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ADJUSTING AND THE YIN YANG OF THE CELL Autophagy, the process of cellular housecleaning and self-renewal, is implicated in neurodegenerative diseases, cancer, and more. Can biotech companies walk the fine line between boosting and inhibiting autophagy to treat disease without causing it? RYAN CROSS, C&EN BOSTON
C R E D I T: CH R I S GAS H
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t the beginning of 2017, Keith Dionne was looking for the next big idea in biotech. He had just joined the life sciences–based venture capital firm Third Rock Ventures, and the investors there were building a company centered on the cellular process of autophagy, the subject of the 2016 Nobel Prize in Physiology or Medicine, announced just months before.
Autophagy literally means self-eating, but the cellular housecleaning it accomplishes is more important than its cannibalistic connotations. Cells build structures called autophagosomes around junk they want to recycle—for example, old or damaged organelles, fat deposits, clumps of poisonous proteins, or even microbial invaders. Once that garbage is bagged, cells break it down into reusable building blocks and nutrients. A year earlier, Beth Levine, an autophagy researcher at the University of Texas Southwestern Medical Center, and a few other experts in the field had come to Third Rock to pitch a biotech start-up that would develop drugs to boost that cellular
housecleaning. The Third Rock investors heard that a wide variety of seemingly unrelated diseases all share an underlying problem: a buildup of cellular garbage. Aggregations of amyloid proteins, for example, are associated with Alzheimer’s disease. An accumulation of lipids in the liver leads to nonalcoholic fatty liver disease. And toxic molecules pile up in a swath of rare genetic enzyme deficiencies. The list kept going, Dionne says. “The thought was if there is a common pathology, then potentially there’s a common solution.” Dionne learned that over the past 2 decades, researchers had shown that boosting autophagy did seemingly everything, from clearing out the toxic proteins that
In brief Autophagy, the cellular equivalent of housecleaning, is now recognized as a crucial process of self-renewal. After the Nobel Prize was awarded for the discovery of autophagy genes in 2016, several biotech companies launched with plans to develop drugs that either boost or inhibit autophagy to treat a bevy of diseases, including Alzheimer’s and cancer. At first glance, drugging autophagy sounds like a panacea, but each approach carries a risk: boosting autophagy might prevent neurodegeneration at the expense of increasing cancer risk, while inhibiting autophagy might kill cancer at the expense of the brain and other organs. Can drugmakers succeed in changing the yin and yang of the cell? JUNE 3, 2019 | CEN.ACS.ORG | C&EN
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Cellular self-renewal
Cancer controversy
Autophagy is a complex cellular process that uses dozens of different proteins for its execution and regulation. In the process, which is initiated by a trigger such as starvation or stress, a membrane called the phagophore forms around cellular debris, organelles, or microbial invaders. When that material is fully captured (A), the newly formed autophagosome fuses to the enzyme-containing lysosome (B), and the contents are broken down (C) and recycled for nutrients (D).
Long before autophagy was linked to human disease, biologists recognized the process as a cellular survival mechanism. When scientists deprived cells of nutrients in the lab, large vesicles would emerge to engulf, digest, and recycle a cell’s innards. Conceptually, this wasn’t surprising. The human body’s ability to survive in periods of extreme, even self-imposed starvation was well known. Inspired by this notion, in 1859 a French physiologist experimented with what he called artificial autophagy, in which he drew blood from malnourished animals and fed it back to them. His intention was to devise strategies for shipwrecked people to sustain themselves while awaiting a prolonged rescue. Upon hearing the gossip, the New York Times called it “cannibalism reduced to a civilized and humanitary institution!” Autophagy, as scientists conceive it today, was first named by Belgian biologist Christian de Duve in 1963. The process that de Duve described in cells, and others later refined, is as follows: during starvation or under other stress, cells begin to form membranes around protein clumps, organelles, or anything else the cell considers to be debris or otherwise dispensable. This developing membrane, called the phagophore, extends around the debris, eventually engulfing it. That newly formed cellular trash bag is called an autophagosome. Finally, an acid-and-enzymefilled vesicle called the lysosome fuses with the autophagosome to dismantle the debris into molecular building blocks and nutrients for the cell to use and build anew. Until 1993, no one knew how this process was controlled. That year, Yoshinori Ohsumi described 15 genes in yeast that were essential for orchestrating autophagy. That work—the basis for his 2016 Nobel Prize—meant researchers could finally study the machinery that made autophagy tick, and perturb it to see what happens when the process is broken. The first connection between autophagy and human disease came in 1999 when UT Southwestern’s Levine serendipitously discovered that a protein called beclin 1 promoted autophagy and blocked cancer growth in human breast cancer cell lines (Nature 1999, DOI: 10.1038/45257). It turned out that some 40–75% of women whose breast and ovarian cancers weren’t driven by an inherited mutation were missing a single copy of the beclin 1 gene. In a subsequent experiment, she showed that mice missing both copies of their beclin 1 genes died as embryos, while mice missing a single copy of the gene lived, only to de-
C R E D I T: KAT E RYN A KO N /S C I EN CE S O U RC E /C & EN
accumulate in neurodegenerative diseases to increasing the life spans of worms and mice. Levine laid out evidence that autophagy could be important for fighting infections and maybe even preventing cancer. It wasn’t just about taking out the trash; it was a process of rejuvenation. Enhancing autophagy, it would seem, could be a panacea. Yet Dionne had known almost nothing about it. “How could I be ignorant of this and yet it have that much potential impact?” Dionne recalls thinking. And why, he wondered, weren’t other researchers trying to develop drugs for this pathway? Actually, a number of scientists had tried. But their therapeutic thesis had run counter to Levine’s: they were all bent on trying to inhibit autophagy in cancers that appeared to hijack the process to their own advantage. For years, that approach overshadowed the idea of enhancing autophagy. Moreover, conflicting and lackluster results in those autophagy-blocking studies eventually soured drugmakers’ interest, and industry retreated from the field. Third Rock, however, was convinced that autophagy enhancement was worth exploring. In May 2018, the firm backed Levine’s broad vision for doing so by launching Casma Therapeutics and funding it with $58.5 million. Dionne, now the startup’s CEO, says that Casma already has six drug-discovery programs to treat diseases afflicting the brain, gut, liver and muscles. And although Casma’s therapeutic goals are particularly large, it’s not the only company designing autophagy-boosting therapies. Autophagy-focused biotech firms are working on treatments for rare genetic diseases, Alzheimer’s, and many other indications. They even think those same drugs could extend our life spans. Yet as investment floods into the field, researchers caution that cellular self-eating has a dark side: autophagy-boosting drugs might have the unintended effect of helping silently lurking tumors sprout and spread. That link to cancer has kept some researchers focused on the older idea of developing drugs that block the process. Despite the lingering controversy of whether it’s better to enhance or inhibit autophagy, the momentum has those who have worked in the field for years excited that autophagy’s time has finally come. “The literature has just grown and grown, and now it’s explosive,” says David Perlmutter, an autophagy researcher and dean at Washington University School of Medicine in St. Louis. “And every single new observation validates how important the system is in our biology.”
ly, permanent autophagy enhancement enhancement,” says Sebastian Aguiar from velop cancer within 12 to 18 months. protects mice from neurodegeneration Apollo Ventures, a venture capital firm foTo Levine, all of this amounted to a cused on the biology of aging. smoking gun: properly functioning beclin 1, and even increases their life spans, but Aguiar is also a cofounder and the chief and perhaps autophagy at large, was a criti- there are scattered reports of increased tumor development. “There is always a yin operating officer of an Apollo-backed biocal tumor-suppression system in the body. and yang here, a positive and a negative ef- tech start-up called Samsara Therapeutics, Several other research teams, however, fect of what autophagy is doing,” says Anwhich is developing drugs that enhance would soon call that model into question. drew Thorburn, an autophagy and cancer autophagy to, among other things, extend One of those teams was headed by Eileen researcher at the University of Colorado life span. In February, the start-up’s four White at the Rutgers Cancer Institute of Denver. scientific cofounders identified a moleNew Jersey, whose lab discovered that Thorburn, who is submitting a paper to cule, called 4,4′-dimethoxychalcone, that cancer cells, particularly ones that refused refute the 2015 Novartis-Pfizer study, still appears to do just that. Found in a Japato die, had suspiciously high autophagy thinks that briefly inhibiting autophagy to nese herb, the compound is consumed on levels. “Our hypothesis was that tumor an island known for its cells were usurping a natsupercentenarians—peoural survival pathway to ple who live to 110 years promote their own survivThe number of autophagy publications has soared in the past decade and beyond (Nat. Comal,” says White, who later and shows no signs of slowing down. mun. 2019, DOI: 10.1038/ started an autophagy-inhibAutophagy papers published per year s41467-019-08555-w). itor company called Vescor 7000 The Nobel Prize also Therapeutics. 6000 seems to have spurred White’s lab showed that investors to go looking for inhibiting autophagy—ei5000 autophagy experts themther with drugs or by deselves. In January, an antileting autophagy genes like 4000 aging conglomerate called beclin 1—weakens cancer 3000 Life Biosciences raised cells and halts their growth, $50 million to fund mulmaking them easier to kill. 2000 tiple subsidiaries that are Meanwhile, University of 1000 each focused on tackling a Pennsylvania oncologist different aspect of aging. Ravi Amaravadi discovered 0 1993 95 97 99 01 03 05 07 09 11 13 15 17 And for help with autophthat the antimalarial drugs Source: PubMed, accessed May 23, 2019. agy, the company reached chloroquine and hydroxyout to Ana Maria Cuervo, chloroquine—already weaken tumors is a good strategy. “Autoan autophagy researcher at the Albert Einknown to inhibit autophagy by neutralizphagy is doing both good things and bad stein College of Medicine. ing lysosomes—made tough cancer cells things,” he says. “And what we’re doing Cuervo has spent most of her career more sensitive to chemotherapy. right now—saying we’ve just got to boost studying a specialized process called Those studies, which were at direct it or inhibit it—is way too crude.” chaperone-mediated autophagy, in which odds with Levine’s hypothesis, spurred individual proteins are flagged and transseveral large pharmaceutical companies to ported one by one into a specialized set of begin developing autophagy inhibitors for lysosomes for destruction. Animal studies cancer. Amaravadi even began testing ansuggest that the process is important for timalarial drugs with anticancer therapies After the Nobel Prize, investors were cleaning up toxic proteins that accumulate in Phase I and II clinical trials. So far, the more receptive to taking meetings with in neurodegenerative diseases. results look encouraging, but they’re not a scientists with ideas for autophagy comIn collaboration with her Einstein colhome run, he says. panies. “There is definitely a growing league Evripidis Gavathiotis, Cuervo has The big pharma programs didn’t last industry excitement around autophagy discovered small molecules that directly long enough for new autophagy inhibiboost chaperone-mediated autophagy tors to be tested in humans. Scientists at without touching any of the other autoNovartis and Pfizer independently conphagy pathways. A protein called retinoic ducted autophagy-inhibition experiments acid receptor α inhibits chaperone-mein dozens of cancer cell lines, and they diated autophagy, and small molecules teamed up to publish a study in 2015 coninhibiting that protein boost the process cluding that the strategy didn’t work (Proc. (Nat. Chem. Biol. 2013, DOI: 10.1038/ Natl. Acad. Sci. U.S.A. 2015, DOI: 10.1073/ nchembio.1230). pnas.1515617113). That paper, which was The work didn’t make much of a splash edited by Levine, has proved contentious. at first. Suddenly, after the Nobel Prize, “It is a very damaging paper for the field,” “the concept became cool,” Cuervo says. says Amaravadi, who is still testing hy“We had at least 10 groups of investors droxychloroquine in people with cancer. talking to us.” To complicate matters further, studies Cuervo and Gavathiotis ultimately in mice have shown that permanently inhibiting autophagy in healthy cells and —Sebastian Aguiar, principal, Apollo teamed up with Life Biosciences to found cancer cells kills tumors but causes death Ventures, and cofounder, Samsara Selphagy Therapeutics, which is currentfrom rapid neurodegeneration. ConverseTherapeutics ly developing the duo’s compounds for
Escalating interest
Getting selective
“Drugs that slow the aging process are the ultimate pipeline-in-apill archetype.”
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degenerative diseases characterized by misfolded protein accumulation. The longterm goal is to treat brain diseases like Alzheimer’s and Parkinson’s, but Selphagy will start by testing its compounds in eye diseases because clinical trials will be cheaper and faster to run. Targeting chaperone-mediated autophagy and other forms of selective autophagy could also provide a means for avoiding the potentially harmful effects of broadly boosting autophagy. Of interest is mitophagy, which uses autophagosomes to remove mitochondria that have been molecularly marked for destruction, and has been implicated in Parkinson’s. Mitochondria, the power generators of a cell, break down and wear out frequently and can leak toxic molecules into the cytosol if not replaced. That makes mitophagy particularly important in brain and heart muscle cells, which stay with you for life. In fact, mutations in genes that regulate mitophagy, PRKN and PINK1, are some of the strongest predictors of early-onset Parkinson’s disease. Translating that knowledge to drug-discovery programs, or simply finding drugs that specifically enhance mitophagy, has been difficult. “There are lots of things that boost mitophagy,” but they do so by damaging the mitochondria, says Roberta Gottlieb, who studies the process at Cedars-Sinai. A few companies are trying, however. In November 2018, a start-up called Rheostat Therapeutics launched with $23 million to boost mitophagy and autophagy for neurodegenerative diseases. Other firms, including Mitobridge and Proteostasis Therapeutics, are designing inhibitors of deubiquitinating enzymes, which remove the molecular markers that destine old mitochondria for destruction. The inhibitors, in theory, could keep pushing mitochondria through mitophagy. Other diseases also feature clear defects in an autophagy pathway. For instance, several rare genetic diseases are characterized by defective lysosomes. And in 2017, neuroscientist David Rubinsztein from the Cambridge Institute for Medical Research discovered that autophagosome formation is impaired in Huntington’s disease and genetically similar conditions. The idea that impaired autophagy is a common thread across so many serious diseases is tantalizing and suggests that a single drug program, in theory, could lead to many cures. “In some cases you are repairing a defect, but I don’t think you need a defect for many of these diseases to justify the strategy,” Rubinsztein says.
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Boost or block? More than a dozen biotech firms are developing small-molecule drugs that either enhance or inhibit autophagy to treat a range of diseases. COMPANY
STRATEGY
Autophagy Neurotherapeutics
Boost
TFEB activator for neurodegenerative diseases
LEAD PROGRAM
Casma Therapeutics
Boost
TRPML1 agonist for Gaucher disease type 3 and Parkinson's disease; undisclosed targets for α-1-antitrypsin deficiency, Crohn's disease, and Pompe disease
Catabasis Pharmaceuticals
Boost
Undisclosed target for cystic fibrosis
Neuropore Therapies
Boost
PI3K inhibitor and cathepsin A inhibitor for neurodegenerative diseases
Petra Pharma
Block
PIP4K2 inhibitor for cancer
PhoreMost
Boost
Undisclosed target for neurodegenerative diseases
Pinpoint Therapeutics
Block
Dichloroquine-based PPT1 inhibitor for cancer
QurAlis
Boost
Undisclosed target for amyotrophic lateral sclerosis
Rheostat Therapeutics
Boost
TRPML1 agonist and mitophagy booster for Parkinson's disease and other neurodegenerative diseases
Samsara Therapeutics
Boost
4,4´-Dimethoxychalcone for undisclosed conditions
Selphagy Therapeutics
Boost
Retinoic acid receptor α inhibitor to boost chaperone-mediated autophagy for degenerative eye diseases
Sprint Bioscience
Block
Vps34 inhibitor for cancer
Vescor Therapeutics
Block
Undisclosed targets in cancer
ACADEMIC CENTER
STRATEGY
LEAD PROGRAM
Alborada Drug Discovery Institute at Boost the University of Cambridge
Inhibitors of PI5P4Ks for neurodegenerative diseases
Autophagy, Inflammation, and Metabolism Center of Biomedical Research Excellence at the University of New Mexico
Block and boost
Infectious diseases, immunology, and prediabetes
Philip and Sima Needleman Center for Autophagy Therapeutics and Research at Washington University in St. Louis
Boost
α-1-Antitrypsin deficiency, Alzheimer's disease, Crohn's disease, and tuberculosis
Sources: Companies and academic centers.
Tantalizing targets As researchers consider how best to control this process, some are looking first to older drugs that have inadvertently been activating or inhibiting autophagy all along. For instance, Amaravadi and University of Pennsylvania chemist Jeffrey Winkler have developed derivatives of hydroxychloroquine that are 1,000 times as potent as the version Amaravadi is testing in the clinic. And they’ve founded a company, Pinpoint Therapeutics, to try to commercialize the work. Repurposing existing drugs holds potential for autophagy enhancement too. Rubinsztein’s group discovered that an antihypertensive called rilmenidine boosts autophagosome formation in mice. More recently, Rubinsztein’s lab found that another antihypertensive drug, called felodipine, enhanced autophagy in three animal models of neurodegenerative disease (Nat. Commun. 2019, DOI: 10.1038/ s41467-019-09494-2). And Cuervo’s lab found that the rare-disease drug lonafarnib helps clear out toxic tau proteins in mice by enhancing lysosome activity
(Sci. Transl. Med. 2019, DOI: 10.1126/ scitranslmed.aat3005). Perhaps the most well-studied autophagy activator, however, is rapamycin—a drug with a storied history and a long list of side effects, including immune suppression. Rapamycin inhibits a protein called mTOR, which is part of a molecular rheostat that controls metabolism. “We know that if you inhibit mTOR, you will induce autophagy,” Casma’s Dionne says. “The challenge is you will induce 20 other pathways as well.” A sizable list of other drugs has also been shown to activate autophagy, including the diabetes drug metformin, the nutritional supplement resveratrol, and aspirin. The problem with many of these molecules, Casma’s chief scientific officer, Leon Murphy, says, is that we have no idea if they are activating autophagy directly, through an indirect pathway, or simply by stressing cells. “We really haven’t been rigorous about evaluating these molecules.” Casma, like many of the recently formed autophagy start-ups, is focused on drugs that more specifically target particular proteins involved in the process. One promising strategy is activating a protein
called TFEB, the master lock for autophagy and lysosomal genes. Studies using gene therapy to permanently boost TFEB in mice show that it can reduce the poisonous proteins in liver and neurodegenerative diseases and even reduce obesity. At least two groups think a TFEB gene therapy could make for a powerful treatment in humans. Harvard University geneticist George Church and a former postdoc from his lab, Noah Davidsohn, have founded a company called Rejuvenate Bio that is testing antiaging gene therapies in dogs. An expansive patent held by the duo includes TFEB and other autophagy-related genes in a table of what might be included in the gene therapy. Church is also working with scientists at Lund University in Sweden to develop a version of gene editing called CRISPR activation that would keep the TFEB gene turned on. By boosting autophagy this way, they hope to reduce the amyloid-β and tau proteins linked to Alzheimer’s disease. Dionne is wary of the gene-therapy approach, partly because some studies show that constant TFEB activation is linked to the development of renal cancer. In contrast, using a small molecule to activate TFEB could allow people to periodically boost autophagy. That may be a safer approach, and there appears to be more than one way to do it. TFEB is a transcription factor that is normally inactive and resting in the cell’s cytosol. During starvation and other autophagy-triggering events, TFEB travels to the nucleus, where it turns on autophagy and lysosomal genes. Several proteins control this process: enzymes called kinases phosphorylate TFEB to keep it in the cytosol, activation of a calcium channel called TRPML1 helps trigger TFEB’s journey to the nucleus, and a protein called exportin 1 expels TFEB from the nucleus. These proteins are all druggable targets for companies looking to boost autophagy.
Pipeline in a pill Research and investment might be proliferating, but at the moment, there are few molecules available to precisely probe autophagy. No drugs specifically designed to boost or inhibit autophagy have entered the clinic, and the first trials from Casma and Selphagy are likely a year or more away. And some think there are still too many unknowns for drug development to move forward. “Some of these things need to simmer for a while,” says Vojo Deretic, who studies autophagy’s role in infections at the University of New Mexico. “Some-
body may get lucky, but I think it is going to be very difficult to do this.” The excitement for basic and applied autophagy science is yielding new resources to make it happen. Last year, the National Institutes of Health awarded Deretic $11 million to launch the new Autophagy, Inflammation, and Metabolism Center of Biomedical Research Excellence. This year, the NIH funded a $37 million effort, by UT Southwestern’s Levine and five other institutions, to develop approaches that use autophagy to fight infections. And in February, Washington University in St. Louis used a $10 million donation to launch the Philip and Sima Needleman Center for Autophagy Therapeutics and Research. “That money is just the beginning,” says Perlmutter, leader of Washington University’s new autophagy center. “We are going to be raising more for the center and investing from an institutional perspective. This is a big priority for us.” And though autophagy-focused biotech companies have ambitions to take on big diseases such as Alzheimer’s and nonalcoholic steatohepatitis, most plan on testing their drugs in rare diseases first. For instance, Casma is working on therapies for a rare brain disease called Gaucher disease type 3. “But there’s no reason to believe that an inducer of TFEB wouldn’t work just as well on a rare genetic disease as it would in a more common disease like Parkinson’s,” Dionne says. Likewise, although Samsara will likely start by testing its autophagy boosters in a rare disease, Aguiar says the company plans on applying the same drugs to multiple age-related conditions. “Drugs that slow the aging process are the ultimate pipeline-in-a-pill archetype,” Aguiar says. Some scientists are already dreaming up ideas for once-a-day autophagy-enhancing pills to clean up the accumulation of toxic proteins in the brain. Rubinsztein thinks that an approach like this could help prevent neurodegenerative diseases in the same way that statins are used to help prevent heart disease. For an idea like that to ever work, however, companies will need to show that long-term autophagy enhancement is safe in humans. For now, that remains an open question. “It has enormously palpable potential,” Perlmutter says. Yet when asked what autophagy’s biggest disease-curing potential is, Perlmutter’s answer is one that is echoed across the field. “Autophagy is important in a lot of things,” he says. “None of them stand out because all of them look interesting.” ◾ JUNE 3, 2019 | CEN.ACS.ORG | C&EN
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