#114 - Eileen White, Ph.D.: Autophagy, fasting, and promising new cancer therapies

Hey everyone. Welcome to the Drive Podcast. I'm your host, Peter Attia. This podcast, my website and my weekly newsletter all focus on the goal of translating the science of longevity into something accessible for everyone. Our goal is to provide the best content in health and wellness, full stop, and we've assembled a great team of analysts to make this happen. If you enjoy this podcast, we've created a membership program that brings you far more in depth content. If you want to take your knowledge of this space to the next level, at the end of this episode, I'll explain what those benefits are. Or if you want to learn more now, head over to peterattiamd.com subscribe Now without further delay, here's today's episode. My guest this week is Professor Eileen White. Eileen is the Deputy Director and Chief Scientific Officer, along with the Associate Director for Basic Research and the co leader of the Cancer Metabolism and Growth Research Program at Rutgers University Cancer Institute in New Jersey. She received her Bachelor's degree from RPI and her PhD from SUNY in Stony Brook and did her postdoc with Bruce Stillman at Cold Spring Harbor Laboratory. Eileen's early work focused on apoptosis, but it was doing some of the work there that she stumbled upon autophagy, and that is the focus of our discussion today. Now, if you're even remotely familiar with this podcast, you'll certainly know that the concept of autophagy has come up on so many previous episodes. It is a fundamental pillar of health and maintenance of health. We talk a lot about it in the context of fasting in particular. I have wanted to sit down with Ilene for a really long time, and I don't think this conversation disappoints, although we certainly could have gone longer in this discussion. We talk about Eileen's career and how it morphed from studying apoptosis into autophagy. We go into describing the the regulation of autophagy, both metabolically and otherwise. And then we spend a lot of time talking about the role autophagy plays in both the prevention of disease and also the treatment of disease. And I think this is where it gets really interesting, especially around cancer. And I think that that's potentially one of the most confusing aspects of the entire discussion on autophagy. And that's actually one of the reasons I really wanted to talk to Eileen was to better understand something that at the surface seems confusing to me, which is that autophagy seems to very clearly protect a person or an organism from getting cancer. Yet once someone has cancer, it appears that autophagy may disproportionately benefit the cancer cell versus the non cancer cell. So we tease this idea apart, along with talking about the amazing work that her lab has done to demonstrate the importance of autophagy in preventing Alzheimer's disease and neurodegeneration, along with the benefits of metabolic health. And of course, we do talk about the age old question that many of you have heard me go on and on about, which is how do we delineate and understand the dosing and frequency of fasting as a tool? In other words, when I talk about doing a fast of three days every month versus seven days a quarter versus five days a quarter, how could we possibly get a handle on what the ideal strategy is? And so we talk a lot about that as well. And I'm actually quite hopeful that from this discussion comes some, some research that can shed light on that. So without further delay, I hope you enjoy my discussion on autophagy with Eileen White. Eileen, thank you so much for extending your trip in San Diego for a day to come and make time to talk with me about this stuff today.

Oh, it's my pleasure. I'm looking forward to it.

I don't know if you remember this, by the way, but David Sabatini introduced us a few years ago. Do you remember? And we had this. I do, yeah. I still have my notes from that ph call. Five years ago I took about 20, maybe not 20, that's an exaggeration. Maybe 10 pages of notes in my sort of journal and probably have gone back to those a dozen times in the last five or six years. So I always appreciate it when people just pick up the phone and talk to total strangers for no reason. So that's greatly appreciated.

Well, you were excited about the science and so nerds like me like to talk about science.

Well, let's actually start from there. Tell me where your interest in science came from. Was it something that was always in you from a young age? Were you just naturally curious?

Yeah, I've been asked that question many times before. I come from a family where there was an interest in science. My mother was an elementary school teacher and my father was a lawyer, but he always wanted to be in science. And all of our discussions were related a lot to new scientific discoveries. So from an early age, I was introduced to science, which was probably unusual. And I then went to college and majored in science and biology and continued from there. And I decided when I was an undergraduate I wanted to get a PhD in biology. And I was very fortunate to go into Graduate school in the department led by Dr. Arnie Levine who discovered P53. And that was an inspirational experience because he has got scientific insight that's absolutely incredible. And then I went on to Cold Spring harbor lab where I was a postdoc with Bruce Stillman and again another incredible scientist. And it was an incredible scientific environment. There were a whole cadre of investigators there that were making major contributions in the field of cancer at the time. And it was just a very thrilling experience to be in an environment where once a week there was some fabulous discovery and everyone was excited about it. There were even, I think two Nobel prizes awarded while I was there. I joined the faculty at Cold Spring harbor after that and then I moved on to Rutgers. I had the fortunate experience of building a cancer center. When I went to Rutgers to be on the faculty, there was no cancer center. But shortly after I arrived, they hired a cancer center director, Bill Hite from Yale and I joined him to help build what's now the Rutgers Cancer center or the Rutgers Cancer Institute, which went from nothing to now. There are multiple buildings, there are 11 hospitals in our health system. We have 240 something members of the Rutgers Cancer Institute and we have, we're a consortium cancer center with Princeton University.

And.

And so it was very thrilling for me to not only have maintain my scientific interest by running a research lab, but also help expand and grow something from nothing where now we're treating large numbers of patients in standard of care and clinical trials and making large discoveries and moving cancer treatment, advancing cancer treatment as fast as we can.

And how many years have you been at Rutgers?

I joined there in 1990, so I've been there for a long time. But I moved about 12 years ago. I moved from one research building to be physically in the cancer center where I could be more directly helpful.

So where in your journey did autophagy pique your curiosity?

It was purely serendipity. So this goes back to when I was a postdoc with Bruce Stillman at Cold Spring Harbor. I was given an oncogene to study. That time they had just sequenced the adenovirus genome. They knew what genes caused cancer in the virus. I was given one of those genes and said figure out what it does. And that was a dream project for a postdoc. And what I found was that this gene was Viral homolog of BCL2. BCL2 is a gene, it's a human oncogene and it functions by blocking apoptosis or programmed cell death. And so that was transformative to me and took other people a while to realize the importance of that. That one novel function of cancer is to evade cell death. That field grew. We and others contributed cloning the other genes that regulated apoptosis. We figured out how it all the mechanism by which it worked. And the pharmaceutical industry started developing inhibitors of BCL2 to promote apoptosis and cancer. And that was the ultimate goal, was to make tumor cells die and have a drug that will do that. And once that happened, the field of apoptosis, I think sort of we accomplished what we wanted to accomplish. We understood everything. And that led to the development of the first of many drugs that were in clinical trials. And in fact, my lab is still involved with taking those drugs and putting them in patients and optimizing their use in solid tumors. So while the field of apoptosis matured to the point where things were being translated, we made a serendipitous discovery. We had engineered tumor cells to be unable to undergo apoptosis. They were refractory to being able to commit suicide.

Can we pause for a second there, Eileen, and just let's explain to folks exactly how apoptosis works, because shortly we're going to obviously contrast this with autophagy. They have common threads, but they're different. So let's go down the the path of what does it take to get a cell to undergo programmed suicide?

So there's a family of proteins called the BCL2 family. They come in different flavors. There are the BCL2 lack proteins which inhibit apoptosis, so they keep tumor cells alive. And BCL2 is a prototypical member of that family and is upregulated and amplified and translocated in many cancers to do exactly that. And are antagonizers of BCl2 and its related proteins. These are called the BH3 only proteins and they are often activated to inhibit BCl2 to trigger apoptosis. And then there's the core apoptotic machinery that triggers apoptosis. And this is backs and back. They reside in the mitochondrial membrane. And when they're triggered to undergo apoptosis, they oligomerize and poke holes in the mitochondrial outer membrane that releases proteins that activate proteases to grade the the cell. And BCL2 and BCLXL, all the anti apoptotic proteins are involved in antagonizing this process.

And what are some of the things that would have to be going wrong in a cell for it to go down that suicidal pathway? So for example, mitochondrial injury, that is irreversible genetic mutation that is unfixable. What are the suite of things that basically take a cell down the path of I can't fix this, and being around here and replicating is going to be dangerous to the host. I got to take myself out of the game.

Right. So mitochondrial damage can certainly trigger apoptosis, but probably the best way to explain it is by using the example of P53. So P53 is a tumor suppressor and a transcription factor. And some of the transcriptional targets of p53 are proteins like Puma and Noxa, which are these antagonizers of BCL2 and activators of BAX and back. P53 is a tumor suppressor. One of the functions is to promote apoptosis to prevent an emerging cancer cell from progressing. One mechanism by which p53 does that is by turning on the transcription of Puma and Noxa, and then that will antagonize PCO2 and initiate apoptosis. Then the question becomes, what activates p53 to do that? That could be a long list of things from DNA damage, from oxidative stress and so forth. You could think of something bad happens to an emerging cancer cell, and then P53 gets activated. And one of the tumor suppression functions of P53 is to turn on these promoters of apoptosis that antagonize BCO2.

Now, loss of function in p53 probably accounts for half of all cancers, correct?

Right.

And I assume that you have to lose both copies of it, or is losing one copy sufficient?

Well, what happens most of the time is not deletion of P53, but rather a point mutation that reduces function. It's more of a dominant negative. So, in fact, there's even evidence that there's a gain of function. So there are hotspot mutations in p53 that are very common in cancer, and p53 functions as a heterodimer. And what these mutant p53s do is that they end up entering into a dimer with wild type subunits, and that interferes with the function of the complex. So, yes, in that respect, it can be a loss of function of the p53 heterodimer, but there's evidence that it not only causes a loss of function, but it actually may do other things as well that are cancer promoting.

That's just a great example of the nuance of evolution. Right? I mean, in med school, the classic teaching 100 years ago for me was P53 loss of function oncogene Gain of function, black and white. Of course, it's never black and white.

Right.

So what is the wreckage of apoptosis? So when a cell undergoes apoptosis to everything outside the cell, inclusive of the immune system, what becomes visible? In other words, does an apoptotic cell, once it dies, elicit any immune respons, or does the process of apoptosis yield sort of an inert body of cellular matter that just goes away?

I'm not so sure I'm the best person to answer that question. I think the whole idea initially was during the process of apoptosis, you would get protein degradation and packaging of pieces of the dead cells into these apoptotic bodies, and then that would reduce inflammation. And then there's evidence that macrophages can then go and take up these apoptotic bodies and that may facilitate antigen presentation and so forth.

So it's possible, for example, that if you have a cell that has become cancerous, either through a gain of function, loss of function, but whatever, there's some mutation that now renders this cell to go down a pathway of cancer. Fortunately, it undergoes the apoptotic transition. The macrophages take it. Is it likely that you get an immune response to that that is protective in the long run against similar mutations? Because, I mean, even though the macrophage is part of the innate immune system, does that ever translate to the adaptive immune system such that you gain some long term immunity from that specific type of mutation?

Yes, I think something like that occurs. And I'm just thinking I'm the wrong person to answer that question. I could give you the better names of people that can do a better job. But the. I think the best way to compare it is to contrast it with necrotic cell death. So in apoptosis you have proteolytic degradation of a cell and packaging it into these bodies. And you say, well, does that limit inflammation? Well, the way to explain how it does is to compare it to a different form of cell death, like necrosis. So necrosis is cells lice, and that is very pro inflammatory. You have nucleic acids released, you have.

Essentially everything is released, including mitochondrial content, which is probably the most immunogenic, given its bacterial origin of the DNA.

Absolutely.

And so apoptosis, what I'm hearing you say is apoptosis is much cleaner than necrosis.

Absolutely.

So now let's talk about autophagy. Let's contrast autophagy with apoptosis.

That's right. Well, before we get to that, I should go back to your Original question of, like, how did we start working on autophagy? Then this sort of bridges us to what you just mentioned. So when we disable apoptosis in a cancer cell, it can't commit suicide. And we're doing that all the time. And we could show that. Then the tumor cells become more tumorigenic. But what we didn't expect was the extraordinary propensity for survival. We could leave the cells out and put them in buffer. They wouldn't die under extraordinary circumstances that we couldn't explain. So why would a cell that just couldn't commit suicide survive in buffer with no nutrients at all?

And just for context, this is the mid-80s. Late-80s.

This is the mid-80s.

Okay.

No, it's probably later than that. It was probably early 1990s. And so it was a conundrum. I mean, just because a cell can't commit suicide doesn't explain how it can be. A cancer cell can just sit in buffer and be fine. And we puzzled over this, like, how can this be? And then we discovered that what these cells had done was turned on autophagy, and we're using that for survival.

Before you go down that path, help me understand something. What did you observe about those cells that were sitting there in the absence of nutrients, surviving? Did you notice any metabolic changes that were unusual? Like, what was your clue that they were able to usurp the environment they were in?

It was an act of desperation. We tried a bunch of things and nothing was informative. And then I told the people in the lab, why don't we just look at these cancer cells under the electron microscope? And that way we can see everything. Because we couldn't understand how they could be surviving a buffer. And when we got the electron micrographs back, we saw something we had never seen before, and all these double membrane vesicles all over the cell. And so that when we finally figured all those are autophagosomes, which we had never, ever seen.

But wait, how did you. I mean, first of all, this is just, to me, one of the beautiful moments in science that I think it's so important for people who don't do science for a living to understand that While science is 99% failure, every once in a while you have a moment.

Like that, A eureka moment.

Yeah. It probably makes up for 10 years of failure.

Yes.

When you realize in that moment you are seeing something that has never been seen before, and therefore this is the cusp of new knowledge.

That's right. That's happened to me a bunch of times. In my career, which is fortunate. And this was one of those moments. And then we started reading, what are these autophagosomes? What do they do? And then when we realized from, when we looked in the yeast literature, they were meant to capture intracellular proteins and organelles and, and bring them to the vacuole or the mammalian lysosome for degradation and recycling. And that this was a mechanism by which yeast survived starvation.

And what was the tumor line or what was the cell line you were doing this in?

At the time, we were using kidney cancer cell lines.

Mice or human, these were mouse. But it was basically the first time this had been seen in mammalian cell line.

No, I think people had seen autophagosomes before. I mean, you got to remember in the olden days when electron microscopes were first available, that's one of the things that people did, was describe all kinds of different processes. So autophagosomes were known to exist, but there was very, very little information, almost no information on autophagy and cancer at the time. So we went into this area where there was almost no information. And so the first question we asked is, okay, well, the yeast data tells us that when you see autophagosomes, that means cells are starved and they're recycling, and they're using this to survive. And we hypothesized that that's what was going on in these cancer cells. And if that was the case, if autophagy was a survival pathway in cancer, that was a game changer. We had to understand it, and we had to demonstrate that that's what was actually happening. And then if that was the case, if cancer cells had usurped the autophagy pathway for their survival, then we needed to inhibit autophagy for cancer therapy. The first thing we did was we looked to see what would happen if we inhibited autophagy in these cancer cells. And the answer was very simple. In many, many circumstances, in many different cell lines that we looked at, when you inhibited autophagy, the survival of the cancer cells was reduced.

Let me interject for a second and ask a question. I don't know if you ever did this experiment, but if you took the kidney line and the kidney cancer line, so basically the same histology from the same tissue from the same animal, but one has the oncogenic properties and one does not, and you put them in the identical nutrient deprived stress, can you quantify the amount of autophagy or the efficiency with which those two cells undergo autophagy? In other words, is cancer Simply preserving the autophagy capacity that it had as a non cancer cell, but not enhancing it or not having any attenuation of it. Is it simply just, hey, this just happens to be something that gets preserved as you go from non cancer to cancer? Or is there some qualitative or quantitative change in the character of autophagy as a cell mutates?

That's interesting. So let me see if I can unpack that. So if you have. The general observation is that normal cells in the Fed state don't have autophagy on, it functions at a very, very low level, and if you starve cells or mammals for nutrients, then there's a massive upregulation of autophagy. What was striking about the cancer setting was that even in the Fed state, autophagy was elevated.

Ah, so there is a fundamental difference there. Yep.

Yes. And then if you stress them, it goes up even further. But the problem is, is that when it's already high, how much higher can it go?

And just to be clear, Eileen, this is in vitro, so you can't even make. When you said that, the first thought that came to my mind was, well, maybe the reason is they're undergoing a different stress, which is, for example, a vascular stress, a hypoxic stress, because, you know, haven't got enough VEGF or they haven't created enough. In other words, the apoptosis is going up despite being fed because there's something else that's impairing them. If what you just said is true in vitro, then that wouldn't explain that, would it? In other words, if they're not limited for oxygen, if they're in a petri dish and this is happening, my hypothesis wouldn't make sense. That would only make sense if what you said was true in vitro.

Right. So in the Fed state, the cancer cells already have elevated autophagic flux, and when you fast them, it does go up, but it only so high it can go.

I see. So this probably. You know, it's really funny. I'm sure you're familiar with the paper that Matt Vanderhyden and Luke Hantly and Craig Thompson wrote in science in 2009, which was, at least to my knowledge, the first time that someone offered an alternative explanation for the Warburg hypothesis, which is, hey, it might not be that the mitochondria of the cancer cells are defective and can't undergo oxidative phosphorylation. It might be that they're optimizing for growth as opposed to metabolism. They don't care as much about ATP as they care about building blocks. And therefore they're deliberately taking an inefficient route of glycolysis to lactate because they want the cellular building blocks. And that might be the explanation here, is that the tumor cell is, is undergoing more constant proliferation and therefore they want more building block.

Exactly right. And in fact, when we look at cancer cells and we study their metabolism, what we've noticed, and this is something that we found, and it's been a common observation, is that nucleotides seem to be rate limiting. And so the metabolism of, of a cancer cell is designed to facilitate de novo synthesis of nucleotides.

So it's really interesting, isn't it, when you really have to think about it. Like you think of all the things that could potentially be rate limiting to a cell, Think of how many phospholipids, for example, they need to build all of those cell membranes, and yet it's the nucleic acid to continue to propagate its DNA that becomes rate limiting. That, to me, is very interesting. I wouldn't necessarily have ever guessed that.

We may learn more as going forward, but that is what seems to be a recurring theme. But it's not just DNA, it's also rna. And you have to remember that RNA and ribosomes make up a huge amount.

They're much greater demand.

Exactly. And think David Sabatini has mentioned this many times that a large amount of the mass of a cell is ribosomal rna. And he had a beautiful paper where he was making the argument that ribophagy, the autophagy of ribosomes, was an important metabolic survival mechanism. And you could think of ribosomes as being a depot, a storage depot for not only nucleic acids, but also protein. And when a cell is stressed or starved, it doesn't need to make protein, so it doesn't really need large numbers of ribosomes. And so the autophagy pathway can cannibalize those ribosomes because they're unnecessary, and then recycle all that protein and nucleic acids to support survival.

Moon ago, you mentioned, of course, that some of this had already been observed in yeast. And the moment we start talking about things that are true in yeast and then true in animals, mammals for example, or higher order animals, we're talking about a billion years of evolution here. So this ranks as one of the few things that seems remarkably conserved over evolution. As a general rule, that makes it very important. Do we have a sense of when this first showed up?

Again, I might Be out of my league. I mean, I know it's certainly a big function in yeast. Prior to yeast. I don't know.

Yeah, but it's amazing. I mean, it's sort of. It's in the category of mtor.

Yes.

Something that is so important that it just doesn't really seem to change over about a billion years. Rule of thumb, it matters.

That's right. And I think when you compare how yeast does autophagy and how mammals do autophagy and what they're using it for, it just looks like mammalian version of autophagy is a little bit more complicated. It's probably. They have probably more different circumstances where autophagy might be necessary, but the basic process is surprisingly the same.

What are some of the other stresses that induce autophagy? And let's maybe just for the moment, even start with just in a normal cell. So let me sort of resynthesize what we've talked about. Clearly, nutrient deprivation is one of the biggest triggers for autophagy. And I mean, maybe just for the sake of time, I'll kind of throw this out there and you can correct me if I'm wrong, but I've always sort of thought of this through three pathways at the sort of mechanistic level. So you have sort of the MTOR pathway, which is mostly sensing amino acids. You have the AMPK pathway, which is mostly sensing energy and ATP in general. And then you have sort of the acetyl COA protein deacetylation pathway, which is also just basically sensing substrate of fatty acid and glucose. Is that sort of a fair way to say that those are three ways that nutrient low nutrients can still trigger the same pathway?

Yeah. And I think you could add on to that stresses that result in organelle damage, such as depolarization of mitochondria or dysfunction of mitochondria, activation of protein misfolding and generation of protein aggregates. So I think there's the things that are directly related to metabolic signaling that you've mentioned. But then there are other stresses that also can tie into the autophagy pathway.

Are there other stresses like the nutrient that are stresses that come from outside the cell to inside the cell? So the protein misfolding, the mitochondrial depolarization, those are things that are occurring as damage within the cell. That stress. Do we know anything, for example, about temperature? Do heat shock proteins stimulate autophagy in extremes of temperature? Does exercise, I mean, which obviously is in the short term quite stressful. How potent is that at inducing autophagy in a normal cell?

So Temperature wise, I would fully expect that temperature extremes would induce protein misfolding and induce autophagy as a remedy for that. But I don't recall any studies on that. In terms of exercise, that's very well studied, that exercise induces autophagy very potently. And you actually need autophagy too, because exercise damages the muscle, and autophagy is one of the processes that helps mitigate the damage that occurs during exercise.

What about hypoxia?

Oh, potently. Hypoxia potently induces autophagy.

Wow.

And in fact, one of the first things we did was when we looked at tumors. Tumors are well known to have hypoxia in the center. When we engineered tumor cells to be genetically deficient for autophagy, and you look at them, they're completely hollow, meaning they.

Have no organelles, Nothing.

Not the cells are hollow. The tumor is hollow.

Oh, wow. The further the cells get from a blood supply, meaning the more susceptible they are to hypoxia. They're dead.

Right. So if you take a tumor, if the middle is hypoxic, that's where the autophagy is most active. And if you genetically ablate autophagy in the tumor, you end up with a hollow tumor because the tumor cells in the middle don't survive.

Yeah. So it does come back to this idea that we talked about earlier about hypoxia being potentially one of the things that autophagy is protecting.

Absolutely.

How easy is it to create an animal model that is unable to undergo autophagy? How difficult is that from a knockout perspective?

Well, it's been done, and we've done it. It can be done different ways. So the original mouse strains that were made were deficient in either of two of essential autophagy genes, one called HEG5 and another one called HEG7. And these mice were developed in Japan. And these mice are born, but they fail to survive the neonatal starvation period. So when mammals are born, meaning once.

They are cut off from an umbilical nutrient source, it's almost like they have a glycogen storage disease. You know, those conditions where you can't produce any glycogen, it's uniformly fatal if not treated the moment you're cut off from an umbilical source of nutrients.

Right. So that neonatal starvation period between the cutoff of the placenta and suckling is a common feature in mammals. And there's potent induction of autophagy during that period. I can't speak for humans, but certainly I would think so. And in Mice, that's exactly what happens. And so these autophagy deficient newborn mice don't survive.

Wow, that really. I mean, again, I think that simply underscores the evolutionary preservation of something. If you knock it out, it is uniformly fatal. Do not pass go, do not collect $200, you're gone.

And then what we did was ask a different question. It was like what happens in an adult mouse? So in a newborn mouse, it's very different situation because any newborn mammal, they don't have any fat, no reserves. And so when the Japanese group did the extraordinary thing of trying to force feed these autophagy deficient newborn animals and they didn't extend their survival very much.

Now was this Yoshinori's group?

This was Noboro Mizushima and the Komatsu group, I believe. I think it was coming from those two labs.

So what happened in that experiment?

They discovered that the mice died shortly after birth. And then they realized that, well, they suspected they had a metabolic problem and they weren't suckling because they were probably too ill to by the time they were, would have been able to. So they force fed them and that allowed them to live for 24 hours, but they still died anyway.

And then are you able to induce an autophagy knockout in an adult?

Yeah, so that's what we did because we realized that the newborn animal is.

It'S just too fragile.

Too fragile. They have no nutrient reserves. And actually in the setting of cancer, we're thinking of you want to treat an adult with a tumor. So what's happening in autophagy in a newborn animal isn't even relevant. And so we engineered mice where we can take an adult mouse and, and give the mice a chemical so that an essential autophagy gene will be deleted throughout the entire animal. So one day they're an adult mouse with autophagy, and then a few days later they're an adult mouse with no autophagy. And these mice were very extraordinary. They lived for two to three months and then they died predominantly of neurodegeneration. Autophagy is very important in the brain over the long term. But if we fasted the mice, they were all dead within 16 hours.

Let's unpack that again. That's pretty remarkable. So you take a normal mouse that's got through the vulnerability period of infancy and you genetically knock out its capacity for autophagy. The first thing you observe is if you fasted for 16 hours, which admittedly is a pretty long fast for a mouse, that might be the equivalent of Fasting a human for a week, but that degree of nutrient deprivation is uniformly fatal. If you continue to feed them well, they only survive another couple of months because they ultimately succumb to neurodegeneration. Suggesting that the role of autophagy in preventing neurodegeneration is essential. And it's really not surprising when you think about the role. Everything you talked about with protein misfolding, I mean, when you start to think about the toxicities that are driving neurodegeneration, using Alzheimer's disease specifically as an example, there's a lot of crap that's basically getting accumulated in neurons. This would be an elegant way to suggest that autophagy is keeping that at bay.

Exactly right. So one other way of looking at it is what tissues are more autophagy dependent than others.

Exactly.

Brain would be really important, and there are a few others. And what we've noticed when we looked at the mouse, the lacked autophagy when we genetically deleted the autophagy gene in the adult mouse, was that there were tissues like the brain that were very sensitive and there were other tissues like the lungs that didn't have any phenotype.

So wait, in other words, when those animals ultimately die of neurodegeneration and you undergo the pathology analysis, obviously the brain is where you see the cause of death. You're saying in the lung it looked completely normal?

Relatively normal.

What about liver?

Liver was very sensitive, so it doesn't lead to the death of the mouse. So if you did a liver specific knockout of an essential autophagy gene, those mice have steatosis and their liver gets huge and whatever, but it doesn't kill them. I mean, they can live for quite a long time.

So you induce fatty liver disease.

Yes.

So again suggesting that autophagy probably plays a role in preventing fat accumulation in the liver.

Exactly right. And also protein aggregate formation. One of the other phenotypes of steatosis is the accumulation of these mallory bodies, which are large protein aggregates composed of a protein called P62. When you lose autophagy in the liver, you're causing accumulation of fat, accumulation of protein aggregates, but the liver manages to tolerate it. The brain, however, is a different story. If you have post mitotic neurons where.

They don't have the capacity to do.

It, they don't have the capacity to do that. I mean, when they accumulate the crap, as you said, then it's game over.

Was there evidence that the brain, in some last ditched effort to survive, was undergoing more apoptosis of neurons?

Yes, that's a common feature of these animals is increased apoptosis in the brain. But before that, you see all kinds of terrible things going wrong. This has been part of a major effort to generate autophagy stimulators as a remedy or as a means to delay neurodegenerative diseases.

I want to come back to this later on in the discussion, but I'll just plant the seed now. Obviously, fasting is one of the most potent stimulators of autophagy. I spend a lot of time thinking about how does fasting fit into our toolkit of longevity? A big part of longevity, in fact, probably the single most important piece of longevity when it comes to the lifespan aspect of it. So, you know, you think of lifespan versus health span, how long you live versus how well you live. On the how long you live front, I think it's very safe to say, based on all of the animal data and frankly, all of the centenarian data, that the key to living longer is delaying the onset of chronic disease. So even when you look at centenarians who are genetically gifted with tools to live longer, if you unpack what the gift is, it's delaying the onset of the disease, not living longer once you have the disease. So the centenarians, once they get cancer, and once they get heart disease, they die at about the same rate over the same duration as the rest of us schmucks. The difference is they get those diseases 20 to 25 years later. And again, that suggests to me that if you want to live longer, you have to delay the onset of these things, not live longer once you have them. And so it's hard to think that fasting doesn't play an essential role in that when you realize the role that fasting plays in the mitigation of Alzheimer's disease and metabolic disease. Of course, what we're going to come back to in a second is cancer, which seems to be this conundrum. This is the needle we're going to want to thread a little later down the line. I'll plant the seed now, but I do want to come back to the idea of ways that we can also induce autophagy sort of pharmacologically or chemically. The first thing that would jump to your mind is anything that mimics fasting. The first thing that comes to mind would be metformin, rapamycin, things like that. That what we talked about earlier, just for the listener to sort of tie this together, we talked about these huge pathways that tell the body nutrients are scarce. So When MTOR activity is down, that's a sign that we're deficient in amino acids. But we can also do that with rapamycin. When AMPK is up, that's the cell being told we're deficient in ATP. Another way you can do that is to give metformin. We haven't talked about sirtuins yet. Maybe I'll pause for a moment. Do we have any sense of what sirtuin activity does in autophagy?

I'm not familiar with that literature.

Okay. I was gonna say, because then you could get into the whole NAD versus NADH ratios and how that might factor into it. So, again, I'm really curious about this through a clinical lens as well, which is what is these suite of products, but almost just saying that out loud. So between the two of us, we remember to come back to this. But I now kind of want to get back to your story, which is, we've got these mice. You've got this much more elegant experiment now, which is you're actually going after the phenotype of interest, which is in an adult in which you inhibit autophagy. What was happening in that animal if it had cancer? So did you ever do the experiment where you had an adult with cancer, then you knock out autophagy?

That's actually one of the reasons we made that mouse. So we had two questions we wanted to answer. One was, if you inhibited autophagy in an adult mouse, what would happen? Because if they died in an hour, then targeting autophagy for cancer therapy would.

Be pointless, especially if you can't do it specifically.

That's right. So the answer was they didn't die in an hour. They died in two or three months, which was actually good news because that meant that there was a potential window of opportunity for inhibiting autophagy, for cancer therapy.

And I'm sorry, Eileen, when they died in two to three months, was it still from the neurodegenerative disease, still have cancer at the time of death?

We had to first make a mouse that lacked where we could switch off autophagy and find out what happened to that mouse. Okay. So we did that, and we saw that they died of neurodegeneration in two or three months, which was good. They died immediately. Then we would have stopped. There would be no point in trying to make cancer in that animal. But we did learn that they were intolerant to fasting, which was perfectly consistent with everything we knew about what autophagy functionally did. So then we moved to the second Step was to do the experiment that you just suggested to make cancer in that mouse, and then after the mouse had cancer, to then shut the autophagy pathway off and then to ask the key question, which died first, the mouse or the tumor? The answer was the tumor died first.

Wow. Okay, so then here's the gangster question. Once the tumor died, could you reactivate autophagy to prevent the neurodegeneration, or is it a one switch direction that required.

A different type of mouse model? So what we were doing was making a mouse with cancer, and then once the mouse had lung cancer, in this case, we deleted an essential autophagy gene in the entire mouse, tumor and all, but the gene was gone. So it wasn't like we could turn autophagy back on in that model. But since then, one of my trainees, in collaboration with a lab in the uk, they have developed a model where they can toggle autophagy off and then back on again. And what they've seen is a remarkable capacity of the normal tissues to restore themselves. So the experiment would be to have a mouse to induce an SHRNA to a specific autophagy gene to down regulate the expression and inhibit autophagy that way, and then later on take that SHRNA away or shut it off and restore normal autophagy in the mouse. And you see a lot of capacity for the tissues to restore themselves back to normal.

So that experiment basically becomes the proof point that says targeting autophagy in cancer makes sense. That's probably the most elegant description you could provide of that.

That's right. I think it would be better if we had specific targeted therapies against some of the enzymes in the autophagy pathway, because these are all genetic experiments and it's not exactly the same.

Right. You might not get the complete penetration.

With a drug or inhibiting a protease is not exactly the same as deleting the gene. But this is all what's called proof of principle that the concept of inhibiting autophagies in cancer is valid.

So what do we know today about what you've just described as it pertains to two things. I want to slice the data across two variables. The first is tissue type or histology of cancer, and the second is underlying genetic mutation. So I know that a lot of what you're describing is clearly true in Kras mutation. What about other drivers?

We and a number of other cancer labs that use genetically engineered mouse models for cancer have been banging away at that for a number of years. And what we've learned Is that Kras driven lung cancer and pancreatic cancer are extraordinarily autophagy dependent. And you do it, you know, make the mouse models and the tumors are very susceptible to the functional loss of autophagy.

Can you briefly tell folks what a KRAS driven cancer does? Like, what is it about the mutation that drives the oncogenesis?

So KRAS is a GTP binding protein that is responsible for activating what's called the MAP kinase pathway. And this pathway is very key in driving cell proliferation. And so cancers have a mutation in ras or mutations in ras that leave it in the GTP bound or on state. So there's perpetual growth signaling through the MAP kinase pathway, which of course is.

The hallmark of cancer, which is it's unresponsive to cell signaling and when you are fixed in the on position, you can't turn off. And basically that is cancer.

That's right. And what's particularly interesting about RAS driven cancers is that we have been very unsuccessful in drugging RAs and there's recent hope that the cysteine, the particular subset of the mutations in ras that involve a cysteine residue, that there are now drugs that target that there's hope after decades of failure.

Is the primary issue in the failure to drug RAs that you can't do it without creating toxicity for other cells that are non cancer, or that it has too many workarounds to whatever you put in place?

I think too many workarounds is a common problem. What they've done is that they said, okay, if targeting RAs is difficult, then let's go downstream of RAS and Trimat kinase. Right, Right. So there are inhibitors of raf, MEK and erk, which are downstream of ras, and those are actively in use in the clinic, but they seem to be not durably effective in RAS driven cancers because of the workarounds.

Yeah. So are there mutations or mutant drivers of cancer that we know are not dependent on autophagy and unresponsive to the autophagy blockade?

Yeah, it seems like there's a spectrum. So rash driven cancers are particularly sensitive. BRAF driven cancers like BRAF V600E is a common BRAF oncogenic mutation. And those cancers are particularly sensitive. And those, the ones that were examined were lung cancers and melanoma. The BRAF V600E mutation, melanoma is very common, homologous recombination deficient breast cancers. And those would be? Well, I mean, those would be models of hereditary breast cancer. Those are very sensitive to loss of autophagy. APC deficient colon cancer is another example.

What about non APC driven colon cancer, which of course is the majority of it. What do we know about that?

I don't think I can remember seeing a paper. I remember the APC deficient model. That's in fact the most commonly used model.

Anything we know about prostate cancer or other hormone sensitive breast cancers.

So prostate cancer is sensitive. We did that work. And hormone sensitive breast cancer, I'm not recalling right now, but there are a long list of cancers that are sensitive. The sensitivity is not all equal. For example, BRAF driven cancers are very sensitive, more so than RAS driven lung cancers. You just compare BRAF lung cancer to RAS lung cancer. The BRAF mutant lung cancer is more sensitive. The most sensitive cancer that we've encountered is in fact RAS driven lung cancer with LKB mutations. And this makes a lot of sense too. One of my trainees, who has her own research lab, hypothesized that we sat down and we thought, what cancer would you predict to be most autophagy dependent? It should be a cancer with loss of LKB1. LKB1 is a tumor suppressor gene that's involved in activating AMP kinase. And AMP kinase activates autophagy as a survival mechanism to low energy. There were a whole class of lung cancers that have lost LKB1, and as a result they can't activate this protective mechanism. This is Dr. Jesse Guo. She made this mouse model rash driven lung cancer without LKB1. And lo and behold, when you delete an essential autophagy gene, you abrogate tumorigenesis. So it makes a huge amount of sense. LKB deficient rash of lung cancer is probably the number one sensitive tumor.

So how do we reconcile these two observations that almost seem to have a difficulty coexisting? So the first is everything you've just stated, which is pretty clear and unambiguously suggesting that autophagy is, at least for a number of cancers, an important part of their survival and proliferation. And we contrast that with an abundant body of literature that suggests that when you combine fasting, which is a potent inducer of autophagy, with chemotherapy, for example, you enhance its efficacy. And we can speculate about why that might be the case. These two things, although not directly comparable, seem a a little bit at odds. How do you think about those things?

I would probably think about it in a slightly different way. So if you want to get at the two different roles of autophagy, one is cancer cells usurping it and turning it on for their own survival, Then the other side of it is when we know that autophagy is protective. We know what happens if you have a mouse without autophagy. Many terrible things happen. It takes a while, but the mice die of neurodegeneration.

Can I interrupt for one second? I'm sorry to do this, but I just. I'll forget this question. I want to come right back to your thought. In those animals that died of neurodegenerative disease after two to three months, did they show an increase in tumor genesis in any other tissue?

No, they don't. But if you make a mouse where you bypass a neurodegeneration by knocking out an essential autophagy everywhere else, okay, but not the brain, then those mice will get benign hepatomas. So benign tumors of the liver. But that makes sense too. Think about autophagy in normal tissues. We know it's important because if you knock autophagy out on a mouse, there's tissue specific but gradual deterioration, ultimately leading to neurodegeneration and you end up with steatosis. Fatty liver disease. The brain phenotype can be explained. As we discussed before, Neurons in the brain need this protein and organelle quality control function. They're post mitotic. They have to have a way of getting rid of the garbage in the liver. What happens when you damage the liver?

It regenerates. It's got infinite capacity.

That's right. But what happens when you.

Unless there's too much inflammation.

Exactly. So you end up with. When you inactivate autophagy and liver, you end up with these chronic cycles of damage repair and chronic inflammation. And that is oncogenic. And that's not, you know, it's particularly.

Obvious in the liver, the pancreas as well, Very, very sensitive to that inflammation.

So I think what this is telling us is something very important. It's telling us that a main function of autophagy in tissue homeostasis is to preserve cellular function, to be normal, to prevent chronic damage and inflammation. And tissues that are susceptible to cancer as a result of chronic damage and inflammation. Autophagy is highly protective, which again, think.

About how complicated this is. Now I'll bring us back to the question I posed a moment ago, but using this example. Why does NAFLD ultimately lead to cancer? Because if you have enough accumulation of fat, you get enough inflammation, you're gonna get hepatocellular carcinoma. Same with pancreatic cancer. This is why alcohol is such a horrible molecule, so toxic to the pancreas, to the liver and you sow those seeds of inflammation and lo and behold, you're increasing this risk of cancer. So on the one hand, we know that autophagy helps ameliorate that, it cleans that up, it buffers that. At the same time, we just realized a moment ago, oh boy, once you do get pancreatic cancer, it's a Kras driven cancer. Autophagy is helping it.

That's right.

So now let's come back to the question I posed a moment ago that I so rudely interrupted you in answering, which was, how do you reconcile these.

I think it's a matter of thinking of the role of autophagy in cancer as being context dependent. On the one hand, functional autophagy can delay the onset of chronic damage and inflammation that are known causes of cancer in particular tissues such as the pancreas and the liver, amongst a few others. So I think that stimulating autophagy through fasting or through pharmacologic means at one point can be thought of as preserving health. But once you have a cancer, I think it's a different ball game. And at that point it's a completely different context. And in that setting, what we've learned is inhibiting autophagy is preferentially damaging to the tumor compared to the normal tissues.

And then going back to the other literature which looks at the efficacy of fasting combined with chemotherapy, which is superior to just chemotherapy, do you think that the reason for that is that the chemotherapy itself, maybe once you. You're rendering the cells more sensitive to chemotherapy and also potentially generating a more durable immune response? Because one interpretation of what you're saying is a person with cancer should never be calorically restricted.

I don't know that's going too far. I would say that I don't know that you can equate caloric restriction with the loss of autophagy or regulation of autophagy, Because I think they're not equal things. Because I think that caloric restriction is limiting tumor nutrients. And so I think what that's doing in the context of cancer therapy needs to be better understood. I'm just not sure that we know what's happening there.

If I'm hearing you correctly, you're saying, look, it might be that we can't necessarily say that fasting isn't helpful in cancer, because while it may be counterproductive from the standpoint of autophagy, that may be offset by other things that are beneficial, such as the reduction of overall nutrients and inflammation that accompany this.

Exactly right.

Yeah. To me of all of the areas of autophagy that have me scratching my head the most, it is this question of, given that fasting is one of our most potent ways to stimulate it, in fact, I would argue it's more potent than metformin, which is an amp AK activator, more potent, probably, than exercise. I mean, it might be the most potent thing we can do to turn this amazing tool on. How do we think about using it in disease prevention and disease treatment? And they aren't necessarily the same thing.

I completely agree. And in fact, I would ask a question of you. So there's multiple efforts in the biotech industry to identify pharmacologic agents that are potent stimulators of autophagy. And I think their idea is in normal, healthy people will take a pill, autophagy will be turned on, and there'll be some fountain of youth type thing.

Yeah, Spermidine is one of the things that people are talking a lot about. Right.

So why not cultivate the use of fasting instead?

I will tell you exactly why, Eileen. And I love how you have fed into. It's almost like you can read my mind and know where I'm going to go with this discussion. I think a big part of it is we don't have the tools to measure the signatures of autophagy. In other words, if a patient comes to me and says, peter, I want to do whatever I can to enhance autophagy, because I have now bought into the idea that it is going to basically protect me from every chronic disease, I would say, yeah, I agree with you. And they say, great fasting seems like a great way to do it. I'd say, you're absolutely right. And they say, well, how long do I need to fast, Peter? Guess what? I get to say? I don't know. And I'll tell you in reality what I say. I say, well, look, I'm really sure that after about seven days of nothing but water, autophagy is fully cranked. And I'm also really sure that if you just go 12 hours without a meal, you probably haven't done anything. Where I struggle is we're between them. Now. I want to share with you some personal experience, and I want you to weigh in on it. And then I think maybe we can pivot off into actually kind of going back to what you and I spoke about over the phone five or six years ago, which is, what would a molecular signature for autophagy look like? And again, I think this is. I put this in the top three most important Translational questions in my field. In other words, as I think about the practice of medicine as it pertains to longevity, it's our inability to understand how to quantify the benefit of nutrient deprivation, in other words, our inability to dose it, that is our greatest certainly among our top three detriments to using this incredibly potent tool. So I fast a lot. I just finished a fast yesterday actually. So I used to do a thing where I fasted seven days every quarter. So four times a year I would just do a water only fast.

How hard is it to do that?

It is not that hard. I'm going to be completely honest with you. I wish I could sit here and say, oh, I'm a real stud. Nobody can do it. No, no, no, anybody can do it. I really think anybody can do it. Which is not to say that there aren't moments throughout those fasts where it's sort of difficult, but you'd also be surprised at how resilient the body is. Is so, yeah, not that hard. You have to make some adjustments. Obviously you have to be very thoughtful about how much water you're drinking and how many electrolytes you supplement. There are a lot of changes that are happening in terms of electrolyte management and things like that. But again, we certainly have the knowledge to know how to manage people through that. But I began to ask the question, right, which is, okay, is seven days, a quarter the right dose? I'm convinced that it's a big bolus of autophagy. But is it frequent enough? What about three days every month? That's about the same number of total days fasted, but it's more frequent, but it's probably less potent. And the reason I sort of decided to try three days a month was I noticed that a couple of things happened at the end of my fasts. So I always check my blood before and after one of these seven day fasts and there's a very predictable set of changes that occurs, some of them that are really obvious. Glucose plummets, insulin becomes unmeasurable, Uric acid goes through the roof, along with beta hydroxybutyrate. Of course, two possible explanations for the uric acid going through the roof. One is the breakdown of nucleic acid. And obviously when I talk, go through, I mean, doubling of uric acid. So much so that I started taking allopurinol during a fast to make sure I didn't get gout. Of course, it could also be, and I've read something that says that uric acid and BHB compete for the same transporter in the kidney. So there might also be of a competitive blockade. But nevertheless, we have at least one possible explanation there. Endocrine function changes dramatically, T3 goes down significantly and reverse. T3 goes up significantly such that the ratio of them changes by four to six fold, which means you basically shut off metabolism. Not surprising, explains why you become incredibly cold intolerant during a fast and also gonadotropin go down. So you have all these really predictable things. Well, what I noticed was I see virtually all of them, though not quite to the same magnitude after three days, but not after two days. So that just got me intuitively thinking in a hand waving way that three days was sort of the minimum dose you needed to really move the needle on a bunch of these other metabolic things, meaning the spike in uric acid, the bottoming out of glucose. So there's another thing that sort of happens. It's as you turn more and more free fatty acid into ketone and turn more glycerol into glucose, you reach an equilibrium where your glucose is pretty much going to stay at about 3 to 4 millimolar, and that takes about two to three days. And again, when we think about it through the lens we've already discussed, AMPK must be up through the roof, MTOR must be through the floor, and protein deacetylation must be off. I think my gestalt is that that takes about three days and obviously it gets greater and greater the more you go. But what would be amazing is if I could draw a tube of blood, send it to you after a three day fast, a four day fast, a five day fast, a seven day fast, a ten day fast, and get some sort of quantification. What is it that we would see? Now, of course it's complicated because you'd probably want to accompany each of those tubes of blood with a muscle biopsy so that you could look at LC3, LC2 and things like that. So I'll stop on my diatribe for a moment and now turn it over to you, which is where would you even begin to look for that signature of autophagy? And let's just start broadly with any tissue. You can have blood, you can have muscle, you can have liver, you can have adipose tissue. How would you now create a dose effect?

Well, we know that what happens in a mouse, let's just take a minute to discuss that. Mizushima lab made a mouse that had a transgenic LC3 EGFP protein expressed and that could use that mouse.

Tell folks what LC3 is, just because we're going to talk about this quite a bit.

So LC3 is the protein that is attached to the autophagous membrane and links to the cargo that ends up in the autophagosome.

So this is a very mechanical thing, right? Like this goes back to your observation in the electron microscope.

Exactly right. So LC3 is one of the key proteins attached to the autophagosome membrane, and you use it to see autophagosomes, because normally when autophagy is off, LC3 is diffuse. But when autophagosomes form, the LC3 protein is attached to them, and you start to see spots where all the autophagosomes are present. And so the Mizushima lab used that to assess autophagy in a living mouse. They fasted the mouse, and they found that they could see the formation of autophagosomes throughout the mouse.

And could they do this in, like, pbmc, out of blood, or do they need to use tissue?

They did it with tissue. What they learned was that, yes, autophagy is turned on during fasting in a mouse, which wasn't surprising, but it seemed to not be uniform across every tissue. So that was interesting. But we don't have a way to do that in people. All we can do, because this involved making a genetically engineered mouse. So the only thing we can do in people would be to look at a tissue section and stain it for LC3 and look to see if there were spots.

In other words, you could not look at LC3 conversion in white blood cells.

You could, but there's another problem in that you could take PBMCs and do a Western blot for LC3, and LC3 gets processed from LC3 1 to 2, and the 2 form is the one that's attached to the otophagosome membrane. So you could do a western blot of PBMCs to measure the conversion of 1 to 2, but then 2 ends up in the lysosome and gets degraded. So the typical measurement of autophagic flux involves measuring the rate at which LC3 1 gets converted to LC3 2, and then the rate at which LC3 2 ends up being degraded in the lysosome. In order to see that flux, you need to block the degradation of LC3 2 in the lysosome with baflomycin or hydroxychloroquine. And so you would only be able by looking at LC3 1 and 2 in PBMCs in a person that was fasting, you would only be able to infer autophagic flux because you wouldn't actually be able to measure it.

It's a clue.

It's a clue. It wouldn't be proof, but it would be, one could presume. I would expect you would see more conversion of 1 to 2 and then 2 going into the lysosome. I don't think that would get you the answer that you need.

Now, what if we had 100 volunteers who were willing to fast and subject to blood draws and muscle biopsies? So you could use the muscle biopsies to actually quantify the flux and establish, let's say you could do it at different time points. So you had 100 people fast for different periods of time, three, four, five, six, seven days, et cetera. You've got tissue and you've got blood. What else could you look for? So again, could BHB be a proxy? Could glucose be a proxy? I remember you once mentioning another organic molecule you had identified. You knew it by how many carbons it had and you thought it was ringed, but you weren't sure what it was yet. Have you figured that out?

Yes, that was something that accumulated when we inhibited autophagy, and that was glucuronic acid. So that would be. I think you're going down the right road. So I think what we can do, and we haven't really done this yet, would be to look at metabolites because metabolism is so drastically changed. So if we can't look at directly measure autophagic flux in humans very easily because we don't yet have the proper tools, we could use metabolites as surrogate markers for the consequences of autophagy.

That's exactly why I call it a signature as opposed to a biomarker, because I think it's basically how do you use machine learning to take many metabolites? There's a bunch of things we know are happening. We just have to integrate them. We know that leucine is going down, we know methionine will be almost unmeasurable. We know what's going to happen to glucose, uric acid, and then there's probably a whole bunch of other small molecules and things in the proteome that we don't yet know that are probably discernible from PBMC directly or indirectly through other things in the plasma. And it just seems like a problem that is so ripe for a machine learning environment where you don't need that many people. Because you know what the gold standard is? You just starve them and Then you have the check, which is the muscle biopsy, which can give you some sort of quantifiable gradation. I mean, do you get the sense that that's something an IRB would approve? It's invasive, you know, it requires biopsies and fasting and things like that. But. Or would they demand that you, hey, first you have to do this in mice?

That's what I would expect. And so one of the things that we have been talking about is we've done some of this, but probably not enough is to do a metabolic characterization of a wild type mouse fasted versus an autophagy deficient mouse fasted. I think that would potentially identify the metabolic changes that were autophagy dependent and I think that would provide some clues as to what to look at in humans. Because the problem with looking at metabolism is you get an enormous amount of data and it's very, very helpful to know what to look for. We may have a list of things that are obvious to look for, but.

Right, but the fine tuning is going to come in the non obvious. It's not going to be a regression model based on five things we know. It's going to be much more complicated.

And I prefer not the under the lamp post science.

Yeah, take an unbiased view and go. And honestly, I get asked about this more than any other translational problem. So the good news is I think there are a lot of people in the philanthropic community that would be interested in this. Even if this is not a question NIH is interested in. I doubt NIH is interested in this problem, although that strikes me as odd given how potent a tool fasting is and yet how we don't know how to dose it. And I think it's worth pausing on that for a moment because that is such a stark statement, if I'm correct. I believe I am. Which is imagine we had the most amazing drug imaginable. Imagine we had a proteose inhibitor for HIV and we knew deep down this could cure hiv. The problem is we didn't know how to dose it. How long would we tolerate that ignorance? Imagine we had a drug that we knew could kill cancer, but we just didn't know how much to give or how often to give it. We wouldn't tolerate that for a minute. And yet in fasting we have arguably the most potent tool, and certainly, if not the most potent, probably one of the three most potent tools in which we can affect human health. And we don't have a clue how to dose it or what frequency with which to use it. And I find that ridiculous. So I'm actually really confident that if there were a really great proposal put together that would go from the animal model to the human model, it would be fundable, it would be fully fundable through philanthropic efforts. And so if nothing else comes of this discussion, I would love to plant that seed with you and think about what would be the right consortium of people to do that work. Obviously there are lots of skill sets that we'd want to have involved in there, but I really believe that could be funded quite easily. And I think that the implication of that is as potent as anything else because again, here I am doing my three day fast every month versus my seven day every quarter versus five day every quarter. We just don't know. And it really is troubling to me. It just drives me insane.

Well, I think for the general community, I think it's an important question, even for practical reasons, because you may be able to control your life to the extent that you can do all this at your own convenience, but a lot of people don't have that flexibility. And so if they can be told that fasting for X amount of time is all you need to do, then the beneficial effects of fasting could be there would be more people that could take advantage of it.

Absolutely. And if you look at the work of someone like Valter Longo, who his assertion is you can get most of the benefit without actually having to be fully fasted. But to do something that is like a fast mimic where you reduce your calories significantly for a period of five days, again, maybe he's right, but we have no idea, we have no idea what the efficacy of that approach is versus a total water only fast for five days. And it would be great to know because if we could demonstrate that you're getting 80% of the benefit doing a fast mimicking diet versus a complete fast, well, that opens the door to many more people who would be willing to do fast mimicry versus an outright fast. And again, I think about this constantly, which is I'm almost willing to do anything. I just want to know what to do.

So I think that now is the right time to ask that question. Let me just digress a little bit to talk about metabolism. So we know a lot about metabolism. Essentially the field of not only just cancer metabolism, but metabolism in general, mapped out all the metabolic, most of the metabolic pathways. But what we lacked the ability to do until fairly recently was to have a thorough understanding of metabolism in a living mammal. Joshua Binowitz And I have invested a lot of effort in developing technology to use isotope tracers. This would be C13 labeled glucose and amino acids and so forth. And to deliver them to living mice, running around and doing normal mice things in a cage. And then look to see how they're used and how different tissues use them and how there's nutrients sharing between tissues. Because when you're fasting, there's many complicated things going on. It's not just like there's no food and you're inducing autophagy and that's.

That, that's the only thing that's happening in a vacuum, right?

So you have dedicated nutrient stores, you have glycogen in your liver that's mobilized and that's dumping glucose into the bloodstream. You have made adipose tissue that starts degrading your triglycerides, and then you end up with glycerol and fatty acids in the bloodstream, which then, you know, are taken up by the liver and so forth. So you have all these, you know, if you're really without nutrients for a long time, then your muscle proteins start being degraded, which is probably undesirable. That's dumping amino acids into the circulation so that you can maintain your survival. We have to understand all of that.

Because it really occurs in different phases. Again, if we just limit it to humans for a moment, where we have a pretty good understanding of this, what's happening in the first 24 hours versus the next 24 hours versus the next 24 hours is very different. And George Cahill's famous fasting study, the 40 day fast on the healthy subjects, really divides it into these phases. What's interesting is by about seven days into a prolonged fast, you pretty much reach a steady state. You've got a pretty consistent flux of triglyceride into free fatty acid out of the fat cell. You reach a steady state level of beta hydroxybutyrate, acetoacetate and glucose, such that basically the sum total of them in millimolar concentrations is about preserved to where you would be non fasting like. And so it begs the question, if we posit that once you reach that steady state of seven to 10 days, you're clearly in a fully turned on autophagy state. What's the switch look like when you're 24 hours or 48 hours or 72 hours into that, are you 80% of the way to the benefit or just 20%? That's a jugular question.

Yes, I think it would be fascinating to understand that. And I think that if you look back over history, almost all cultures have fasting as part of their history. And I'm thinking that that is not by accident. I think they must have learned by trial and error that this was a healthy thing to do. And so I think that autophagy, I would expect, is playing a major role in promoting health in response to fasting. But I really think, well, maybe I'm sticking my neck out, but I think using fasting, as opposed to trying to find some pill you could take is something that's easy to do.

And of course, it speaks to the irony of it, which is if you took probably 1% of the budget that is being dispensed to find pills that stimulate autophagy, we would actually be able to answer this question clearly.

Absolutely.

And actually just have a dose response. And look, that doesn't mean these things can't coexist. I'm all for it. And I wanted to ask you, of course, about some of the other pills like rapamycin and metformin, and the role that they might have and how we might be able to measure that. But again, this just strikes me as the most obvious question in the space of how to prevent disease. And it's like you have this beautiful, beautiful tool, and you don't know what the dose is, and you don't know what the frequency is.

If someone knows that they're susceptible to a neurodegenerative disease, has anyone looked at those people to see if they engaged in some sort of fasting regimen, whether that was helpful or not?

Indirectly, I mean, it's possible that that's been done directly, and I just am not familiar with it. I think what we've seen indirectly is dietary restriction, as opposed to just pure caloric restriction, where you improve the quality of macronutrients, specifically around improving glycemic control. You can take people that are in an early stage of cognitive impairment and delay it and or reverse it through that type of nutritional intervention. Now, of course, that doesn't necessarily say autophagy is playing a role because that's doing a lot of other things. It's improving glucose and insulin signaling in the brain. It's doing a lot of other things, and we're really starting to see the impact of metabolism in the brain. So that's not entirely clear. Indirectly, I would say there are, I think, some pretty interesting, compelling pilot data that suggests that rapamycin is neuroprotective. And again, rapamycin, a very potent inhibitor of mtor, would presumably on some level, induce autophagy I think it's a very interesting question as to what is it about rapamycin that induces a longevity phenotype. Rapamycin, to me, is the most interesting molecule out there because it is, I think, the only molecule that has demonstrated a longevity benefit across all four models of eukaryotic cells. So that's a really big deal that can't be ignored. But how much of that benefit is through autophagy? I'd like to turn that question to you. How much of it is through inhibition of senescent cells, reduction of inflammation? Again, so it's very indirect and it speaks to. Again, again, I don't mean to sound like a conspiracy theory guy, because I'm not, but it is a little frustrating that we have these amazing tools, but because they're not particularly profitable, you don't really have somebody that's interested in, in answering them. And that's why I again come back to A, I think these are answerable questions. B, I don't think they are billion dollar questions. I think they are really questions that are amenable to the philanthropic community. And I think from an ROI perspective, it's hard to think of examples of where you could put dollars to work in research that would have a greater impact on human life.

Right. And I think that our biomedical community is mostly focused on putting out fires rather than disease prevention. Although I've seen a change. I mean, I see at the nci, the National Cancer Institute, a bigger interest in cancer prevention. So I think people are coming around to realizing that making people healthier longer is probably more important than once they discovered to have stage 4 pancreatic cancer. What can we throw at it to make them live another two months?

Yeah. Again, it is sort of amazing to me how lopsided our resource allocation is with respect to that problem, because you're absolutely right. We have spent probably a quarter of a trillion dollars in the last 40 years on the second question, which is once you have metastatic cancer, how do you live longer? And we've done an analysis on this. So for the quarter of a trillion dollars that has been spent on that problem, on average for solid organ tumors, we have extended median survival by less than about a year since 1970. So almost 50 years. That's pretty sad when you think about the fact that there's not much evidence we've reduced the arrival of cancer. In fact, all we've done is basically come up with a second leading cause of cancer in terms of modifiable behavior, which is after smoking, it becomes down to diabetes, insulin resistance and all of the metabolic dysregulation. So, yeah, I'll get off my soapbox now. But again, this is in many ways just a sort of a plea for help, which is, I think there's just an amazing opportunity to understand this.

So there's another component to this that worries me greatly. So what we've seen, you know, so a lot of this you're talking about controlling metabolism to preserve health through implementation of fasting and understanding fasting. But look what's happened to the American diet. I mean, there are people now that don't even recognize vegetables in the supermarket. There are people that only eat prepared food. And so what we've seen is on the one hand, you're talking about preserving health and all that, but on the other hand, the overall health of Americans is deteriorating. Obesity is greatly increased and has no sign of abating. The diet of Americans loaded with high fructose corn syrup and diets that are disproportionate with prepared food. So the only way to globally improve the health of Americans, or anyone else for that matter, is to deal with both of these problems at the same time.

And to me, that's probably the greatest line of reasoning that says fasting is probably protective against all chronic disease. Because if you look at the three main chronic diseases that account for, to our last analysis, 82% of deaths above the age of 50 in the United States, excluding COPD. So if you take out the obvious smoking related death of COPD, 82% of death is attributable to cardiovascular disease, cancer, Alzheimer's disease and complications of diabetes. That's pretty stark. There's no question that when you improve metabolic health, which you can do through fasting, you reduce the risk of all of those significantly. Of course, the question becomes, how much of a role does autophagy play in that? Specifically, you look at the example you gave of neurodegeneration, that makes a very compelling case for it, probably also in cancer. What do we know about cardiovascular disease, by the way?

Nothing comes to mind. I don't recall seeing anything in that area.

Yeah, I mean, my take on the literature is that the benefits of fasting in cardiovascular disease are primarily mediated through the metabolic health benefits of it. Lower glucose, lower insulin, lower homocysteine in time, lower inflammation primarily, as opposed to something that directly pertains to APOB or the inflammatory response to that. But again, maybe it's just there and I haven't seen it. What else do you think is going on? What travels with Autophagy. Can we talk about senescence for a moment? Do we understand a sense of what's happening when an animal or a human is undergoing autophagy with respect to either SASP or just the overall senescent cells?

Yeah, there seems to be complicated roles for autophagy and senescence. There's evidence from the Narita lab that sasp, the secretion of inflammatory factors that occurs during senescence is facilitated by autophagy. But then in the cancer setting there's also examples where loss of autophagy limits senescence. So I think in the senescence area it's still a little bit confusing and maybe a little bit context dependent as to what's happening.

And then going back to the question about molecules, what do we know about metformin and autophagy? Do we know that in a fed state, if we give an animal or a human metformin, we can still induce atophagy all things equal through just the AMPK activation?

Yes, and I think that that's true, but I think that we don't understand over the long term what happens and what the consequence of having autophagy or not having autophagy is. So for example, we've never given metformin to our mice that don't have autophagy. That might be an interesting thing to do. What is the consequence of autophagy induction by metformin? It actually might be better to do that in a system where autophagy could be wasn't completely gone but could be toggled up.

Do you have the ability to turn automophagy into an analog versus a digital meaning where you can actually use gradations versus just on or off?

Yes, I think the mouse model where autophagy expression of a central autophagy gene is controlled by an SHRNA might be the way to do that experiment.

And then what about rapamycin? What do we know about the use of rapamycin which has been studied so liberally across again, everything from yeast, flies, worms, mammals uniformly extends life potent inhibitor of mtor which would signal autophagy all things equal. Where do we see that relationship?

So there's been a lot of discussion about using rapamycin or rapalogs as autot autophagy stimulators. But it's like what you said before, it does many other things and it's.

Also immune suppressive, although that also depends on the dose. Right? I mean the Everolimus data suggested that it was actually immune enhancing when given intermittently.

Also have to remember that if you have A small molecule like rapamycin, and you want to use it to preserve someone's health, you have to make sure that it's safe. And I think that's when everyone backs away. Because if you've got cancer and they want to try an experimental drug and you've got no other hope and you're going to be dead in a short period of time. There's a bit of latitude in what can be done clinically to test whether or not there's a small molecule or some sort of drug that will be safe and possibly have efficacy. But when you're talking about prevention, it's a big problem. The drug companies are not interested in it because the amount of time it would take in the risk of.

Right. Just a much narrower margin, by the way, that's exactly the reason we're in the situation we're in. So just to reiterate, we have a situation where I am not convinced that longevity as a game is going to be won on the back of extending the time you have a disease. I have never seen a shred of evidence to suggest that that is the answer. Everything in humans and animals points to the opposite end of the spectrum. Longevity is about delaying the time it takes until disease comes. The implication of that is prevention is the single most important tool in the longevity toolkit. How do we reconcile that with what you just said? All of our pharmacologic efforts, the trillion dollars we spend on drug development is all on the wrong side of the equation. It's on the how do you live longer once you have a disease. And I understand why that's the case for all the reasons you just said. And if that doesn't make the most compelling case for taking the best and safest drug of them all, which is fasting and understanding how to dose it and what frequency to dose it, I don't know what makes a better case. Along with exercise, by the way, I put exercise in that same category, which is it bothers me that we don't really know how to dose exercise either. I mean, it's less of a problem, I think, because for most people the issue is do more. But it would be really nice to know what the dose response is on different types of exercise, especially for people who want to do the minimum effective amount of. So both from an exercise perspective and a nutrient deprivation perspective, there's no more low hanging fruit in terms of minimizing human suffering than understanding how these things work.

You should start a biotech company to do this.

David Sabatini and I have talked about this at length and he has constantly told me to do this. And the way we've thought about it, it's not going to happen because I won't do it. So someone else will have to do it. The way David describes it very eloquently is right now, metabolic response to nutrients is a black box. And what David thinks, and I think he's right, is there's a multi billion dollar opportunity in decoding the black box. In other words, when we understand exactly what the response is to each different type of nutrient in every different dose and frequency and we can decode that in the way that we can do with so many other biochemical processes, you can do everything because then you could actually develop drugs probably that could actually do something. So there's a drug development platform that comes out of that. And then from my standpoint, what I'm really interested in is simply just on the front lines as a sort of knuckle dragging doctor. How do you even just put this into clinical practice? But again, it's high risk. It's a lot of effort to do part of that. I think on the drug development side, there's a lot there. I don't think it's very high risk. On the question I posed to you earlier, I think the, let's do the mouse study and identify the 50 metabolites, proteomic signature things that are generally going up with autophagy and then let's shotgun that in an unbiased way against human subjects. I feel like that's a project in the tens of millions of dollars, not even the hundreds of millions of dollars. And again, if you add one year of life to each human as a result of that, that's kind of staggering.

Right. And I think if you couple understanding mechanisms of metabolic delay of damaging diseases with surveillance for risk factors, I think those two things really need to be coupled together because you can. Even if you define what the optimal diet and exercise and fasting regimen is for delaying the onset of disease, there are still unlucky people. And I think that we can't forget about them in the context of health and longevity and well being, because you could have the healthiest diet and do everything. Right.

Vogelstein has written about this. Right. I mean, there's clearly a component, there's a stochastic component to this and. Yeah, so absolutely. And again, I don't think there's any reason to believe that we couldn't be addressing both of these.

Yes. I mean, I think that there's no reason to do everything. One always has to bear in mind that there is this other risk factor, that no matter what happens with understanding metabolism and fasting and good health practices, that other thing is still going to be there. If you have a BRCA1 mutation, that's a compounding and separate issue.

So in 2016, the Nobel Prize was awarded for basically the genetic elucidation of autophagy. What are the salient features of that award? What was it about oh Soomi's work that led to that award?

So what Osumi did was quite profound and very creative. He developed an assay. Well, he asked, what are the. I mean, yeast requires nitrogen for survival. And he asked, what are the genes that are required for nitrogen survival? And he identified the autophagy, essential autophagy genes. And I always wondered whether, if he was in the United States, would work like that be funded, because it just seems like it was an important question.

But it didn't have such a clear application down the line.

That's right.

It was a bit too high risk to fund.

Right. And also the clear disease connection wasn't there and so forth and so on. And nonetheless, he did that. And I guess the point is, is that sometimes scientific discoveries are so basic that you can't ever anticipate what it would ultimately lead to. And in this case, it led to something very extraordinary. But he probably had no idea at the time. And once they discovered these essential autophagy genes in yeast, then it was apparent that there were homologues in mammals and so forth and so on.

How conserved are those genes between yeast and mammals?

If you do a blast search, you can see them. I mean, not all of them, but the amino acid homology was compelling.

Wow. So when you sort of think about the future of this, I think so much of what we've talked about kind of feeds into what your optimism is. But how do you want to spend the next 10 years of your career? What are the questions you want to probe?

I think I would like to translate what we've learned about the role of autophagy in cancer. And that involves developing small molecule inhibitors to inhibit autophagy for cancer therapy. And Alec Kilmen and I started a company to do that. And what we're focusing on now is defining at the molecular level what the functional requirements for autophagy are in individual cancers. And this involves understanding the metabolic role of autophagy, why one cancer needs autophagy more than another. And then the newest connection is the connection to inflammation when you inactivate autophagy. You stimulate inflammation. And this is what we've talked about earlier in the context of cancer, that could be a really good thing, because the game changer in cancer therapy now is immune checkpoint blockade. In fact, what I was just talking about at the ASCR meeting yesterday was the particular patient that came to our cancer center and went through surgery, radiation, chemotherapy, and it all failed. And her body was riddled with tumors, and she went on a clinical trial for immune checkpoint blockade, and all the tumors melted away. And that was five years ago, and she's perfectly fine. So what we have to do is make that work for everybody. And if inhibiting autophagy activates the immune response and can facilitate not people who wouldn't respond to immune checkpoint blockade to respond, then that would be critically important to do.

I mean, let me think about that for a second. So when we think about the patients that are responsive, and we really have two big targets, right? CTLA 4 and PD1. Melanoma obviously is a huge success story here because it is so mutagenic. It's interesting. I have a friend who has lynch syndrome. So that's a familial syndrome where people are predisposed to cancer. He developed colon cancer when he was quite young, went on to develop pancreatic cancer, adenocarcinoma of the pancreas, which is uniformly fatal. Almost without exception, he presented with an advanced state, so he was not even a surgical candidate. So the tumor had completely engulfed his mesenteric artery and vein, which meant he couldn't even undergo the surgical procedure, though it wouldn't have done much anyway. I had just read a paper six months earlier in the New England Journal of Medicine about. I forget what the paper was exactly about, but it made me think that because he had lynch syndrome and he has so many mutations, he might be a candidate for a checkpoint inhibitor. So we went back to his oncologist and said, hey, can we get him on keytruda? They said, which is an anti PD one? They said, no, there's no standard for that. But we found a clinical trial actually got him in. He got keytruda. That was five years ago. He's disease free. So you go from unresectable pancreatic adenocarcinoma to know pancreatic cancer. Pretty remarkable. Now, the question is, when I think about how broadly extendable that's going to be, it really comes down to how many shots on gold do you get? How many mutations do you get such that you can activate these checkpoint inhibitors? And so tell me how autophagy fits into that because I think I'm missing the link of why enhanced immune non specific immune response would factor into that. I know there's a link, but I need you to explain it to me.

So that's exactly where we're going with research. And should.

I should have been at the ARC yesterday. Yeah.

So we know that tumors with a very high mutation burden respond better to immune checkpoint blockade, but it's not that simple because there are patients with tumors that do have a high mutation burden that don't respond, and we don't know why. It could be they've upregulated some other checkpoint that we can't yet inhibit, or it could be some other reason they don't express Class 1. Or that the immune system can't see the tumor for some various reasons. And then we also know there are tumors that have a low mutation burden that do respond.

That do respond. Yeah.

And so a big part, in fact, we just got an NIH grant to study this is to make mouse models of cancer with low, medium and high mutation burden with which to.

And then you can bang out autophagy and.

Exactly. We haven't done the autophagy part yet. We're just doing the. Generating the models using proofreading mutations and polymerase epsilon and delta to generate mice with cancer with various levels of mutation burden in their tumors. This is so cool, because we'll be able to ask basic questions like how many mutations do you need when you have a low mutation burden, what can you do to make the immune system see that tumor? Do you need only mutations in the nuclear genome? What about mutations in the mitochondrial genome? So one of the mouse models we made was to generate a mutator phenotype in the mitochondrial genome. There are human cancers that have a high level of mitochondrial genome mutations. Whether that has any effect on anything is completely unknown. There's no reason why they couldn't be presented as tumor antigens.

And you'd think, if anything, they would be more immunogenic. I mean, they should be all things equal just because of their bacterial origin. That should elicit a much greater immune response.

Exactly right. This is what this grant is designed to do to generate mutator phenotypes in mouse models of cancer. So we can have a spectrum from low to really high mutations in the nuclear genome as well as the mitochondrial genome. Then to figure out the mechanism of response to immune checkpoint blockade to make cold tumors hot. That's essentially what the loss of autophagy is doing by promoting inflammation. It's Taking a tumor that is not killed by T cells that does not respond to immune checkpoint blockade and rendering that tumor responsive.

And where do you put this in the hierarchy of optimism for the future of cancer therapy? I mean, to me, the interesting stuff in cancer therapy is getting more and more targeted and stacking more and more therapies on top of each other. So this is an elegant example of stacking something that is clearly going to become a pillar of oncology, which is immune based therapy, with something that frankly is partially metabolic and frankly, partially more complicated than just metabolic therapy. So you've got, you almost add this to the layer of pieces of Swiss cheese you start to stack on top. If you have enough of them, you're not going to be able to drop a pencil through. The cancer doesn't survive.

That's right. So one of the limitations that we have with immune checkpoint blockade is some of which I've already mentioned, we can identify who's going to respond and who's not going to respond, and we have to extend the responder pool. But we have to be able to model that because it's very clear that a single agent is going to be Immune checkpoint blockade therapy is not going to help most of the patients. So how do we go about optimizing this treatment and having models where you can combine immune checkpoint blockade with other therapies to evaluate what is the optimal response is critical. And that's what we're doing. And whether fasting influences this is completely unknown.

What's the role of autophagy in the immune cell itself? So either adaptive or innate? I mean, maybe both.

So we know that one of the things we did was to turn autophagy off in a mouse and ask how that affected basic immune responses. And the answer was everything in the short term appeared to be completely functional. And if anything, the T cells were more anti tumorigenic. But if you go into the long term, if you knock out an essential autophagy gene only in T cells, for example, and look nine months later, I think those T cells are not going to be very functional. But it really depends on how you design the experiment. For cancer therapy, we want to know what happens acutely when you're inhibiting autophagy. You're going to be looking at things in the short term, not in the long term. So there's still a lot we need to do in that area. But in the short term, for cancer therapy, the immune system seems to function well, if not better in the presence of autophagy inhibition.

This just doesn't stop getting confusing because again, you would think that given the benefits of autophagy and preventing cancer, one of them, you would think that that would only enhance innate immunity because of the role innate immunity plays in cancer screening, which again, I think just speaks to. We are still really scratching the surface of all of the different tentacles that come out of these tools. Something like fasting seems very simple, and it's simple, of course, to do, but it has such a set of pleiotropic extensions and benefits that it's very unlikely that it's about all or none. There's nothing black and white here. It's really all these shades of gray that it's not intuitive when you look at them, what the net effect is, because it's a little bit of this, a little bit of that, more of this than that. It's the balance of this versus that.

Yes, I would agree. And I think context is important too.

When you think of all the big chronic diseases, just based on what you've talked with us today about, I think we have to be really excited about Alzheimer's disease based on the model you've shared. I mean, that strikes me as an amazing opportunity because one, we don't have a single tool once somebody has Alzheimer's disease. I'm sure you saw the most recent. I don't know if you follow that literature, but we just saw two enormous failures in the anti amyloid beta drug trials. So we're back to kind of square one, which is not a single drug that works for this condition. If any disease demands prevention, it has to be this one. It's hard to make the case that fasting isn't going to play a beneficial role there, isn't it?

I believe you. I'd love to.

I mean, we have to test this now.

It has to be tested. But I think that when you look at the dramatic failures in preventing or delaying Alzheimer's disease, you have to ask the question, what is the root cause of that? And if you look at the approaches, all the approaches are designed to ameliorate a symptom. And the research has not yet gotten to the root cause. And I think that that is the reason for these spectacular failures is they're trying to treat a symptom of the disease rather than the cause of the disease. And when you look at the genes that are involved in neurodegeneration in general, they fall into a broad array of different categories. And so my thinking is that they're all doing different things, but there's some common denominator that has yet to be identified. And I think that until the root cause of disease is identified, just finding means to ameliorate the symptoms is not going to be productive.

Eileen, you know, I could sit here and talk about this for hours and hours. I want to be respectful of your time because I know you've stayed an extra day to have this discussion with me, which I really appreciate. Is there anything else you want to talk about today either as it pertains to your work, something you're excited about in the future, or anything else that pertains to autophagy?

I mean, I think that understanding in greater detail how autophagy impacts metabolism, we've done some of that, but I think there's way more to do. We have the technical ability to examine metabolic flux in a mouse in vivo, in normal and starvation conditions and in response to different diseases. And we're only at the beginning of doing that. And I think that that's something that we will continue to do and hope that we can identify new targets for anti cancer therapy or signatures of metabolic problems. And we will continue to do that. But again, I want to return to the immunotherapy. I think in the field of cancer, building on that huge gain, we can't take our eyes off that ball.

Those are both very interesting and of course the former, it sounds like you really agree with David Sabatini, which is there's an enormous opportunity to decode metabolism in a way that we should have done 20 years ago.

I think we lack the technology. I mean, I think that. So maybe I could take a minute to explain what we do and that by using C13 or N15 labeled tracers, you can put them into a mouse, infuse a mouse with these tracers and by looking at where they go by mass spectrometry over different time, over different time points, you can see metabolism. And that was something that was never possible before.

What is it? Because we've been able to label these things forever, what was it the mass spec didn't have the resolution before? Or what is it that. Why is it we couldn't do this 20 years ago?

I guess I think they could do it with radioactive material and they could do it somewhat with the technology they had way back when. But I think that the technology now is far more sophisticated. By using heavy isotopes, you don't need radioactivity. Now there are more mouse models of disease. You can even do this in humans. Ralph D. Baradinis at UT Southwestern is infusing these isotope traces into humans with cancer and actually measuring the metabolism of human tumors.

Wow, that's interesting. I did an experiment once on myself with doubly labeled water, which of course is a very simple version of doing that sort of thing to examine energy expenditure. I had a field day doing that.

Oh yeah. But I think that in fact next week I'm supposed to go to a keystone tumor metabolism meeting where all the experts in this area will get together and talk about this in detail. But this is a growing field, it's very exciting, and understanding metabolism in mammals at a level we've never seen before in various disease states is tremendous.

And then lastly, is it safe to say that should there be a strong enough public demand and a philanthropic demand to go after that question we talked about earlier about sort of decoding the dose effect of fasting? Is that the sort of thing you'd be interested in working on?

Yes. I was fascinated by what happened when mice didn't have autophagy and they died when they were fasted. And I am very much interested in fasting as a way to preserve health. I'm also interested in, as I mentioned earlier, the dietary part, because I think all of this has to go together. It's not just how many calories you eat or how often you eat them, it's what they are. So yes, that's something that's very important to me.

Well, Eileen, thank you very much. This was super, super interesting and I know that folks are going to this will probably pose a few more questions than even we had time to go into, but that's great. Your work is fantastic and I appreciate your generosity.

Oh, this has been a lot of fun. I'm glad I took the time to do this.

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Sam.