In the ongoing tug of war between drugs and cancer, the 2001 approval of the first targeted kinase inhibitor allowed the pharmaceutical side to make a forceful heave. Since then, however, cancer has been gaining ground as more and more patients develop resistance to these drugs.
Recently, the lab of George Daley has tugged back with work that characterizes the most intractable mutation behind resistance to the kinase inhibitor imatinib (Gleevec). Insight stemming from this work may help direct the development of new drugs to treat resistant cancer. Moreover, the findings may be generally applicable because several different kinases have evolved resistance to inhibitors through this same mutation.
In 2001, Daley, an HMS associate professor of biological chemistry and molecular pharmacology and of pediatrics at Children’s Hospital Boston, and first author Mohammad Azam, an HMS instructor in pediatrics at Children’s, began investigating the mechanisms behind resistance to imatinib, used to treat chronic myelogenous leukemia (CML). The first clinical cases of resistance involved a mutation labeled T315I, called the “gatekeeper” mutation. This change occurs in the BCR-ABL fusion protein targeted by the drug. Because the mutation adds bulk to an important residue in the kinase ATP-binding site, scientists speculated that the alteration might confer resistance by physically obstructing drug binding.
Azam reasoned that evolution of resistance would not end with the gatekeeper mutation. So he performed an experiment to introduce random mutations in the BCR–ABL oncogene using a specialized bacterial strain. The resulting mutations “looked random,” said Azam, but structural modeling revealed two classes of resistance mutation.
Since then, Azam and Daley have been searching for ways to battle these two classes of resistance.
In 2006, Azam identified a class of drugs that suppresses the emergence of one class of resistance mutations. These alterations affect the edges of the kinase domain and push it into an active state. This bodes poorly for imatinib, which works by binding to the kinase in its inactive state and trapping it in that state, thereby shutting down the activity that causes cancerous cell growth and proliferation.
In contrast, the new compounds bind to the active conformation of the kinase domain. These new drugs, however, proved ineffective against the second class of resistance mutations. The modifications affect the active binding site of the kinase and include the gatekeeper mutation, which Daley has called “the mutation from hell.” So far, no drugs have been approved to treat it although several candidates are in development.
Though the idea that the gatekeeper mutation physically obstructs imatinib has persisted, Azam recently had a hypothesis-altering breakthrough. He introduced the T315I mutation into the nonmutated c-ABL kinase and discovered that the gatekeeper mutation activates the kinase on its own. It was important to do this in the context of c-ABL and not BCR-ABL because BCR-ABL also deregulates kinase activity, obscuring activation that might result from the gatekeeper mutation. This finding suggests that the gatekeeper mutation may be involved in cancer development, said Azam.
This discovery inspired a new hypothesis that the gatekeeper mutation confers resistance to imatinib by tilting the kinase into a hyperactivated state. Azam found evidence supporting this proposition using several different approaches, described in the September 2008 Nature Structural & Molecular Biology.
He first verified that the gatekeeper mutation introduced in the c-ABL kinase was sufficient to cause leukemia in mice via injected test cells. Then co-authors from the University of California, Berkeley, crystallized the related SRC kinase carrying the gatekeeper residue. The crystal structure confirmed that the gatekeeper mutation stabilizes the active kinase conformation.
This work dovetailed with a separate 2007 study showing that kinases have a structure called a hydrophobic spine. When assembled, the spine flips the kinase into the active state. In the inactive state, the spine is dismantled. The crystal structure Daley and Azam studied revealed that the gatekeeper mutation, which swaps a threonine residue for a bulkier isoleucine residue, shores up the hydrophobic spine and sways the kinase into an active state.
Azam and Daley then collaborated with co-author Nathanael Gray, HMS assistant professor of biological chemistry and molecular pharmacology at the Dana–Farber Cancer Institute, to find a pharmacological way to break down the stabilized spine. If a stabilized spine activates the kinase, they reasoned, destabilizing the spine should inactivate it. The team tested a series of compounds. One of them, called compound 14, “facilitates the destruction of this spine,” said Gray. The drug inhibits the mutant and shuts down kinase activity.
Compound 14, like imatinib, binds to the kinase only in the inactive state. Yet it “has significantly more binding energy,” which allows it to overcome the hyperactivated state of the BCR-ABL kinase with the gatekeeper mutation, said Gray. The difference in binding energy can be likened to the difference between a large and a small child on a playground seesaw; the larger child is able to lift more weight. Though compound 14 is effective against the intractable mutation, it is not a candidate for clinical use because it lacks selectivity.
This study does not point directly to a new drug to combat resistance, but it does suggest a strategy for designing inhibitors of the gatekeeper mutation. “If you have this gatekeeper mutation, you are going to need a very specific kind of drug,” said Daley.
Medical chemists, according to Gray, use the kinds of structural, crystallographic, and biochemical analyses from Daley’s lab to guide the development of new compounds. “Kinase-inhibitor design has matured from an empirical make-it-and-test-it paradigm to one where you are doing a lot of structure-based design and molecular modeling,” said Gray. “You still have to make it and test it, but you can be much more sophisticated about what you make.”
This design strategy could guide the development of drugs that work for multiple diseases because the gatekeeper mutation appears to be generally applicable to many kinases. “In kinases, many inhibitors make interactions with the gatekeeper, so this mechanism for resistance is likely to be a recurrent theme,” said Gray. The team also investigated the consequences of the gatekeeper mutation in several other kinases involved in cancer, including the cKIT cytoplasmic kinase and the EGF and PDGF receptor kinases.
Drugs that target specific classes of mutations may also lead to increased genetic screening in the clinic. According to Daley, for CML and some other forms of cancer, drug targets are already being sequenced, particularly if patients show signs of resistance. “We are evolving toward a paradigm where more and more patient tumor samples are sequenced in order to define drug sensitivity,” he said.
Conflict Disclosure: The authors declare no conflicts of interest.
Funding Sources: The National Institutes of Health, the Leukemia and Lymphoma Society, and the Thomas Anthony Pappas Charitable Foundation
