Velcade: First in a New Class of Cancer Drug

Proteasome Inhibitor Emerges from Currents in Science, Industry, Government

Forty years ago, Alfred Goldberg, now a professor of cell biology at Harvard Medical School (HMS), had many questions in mind that led him to abandon his medical studies. They were getting in the way of his education, he said about his decision to devote himself to basic research.

Found in Translation: The Tale of the Cancer Drug Bortezomib (Velcade)

One question he was pondering—How do cells destroy proteins they no longer need?—brought many more questions and unforeseen paths of investigation. With collaborators and colleagues along the way, Goldberg played a central role in discovering the proteasome, a large intracellular protein-destroying machine that exists in every cell of the body. The research, through sustained federal grants, foundations, and private funding, led to the development of a small molecule to shut down this organelle. In doing so, the work revealed a new way to treat certain cancers and brought the first proteasome inhibitor, bortezomib—known commercially as Velcade—to more than 50,000 multiple myeloma patients. Second-generation inhibitors that target the same sites are now in human trials.

Pinpointing the Proteasome

“People interested in protein breakdown were few and far between,” in the 1960s, ’70s, and even into the ’80s, said Goldberg. Ever since the 1950s, scientists thought they knew how cells degrade proteins: specialized vesicles called lysosomes engulf and digest them. But the process was not deemed very important. Most researchers focused on the more exciting areas of protein synthesis and gene expression. The few scientists investigating protein breakdown, however, recognized that this process must also be of major importance in cell regulation and realized that many intriguing mechanistic questions remained to be answered.

The lysosome theory of protein degradation began to crumble in the early 1970s as Goldberg accumulated evidence that cells contain another, unknown system for the job. This system was acting as the cell’s quality control mechanism, destroying mutated or damaged proteins and helping cells adapt to new nutritional conditions that Goldberg did not think the lysosome could carry out. “We became interested in the existence of a non-lysosomal pathway because the lysosome didn’t seem to have exquisite selectivity,” he explained. Further evidence came from NIH-funded studies by Goldberg and colleagues on organisms lacking lysosomes—bacteria and immature red blood cells called reticulocytes. In both cases, these cells lack the machinery thought to do the job, but highly selective protein breakdown still occurred.

Tracking down this intracellular chop shop required extensive detective work. Goldberg’s lab and others showed that the mystery system uses energy in the form of ATP, which was very surprising and clearly differentiated this process from known protein-degrading enzymes. His lab eventually showed that outside the lysosome, there was an enzyme system that could destroy misfolded proteins if provided ATP.

Analyzing this system and solving the puzzle of what accounts for this energy requirement led to a Nobel prize–winning discovery by Avram Hershko, Aaron Ciechanover, and Irwin Rose. They first explained in the late 1970s that cells tag proteins slated for breakdown with the tiny protein ubiquitin. Attaching this flag to a protein in a series of three enzymatic reactions requires ATP and leads to the protein’s rapid destruction.

A second use of ATP, independent of ubiquitin tagging, was identified by Goldberg and his colleagues. Supported by the National Institute of Neurological Disorders and Stroke (NINDS) and despite focusing on protein degradation in skeletal-muscle atrophy, their basic research relied on the common bacterium E. coli since it offered many technical advantages. “We discovered in bacteria, which lack ubiquitin, a new kind of enzyme—now called ATP-dependent proteases—responsible for protein breakdown in these organisms,” Goldberg said.

He and his colleagues then investigated whether analogous enzymes in the degradation process are at work in mammalian cells operating after proteins are tagged with ubiquitin . As it turned out, ATP-dependent proteases were important across species. In mammalian cells, there is one large ATP-dependent protease, the 26S proteasome, which Goldberg named. The two research groups had found complementary steps in a selective protein-degrading system.

Details became clearer in the late 1980s, as Goldberg, supported by NINDS and the National Institute of General Medical Sciences (NIGMS), and University of Utah researchers led by Martin Rechsteiner independently homed in on this proteolytic machine. Within all eukaryotic cells are tens of thousands of these degradative organelles. Each is a hollow, barrel-like enzyme complex with a cap on either end, gatekeeping the inner chamber. Once a ubiquitinated protein dissociates from the proteasome cap, the ubiquitin tag is removed and the protein transported into the interior, where it encounters enzymatic active sites lining the proteasome’s internal walls that gnaw the protein into small pieces.

Researchers learned in the late 1990s that the proteasome sits at the center of many cellular processes—its degradation of proteins plays a crucial role in regulation of cell growth and cell division, fields of interest to many more scientists and central to understanding cancer.

“Suddenly, my telephone rang much more often. Those of us in the field were not wallflowers any more,” Goldberg noted.

Working with his grants from the National Institutes of Health (NIH) and other funding sources, especially the Muscular Dystrophy Association and the National Space Biomedical Research Institute (the national academic basic science consortium of NASA), Goldberg continued to pursue his early questions about how cells dispose of unwanted proteins and how they can use protein breakdown to mobilize amino acids in processes like fasting or disease. These investigations revealed more clues about the proteasome’s additional functions.

“Muscle atrophy—as seen with disuse, nerve injury, fasting, and many diseases including cancer, AIDS, and TB—has a single basic mechanism involving excessive activation of the ubiquitin–proteasome pathway in the muscle,” Goldberg explained. This loss of muscle mass and strength is a debilitating feature of many chronic diseases. The surprising role of the proteasome in this process, in addition to its protective role in the destruction of malformed proteins and control of regulatory proteins led him to wonder whether he could reduce muscle breakdown by slowing the proteasome’s activity. The search for proteasome inhibitors was on.

Putting the Brakes on Protein Breakdown

Goldberg gathered a group of fellow researchers to find ways of controlling the powerful destructive appetite of the ubiquitin–proteasome pathway. “At the time, there was no opportunity for biochemists like us to work with chemists and biologists and pharmacologists, in a team effort in the academic setting. So we started a company,” said Goldberg. Founded in 1993, this company, initially called MyoGenics (because of its goal in building up muscle), synthesized the first proteasome inhibitor, MG132, as a lead compound for further drug development and a tool for research on proteasome function.

“At the time, we didn’t know anything about the proteasome’s structure or mechanism,” he said, “but we knew something about the substrate specificity of its active sites and what kind of peptides could enter cells. Based on this information, we built the first inhibitors as analogs of the peptide substrates of one of these sites.”

There are three types of active site within the cavernous interior of the proteasome, each one able to sever a protein’s amino acid chain at a point following one of three general types of amino acid, hydrophobic, basic, or acidic. The proteasome inhibitors that the scientists at MyoGenics created block one of these active areas—the site that cleaves proteins after hydrophobic groups, thus slowing protein degradation, though not preventing it.

MG132 found frequent use as a laboratory research tool to examine the consequences of blocking the ubiquitin–proteasome pathway. To date, more than 2,000 scientific publications have mentioned use of the compound, said Goldberg, and it has led to identification of many novel functions of the proteasome. One of the key roles discover ed by Goldberg and Kenneth Rock, then an associate professor of pathology at Dana–Farber Cancer Institute (DFCI) and now chairman of pathology at the University of Massachusetts Medical School, was the role of the proteasome in antigen presentation, which is critical in allowing the immune system to protect against viruses and cancer. The inhibitor’s applicability beyond the laboratory developed with these insights about the organelle itself.

The NF-kappa B System

Across the Charles River in Cambridge, Tom Maniatis, currently the Thomas H. Lee professor of molecular and cellular biology at Harvard University, pursued a seemingly unrelated line of research. In the early 1990s, his laboratory group studied a transcription factor, nuclear factor-kappa B (NF-kappa B) and its inhibitor, I kappa B, but did not know the signal transduction pathway controlling the two molecules. Their experiments yielded one hint: the pathway consumes energy, and “the only other ATP-dependent processing known at the time was through the proteasome,” said Maniatis.

Vito Palombella, then a postdoctoral researcher in Maniatis’s lab, in collaboration with Goldberg, confirmed that suspicion: the proteasome controls activation of NF-kappa B by regulating I kappa B destruction. In a healthy cell, I kappa B acts as a brake, holding NF-kappa B inactive in the cytoplasm. In a stressed cell, a series of enzymes tags I kappa B with ubiquitin, marking it for proteasomal destruction—in effect, releasing the brake. The freed NF-kappa B escapes to the nucleus, where it triggers transcription of specific genes. Their products are involved in the inflammatory response and growth of immune cells that contribute to all chronic inflammatory diseases. In other cells, these genes promote angiogenesis, survival and anti–programmed cell death, cell adhesion, and cell growth, processes that go awry in cancer . This NIH-funded work changed remarkably our understanding of inflammatory diseases, said Goldberg.

Some of Palombella’s initial experiments used the proteasome inhibitor MG132, which blocks activation of NF-kappa B by protecting I kappa B from destruction. These investigators realized that blocking the proteasome to prevent NF-kappa B activation could inhibit the body’s activation of inflammatory responses. Of particular interest, proteasomes in certain instances overzealously digest I kappa B, allowing NF-kappa B to remain continually active, helping cancer cells to survive. A proteasome inhibitor could strip the mutinous cells of these defenses, making them vulnerable to treatment.

“Through this study, we opened up a whole new direction that had clinical applications. It serves as a really beautiful example of how fundamental research can lead to breakthroughs with tremendous clinical applications,” said Maniatis, one of the co-founders of Goldberg’s company. The company changed its name to ProScript, denoting a change in direction to investigate proteasomes and transcription.

Drug Development Hurdles

Though useful at the laboratory bench, the initial proteasome inhibitors like MG132 lacked specific properties needed in a drug molecule, such as stability in the bloodstream and increased potency. Goldberg and colleagues recruited from the pharmaceutical industry a talented medicinal chemist, Julian Adams, to lead the refinement of the drug. Adams introduced a boronate group to the small peptide backbone of MG132, creating a more powerful molecule, which was eventually modified further to yield the proteasome inhibitor bortezomib.

The small company had given its earlier versions of proteasome inhibitors to many outside scientists for their experiments to advance understanding of this pathway. It gave bortezomib to the National Cancer Institute to screen it against multiple forms of human cancer. The NCI screen showed the compound had efficacy against a unique range of cancers, and the NCI committed its resources to help develop it as an anticancer agent.

“Most scientists do not realize how the progress of drug development depends so much on non-scientific issues and random events—and luck,” commented Goldberg. Though potential as a cancer treatment hovered around bortezomib, an obstacle-strewn path lay ahead. The drug bounced through an alliance with a major pharmaceutical company and saw spurts of progress—such as almost entering human trials for arthritis—but wound up “on someone’s shelf headed for obscurity,” Goldberg recounted. Concerns about the drug’s possible toxicity and its novel mode of action unsettled many pharmaceutical companies that were considering development because ProScript lacked substantial funding and support from partners. Eventually, when partnerships did not pan out, the company Goldberg had founded was taken over by a larger biotech company, which was taken over by an even larger company, Millennium Pharmaceuticals.

Julian Adams relentlessly watched over bortezomib, shepherding it through these transitions and keeping the compound from fading into obscurity. “Julian Adams deserves a great deal of credit for championing the drug,” said Maniatis. Ultimately, at Adams’s insistence, Millennium brought the drug to market based on its continuing success in preclinical and clinical trials.

A Drug’s Debut

In May of 2003, the year that culminated the five-year doubling of the NIH budget and continued the steady support of Goldberg’s work on protein degradation, an expedited review by the Food and Drug Administration ultimately approved bortezomib just four months after its submission to the agency for treating multiple myeloma.

“It is extremely unusual for such rapid bench to bedside translation,” said Kenneth Anderson, the Kraft family professor of medicine at HMS and DFCI, who is also chief of the Division of Hematologic Neoplasia and director of the Jerome Lipper Multiple Myeloma Center at DFCI. The FDA first approved the drug, marketed as Velcade, for use in patients whose multiple myeloma had relapsed after two prior treatments and who had demonstrated resistance to their last treatment.

Many early laboratory studies had found the drug killed diverse types of cancer cells, but phase I studies in patients suggested that myeloma cells were particularly sensitive. Preclinical work by Teru Hideshima in Anderson’s lab confirmed that bortezomib made multiple myeloma cells self-destruct, inhibiting their growth and causing programmed cell death, or apoptosis.

Multiple myeloma is a cancer of the immune system’s plasma cells. These cells originate in bone marrow, normally developing from B cells. In myeloma, aberrant B cells instead produce myeloma cells—dysfunctional plasma cells—that reproduce uncontrollably and accumulate in the bone marrow. There, these cancerous cells activate NF-kappa B, which generates large quantities of cytokines, such as interleukin 6, a signal that promotes the myeloma cells’ own growth, and interleukin 1, which enables the tumors to destroy the surrounding bone. The American Cancer Society estimates that in 2006, 16,570 Americans received a diagnosis of multiple myeloma, and 11,310 died of the cancer.

“As the development of bortezomib shows, you often can’t predict the real benefits of basic research,” Goldberg said. “What is tragic today is that cutbacks in funding new grants are stifling biomedical innovation. Many good ideas are not being supported.”

It is now recognized that certain characteristics of multiple myeloma make it particularly susceptible to bortezomib. The cancerous plasma cells normally crank out many copies of a specific antibody. This constant high-speed manufacturing requires vigilant, rapid quality control to keep the protein assembly lines running smoothly. These cancerous cells “make an awful lot of misfolded proteins, so they are carrying out enormous amounts of quality control, which means lots of protein destruction by the proteasome. They have a garbage collection problem to begin with, and when you add the proteasome inhibitor, they’re completely overwhelmed,” Goldberg explained. “In addition, these cells depend on NF-kappa B for growth. Thus, myeloma cells are really primed for killing by a proteasome inhibitor because of this quality-control problem.” Many of these details came to light only “ex post facto, added Goldberg, after myeloma patients received the drug in the clinic and were found to respond well. It was a “surreal bit of luck” that finally brought bortezomib and multiple myeloma patients together.

Phase I trials tested bortezomib in cancers of the blood and solid-tumor cancers. Usually, phase I trials do not expect to determine efficacy, but to test only for toxicity. A multiple myeloma patient happened to enroll in one of these first clinical studies, said Goldberg, and the use of bortezomib revealed an efficacy in this patient that stood out from other cancers in the study. Additional phase I trials evaluated more multiple myeloma patients and went on to establish the drug’s relatively low toxicity compared to many other cancer drugs, coupled with high efficacy, while also defining appropriate dosing schedules.

Phase II studies focused on patients with relapsed or refractory multiple myeloma. In one study led by Paul Richardson, clinical director of the Jerome Lipper Multiple Myeloma Center at DFCI and an HMS associate professor of medicine, multiple myeloma patients had tried an average of six previous treatments. Thirty-five percent saw a complete, partial, or minimal response with use of bortezomib.

The FDA approved bortezomib based on phase I and II studies, which is very unusual, though phase III studies continued postapproval, further gauging how doctors could most effectively use the drug. A pivotal phase III trial compared bortezomib to dexamethasone in a large, randomized international trial. The study enrolled relapsed multiple myeloma patients who had received between one and three prior therapies. The response rates, time to progression, and survival of patients were all significantly prolonged with bortezomib. This study’s results prompted an extension of the FDA-approved indication in 2005 to include cases of myeloma in which just one prior therapy had been tried.

The speed of the drug’s development and approval, from preclinical work and phase I trials in 2000 to approval in 2003 and to the expanded indication in 2005, “really is a remarkable tribute to a collaborative effort between academia, the National Cancer Institute, the Food and Drug Administration, the pharmaceutical industry—in this case, Millennium—the patients, and advocacy groups. This team approach to science remarkably shortens the time of drug development,” said Anderson.

Adding to bortezomib’s clinical success, it was approved in 2006 for another blood cancer, mantle cell lymphoma, and its approval for other cancers in the future appears likely.

On the Horizon

As clinical use of bortezomib grew, doctors found that the cancer can develop drug resistance. Scientists, however, are finding ways around this complication with research on combination treatments, new therapeutic mechanisms of action, and second-generation proteasome inhibitors, Anderson explained.

One potential solution is using bortezomib in combination with other novel agents. Constantine Mitsiades, in Anderson’s lab, showed, for example, that using one drug to block heat shock protein 90—essential for folding many cell proteins—coupled with bortezomib’s blocking of the proteasome, attacks the cancer cells on two fronts. In fact, as many basic studies in the 1980s and 1990s showed, heat shock protein 90 is a “molecular chaperone,” one of the cell’s key defenses against the kinds of misfolded proteins that accumulate when the proteasome is inhibited. Clinical trials revealed that this combination can sensitize myeloma cells to anticancer drugs and overcome resistance to proteasome inhibitors. This approach might allow for better treatment of myeloma as well as expansion of bortezomib against a broader range of diseases.

Much of the understanding of how bortezomib works against cancer and its lack of general toxicity despite the presence of proteasomes in all cells did not come to light until after the drug’s approval. And scientists are continuing to discover new information about the proteasome and its inhibition. Through continual support from the NIH, Goldberg’s laboratory currently studies the simpler proteasomes from archaebacteria to gain insight into the more complex workings of the human 26S proteasome and, specifically, to determine how cells inject certain proteins into the proteasome while preventing inappropriate degradation of normal cell constituents.

A recent publication on NIH-funded work from postdoctoral fellow David Smith in Goldberg’s lab reveals that the proteasome uses ATP as a gating mechanism to assist in the unfolding and injection of proteins into the proteasome’s digestive chamber (see Molecular Cell, Dec. 9, 2005). Another more recent publication from the Goldberg lab explains why proteasome inhibitors are not more toxic—the inhibition of one specific enzymatic activity within the organelle eliminates just a fraction of protein breakdown. At therapeutic levels, this process may be inhibited by only 30 percent. Normal cells can function despite this decrease in protein breakdown, but cancer cells are crippled by this partial inhibition. “It’s very satisfying that the same problems we’ve been thinking about for 35 years, we finally think we’re understanding,” said Goldberg.

“All of that sounds a long distance from muscle wasting, which we started to investigate,” Goldberg said, but he notes that proteasome-inhibitor research has come nearly full circle, with new research also finding that bortezomib may retard muscle atrophy and proteasome inhibitors emerging as extremely valuable tools in better dissecting this debilitating process as well as many other cellular processes important in human disease.

The story of bortezomib, said Goldberg, “is a wonderful example of the unpredicted benefit of basic research. No one could have predicted where this would go. We realized protein degradation was a fundamentally important process, but treatment of myeloma was not imagined at the time.” It is this potential that confers such importance to funding for basic science like that received by Goldberg since early in his career from private foundations as well as the NIH.

“Clearly, basic science is the source and power from which developments derive, improving diagnosis, prognosis, and treatment of patients,” Anderson said. The magnitude and scope of support for research ultimately determines the prospects for medical applications and advances. When one researcher sits down at the lab bench and asks one question, the eventual benefits can utterly defy prediction.