A small-molecule drug named ISRIB quickly and safely restores to old mice youthful levels of cognitive function. Alphabet Inc,’s Biotech Firm Calico holds the license to ISRIB.
The anti-aging properties of ISRIB (pronounced “iz-rib”) were discovered in a collaboration between neurocognitive researcher Susanna Rosi and biochemist and biophysicist Peter Walter, both of the University of California, San Francisco. In a previous project Rosi and Walter showed that ISRIB could reverse memory failure and restore normal cognitive function in mice after traumatic brain injury — and it could do so even when it was given to the mice weeks after the injury. Prior to his work with Rosi, Walter and other collaborators demonstrated that ISRIB enhances memory in healthy mice and in mice with Down syndrome, and causes some treatment-resistant prostate cancer cells to self-destruct. Other research teams working with ISRIB have shown it to protect mice’s inner ears against noise-related hearing loss.
SUSAN MERRELL, COURTESY UCSF
In all studies, the researchers have observed no serious side effects.
The new study on the reverse of age-related cognitive decline was published on December 1 in the journal eLife. According to the eLife paper, ISRIB rejuvenates brain cells and cognitive function by interfering with the Integrated Stress Response (ISR), which is a set of signaling pathways in the brain that call key protective processes into action.
As Rosi explained in a Zoom interview, “One way to think of how ISRIB rejuvenates brain cells and cognitive function is that, when the ISR protectively blocks production of proteins, it’s as though it’s setting a switch to ‘off.’ Ideally, that would be a temporary change only. What we’re learning from our work is that sometimes the ISR gets ‘stuck’ and doesn’t let the switch move back to ‘on.’ That can make the cognitive deficits that were caused by the ISR itself seem to be a result of permanent damage — when they’re not.”
In turning the protein production switch back to “on,” ISRIB activates a specific enzyme that the ISR had blocked. Re-activated, that enzyme triggers resumption of normal protein production.
STEVE BABULJAK, COURTESY UCSF
The lab experiments central to Rosi and Walter’s demonstration of rejuvenation in the brain cells and cognitive function of old mice were led by Karen Krukowski, a research physical therapist with a special interest in the impact of aging and traumatic brain injury on cognitive decline. In a test of the mice’s spatial memory and ability to learn, for two days Krukowski’s team trained mice to escape from a maze. The task required finding a platform hidden under water that had been made opaque. Young mice typically required only two tries to escape the maze. Old mice that weren’t treated with ISRIB typically required four tries. On average, old mice given a few daily doses of ISRIB required only three tries.
To specifically test spatial memory, one week later the experimenters had old and young mice escape the same opaque water maze as before. The mice did not receive any additional training or ISRIB treatment for this test. The old mice that had originally been treated with ISRIB remembered about as well as young mice how to escape the maze. The untreated old mice did not.
To test episodic and working memory, eighteen days after their last ISRIB treatments, the old mice were trained every day for four days to use visual cues to locate a single escape tunnel on a platform with 40 holes. By the fourth day the old mice were finding the tunnel on average 20 seconds faster than the old mice who had never been treated with ISRIB.
The researchers studied the cells of the hippocampus, a part of the brain necessary for learning and memory. When they did, they found that the common markers of aging were largely absent and that the connectivity and electrical responsiveness of the cells were improved.
“The signs of aging diminished overnight,” Rosi explained in the interview. “For the first time we saw that the cognitive decline of old age is not necessarily a permanent loss of capacity or mental resources. Rather, the cells may just get caught up in a cycle of stress. ISRIB seems to break the cycle and re-boot the brain.”
Walter first identified the therapeutic potential of ISRIB in 2013. He and UCSF together hold the patent. In 2015 they licensed ISRIB to Calico.
Memory-Boosting Chemical Is Identified in Brains of Mice
UCSF Cell Biologists Find Molecule Targets a Key Biological Pathway
Memory improved in mice injected with a small, drug-like molecule discovered by UC San Francisco researchers studying how cells respond to biological stress.
The same biochemical pathway the molecule acts on might one day be targeted in humans to improve memory, according to the senior author of the study, Peter Walter, PhD, UCSF professor of biochemistry and biophysics and a Howard Hughes Medical Institute investigator.
Peter Walter, PhD
The discovery of the molecule and the results of the subsequent memory tests in mice were published in eLife, an online scientific open-access journal, on May 28.
In one memory test included in the study, normal mice were able to relocate a submerged platform about three times faster after receiving injections of the potent chemical than mice that received sham injections.
The mice that received the chemical also better remembered cues associated with unpleasant stimuli – the sort of fear conditioning that could help a mouse avoid being preyed upon.
Notably, the findings suggest that despite what would seem to be the importance of having the best biochemical mechanisms to maximize the power of memory, evolution does not seem to have provided them, Walter said.
“It appears that the process of evolution has not optimized memory consolidation; otherwise I don’t think we could have improved upon it the way we did in our study with normal, healthy mice,” Walter said.
Identifying the Chemical that Enhances Memory
The memory-boosting chemical was singled out from among 100,000 chemicals screened at the Small Molecule Discovery Center at UCSF for their potential to perturb a protective biochemical pathway within cells that is activated when cells are unable to keep up with the need to fold proteins into their working forms.
However, UCSF postdoctoral fellow Carmela Sidrauski, PhD, discovered that the chemical acts within the cell beyond the biochemical pathway that activates this unfolded protein response, to more broadly impact what’s known as the integrated stress response. In this response, several biochemical pathways converge on a single molecular lynchpin, a protein called eIF2 alpha.
Scientists have known that, in organisms ranging in complexity from yeast to humans, different kinds of cellular stress – such as a backlog of unfolded proteins, DNA-damaging UV light, a shortage of the amino acid building blocks needed to make protein, viral infection, iron deficiency — trigger different enzymes to act downstream to switch off eIF2 alpha.
“Among other things, the inactivation of eIF2 alpha is a brake on memory consolidation,” perhaps an evolutionary consequence of a cell or organism becoming better able to adapt in other ways, Walter said.
Turning off eIF2 alpha dials down production of most proteins, some of which may be needed for memory formation, Walter said. But eIF2 alpha inactivation also ramps up production of a few key proteins that help cells cope with stress.
Study co-author Nahum Sonenberg, PhD, of McGill University previously linked memory and eIF2 alpha in genetic studies of mice, and his lab group also conducted the memory tests for the current study.
Potential for Human Drug Development
The chemical identified by the UCSF researchers is called ISRIB, which stands for integrated stress response inhibitor. ISRIB counters the effects of eIF2 alpha inactivation inside cells, the researchers found.
“ISRIB shows good pharmacokinetic properties [how a drug is absorbed, distributed and eliminated], readily crosses the blood-brain barrier, and exhibits no overt toxicity in mice, which makes it very useful for studies in mice,” Walter said. These properties also indicate that ISRIB might serve as a good starting point for human drug development, according to Walter.
Walter said he is looking for scientists to collaborate with in new studies of cognition and memory in mouse models of neurodegenerative diseases and aging, using ISRIB or related molecules.
In addition, chemicals such as ISRIB could play a role in fighting cancers, which take advantage of stress responses to fuel their own growth, Walter said. He already is exploring ways to manipulate the unfolded protein response to inhibit tumor growth, based on his earlier discoveries.
At a more basic level, Walter said, he and other scientists can now use ISRIB to learn more about the role of the unfolded protein response and the integrated stress response in disease and normal physiology.
Additional UCSF study authors are Diego Acosta-Alvear, PhD, Punitha Vedantham, PhD, Brian Hearn, PhD, Ciara Gallagher, PhD, Kenny Ang, PhD, Chris Wilson, PhD, Voytek Okreglak, PhD, Byron Hann, MD, PhD, Michelle Arkin, PhD, and Adam Renslo, PhD. Other authors are Han Li, PhD, and Avi Ashkenazi, PhD, from Genentech; and, Karim Nader, PhD, Karine Gamache, and Arkady Khoutorsky, PhD, from McGill University.
The study was funded by the Howard Hughes Medical Institute.
Research Finds ‘Achilles Heel’ for Aggressive Prostate Cancer
Treatment-Resistant Cancers Self-Destruct When Exposed to Experimental Drug
UC San Francisco researchers have discovered a promising new line of attack against lethal, treatment-resistant prostate cancer.
Analysis of hundreds of human prostate tumors revealed that the most aggressive cancers depend on a built-in cellular stress response to put a brake on their own hot-wired physiology. Experiments in mice and with human cells showed that blocking this stress response with an experimental drug – previously shown to enhance cognition and restore memory after brain damage in rodents – causes treatment-resistant cancer cells to self-destruct while leaving normal cells unaffected.
The new study was published online May 2 in Science Translational Medicine.
“We have learned that cancer cells become ‘addicted’ to protein synthesis to fuel their need for high-speed growth, but this dependence is also a liability: too much protein synthesis can become toxic,” said senior author Davide Ruggero, PhD, the Helen Diller Family Chair in Basic Cancer Research and a professor of urology and cellular and molecular pharmacology at UCSF. “We have discovered the molecular restraints that let cancer cells keep their addiction under control and showed that if we remove these restraints they quickly burn out under the pressure of their own greed for protein.”
“This is beautiful scientific work that could lead to urgently needed novel treatment strategies for men with very advanced prostate cancer,” added renowned UCSF Health prostate cancer surgeon Peter Carroll, MD, MPH, who is chair of the Department of Urology at UCSF and was a co-author on the new paper.
Prostate cancer is the second leading cause of cancer death for men in the United States: More than one man in 10 will be diagnosed in his lifetime, and one in 41 will die of the disease, according to data from the American Cancer Society. Tumors that recur or fail to respond to surgery or radiation therapy are typically treated with hormonal therapies that target the cancer’s dependence on testosterone. Unfortunately, most cancers eventually develop resistance to hormone therapy, and become even more aggressive, leading to what is known as “castration-resistant” disease, which is nearly always fatal.
As part of a “growth first” strategy, many cancers contain gene mutations that drive them to produce proteins at such a high rate that they risk triggering cells’ built-in self-destruct mechanisms, according to studies previously conducted by Ruggero and colleagues. But aggressive, treatment-resistant prostate cancers typically contain multiple such mutations, which led Ruggero and his team at the UCSF Helen Diller Family Comprehensive Cancer Center to wonder how such cancers sustain themselves under the pressure of so much protein production.
Deadliest Cancers Throttle Excess Protein Synthesis
To explore this question, Ruggero’s team genetically engineered mice to develop prostate tumors containing a pair of mutations seen in nearly 50 percent of patients with castration-resistant prostate cancer: one that causes overexpression of the cancer-driving MYC gene, and one that disables the tumor suppressor gene PTEN. They were surprised to discover that the highly aggressive cancers associated with these mutations actually had lower rates of protein synthesis compared to milder cancers with only a single mutation.
“I spent six months trying to understand if this was actually occurring, because it’s not at all what we expected,” said Crystal Conn, PhD, a postdoctoral researcher in the Ruggero lab and one of the paper’s two lead authors. But she saw the same effects again and again in experiments in mouse and human cancer cell lines as well as in 3-dimensional “organoid” models of the prostate that could be studied and manipulated in lab dishes.
Conn’s experiments eventually revealed that the combination of MYC and PTEN mutations trigger part of a cellular quality control system called the unfolded protein response (UPR), which reacts to cellular stress by reducing levels of protein synthesis throughout the cell. Specifically, these mutations alter the activity of a protein called eIF2a (eukaryotic translation initiation factor 2a key regulator of protein synthesis, by turning it into an alternate form, P-eIF2a, which tunes down cellular protein production.
To assess whether levels of P-eIF2a in patient tumors could be used to predict the development of aggressive, treatment-resistant disease, Conn collaborated with Carroll, who holds the Ken and Donna Derr-Chevron Distinguished Professorship in Urology, and Hao Nguyen, MD, PhD, an assistant professor of urology, to examine 422 tumors surgically extracted from UCSF prostate cancer patients. They used a technique called tissue microarray to measure the levels of PTEN, Myc, and P-eIF2a proteins in these tumors, then asked how these biomarkers predicted patient outcomes using 10 years of clinical follow-up data.
They found that P-eIF2a levels were a powerful predictor of worse outcomes in patients with PTEN-mutant tumors: Only 4 percent of such tumors with low P-eIF2a continued to spread following surgery, while 19 percent of patients with high P-eIF2a went on to develop metastases, and many eventually died. In fact, the presence of PTEN mutations and high P-eIF2a levels in prostate tumors outperformed a current standard test (CAPRA-S) used to assess risk of cancer progression following surgery.
ISRIB Selectively Kills Aggressive Prostate Cancers
Next, the researchers examined whether blocking P-eIF2a‘s suppression of protein synthesis might effectively kill aggressive prostate cancers, said Nguyen, who was co–lead author on the new paper. “Once we realized that these cancers are activating part of the UPR to put the brakes on their own protein synthesis, we began to ask what happens to the cancer if you remove the brakes,” he said.
The researchers collaborated with UCSF biochemist Peter Walter, PhD, whose lab recently identified a molecule called ISRIB that reverses the effects of P-eIF2a activity. (Walter and UCSF neuroscientist Susanna Rosi, PhD, have shown that ISRIB can boost cognition and restore memory after severe brain damage in rodents – likely by restoring the production of proteins needed for learning in injured brain cells.)
Conn tested ISRIB on mice with prostate tumors and in human cancer cell lines and discovered that the drug exposed aggressive cancer cells carrying combined PTEN/MYC mutations to their full drive for protein synthesis, causing them to self-destruct. Intriguingly, she found that the drug had little effect on normal tissue or even on less-aggressive cancers lacking the MYC mutation. In mice, PTEN/MYC prostate tumors began to shrink within 3 weeks of ISRIB treatment, and had not regrown after 6 weeks of treatment, while in contrast, PTEN-only tumors had expanded by 40 percent.
To further investigate the potential use of ISRIB against aggressive human prostate cancer, Nguyen implanted samples of human prostate cancer into mice, a research technique called “patient-derived xenografts” (PDX) that has historically been unsuccessful in studies of prostate cancer.
In one experiment, the researchers transplanted different groups of mice with cells from two tumors extracted from the same prostate cancer patient: one set of cells from the patient’s primary prostate tumor and another from a nearby metastatic colony in the patient’s lymph node. They found that mice implanted with cells from the metastatic sample – which exhibited the expected “aggressive” proteomic profile of high MYC, low PTEN, and high P-eIF2a levels – experienced dramatic tumor shrinkage and extended survival when treated with ISRIB, while mice implanted with cells from the less-aggressive primary prostate tumor experienced only a temporary slowing of tumor growth.
The authors used a third PDX model of metastatic prostate cancer to assess whether blocking the UPR could effectively treat advanced castration-resistant disease: they showed that transplanted tumors, which typically spread and kill mice within 10 days, were significantly reduced and the animals’ lives significantly extended under ISRIB treatment.
“Together these experiments show that blocking P-eIF2α signaling with ISRIB both slows down tumor progression and also kills off the cells that have already progressed or metastasized to become more aggressive,” Conn said. “This is very exciting because finding new treatments for castration-resistant prostate cancer is a pressing and unmet clinical need.”
The researchers hope that this discovery will quickly lead to clinical trials for ISRIB or related drugs for patients with advanced, aggressive prostate cancer. “Most molecules that kill cancer also kill normal cells,” Ruggero said. “But with ISRIB we’ve discovered a beautiful therapeutic window: normal cells are unaffected because they aren’t using this aspect of the UPR to control their protein synthesis but aggressive cancer cells are toast without it.”
“The only side effect we’re aware of,” Conn added, “is that this drug might make you smarter.”
Additional authors on the paper include: Yae Kye, Lingru Xue, Craig M Forester, MD, PhD, Janet E Cowan, Andrew C Hsieh, MD, John T Cunningham, PhD, Charles Truillet, PhD, Michael J Evans, PhD, Byron Hann, MD, PhD, and Peter Walter, PhD, of UCSF; Feven Tameire and Constantinos Koumenis, PhD, of the University of Pennsylvania Perelman School of Medicine; and Christopher P Evans, MD, and Joy C Yang, PhD, of the UC Davis School of Medicine.
The research was supported by the US National Institutes of Health (R01-CA140456, R01-CA154916, and P01-CA165997), the US Department of Defense (W81XWH-15-1-0460), the AUA-SUO-Prostate Cancer Foundation (16YOUN14), the Urology Care Foundation (A130596), the American Cancer Society (PF-14-212-01-RMC), an American Association of Cancer Research (AACR)-Bayer Prostate Cancer Research Fellowship (17-40-44-CONN), the Campini Foundation, the Leukemia and Lymphoma Foundation, the American Cancer Society (130635-RSG-17-005-01-CCE), Calico Life Sciences LLC, the Weill Foundation, the Howard Hughes Medical Institute (HHMI), the UCSF Department of Pediatrics (5K12HDO72222-05), and the Goldberg-Benioff Program in Translational Cancer Biology.
UC San Francisco (UCSF) is a leading university dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care. It includes top ranked graduate schools of dentistry, medicine, nursing and pharmacy; a graduate division with nationally renowned programs in basic, biomedical, transitional and population sciences; and a preeminent biomedical research enterprise. It also includes UCSF Health, which comprises three top-ranked hospitals, UCSF Medical Center and UCSF Benioff Children’s Hospitals in San Francisco and Oakland, and other partner and affiliated hospitals and healthcare providers throughout the Bay Area.
Gene Plays Critical Role in Noise-Induced Deafness
Experimental Drug Prevents Inner-Ear Damage, Protects Hearing
In experiments using mice, a team of UC San Francisco researchers has discovered a gene that plays an essential role in noise-induced deafness. Remarkably, by administering an experimental chemical – identified in a separate UCSF lab in 2013 – that acts on the pathway linking this gene to hearing loss, they found that they could prevent noise-induced deafness in the mice, a condition that affects tens of millions of adults and about 17 percent of teens in the U.S.
The researchers said a similar drug to the one used in their study might one day be used as a hearing-protection regimen before working in a noisy environment or going to a loud concert. They’re also looking at whether it could be taken shortly after exposure to loud noise to protect hearing, such as when soldiers are exposed to explosions on the battlefield.
Whether of environmental or genetic origin, hearing loss is almost always caused by damage to sensory “hair cells” in the cochlea, which detect sound waves and transmit auditory information to the brain. Unlike many types of cells in the body, if hair cells die, they cannot regenerate.
“It doesn’t take very long for hair cells to die and for the cochlea to basically become a scar – the whole structure of the cochlea changes,” said Dylan Chan, MD, PhD, an assistant professor of otolaryngology at UCSF and co-senior author of the new paper. “To restore hearing would not only require getting hair cells to regenerate, but also reproducing this finely tuned mechanical structure. The better option is to try and prevent the death of hair cells in the first place.”
As reported in the Oct. 15, 2018, issue of The Journal of Clinical Investigation, researchers led by Elliott Sherr, MD, professor of neurology and of pediatrics at UCSF, were interested in a gene called Tmtc4 for its potential role in brain development. When the team deleted the gene in mouse embryos, they were surprised to discover that the mice became almost completely deaf by one month after birth.
“The observation that these mice were going deaf was a fortuitous one, so we took it and ran with it and really landed right on the biology,” said Sherr. “A lot of times when you do experiments you get hints that you’re heading in the right direction, but literally every experiment we did pointed us in the same direction. We were able to go from observation to mechanism to treatment in one window of time, which was really exciting.”
The Tmtc4 protein normally plays a role in the endoplasmic reticulum (ER), a structure inside the cell that helps regulate the majority of protein production. Working with Chan, Sherr’s group found that the loss of Tmtc4 in cochlear hair cells distorted the delicate balance of calcium that ordinarily exists between the ER and the rest of the cell and triggered the unfolded protein response (UPR) – a quality control system that causes a cell to self-destruct if it’s producing faulty (and perhaps dangerous) proteins. The UPR triggered hair-cell suicide, ultimately leading to total deafness.
Because the mice could hear normally after they were born, the researchers realized that they weren’t dealing some form of congenital deafness. Instead, they suspected that the rapid hearing loss observed in Tmtc4-deficient mice was due to a heightened sensitivity to normal sounds. To test this hypothesis, the researchers exposed normal mice to loud noises and found that this triggered hair-cell death triggered by the UPR, just as was seen in the mice lacking Tmtc4.
“No one had shown that noise-induced hearing loss involved the unfolded protein response,” said Chan. “It really opens up a lot of potential for identifying different therapeutic options and exploring how targeting the UPR could be effective for lots of kinds of hearing loss that we really don’t have any treatments for.”
To avert noise-induced hearing loss in the mice, the scientists needed to somehow block the UPR and prevent hair cells from self-destructing. Fortunately, they were right down the hall from Peter Walter, PhD, a professor of biochemistry and biophysics at UCSF whose lab identified a drug in 2013 that does precisely that. The compound, known as ISRIB (for Integrated Stress Response Inhibitor) inhibits part of the UPR; it has been shown to reverse memory failure caused by traumatic brain injury in mice and to kill aggressive prostate cancer cells.
Giving the mice ISRIB before they were exposed to loud noises prevented hair cell damage and noise-induced hearing loss, and Sherr says the researchers are now looking to see if the treatment would be useful to prevent other types of hearing loss, including age-related hearing loss.
“People who become deaf as they get older become very isolated. There’s a lot of associated depression and an increase in the risk of developing dementia, so there are lots of good reasons for us to try to prevent hearing loss later in life.”
Authors: Additional authors on the paper include Jiang Li, MD, PhD, Omar Akil, PhD, Stephanie Rouse, Conor McLaughlin, MD, and Ian Matthews, of UCSF, and Lawrence Lustig, MD, of Columbia University.
Funding: This work was supported in part by the National Institute of Neurological Disorders and Stroke (2R01NS058721), the National Institute on Deafness and Other Communication Disorders (R03DC015082), and the National Center for Advancing Translational Sciences (UL1TR001872).
Conflicts: Sherr and Chan are founders and shareholders in Jacaranda Biosciences. Sherr is listed as an inventor on a published patent application, Novel Methods of Treating Hearing Loss (PCT/US2016/058348).
UC San Francisco (UCSF) is a leading university dedicated to promoting health worldwide through advanced biomedical research, graduate-level education in the life sciences and health professions, and excellence in patient care. It includes top-ranked graduate schools of dentistry, medicine, nursing and pharmacy; a graduate division with nationally renowned programs in basic, biomedical, translational and population sciences; and a preeminent biomedical research enterprise. It also includes UCSF Health, which comprises three top-ranked hospitals – UCSF Medical Center and UCSF Benioff Children’s Hospitals in San Francisco and Oakland – as well as Langley Porter Psychiatric Hospital and Clinics, UCSF Benioff Children’s Physicians and the UCSF Faculty Practice. UCSF Health has affiliations with hospitals and health organizations throughout the Bay Area. UCSF faculty also provide all physician care at the public Zuckerberg San Francisco General Hospital and Trauma Center, and the SF VA Medical Center. The UCSF Fresno Medical Education Program is a major branch of the University of California, San Francisco’s School of Medicine.
