what appears to be another serendipitous twist, HMS researchers have discovered what is responsible for the inability of some patients with chronic myelogenous leukemia (CML) to respond to Gleevec (formerly STI-571). They have found a version of the rogue enzyme, Bcr-Abl, that resists Gleevec's arrows. "We weren't looking for Gleevec-resistant mutants," said Richard Van Etten, HMS associate professor of genetics (medicine) at the Center for Blood Research. Van Etten along with Sergei Roumiantsev, Bradley Brasher, and colleagues report the discovery in the Aug. 6 Proceedings of the National Academy of Sciences. Varying Mutant BehaviorsThough the Van Etten group was not in direct pursuit of them, Gleevec resistance-conferring mutants had been suspected soon after the initial clinical trials ended in 1999. The drug was stunningly successful in patients at early stages of disease but quickly stopped working in most patients with more advanced forms of CML. Last year, researchers at UCLA headed by Charles Sawyers found that some resistant patients carried a mutant version of Bcr-Abl. The original mutation, T315I, confers resistance by altering the enzyme's physical structure to block Gleevec from binding.The new mutation, which the HMS researchers discovered while investigating normal versions of Abl, differs from T315I in several intriguing ways. To begin, the genetic defect, Y253F, has been found as frequently as T315I in Gleevec-resistant patients. Though it helps cells resist Gleevec's assaults, Y253F does not make them entirely immune, which T315I does. Finally, although the precise mechanism of resistance is not known, the mutation appears to confer resistance not by physically blocking Gleevec but by weakening its bond with Abl. These findings--relatively high frequency, lower resistance, a possibly weaker mechanism--could spell good news for some patients with advanced forms of the disease.
Sergei Roumiantsev, Richard Van Etten, and Bradley Brasher (l to r) have found a mutation in some cancer patients that confers resistance to the new drug Gleevec but suggests that boosting the dose might help. (Photo by Steve Gilbert) "Because the resistance conferred by the new Bcr-Abl mutation is intermediate, it is plausible you could overcome it," Van Etten said. In fact, his team found they could inhibit Y253F-bearing cells by exposing them to higher levels of Gleevec, though this was not true of T315I-bearing cells. The same might be true of advanced CML patients who relapse on Gleevec treatment. Those with the Y253F mutation might respond to higher doses. "In theory, there is a lot of room to escalate the dose of this drug in patients," he said. An even more effective approach to combating resistance in CML would be to combine Gleevec with drugs that target other aspects of the disease process, as is done in the treatment of HIV, said Van Etten. "The efficacy of combinations of molecularly targeted drugs in HIV disease shows us what we should do in the treatment of chronic myelogenous leukemia." Characterizing AblDogged pursuit rather than serendipity characterized Van Etten's earliest forays into CML research. The disease was first traced decades ago to a shortening of the already diminutive chromosome 22. The shortening was subsequently shown to be due to a translocation with chromosome 9. Located on the small bit of genetic material acquired by chromosome 22 was a gene, ABL, that teamed up with an adjacent gene, BCR, to form a fusion protein. The fusion protein, Bcr-Abl, was thought to play a role in disease, but researchers were unsure if it was acting alone--and they had no mouse model to explore the question.In the late 1980s, Van Etten and George Daley, now at MIT's Whitehead Institute, infected mouse bone marrow cells with a retrovirus carrying a cloned BCR-ABL gene. They then used these altered cells to repopulate the bone marrow of radiation-treated mice. After several false starts, they produced mice that developed the symptoms of CML. Having demonstrated that Bcr-Abl was acting alone--and developing the first mouse model of chronic myelogenous leukemia in the process--Van Etten spent much of the 1990s trying to characterize the activity of the normal c-Abl protein, which is present in every cell, as a prelude to understanding how it goes awry in Bcr-Abl-bearing white blood cells. Normally, cells keep the kinase activity of c-Abl under very tight control. Van Etten and his colleagues suspected that they might be doing so in part through an inhibitory protein that binds, and essentially silences, c-Abl. To explore this hypothesis, the researchers looked for a mutant that resisted binding by the inhibitor. It turned out, a British group had years earlier generated a c-Abl version, Y253F, with the properties expected of such a mutant. To purify the enzyme in its unphosphorylated state, Brasher, then a graduate student, exposed it to Gleevec, which traps deactivated Abl--"a fortuitous decision," said Van Etten. To their surprise, the Y253F mutant resisted Gleevec's assaults. "Brad was having a devil of a time turning it off," he said. The question immediately arose--were any of the patients who had relapsed in the 1999 clinical trials carrying the Gleevec-resisting mutation in their Bcr-Abl fusion protein? Working with Sawyers's team at UCLA, the HMS researchers found the mutation in five out of a cohort of 20 patients, making it about as frequent as T315I. The differences between the mutations were exciting; for example, in his original experiments, Brasher had a hard time turning off the Abl Y235F. With the T315I mutant, however, he and Roumiantsev, a postdoctoral fellow, found it was impossible. The Defect's MechanismInitially, the researchers thought Y253F might confer its intermediate brand of resistance by increasing Abl activity, since one of the effects of the mutation is to dysregulate the enzyme. But they found other mutations that dysregulated Abl did not confer resistance the way Y253F did. In fact, they had the opposite effect--they made cells more sensitive to Gleevec.They explored another possibility. Earlier studies had suggested that activating Abl might make it more resistant to Gleevec. But the researchers found that while phosphorylation increased the resistance of both wildtype and mutant versions of the enzyme, even unphosphorylated Y253F enzyme was resistant. So the question remains--how does Y253F confer intermediate resistance? One possibility is that the mutation, which is found in a different region of the Abl kinase domain than T315I, called the nucleotide binding loop, or P-loop, may weaken the bond between Gleevec and the Abl protein without eliminating it entirely (see figure). There is an even larger puzzle: why are patients with advanced CML so likely to develop Gleevec-resisting mutations in the first place? Van Etten believes that patients may carry some resistant mutants at relatively high frequency even before they are given Gleevec, which could help explain why they relapse so quickly--after only one or two months. "That seems too fast," said Van Etten. "It suggests that some mutants may be selected for during the biological progression of the disease. They might actually be driving progression." To understand how this might happen requires going back to Van Etten's original hypothesis--that c-Abl is repressed by a cellular inhibitor that may also impair the activity of Bcr-Abl in leukemic cells. Say a mutation arose that weakened the bond between Abl and its inhibitor, increasing Abl activity and cell proliferation. That same mutation might weaken the bond between Abl and Gleevec. Van Etten and his colleagues have identified a candidate Abl inhibitor and will be testing it to see if it binds normal Abl differently from the Y253F mutant. "But at the moment," he said, "this is still speculation." --Misia Landau |
