Scientists at the University of Michigan's Comprehensive Cancer Center are the first to study the effect of these drugs, called HDAC inhibitors, in laboratory mice with cancer after the mice received a bone marrow transplant. Results of the U-M study were published online in this week's early edition of the Proceedings of the National Academy of Sciences.
Other studies have shown that high doses of HDAC inhibitors can be an effective anti-tumor agent in mice and humans. Now, U-M scientists have found that low doses of the same drugs have a powerful anti-inflammatory effect. This prevents the production of proteins called inflammatory cytokines, which cause the extensive cell damage seen in GVHD patients. "What's so exciting is that HDAC inhibitors already have been tested as a chemotherapeutic agent in people," says co-author James L.M. Ferrara, M.D., director of the U-M's Blood and Marrow Transplant Program and a professor of internal medicine and pediatrics in the U-M Medical School. "They have relatively little toxicity and the doses required to generate an anti-inflammatory effect are 50- to 100-fold lower than doses needed to kill cancer cells."
If HDAC inhibitors work as well in cancer patients as they did in mice in the U-M study, they could reduce the risk of death, hospitalization and serious side effects associated with bone marrow transplants used to treat leukemia and other related cancers.
More than 5,000 Americans receive allogeneic bone marrow transplants annually. An allogeneic donor is someone other than the patient or the patient's identical twin. Between 500 to 1,000 Americans die from graft-versus-host disease each year
"An allogeneic bone marrow transplant is a double-edged sword," says Pavan Reddy, M.D., an assistant professor of internal medicine in the U-M Medical School and corresponding author of the PNAS paper. "The good side is the graft-versus-leukemia (GVL) effect, which means that T cells from donated bone marrow, called the graft, will attack and kill any cancer cells remaining in the patient. GVL represents the most potent known form of immune therapy against malignant diseases. Without GVL, the cancer will most likely return.
"The bad side is graft-versus-host disease. In GVHD, T cells from donated bone marrow, in concert with inflammatory cytokines, attack the patient's skin, liver and gastrointestinal tract," Reddy explains. "The key is to block the inflammatory cytokines, which exacerbate GVHD, but leave the cancer-killing donor T cells untouched, since they are vital for an effective GVL response."
In previous research published in the June 2002 issue of Nature Medicine, Ferrara and Reddy, with colleagues from the U-M Cancer Center, discovered that inflammatory cytokines are the major cause of GVHD-induced cell damage. Since then, they have been searching for ways to neutralize cytokines or block their production. Their current work with HDAC inhibitors is an extension of this earlier research.
"No one has looked at this class of drugs in a post-bone-marrow-transplant setting before, but two other groups have found similar anti-inflammatory effects in mouse models used for research on lupus and sepsis," says Reddy. "So now there are several studies showing that HDAC inhibitors are more than just anti-tumor agents."
Reddy and his U-M research colleagues conducted two sets of experiments with strains of laboratory mice commonly used in research related to bone marrow transplants. In the first experiment, three groups of mice were given standard bone marrow transplants. Mice received bone marrow from either allogeneic (genetically dissimilar) or syngeneic (genetically identical) donors.
Between day 3 and day 7 after the transplant, U-M scientists gave low doses of an HDAC inhibitor called suberoylanilide hydroxamic acid (SAHA) to one group of experimental mice that received allogeneic transplants. When researchers compared results in these mice to results in mice given the same type of bone marrow transplant, but not the HDAC inhibitor, they found:
- The drug improved survival rates in post-transplant mice by 60 percent.
- Although some SAHA-treated mice still developed GVHD, they had milder symptoms and less intestinal damage than mice that did not receive the drug.
- SAHA had no effect on how donor T cells responded to host antigens by binding to cancer cells and killing them.
"From earlier studies in these mouse models, we knew that donor T cells need about 72 hours to interact with antigens on host cells and become activated," Reddy explains. "We also knew that production of proinflammatory cytokines peaks at day 7 after the transplant. This is why we administered the drug between day 3 and day 7. The idea was to give T cells enough time to be activated by host antigens, while blunting the flood of proinflammatory cytokines."
To find out whether the drug or the graft-versus-leukemia effect was responsible for tumor-free survival of mice in the study, Reddy conducted a second series of experiments. This time, he gave experimental mice a lethal dose of leukemia cells, in addition to either an allogeneic or syngeneic bone marrow transplant.
All mice given a syngeneic transplant and SAHA died, while 50 percent of mice receiving an allogeneic transplant and the drug survived. "In syngeneic transplants, the donor cells are genetically identical to the host, so they won't react to foreign antigens on host cancer cells," Reddy explains. "The fact that all the syngeneic transplant mice died of cancer suggests that donor T cells and GVL -- not the drug -- were killing the malignant cells."
Scientists know that HDAC inhibitors like SAHA are involved in a biochemical process called acetylation and deacetylation, which affects the cell's ability to transmit coded genetic instructions from DNA to cellular proteins. DNA spends most of its time wrapped around proteins called histones in the cell's nucleus. Scientists call this DNA-histone package chromatin. Enzymes that promote acetylation relax and unwind chomatin making the DNA accessible for transcription. Enzymes that promote deacetylation, on the other hand, make chromatin wind tighter around histones. This makes the DNA unavailable for transcription.
"HDAC inhibitors prevent deacetylation by blocking a binding site the deacetylation enzymes need to keep the chromatin tightly coiled up," Reddy explains. "This means the chromatin stays relaxed and open for gene transcription."
In the U-M study, Reddy, Ferrara and their colleagues examined histones from spleen cells harvested from mice seven days after the mice received an allogeneic bone marrow transplant. They found evidence of increased histone acetylation in the SAHA-treated mice.
"We believe that this class of drugs may be increasing the expression of tumor suppressor genes or genes that trigger suicide in cancer cells, while inhibiting expression of genes that produce inflammatory cytokines," Reddy says. "But clearly more research will be needed to know exactly how they work."
Scientists know all too well that what works in mice doesn't always work in people, but Reddy is cautiously optimistic. "When HDAC inhibitors were tested in mice and in clinical trials with leukemia patients, researchers found the same anti-tumor effect," he says. "The mice we selected for this research are known to be good models for what happens in humans after a bone marrow transplant. So there are reasons to believe that we may see the same anti-inflammatory effects in people, as well. But this will need to be determined in well-designed clinical studies."
"We are very excited about what this kind of therapy could mean to patients with blood- and marrow-related cancers who need a transplant, but have a high risk of developing graft-versus-host disease," Ferrara says. "Only about 25 percent of BMT patients have a perfectly-matched sibling donor, so this is good news for the other 75 percent."
Ferrara adds that he has discussed human clinical trials of post-transplant HDAC-inhibiting drugs with officials at the National Cancer Institute, the federal agency that funds the research. "We have proposed to test this class of drugs in BMT patients, and the idea has been endorsed enthusiastically by a scientific review panel from the National Cancer Institute," Ferrara says. He estimates that such a trial is still several years away, however. "We need to complete more work in our mouse models first to work out the best timing and dosage for the drug, and find any toxicities we might have missed."
Other U-M Medical School researchers who assisted in the research study were Yoshinobu Maeda, M.D., a research fellow, and Kevin Hotary, Ph.D., a research investigator in internal medicine. Additional collaborators included Chen Liu, from the University of Florida College of Medicine; and Leonid L. Reznikov and Charles A. Dinarello, both from the University of Colorado Health Sciences Center.