Inducing Indigestion: Companies Embrace Autophagy Inhibitors (JNCI)

By Ken Garber

Journal of the National Cancer Institute, May 4, 2011

Autophagy, the process of “self-eating” within cells, has been controversial in cancer since it was first linked to the disease in 1999.  Because autophagy can either suppress or promote cancer, depending on the context, the field has been marked by both confusion and an unwillingness by drug companies to get involved in therapies.

But that’s changed.  A consensus is emerging that it’s the tumor promotion role of autophagy that’s important in established tumors.   This consensus has helped justify more than a dozen ongoing clinical trials of a commercially available autophagy inhibitor in a variety of tumor types.  And drug companies are now rushing to start their own autophagy programs, actively looking for new compounds that potently and specifically block autophagy pathways.   “It’s been a total turnaround,” said Eileen White, Ph.D., an autophagy researcher at the Cancer Institute of New Jersey in New Brunswick.

While hopes are high for such drugs, doubts still persist about the wisdom of inhibiting autophagy, at least in some situations.   “Autophagy is, as is commonly said, a double-edged sword,” said Daniel Klionsky, Ph.D., an autophagy researcher at the University of Michigan in Ann Arbor.  Evidence for widespread activated autophagy in human tumors, as opposed to cell line models, is growing but still frustratingly deficient.  And the field awaits its first unambiguous clinical success.  But autophagy, for the moment at least, has become a hot field for translational cancer research.

From Tumor Suppressor to Tumor Promoter

Autophagy, first reported in 1963, is a remarkable process common to all eukaryotes.  In autophagy, a membrane called the phagophore forms and elongates and eventually encircles damaged or unneded proteins and organelles in the cell cytoplasm.  This new organelle, called the autophagosome, later merges with (and deposits its contents into) the lysosome, an organelle that contains enzymes for breaking apart cellular debris.  Autophagyfunctions as a garbage disposal for cells, which use the breakdown products to fuel their energy production and to replenish building blocks for proteins and other essential molecules.

In 1999, Beth Levine, M.D., at Columbia University in New York showed that beclin-1, a protein required for autophagy induction, functioned as a tumor suppressor gene.   Between 40-70 percent of human breast, ovarian and prostate cancers show loss of one copy of beclin-1, suggesting that autophagy plays an important role in preventing these tumors.

But whereas autophagy clearly protects against at least some tumors, it can promote others.   About six years ago, White’s group found that tumor cells lacking the cellular machinery for apoptosis, or programmed cell death, were able to survive long periods when starved of nutrients.   How they survived was a mystery.  In 2006, White reported that autophagy was the survival mechanism, and that inhibiting autophagy caused the cells to die.  This and other accumulating evidence suggested that tumor cells use autophagy to survive conditions of metabolic stress, such as hypoxia, commonly found in tumors.  Gradually the field embraced the tumor promoting function of autophagy.

The dominant view now is that autophagy suppresses growth very early in tumor development but promotes growth later.  “The [tumor cell] protective role of autophagy is relatively well established, I think, at this point,” said Ravi Amaravadi, M.D., an oncologist at the University of Pennsylvania in Philadelphia.  “The field is now fully on board with that… Once tumors are established, especially in the tumor microenvironment, and especially in the context of cancer therapy, autophagy is a tumor promoting mechanism.”

Exactly how autophagy promotes tumors, though, has been a mystery.   Two papers published this year in Genes & Development provide at least a partial solution.  In the first study, White’s group introduced the Ras oncogene into cells with normal autophagy and defective autophagy.  The autophagy-competent cells, put into mice, developed into much faster-growing, more aggressive tumors than the autophagy-deficient cells.  The autophagy-deficient cells displayed damaged mitochondria and diminished oxidative energy production, or respiration (which takes place in mitochondria), suggesting a major role of autophagy in supporting energy metabolism.   Based on these experiments, White thinks that autophagy is promoting tumor growth in these cells by both freeing up the components, or substrates, of oxidative energy production and disposing of defective mitochondria.

That’s the same conclusion reached by Alec Kimmelman, M.D., Ph.D., at the Dana Farber Cancer Institute in Boston in the second Genes & Development paper, published online in March.   Kimmelman’s group used drugs and RNA interference to block autophagy in pancreatic cancer cells, slowing cell proliferation and decreasing oxygen consumption. Adding a compound that’s a key energy production intermediate to these autophagy-defective cells led to renewed growth.  Autophagy, Kimmelman concluded, allows the cell to grow by enabling continuing energy generation.

Kimmelman also found abundant markers of autophagy on primary human pancreatic tumor samples, markers that were absent in early precursor lesions, “meaning that autophagy was activated late in the process of malignant transformation,” he said.  This, too, fits the current model of autophagy in cancer.

Lagging Science?

The biggest surprise in both studies was that Ras-transformed cells showed very high levels of autophagy even in the presence of abundant nutrients.   (Normal cells show little if any autophagy under such conditions.)  This suggests that Ras itself, not external stress, is causing the autophagy, through an unknown mechanism.   White thinks that Ras-triggered cell growth itself is a stressor that requires the autophagy response, and that such tumor cells are in a state of “autophagy addiction.”  The implication is that targeting autophagy in Ras-driven tumors (about a quarter of all tumors) should be especially effective.

That hypothesis is driving a phase II clinical trial at Dana Farber in pancreatic cancer.  (About 80% of pancreatic tumors are driven by activated Ras.)  Patients who have failed standard chemotherapy treatment are given single agent hydroxychloroquine (HCQ), a commercially available anti-malaria drug that inhibits autophagy at the lysosome stage.  HCQ, because it’s cheap and relatively nontoxic, has become the autophagy inhibitor of choice for clinical trials.  At least sixteen phase I and phase II trials, most in solid tumors using HCQ with other treatments, are underway.  Companies involved include Pfizer, Novartis, Millennium Pharmaceuticals, and Merck.

However, there is still concern that the science is lagging.  Studies implicating autophagy in tumor promotion have mainly come from cell line models.   “There is very limited information in animal models and from primary human tumor samples,” said David Gewirtz, Ph.D., who studies experimental cancer therapeutics at Virginia Commonwealth University in Richmond, adding that such studies should be a focus of research.   For technical reasons, however, it’s very hard to track autophagic flux—the autophagy process–in human tumor samples, or in vivo.   Static markers of autophagy can be misleading.  “There aren’t many assays that can be used to follow autophagy,” said Klionsky.  “In vivo, there’s almost nothing.”

But researchers are now starting to venture beyond cell lines.  Good antibodies for autophagy markers are finally available, making immunohistochemistry (the staining of tumor cells with antibodies to highlight individual proteins) feasible, although it’s still hard to track autophagic flux.  And Amaravadi’s group has developed a semi-quantitative approach for looking at autophagy in tumor samples using electron microscopy.  In studies published online in February in Clinical Cancer Research, Amaravadi’s group tracked autophagy this way in human metastatic melanoma samples.  “We found these astronomically levels of autophagy, better than we could ever create in the lab,” Amaravadi said.  “The higher the level of autophagy in your tumor, the worse you did as a patient.”  As for animal models, White’s group has crossed genetically engineered mouse cancer models with autophagy conditional mutant mice, enabling future studies of autophagy and cancer in vivo.

Another worry with autophagy inhibitors is that combining them with chemotherapy could be counterproductive, since certain chemotherapy drugs may work by inducing autophagic cell death.  Thus, blocking autophagy may protect tumors from the treatment.  That’s not a reason not to go forward with trials, said White.  “The evidence for autophagic cell death is still very, very weak,” she said.  “Until someone explains what it is, and demonstrates that it can occur in a physiological condition in vivo, I think conclusions that you can get autophagic cell death are going to be hard to substantiate.”

Gewirtz, though, considers autophagic cell death real, and believes that some chemotherapy drugs induce it in patients.   “There’s extensive preclinical literature, with a variety of agents, showing that autophagy can kill tumor cells,” he said.  He acknowledged that in vivo evidence for this is scant, but pointed out that the same is true for autophagy’s protective role in chemotherapy.  “I’m not sure that the literature completely supports autophagy as being protective of tumors in animal studies of chemotherapy either,” he said.

New Trials, New Drugs

Meanwhile, HCQ trials are proliferating, partly thanks to a  2006 randomized, placebo-controlled glioblastoma clinical trial published in the Annals of Internal Medicine.  Researchers at the National Institute for Neurology and Neurosurgery in Mexico City added chloroquine (a somewhat more toxic version of HCQ) to standard chemotherapy following surgery resulted in a more than two-fold increase in survival.  Due to low patient numbers, the results did not reach statistical significance, but news of the trial spread broadly, prompting many oncologists worldwide to give chloroquine or HCQ to their glioblastoma patients.  The NCI is sponsoring a phase I/II trial of HCQ in glioblastoma, with two-year survival as an initial endpoint.

Another important combination trial, at Penn, is a phase I study of HCQ plus temsirolimus, an mTOR inhibitor.  Temsirolimus is approved for renal cell carcinoma, but responding patients inevitably relapse.  Response rates in general for mTOR inhibitors in clinical trials have been disappointing, given that mTOR, a kinase that basically acts as a nutrient sensor, is an important driver of cell growth.  Since mTOR is a negative regulator of autophagy, inhibiting mTOR  with temsirolimus and similar drugs could be upregulating autophagy and keeping tumor cells alive.

Adding an autophagy inhibitor should overcome this resistance.  In animal models, mTOR inhibitors mostly block tumor cell growth but don’t kill the cells outright, and HCQ shows similar effects.   “But if you put the two together there’s this massive synergy and you get cancer cell death,” says White.

It’s this potential to overcome resistance to kinase inhibitors, said White, that is driving much of the drug company interest in autophagy.  For example, virtually all major pharmaceutical companies are developing PI3 kinase inhibitors for cancer, and these drugs, like mTOR inhibitors, upregulate autophagy, so companies see the logic of combination treatment.

But HCQ is not the ideal autophagy inhibitor.  Amaravadi, monitoring drug effects in the glioblastoma clinical trial, found that the drug did not always inhibit autophagy.   “So it may be that HCQ needs to be at very high doses to block autophagy, and that might not be possible with many anticancer regimens in combination,” Amaravadi said.

White agreed that better autophagy inhibitors are needed.  “All of us in the field don’t think that [hydroxy]chloroquine is the be all and end all,” she said.  “We are using it now because we can, and we’re hoping that preliminary data looks encouraging and that we will learn from this approach.  But there will be better drugs.”   In fact, many drug companies and some academic groups are now screening for specific small molecule autophagy inhibitors.  At least 35 “autophagy-related proteins” have been identified to date, with several considered highly druggable using small molecules.  “There are a number of good targets,” says Amaravadi.   With drug companies now fully committed, the hypothesis that autophagy protects established tumors will soon be put to a definitive test in the only setting that really matters: people with cancer.

Dr. Amaravadi’s laboratory receives research funding from Millennium Pharmaceuticals and from Pfizer, and clinical trial funding from Millennium and Novartis.  Dr. Amaravadi was paid an honorarium from Millennium for a lecture.  Dr. White’s laboratory receives research support from Johnson & Johnson.

© Oxford University Press 2011.

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