#110 - Lew Cantley, Ph.D.: Cancer metabolism, cancer therapies, and the discovery of PI3K

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. I guess this week is Lou Cantley. Lou is the Meier Director and Professor of Cancer Biology at the Sandra and Edward Meyer Cancer center at Weill Cornell Medical College in New York City. Lou's research has made significant advances in cancer research, stemming primarily from his discovery of the signaling pathway for pi3kinase, or pi3k in the mid-80s. And his results here have really touched multiple fields, including cancer first and foremost, but also diabetes and autoimmune disease. Lou is a very decorated scientist, to put it mildly. It would take me too long to rattle off all of the awards that he's won, but I'll leave it to the four most prestigious the Gardner Award, the Breakthrough Prize in Science, the Wolf Prize, and most recently, the Horowitz Prize. Arguably, the only two prizes that are not on that list would be the Lasker Prize and the Nobel Prize. And I think that many people suspect that Lou will ultimately be awarded at least the Nobel Prize, if not both. This is a podcast that at times gets very technical, but most of the technical stuff falls at the end of the podcast. It's actually what we started with, but much like we did in a previous podcast, we're going to move that to the back end of the podcast so that we can jump right into the more concrete stuff that I think people want to hear about with respect to science. And we'll treat what was the first 45 minutes of this interview as a technical tour de force of the discovery of pi3k, and we'll move that as basically an appendix. So if you're listening to this and you really want to understand science, and you really want to understand how discovery is done, stuff that I really like to hear about, by all means you'll just listen to this as it's laid out. And if you really just want to hear about the implications of Lou's work as it pertains to cancer, then you will probably just listen to the first hour and 45 minutes or so of this and not the remaining 45 minutes. Before we jump into this, there are just two things I want to say. The first is I want to note Lou's disclosures. Lou is the co founder of two pharmaceutical companies, Agios and Petra, and he is also the co founder of a company that is just getting off the ground called Faith. That's F A Fayeth is a company that hopes to pair nutrition with pharmaceuticals. And that will all make sense during the podcast. These are all really interesting companies, but I think it's important obviously to list those disclosures at the outset. Secondly, I want to be sure that everyone understands that we have a discussion here that is very detailed at times and very tactical at times with respect to cancer treatment. And certainly every time I talk with Lou, I come away with a new way of thinking about the care of patients with cancer. Please do not mistake what we are talking about here as medical advice. It is impossible to provide medical advice to someone with whom you do not have a doctor patient relationship. And so, unfortunately, I hate saying that, but it is an obligatory disclaimer that I must provide. And whatever we talk about here, the combinations of therapies with respect to drugs for diabetes, drugs for cancer, and of course, nutritional therapies really needs to be had in discussion with your physician. Actually, I lied and I said two things. There's one other thing I want to talk about, which is there is a cancer trial that is about to take off that is a really interesting and exciting prospect through one of Lou's companies. The enrollment for that trial is just kicking off now, and we will list to that should anybody listening to this podcast either have the type of cancer that's being studied or know somebody who does. And it would be great to know that people are able to more readily get enrolled in that clinical trial. So without further delay, please enjoy my conversation with Lou Hanley. Lou, thank you so much for coming over.

It's great to be here.

Peter, you live like 10ft from me here and yet this is the first time you've been here, I think, right?

Yeah, it took me five minutes to walk here.

Yeah, it's great to see you always. You're definitely one of the most generous people with his insights and with the work you've done. And I consider you an absolute mentor in how I think about these problems in biology. So it's. We've been trying to get on this podcast together for a year. But it's a testament, I was going to say to how busy I am, but it's. No, it's to how busy you are. I'm busy, but you are even busier than me.

I do keep pretty busy.

Well, there are a lot of things I want to talk about today. I think to set my own expectations. We probably won't be able to get through them all, which speaks in some ways to the breadth of your work. We're going to talk so much about cancer and metabolism and things here. But there's what we know today about Pi3k, which I think would be a nice thing to spend a moment on. But I also love this idea. Like I have this idea, Lou, which is we should be teaching kids science, not the way you were taught biology as a 16 year old, which was rote memorization. We should simply teach science through the lens of discovery. So in other words, every biology class, even at the high school level and even younger, should be less about memorizing what it is that you see. Look under this microscope and memorize all the organelles or something like that. It's more of let's tell the stories of science. Because one, if our goal is to train scientists, well, they're going to go and become experts in whatever they're going to become expert in. We don't have to worry that in high school they're not going to learn enough content. But if we're trying to screen for people who are interested in science, what you have to be screening for is the process, is the discovery, is the game, is the thinking that went into the experiment. It's the blind luck, it's the bad luck. And that comes out in the story of everything. I've yet to hear a single world class scientist and I have the privilege of sitting down with so many of them, tell their story without some element of good luck and bad luck combined with serendipity.

Yes, serendipity in science is absolutely critical that when you get a result that's unexpected, you should be laser focused on understanding at the biochemistry, at the chemical level why you got a different result than you expected to get.

And following these journeys is to me a better way to weave the narrative arc of science. I mean, I think frankly, it's not that there's no value in knowing that DNA is a helical structure and it has these base pairs and it has this backbone. But that shouldn't be the focus. To me actually walking through the fumbling of how those guys got there, that's a very interesting story. And that probably will serve a non scientist better when they're done with science class because they'll have an appreciation for the process. And now I'm getting on my soapbox about it would be better if policymakers understood that that's how science worked as opposed to trying to remember what they learned in a biochemistry class 30 years earlier. And of course, for the people who are going to go into science, I still think they're going to go back and learn the basics anyway so we don't have to through rote memory get that stuff into them. But it's this process of thinking and collaborating and walking down the hall and seeing the result, as you said, that doesn't quite make sense. And saying, well, wait a minute, there's something in there, there's a story. I mean, science is basically just one big mystery. It's really about curiosity. That's what I'm hearing in your story is it's less about your innate brilliance and more about your obsessiveness, your ability to make an observ observation and not let it go.

Yeah, if I get a result that suddenly doesn't make sense to me, that's more exciting. It means there's something more complicated going on than the simplest explanation for what I'm seeing. If the simple explanation works, then fine, you publish it in some second rate journal. But if you've broken up a whole new field because your simple explanation doesn't work and you figured out why it didn't work and what was wrong, then that's where most breakthroughs come from.

When did cancer become such a high focus? So we met in 2011, two years after you, Matthew Vanderheiden and Craig Thompson wrote what I thought was sort of one of the most interesting papers I'd read on cancer in the journal Science. It's hard to believe we're 10 years away from that paper. What was the lead up to your collaboration in that paper? And less about the paper, but more about thinking about cancer metabolism. Because today when I think of Luke Hantli, I think of cancer metabolism. When did that transition occur?

It was a postdoc. Joined my laboratory, Matt Vanderheiden, who had trained with Craig Thompson. Matt's an MD, PhD now on the faculty at MIT, but he had been obsessed about glucose metabolism as a graduate student with Craig. And in fact I'd gotten one of his papers to review, which I was very impressed by. And so he suddenly showed up at my office one day saying that he Wanted he was interested in doing his postdoctoral work as he finished his clinical training. And at that time, a graduate student, Heather Christoph, had been working in my laboratory. And she'd made the observation that if she used tyrosine phosphorylated peptides to pull down a degenerate sequence context, it wasn't a single peptide. It was a whole mixture of billions of peptides all of the same length, biotinylated. So you could pull down anything they bound to and then use mass spec to identify all the things that came down. First of all, she rediscovered all the proteins that were known to bind to, that have SH2 domains. This is a domain that many proteins, including pi3 kinase have, that allow them to bind to tyrosine phosphorylated proteins. And so in that one experiment she rediscovered everything that had been published of all the things that bind to tyrosine phosphorylated proteins. But she discovered the thing that bound better than anything else in the cell or that was most massively pulled down was an enzyme in metabolism, pyruvate kinase. And that was shocking. And I remember her first response to that was, well, the only novel thing I found here that wasn't already known was this, this affinity for pyruvate kinase, pyruvate kinase. And she said, well, that's a metabolic enzyme and they're all boring, so I don't think I really want to work on that. And I said, no, no, that's whenever.

You get the result, you pulled a loop.

The totally unexpected result, that's the thing you should focus on. And so she did. And at the time that Matt was interviewing with me, I told him about her results and he got very excited about that. And the two of them worked together to show how that was working.

So this is early 2000s, right?

This was 2000, mid 2005. 2005, maybe.

Matthew Vanderheiden was a classmate, I believe. I've obviously met Matt since, but I believe he was a medical school classmate of one of my close friends, Ted Schaefer, who I actually had on the podcast. And Ted also was an MD, PhD, did his PhD in Harold Varmus lab. So it all kind of comes full circle. A lot of smart folks came out of the University of Chicago.

So anyway, so he, Matt and Heather ended up publishing two back to back papers in Nature that both explained structurally how phosphotyrosine peptides or proteins binding to pyruvate kinase could turn off its activity. And it also explained why turning off the activity of pyruvate kinase was important to increase anabolic growth. And that is kind of the basis for why we decided to start agios, that Craig was interested in how metabolism was regulated in cancer cells. And we had made this observation that pyruvate kinase regulation was important in oncogenic regulation, anabolic processes in cells. And so we proposed pyruvate kinase activators, rather than inhibitors, as a way to reduce tumor growth as one of the targets to go after at Agios as we were starting the company. So that review, which I should say that Matt played a much bigger role in writing that review than Craig or.

I did, he was the first author. So yeah, that was pretty clear.

Yeah.

When did the Warburg effect, when did you start paying attention to it? I mean, obviously it was around before you were born.

So actually I mentioned F. Racker earlier. F. Racker knew Warburg quite well, and they overlapped in Germany. He was obsessed by understanding the Warburg effect. The Warburg effect being a result published by Warburg back in the 1921 or so, almost 100 years ago, that if you chemically induce cancer in rats, the tumor that evolves takes up glucose and. And metabolizes it in an anabolic way much, much faster than the tissue of origin prior to transformation. This was chemically induced cancer formations.

And this also, this observation was that even when there was sufficient cellular oxygen to generate ATP much more efficiently through the mitochondria, it was as though the cancer cell elected to go this faster, less efficient route.

That's right. The surprising result was, well, there's plenty of oxygen. We've taken the tumor out, we are in an oxygen environment. Why doesn't it make ATP through mitochondrial oxidative phosphorylation instead of through glycolysis?

Why do you think the Warburg effect was largely forgotten in the 40s and 50s and 60s?

I mean, it wasn't. It fell out of favor, really not until late 60s, early 70s. And what was taking over at that time was evidence for virally induced cancers. Vsarc, Harold Varmus and Mike Bishop. So once it was shown that viruses could turn on cancers, the idea that it was just an altered metabolism fell out of favor. Of course, the discovery of oncogenes, of which VSARC was the first clear oncogene, because it told us that we've met the enemy and it is us, it is our own genes that are getting altered in cancer. And so proto oncogenes were being picked up by retroviruses.

Did anyone at the time think these two Mechanisms aren't mutually exclusive. The expression of or the amplification of, or the mechanism by which this viral injury to the genome can perpetrate and can perpetuate could be through this defective metabolism.

I think people who thought about it deeply and who were trained as biochemists, who were doing metabolism, which F. Racker was one of the prime examples, continued to realize you have to alter metabolism to do cancer. You need to switch a cell from a quiescent, not only from a quiescent state to a dividing state, but in order to divide and make a bigger cluster of cells, you have to drive glucose and amino acid uptake and anabolic processes, converting those to proteins and lipids and DNA and rna. So there was no doubt you had to turn up metabolism. The question was, at what level did metabolism play the role? Was it just a consequence?

Yes, exactly.

Of the viral transformation or the oncogenic transformation, or did they work cooperatively in some more complicated way? So our paper on pyruvate kinase suggested, yes, there is some clear cooperation where the active tyrosine phosphorylating proteins was directly regulating the activity of the metabolic enzyme, rather than just turning up the expression, for example, through a transcriptional cascade. So that's why shifted the emphasis back to there may be targets in metabolism that could give you an efficacy toxicity ratio for treating cancer. The truth is, chemotherapy, as you know better than I, is really targeting metabolism. You're blocking DNA synthesis or other steps in metabolism as a way of killing cancer cells. So that's, in a way, targeting metabolism is our first approach to curing cancer. And now we're back to doing it in a more serious way today.

What was the point that you guys made that was so novel in that 2009 paper? Because it's a subtle point that I think you can easily miss. You could easily look at that paper and say, oh, this is just another paper discussing the Warburg effect. But you came up with a very clear explanation for why the Warburg effect might exist that was different from the explanation that I think I had looked at before, which was the mitochondria are defective. So I think the easy explanation for the Warburg effect is the mitochondria don't work, therefore the cancer cell has no choice but to undergo anaerobic glycolysis. You guys propose something different?

Yeah, we proposed that you could, even with totally functional mitochondria, you could divert intermediates in glycolysis into anabolic processes by regulating steps in glycolysis. And so the biggest question we know that if you have high ATP in the cell, it will shut off glycolysis. And that's a feedback that there's high citrate and high ATP. Either one of those directly bind to phosphofructokinase, shut off its activity, and the remaining glucose that comes in, that gets phosphorylated either goes to the pentose phosphate shunt, or it goes to glycogen storage, or it just goes back out of the cell again. So there's a break right there. And so Frakr was obsessed by how you relieve that break, because how do you get a 10 to 50 fold increase in glycolysis if you're making so much ATP? It should shut it should shut it off, shut it back off again. So the idea that it was defective mitochondria was a simple explanation. There's no other way to make ATP, so you have to make it through glycolysis. But the idea that there was something more complicated than that was really what we were discussing in that review, that there was a way to regulate steps in glycolysis that would divert intermediates in glycolysis from making ATP to the carbon atoms of glycolysis would go into lipid synthesis, serine synthesis, glycine synthesis.

So it was sort of like a mass balance argument. It was an energetics for construction argument. It was basically saying, look, the way to really think about this is cancer's growing like crazy. It's easy to talk about the energy requirement, but just think about the carbon requirement of growth.

Right? So one analogy I like to make is imagine you have a river flowing through a valley, and you want to flood some fields in order to get enough water to grow plants. So if you put a dam here, then those other little canals that you've generated, the water level will go up to flood into those other canals and allow you to grow plants. That's sort of what happens if you turn off pyruvate kinase. Then you slow down ATP production. That means there's less ATP made. That means glycolysis, upper part of glycolysis continues, but instead of going to make more ATP, it goes off to make serine and glycine and ribose and lipids. And that gives you all the molecules.

You need to grow without shutting the system off, because you don't let it go all the way to ATP.

Right?

Yeah.

And that allows the mitochondria to make whatever ATP you need. And glycolysis is now used to do anabolic processes Instead of make ATP, that still makes some ATP, but you can now rebalance that whole equation so that you can grow better rather than just make ATP.

Now, this had a profound impact on the way you thought about your life. You're one of the. I don't want to say the few, but. But there are a lot of people who, a lot of great scientists, frankly, who their professional work and their personal. The way they go about conducting themselves in their own choices around food or whatever, there seems to be a disconnect there. Not the case with you. I remember the first time we had a meal in probably 2012, you were quite particular about what you ate and what you didn't eat. And the thing that you were most careful about was fructose. So what is it about the work that you were doing in the 2000s, the early 2000s, into this decade we're in today, the beginning of the decade we're in today, that had you start to think about nutrition in that way.

So I have to say that I made my decision about what to eat and not to eat before I knew anything about metabolism. And it came from a simple empirical observation. So I grew up, I mentioned earlier, I grew up West Virginia, right backwoods of West Virginia. In retrospect, I realized that as I was growing up in the 50s, we were eating incredibly healthy meals. In those days, the most expensive thing to buy at the grocery store was sugar. And my grandparents, who I spent a lot of time with working on their farm, raised everything they ate. They were substance farmers. They had essentially no income. They sold some strawberries in the spring. My grandfather would plow fields for people to make additional money. But they had very minor income. But they lived off of everything they grew. The one thing they couldn't grow for a while, they grew some sugar beets. But it was hard to get enough sugar. And so when my grandmother went shopping, essentially the only thing she would buy was flour and sugar. And one 5 pound bag of sugar had to last several years. And so when she would make what she called a cake, we would call it a biscuit today, it had essentially no sugar in it because it had to be sprinkled in very small amounts. And so that as a consequence, we were all very healthy. Nobody in my family, in fact, I didn't know anyone who was overweight. Western coast, most people lived like my grandparents. And that was in the 50s. What happened in the 60s was really what was tragic. It was all triggered by Castro, as you know, the story and Castro's taking over Cuba and US Boycotting sugar from Cuba. In the anticipation the cost of sugar would go up even higher. A German scientist had worked out a way to convert cornstarch into high fructose corn syrup which could be made much, much more cheaply than raising cane.

It's funny, I always thought it was a Japanese scientist. I didn't realize it was a German scientist.

I could be wrong on that.

I would take your word over mine.

We'll blame somebody from World War II.

The irony of it is it's always someone that got hurt in World War II. And the joke is this was their payback.

Exactly. And then Castro's payback. So Castro did this to us, we did it to ourselves is the truth. And we strolled doing it as long as I was where the primaries are held first. Primaries and where nobody's going to cut off taxpayer support for cornstarch growing corn.

But you had this profound belief. I mean, I guess you got to experience something very few people do, which is a natural experiment.

Yeah. So I watched West Virginia go from what I would argue in the 50s was the skinniest state in America. I never saw anyone overweight. None of the kids in my school, none of my relatives, none of them were overweight in the 50s. Most of them were very thin. And then along came high fructose corn syrup. And suddenly the cheapest thing you could buy were sodas that Normally at a 6 ounce coke would be my total allowance for a week to buy one 6 ounce coke. And now suddenly you fought 10 times as much for that cost. And so people advent of very sugary drinks. People had started consuming 10, 20, 50 times as much sugar as they were consuming before and over from the mid-60s to mid-70s. I watched. When I went back, I was at that time at Cornell getting my graduate degree and didn't go back to Western Union that often. But when I did go back, I every time noticed a dramatic change in that all my friends and relatives were suddenly becoming obese. I just watched what they ate and I realized they were consuming massive amounts of sugar. Many of them were consuming diet drinks. But then at the end of the day, because of their addiction, the sweets would eat like a gallon of ice cream per person. When I asked them how can you eat a gallon of ice cream? They would tell me if I don't eat a gallon of ice cream, I'll wake up either two hours later and go right to the refrigerator. So I can't sleep through the night without eating a massive amount of sweets. So at that point I realized this is A real addiction. And I cut out drinking anything that was sweet, and I cut out eating dessert. So that was 1975. Didn't know anything about the metabolism of fructose or glucose. Just empirically, I saw the change in diet and the consequent massive change in obesity.

So you basically have these two sort of separate stories, which is this is this observational journey of your life, and then you have the work of your career that is bringing you closer and closer to cancer metabolism. When did you finally realize in the laboratory that this observation of yours had mechanisms to it that could go far beyond obesity, but also play a role in metabolic diseases inclusive of cancer?

What really brought it all together? And this is why I keep emphasizing insulin as a postdoc and ultimately starting my own lab. I kept coming back to how does insulin work? And whenever we managed to see this PI kinase activity co precipitating with SRC and insulin receptor, I realized that the same enzyme was mediating the effects of the oncogenes and also effective normal insulin signaling. And so that by 1990, it was very clear in my mind that insulin was triggering cell growth in exactly the same way that oncogenes were. They were both activating pi3 kinase. And that suggested then a correlation between the two. And particularly as we began to notice that the mutations so many years later, mutations in PI3 kinase were identified. It was more than 15 years after we discovered the enzyme. The mutations were picked up from Bert Vogelstein's laboratory. And as we began to explore what those mutations did, we realized they increased the ability of insulin to activate the enzyme. So this whole connection between everything that insulin does goes through pi3kinase. And most oncogenes managed to activate pi3kinase by either directly binding or indirectly activating it, not necessarily through insulin, but they were bypassing the need for insulin.

We're going to come back to that very important observation in a moment. Right?

Yes.

You know exactly where I'm going with the next level. I'm questioning.

Okay, Right.

Yep.

But it also raised the possibility that being insulin resistant, which results in elevated serum insulin because the pancreas has to generate more insulin. If your liver and muscle and fat cells are failing to respond to insulin, then more insulin has to be made in order to bring glucose back down. So insulin resistance is pretty much a silent disease because you don't really know it. If you go for an overnight fasting glucose measurement in the urine, your glucose is okay. But if you do a glucose tolerance test, which very few people do, you realize that the Glucose goes up much higher, takes longer to come back down, and the insulin level is much, much higher than normal during that process of adapting. So that's how insulin resistance is defined. But I realized that high level of insulin, if we add it to our cells and culture cancer cells and culture it, makes them grow better. And we've known that since the 1960s and 70s. We use fetal calf serum to make our cancer cells grow in culture. And if fetal cath serum doesn't work, we either order another batch, you know, another lot from the company, or we just add insulin. And adding insulin always makes it work again. And so we've known for a long time that if you have high levels of insulin, you can make almost any cancer cell grow better than just pure fetal capsidin, which already has some insulin. And it's variable from batch to batch. So in a way, it's been staring us in the face for years that insulin will drive the growth of cancer cells. And cancer cells tend to have more insulin receptor than the tissue from which they emerged. So in the process of tumors growing out, evolving, they turn up the expression of the insulin receptor, and that allows them to respond to insulin better. So as I kept seeing more and more data on this over the last 15 years or so, I became convinced that the act of being insulin resistant or the state of being insulin resistant in humans, sets them up to accelerate tumor growth.

When I interviewed Sid, gosh, I don't know how long it's been, probably six, eight, nine months ago, he shared something with the listeners that I think, for many people, is still hard to believe, which is smoking is the leading environmental cause of cancer. Obesity is second. In fact, I think he even mentioned that in the documentary that was made after his book. You were in that documentary as well. Great documentary. It was not named after the book, was it? Was the documentary also called the Emperor of all Maladies?

Yes, it was.

Okay, okay. Phenomenal. We'll link to it in the show notes because it's a real gem. I mean, it's worth every penny. You gotta pay to download on Apple and watch that PBS special. Ken Burns, of course, is a. A master. But Sid made the point there as well. And hearing you say what you're saying makes you think it's really less the obesity and more the hyperinsulinemia that accompanies obesity in 80% of cases, which is high enough that you could easily, just from an epidemiologic standpoint, identify obesity per se as the trigger, meaning excess adiposity. But my belief, and I Think yours is that? No, it's probably the hyperinsulinemia, which means if you're lean and hyperinsulinemic, you're worse off. And I think those data are becoming abundantly clear.

There was a paper published a few years ago out of Montessori, Albert Einstein showing that in fact, if you take breast cancer patients and separate them into overweight, slash obese, insulin sensitive, because a lot of people can, if you put on peripheral fat but don't have visceral fat.

Don't have visceral fat.

Yeah, you don't necessarily become insulin resistant with overweight. So body mass index is not.

Yeah, it's not enough. It's a first order term.

That subset of overweight women who were insulin sensitive did not have an increased risk of breast cancer, but the ones that were insulin resistant did. So that says a lot. And that paper also had data on lean women who got breast cancer and that correlated with insulin resistant lean women. And in many nationalities, particularly Asians, they may look very lean, but still have a lot of visceral fat. And so that I think we, our current mechanism of using body mass indexes, our correlation is not a good one.

Yeah, it's just not good enough. And Mitch Lazar in 2013 I believe, published a paper showing basically the 2 by 2 of lean and not lean, metabolically healthy and not as a proxy for hyperinsulinemic. And it turned out that it wasn't the body mass that was the thing that tracked with health. Now, a moment ago you gave this very eloquent explanation of the role of insulin in cancer. Now four months ago in science you published a really interesting paper. What did that paper show?

So this is a paper in which was inspired by Mayor Bloomberg's attempt to ban the 40 ounce coke. So Ga Young, a postdoc in my laboratory who migrated with me from Harvard to Weill Cornell 2012 13, decided that she was inspired by Mayor Bloomberg and she would try to test the possibility that high sugar consumption would increase colorectal cancer. She had worked with Bert Vogelstein as a graduate student, so she knew a lot about colorectal cancer and was working on mouse models of colorectal cancer as she migrated into my lab. And so we decided that we would design an experiment to test whether there was higher risk for colorectal cancer with high sugar consumption. And so there was really two questions there. And my hypothesis going into this was the insulin level, that if you go on a high sugar diet, that you'll eventually become insulin resistant and that will drive High levels of insulin, which may accelerate the growth of colorectal cancer. And in fact, we had data that said that what I just said was true, that in fact, yes, if we allowed the mice to have sugar in their drinking water so they could adlib feed as much sugar as they wanted on top of their normal chow diet.

How much sugar was in the chow?

So the normal child diet, I don't remember exactly.

Normal Chow might be 10%, but you can buy these really high sugar standard American chows that are.

Yeah, so we just gave them the normal chow diet. And so because they were consuming massive amounts, they could drink as much of this sugary water as they wanted. They drank three to four times as much water, sugary water as if they just had as the controls without the sugar. And a consequence, they became massively obese. And they had accelerated polyp formation in the context of having knockout of the APC gene. So APC is the first gene in human cancer that gets lost to an initiate the process of colorectal cancer development. As Bert Vogelstein.

So the controls drinking normal water versus the sugar drinking waters both had the APC knockout.

They both had the APC knockout.

And what was the difference in polyp formation?

So in that three or more fold size of polyps, they were much more aggressive.

And what about the cancerous transformation?

Well, these mice, the polyps became so big that we had to sacrifice the mice because they literally could not digest food anymore. They didn't go into metastatic disease. So we couldn't call it a progressive other than the fact that they were massive and it was beginning to occlude. They call it polyps now.

In this experiment, you also tried to figure out what was playing the role in this. And you looked at glucose. So you could do this experiment with glucose water and fructose water, Correct?

That's right. But let me get to the next step first. Okay, so that was nice and fine, you know, was consistent with my idea. But GA wanted to challenge me on the idea that maybe the sugar was directly feeding the growth rather than just making the mouse insulin resistant and obese.

Oh, I'm sorry, because at this point your hypothesis was all still through the insulin. This was all being mediated through insulin.

Yeah, that was my hypothesis. So in order to try to always challenge people in my lab to prove me wrong rather than prove me right. And because it's always learn more if my simplest idea is wrong.

Another great tenet of science.

So she challenged me on this. And Marcus Goncalves is the other postdoc in my lab that worked with her, and the two of them worked together, and they designed a diet that would not allow the mice to become obese. In other words, they were taking the total amount of sugar they were consuming was the equivalent of a 12 ounce cola, sugary drink. And so on that diet, at least over the six months or so that they were on the diet, they really didn't gain weight, maybe 5% increase in total calories consumed. So they did not gain weight, they did not become insulin resistant, we didn't see elevation in C peptide, glycosylated hemoglobin. So by all characteristics, those mice were normal, they were not insulin resistant, and yet they still had increased polyps by two to threefold size. So at that point, that raised the possibility that the sugar might be directly feeding the growth of the polyp. And so we characterized that by using either radioactive glucose or radioactive fructose or carbon, 13 labeled a non radioactive but traceable form of carbon atoms in fructose or in glucose. And we also tried feeding them all only glucose, the same number of calories of glucose or the same number of calories of fructose versus the mixture of glucose plus fructose. And they had to have both sugars, fructose and glucose, before the polyps would grow faster. So that was really surprising.

And do you think that that could be because the glucose provides the insulin and the fructose is. What do you think the fructose is doing in there? Because based on everything you said earlier, a glucose water alone, just a pure dextrose solution, should have been sufficient, right?

That's right. So that turned out not to be the case. And the way to figure it out is again, using carbon tracing, either by radioactivity or heavy atom. So we did both. And first of all, does the fructose or glucose get all the way to the colon? Question number one.

It would seem highly improbable, but it.

Turns out if you add, if you have glucose plus fructose, the glucose competes for the fructose for entry into the small intestine. And as a consequence, the glucose is not as efficiently taken up and it makes its way all the way to the colon. If you give it in a sugary drink.

I was just about to say, Lou, does this have something to do with it being in a liquid, so the transit speed is quicker?

That's right. So if you gave the same amount in a solid food, it would never make it to the colon, but because it was in a watery mixture, it would transit the Intestines fast enough that there was still fructose left.

Your experiment was elegant because you didn't have to do what my next experiment would have been, which is why I'm not a postdoc in your lab, is I would have done colonic lavage, I would have gone retrograde and seen if I could bathe the polyps and not even deal with the absorption. But I would have missed this detail.

We wanted to replicate what.

What's physiologic? Of course. Yeah.

The condition which humans and other labs had already shown, that again, giving a fructose glucose mixture as a liquid at a certain volume, the fructose would make it all the way to the colon. So what we found that the fructose made of the colon, the glucose got absorbed in the small intestine, but there was some local increase in glucose level in the bloodstream. That is.

This is the first time I've ever seen this elegant. A demonstration of how you could miss in blood sugar a problem because by definition, the faster it's going through you, the faster the glucose and fructose are getting through and bypassing the small intestine, the more likely you'll get actual colonic content of them and the less likely you'll see glucose in the blood sugar. Which is why the group that in the second experiment, who were not being given ad libitum access to it, didn't get obese and didn't develop hyperinsulinemia, but still developed the polyphenotype.

Yeah. As a side issue along the lines, you're saying we actually found in a case where we had the water fed rather than a fixed amount to 12 ounce soda equivalent, that the subset of mice that had the APC mutations actually were protected from insulin resistance. Polyps were eating so much of the sugar that it was protecting the mouse from becoming insulin resistant.

That is unbelievable. And at some point you'll blow through that and they'll become quite insulin resistant and get hypertrophic polyps.

Yeah, yeah. But in this case, we weren't allowing that to happen. It was a very small amount of sugar equivalent of a single drink a day. 12 ounce sweetened drink. The real surprise was when we got to the molecular mechanism and that was that you had to have fructose and glucose because the actual carbon atoms that were being used to grow the tumor were coming from the glucose and not from the fructose. And what the fructose was doing is when it went into the polyp, it was converted to fructose 1 phosphate by an enzyme called ketohexokinase, also called fructokinase. That enzyme is only found in three tissues in any significant concentration. The liver, the kidney, and the gut. And so these polyps, like the normal gut cells, have that enzyme. That has been something of a mystery of why that enzyme exists at that location. But in any event, it does, what happens is the fructose goes in, it gets phosphorylated by that enzyme, and it happens very rapidly. It's a very active enzyme. And that drops the ATP level in the cell because you're consuming ATP to phosphorylate that fructose that's coming in. Now, I mentioned earlier that the thing with the Warburg hypothesis is that in order to get glucose to flux at a high rate into a cell, you have to drop the ATP level. Well, this is a way to drop the ATP level. Instead of consuming it in some other way, you're consuming it by phosphorylating the fructose. There's an additional complication, or not complication, but intervention, that is also important, which is once you start doing glycolysis at a higher rate, process of doing glycolysis incorporates an inorganic phosphate to make that doubly phosphorylated glycerol and 1,6 bisphosphate. And so that additional consumption of inorganic phosphate drops a negative regulator of inosine deaminase, and that drops the ability to keep ATP synthesis going on in the cell. So the combination two drops the ATP level dramatically. And now the glucose that's coming in is flooding through glycolysis, but it's going into all these anabolic processes. It's being used to make lipids. We see all the label from glucose going into fatty acid synthesis and serine synthesis and nucleotide synthesis going up five or tenfold. It's really quite dramatic what happens. But if you leave the fructose out, even though the glucose gets in the cell, it can't go through glycolysis at a high rate. And so you don't get growth. You have to have both molecules. The carbon atoms are coming from the glucose, the fructose.

The fructose is basically driving the kinetics.

Exactly.

When you describe it this way, you sound crazy. I mean, you're the guy nobody wants to talk to at the party. Your wife must be annoyed senseless by her crazy husband who's got this hypothesis that now has so much emerging data behind it that says if you wanted to create a molecule to kill people, it's not Just glucose. It's not even just fructose. It's put the two together. And guess what? Nature did that. Nature came up with a 50, 50 mixture of this thing. And if that weren't enough, she made it taste so good.

Right.

How do we reconcile this?

Yeah, so that's the part I like best, because I think about everything in regard to evolution. Why do things evolve? Why do we evolve to be addicted?

Because you're ruining the party right now.

Exactly. So I think this allows. If you think about humans 100,000 years ago, our metabolism hasn't changed in the last hundred thousand years. So 100,000 years ago, how often during a typical year in a temperate climate would you have available high amounts of fructose and glucose?

I mean, maybe in the fall, A month.

A month or so. So you would get berries ripen, apples ripened. Keep in mind, these are very small berries, very small apples. This is before domestication plants. And so as a consequence, what that means is at the end of the growing season is when most of the sugary producing fruits are being produced. If you could eat enough of those and you could keep your appetite up enough to just keep consuming anything in place, anything available, you could put on.

Weight as you go into the cold season.

As you go into the cold season. And if you put on enough weight, then you might actually survive to the next spring period when there's actually some roots to dig up to eat and keep going. And anyone who, probably almost anyone 50,000 years ago who didn't put on £50 or so in the fall would not be alive for the next spring. So very strong requirement to gain weight in order to survive a period of time when no food is available.

Yeah. Rick Johnson has written so eloquently about this, and I'm blanking on the name of his collaborator who's an anthropologist, and they even trace it back to which primates probably developed this first. And they were primates that had left Africa, gone to northern Europe, and most of them die off. It was only the primates that could develop this mutation. And I believe it was a mutation in both uricase and fructokinase. I could be wrong on that. And I've interviewed Rick, and so we can go back and listen to that. But you had to develop this mutation or else you wouldn't survive the winter. And then what happened was tens of thousands, if not longer, of years of strengthening, that is what allowed those primates to then come back to Africa to basically become our descendants. So we as humans, as a Species have these mutations that would have served us well when glucose and fructose combined were provided just at the right level.

Yeah. So if you didn't anticipate that you needed to gain weight, your body would tell you to do it. Basically how it came about and so makes perfect sense. The extreme example is the hibernating bear. We call it the honey bear often. Right. Because in the fall, not only do they eat every berry they can find in the woods, but they also climb up the trees and get to the honey from the bees and they put on 150, maybe even £200 within a period of two or three months. And then they go into extreme insulin resistance and they hibernate, they fall asleep. But because of being insulin resistant and having metabolic syndrome, their ability to break down fats is impaired. And that keeps them from quickly burning up.

All their metabolism slows greatly. They preserve the little bit of glucose they have for their brain, and they feed their body off ketones and fats.

I assume for a long period of time, and then they survive. So we don't hibernate, but we probably became insulin resistant every fall 100,000 years ago in order to survive these periods of starvation.

I mean, you've touched on this briefly from a public health perspective. I know you're not a policymaker, you're a scientist. Many would say it's impossible. Like we're just never going to see the day when sugary beverages go away. But the data are becoming harder and harder to ignore that there is something uniquely toxic, chronically toxic. People don't like the word toxic because they think it only has an acute implication. Like ethanol can be acutely toxic. But I'm talking about the chronic toxicity. But the chronic toxicity associated with sugar sweetened beverages, inclusive of juices and things like that. Right. It's not just a Coke. These data are becoming almost impossible to ignore, yet they still proliferate. I believe their consumption is down. I don't believe people consume nearly the amount of sugar sweetened beverages that they consumed 20 years ago. I believe we peaked at around the turn of the century, but we're nowhere near what you consumed growing up in West Virginia. Is it possible to get back to that? Is there a solution or is it simply something that each person must be accountable for? Do you have a view on that?

Even how you reverse the policy? I think changing cigarette smoking, there's a lesson to be learned there. What was the incentive? How did the government make this happen? Reducing advertisement is one way to do it. I mean, if you look at TV commercials, almost every commercial about food is about sugary food. And there are many of them tailored to young children to get them to buy the sugary cereals, to get them addicted. To young children, you've got them for life. Just like cigarette smokers, you get them addicted. 15, 14. They're addicted for life. And there's a market for you. So I think that's the appeal we have to make. And the question is, how do you do this? One way taxing also helps. If sugar suddenly becomes like it was in the 50s, the most expensive thing that you bought at the grocery store, then maybe you would quit buying it. But of course, most people don't buy sugar anymore. They buy processed foods that have sugar already added to them.

By the way, looking at your experiments, was there any distinction between if you used sugar as the substrate versus high fructose corn syrup?

No, sugar. Sucrose. By sugar, we typically mean sucrose from cane sugar, which can be crystallized because it's a pure molecule, while high fructose corn syrup is a mixture of fructose and glucose. That's 60, 40 rather than 50, 50. But in our experiments, the difference between those two doesn't make a whole lot.

The fact that you need to. If it's sucrose, you have to hydrolyze the bond is irrelevant to the kinetics that you describe.

That happens very quickly.

So you don't buy the argument that some. I don't buy it either, by the way, but that you should eat your sugar in natural form, which is. It should say cane sugar or beet sugar or something on the ingredient. And there's this whole group of folks who believe that high fructose corn syrup is horrible, but quote, unquote, naturally occurring sugar is not. There's so much confusion around this. But I just want to know if experimentally you see any difference.

No, I think whether sucrose. What shows the difference is whether the sugar is embedded in a fibrous fruit as opposed to being pure in a water.

There's something about that liquid that is devastating.

There are two things. One, for colorectal cancer, it is, as I said before, the fact that having it in a sugary water drink gets it all the way to the colon. If you had the exact same amount of sugar, but it was embedded in a fibrous fruit like an apple, then none of that fructose would make it to the colon. The transit through this intestine would be so slow that the sugar would get absorbed.

But does that increase the Risk of cancer in other organs?

No, I think this is absolutely unique to colorectal cancer. But I would argue, on the other hand, the difference between eating an apple and eating apple juice not only is whether the fructose makes it all the way to the colon, but whether or not your glucose levels spike after consuming an apple, a whole apple, eating it as opposed to apple juice. And the answer is, with apple juice, you're going to get a glucose spike for sure. Eating an apple, you may get hardly any change in your glucose because it takes so long to break it down that the absorbance is slow.

Well, and you might. You know what I've observed? I don't drink apple juice. I don't drink beverages that have sugar in them. But what I notice is nothing has ever spiked my blood sugar more than a raisin. On a per mass basis. You took a fixed mass of raisins and you contrast it with a fixed mass of anything else. It's pretty stark because it doesn't have the water, it doesn't have the fiber, it doesn't have anything else. And even if you ate, say, call it compare apple to apple juice, if you did equal grams of glucose, you might even get a similar area under the curve. But where it's going to look different is the apple juice, which will have, you'll get a big spike, and the apple, you'll get a gradual. You're going to have different insulins in response to those. So the glucose auc could be the same, but the insulin response could be quite different.

That's the point.

Point.

And it's, in my opinion, it's all about insulin. Most endocrinologists worry about the glucose, I worry about the insulin.

So really there's three things you're worried about. You're worried about glucose, fructose and insulin, but in how they coexist.

Right. So colorectal cancer is unique and can feed directly off the fructose if you get it all the way to the.

Colon through, but it has to be in liquid form.

But the difference between liquid form apple and in apple juice is, as you say, the area in the curve is the same, but the fact that the glucose isn't going up as high means you don't get as much insulin release. And of course, if digested very slowly, the rate at which you're ambiently burning glucose in your brain and muscle may be almost equal to the rate at which it gets absorbed in the bloodstream, in which case you hardly see any increase at all. And that means no spike in Insulin whatsoever. So that's the ideal situation, is to never allow that insulin to get high.

Now, before we go into the why, I want to go back to some experiments, some drugs, and something you said earlier. Let's go back to pi3kinase. Everything you said about pi3kinase would make someone listening to this think, if you could block it with a drug, could you block cancer? Well, obviously you've thought of that. So tell us about drugs that block pi3k inhibitors when given to cancer patients.

Okay. So by 1990, I was quite sure that everything insulin did went through pi3kinase. And it was also quite clear to me that many cancers have emerged because of activation of pi3kinase. Some pharmaceutical companies came to me and said, should we develop a PI3 kinase inhibitor? And I said, well, I can't imagine how you would be able to thread that needle of being able to inhibit PI3 kinase without causing diabetes, without causing severe diabetes. And unless insulin went up high enough to offset the inhibition of PI3 kinase, you would have to go off the drug because of a hyperglycemia. If you raise the insulin level, it could override the pi3 kinase. It's also going to activate the tumor to grow more.

So it could be worse.

Yeah. So I was skeptical and never in spite of all of our data saying that pi3kinase was driving a lot of cancers, I never pulled the trigger in starting a company or even encouraging anyone else to start a company to make inhibitors of pi3 kinase. The fact that mutations in pi3k were picked up and Bert Vogelstein's laboratory in colorectal cancer and ultimately shown to be in many types of cancers, led the charge to actually develop an inhibitor. Because by then it was clear that other mutant tyrosine kinases, when they were hyper mutated or hyper produced in cells, could be drugged, and those would be effective ways to block cancer growth. So with those observations, numerous companies, probably 15 companies, went after projects developing PI3 kinase inhibitors. I was still skeptical that you would be able to manage the insulin problem, but agreed. In fact, applied for and got funding from stand up to cancer American association of Cancer Research to put a team together to try to figure out how to most effectively use those inhibitors as they went into the clinic. And I remember after we received that money, several pharmaceutical companies contacted me and said, will you work with us? We have a PI3 kinase inhibitor in human phase one trials. And they said, what's more, it doesn't cause hyperglycemia, so it's going to be a safe drug. And I said, well, if it doesn't.

How does that work?

If it doesn't cause hyperglycemia, it's not hitting pi3 kinase. So, no, I'm not interested in working with a drug that doesn't cause hyperglycemia. Ultimately, we decided to work primarily with Novartis that had a drug that we. Several drugs actually, that clearly called hyperglycemia. So I was convinced they were hitting pi3 kinase and that that was an on target effect. And the question then is, can you thread that needle? Can you manage the glucose level without getting insulin levels so high that they would reactivate pi3 kinase in the tumor? And that had been the challenge. So we suspected, I should say that in working with Novartis, we got them to agree that when the patients got hyperglycemia that the endocrinologist would judge was not tolerable, that they would ensure that if the patient could not be managed on metformin that they would have to go off to trial. In other words, I didn't want them.

You didn't want them being given insulin?

I didn't want them to be given insulin or an insulin secretogue which also raises serum insulin. And I should say that the endocrinologists who were called in and consult for these patients who were hyperglycemic on the trial disagreed with me. They said insulin is totally safe, they should be able to get insulin and we should do it that way. But I was quite adamant. I said, well, I won't work with you on these trials unless you exclude patients who have to be managed on insulin or insulin secretogues. So Novartis kind of reluctantly agreed to do that and went all the way through their approval trial with that as their requirement. Anyone who couldn't be managed, who would need an insulin or insulin secretagogue, would be off the trial. And their drug got approved just in May this year. It took a long time because roughly half the patients who tried to enroll could not be managed on metformin.

Could you also use SGLT2 inhibitors?

So at that time, the SGL2 inhibitors had not yet been approved. So we're talking about 10 years ago when the phase 1b trials that were the lead in to the approval trial where we worked with Novartis on that, but Novartis stuck with the initial requirement through their approval trial that everything had to be managed on metformin, otherwise they had go off the trial. And that's why it took them a long time to enroll enough patients to complete the trial. Now, the SGL2 inhibitors are approved sodium glucose co transporter inhibitors that reabsorb glucose from the ultrafiltrate in the kidney. If you block that, then the glucose ends up in the urine. And so that's a good way to lower glucose and thereby lower insulin. So that tool wasn't available at that time. It's now available today. So we did a study over the last four years. It took a long time to do this. Lots and lots of mice and lots of experiments to test whether in a mouse model for cancers, a variety of cancers, 12 different types of cancers. If you give a PI3 kinase inhibitor and use either a sodium glucose co transporter inhibitor or put the patients on a ketogenic diet, which means that they only have about 8% of their total diet as carbohydrate, and even that is slow release carbohydrate. The rest, 80% is fat and 12% is protein. And we ask now, comparing metformin versus insulin versus sodium glucose CO transporter inhibitor versus a ketogenic diet, what works best in getting the PI3 kinase inhibitors to shrink the tumor?

And I want to just pause for a moment. This is something you alluded to in passing 30 minutes ago, which is very important, which was insulin can go around the PI3K inhibitor when you block it, and that's why just blocking pi3k alone wasn't going to be enough. Is that correct?

More accurately, we know that if you take a tumor that has PI3 kinase mutations, for example, ex vivo. So we did a lot of this work in organoids from our human patients at Weill Cornell, and we would take an organoid endometrial tumor, for example, and give it a PI3 kinase inhibitor at the therapeutic dose, and we could kill every cell in that organoid. But if we now added back the level of insulin that would be in.

The bloodstream at the level of glucose that they would have experienced, and the.

Level of insulin that we actually measure in the blood of the patient 30 minutes after giving them the inhibitor, how.

High is an insulin level typically in that patient?

So 10 nanograms per per mil is roughly what we see within 15 to 30 minutes. And by 90 minutes, it's up to twice that, 10 to 20 to 30 nanograms per milliliter of insulin. And by two hours, the insulin level is high enough that the glucose now in the bloodstream starts to drop. So that tells us that that massive amount of insulin is enough to reactivate pi3 kinase in liver and muscle. And so it's not going around pi3kinase. It's just reactivating it in spite of the presence of the drug.

Because you can't develop a complete inhibitor for physiologic reasons.

That's right. At the dose that you can tolerate.

Yeah, yeah.

Fighting this battle.

I see, I see.

If you completely turn off pi3 so.

The insulin still has to go through pi3k no matter what. You're putting up a porous dam. Because a complete dam would kill the patient.

Exactly.

And insulin is tougher, and in the end, it's going to win.

So let's suppose you're giving an estrogen receptor antagonist and you start getting toxic effects of losing estrogen. Would you decide that you would now give estrogen.

Yeah.

To correct that problem? And so that's what the endocrinologists say. We want to give them insulin to bring their glucose down.

You're like, that's the whole thing we're trying to stop.

That's what we're trying to stop is insulin, because insulin's what's driving the activation. Those mutations in PI3 kinase will not activate PI3 kinase unless insulin is added to the tumor.

And I recall, I think I even told this story, I may have told this story when I was talking with Sid on the podcast, but we were having dinner one night and I talked about a friend of mine with breast cancer who was in a trial and she was in an arm in one of the Boston hospitals, and she was on a pi3k inhibitor, and I think it was a phase two. She was the only woman who survived. She's still alive today. The only woman with metastatic breast cancer that survived, who on her own went on a ketogenic diet. And I remember telling you this out of curiosity, like, Lou, what do you think of this? Do you think this is just an odd coincidence? And that was the time that you and Sid were starting these experiments, as Sid described it, that was sort of maybe the final clinical, the little pearl that sort of made you guys go off and do this. And that paper was published a year ago, right? That was published was last summer. Last July.

Last July, yep.

So what was the efficacy of a ketogenic diet versus an SGLT2 inhibitor and metformin? Were they equal?

So if you did a run in to the ketogenic diet, so they have an entire week on the diet and then they're given the inhibitor, it's incredibly effective because by then you've depleted all the glycogen, all the relevant tissues. If you just start on a ketogenic diet a day before you give the first PI3 kinase inhibitor. It's still pretty effective, but not as effective as the whole week run in to deplete glycogen.

Is there any reason not to combine these ketogenic diet? Metformin, SGLT2 inhibitor, PI3K inhibitor as a FU to cancer for cancer's dependent on this pathway, of course.

Well, let me address the sodium glucose co transporter inhibitor first because that is almost as effective as a ketogenic diet. In our mouse models, it keeps the glucose level not quite as low as a ketogenic diet, but pretty damn low. And the importance of course, is in both cases it brings the insulin. Ambient insulin in the serum drops dramatically. But the important thing is that every single 12 different types of cancers, some of them had PI3 kinase mutations, others didn't. Some were RAS mutant tumors, didn't matter. Some were. One was AML and Sid's study.

That's right. I remember talking about that that night.

And I was skeptical that AML would have because you never see a pi3 kinase mutation in AML. But the bottom line was there was a benefit in every single cancer. We looked at the reason we did 12 different types. We were trying to find one example.

Of a cancer that would violate this principle.

That would violate this principle. The bottom line is I think all cancers require some amount of pi3 kinase to survive. And so if we can keep the insulin level low, and insulin, of course, is the best way to activate Pi3 Kinase. If you can keep the insulin level low and now turn off pi3kinase, you don't get that rebound of insulin and the tumors just go away. So ketogenic diet, if you can stay on it would be my recommendation. If you have cancer and you're going on a PI3K inhibitor, sodium glucose CO transporter inhibitor, however, is easier for most people to take. It's a pill. And that's, I would say, the second best.

And of course, again, it's hard because we're sitting here, we can't give medical advice to people. I get asked this question all the time. It's a very difficult question to answer because I'm not managing the given patient. But cancer patients are among the most motivated patients you'll ever meet. I mean, you know this, you've been involved in these trials. They will do anything they believe has some modicum of science behind it that can increase their odds of survival. Tragically, we're still kind of living in a world where the mainstream approach to this is a little bit convoluted. Obviously, one of the most significant concerns that many oncologists have, well meaning is that patients are going to lose weight. I don't know how bad it is today, but certainly back when I was on the wards, we would force feed cancer patients with ensure. I don't know if you've ever had ensure, Lou, but let me ensure you it is full of sugar. So now we're back to force feeding patients liquid sugar when they have cancer because we're afraid of them losing weight.

Exactly. So one caveat or one concern which came out of SIDS part of our trial is, is that at least in the mouse models of aml, ketogenic diet.

Alone, it was actually negative, wasn't it?

Ketogenic diet alone in most of the tumors doesn't have too much. So just switching to a ketogenic diet, I think it's rare that that alone is going to work. Maybe in some cases, but in the case of the aml, there was some evidence of some toxicity that came in the context. Not normal mice, ketogenic diet. They're probably perfectly healthy. They live a normal lifespan, in fact, probably live a little longer. But in the context of this AML model that Sid's lab was using, there could be some adverse effects. So we're cautioning people don't automatically go on a ketogenic diet if you have cancer unless you have somebody paying attention to potential side effects.

Well, I had the luxury of sending, I mean, you've been so generous and I've sent many tissue specimens to you from patients or friends, relatives of patients, and you've got your own CLIA developed assay to look for insulin receptors. And we've even made clinical decisions based on that, which is this is a patient who has lots of insulin receptor on their tumor. This is a favorable approach to cancer. This is a patient who doesn't might not make that sense. How far is that type of an assay away from being a mainstream assay that any physician can use as part of their toolkit to start to customize.

Cancer therapy, to reveal my conflicts here. I do have a company I've started Petra Pharmaceuticals that is beginning a PI3 kinase inhibitor trial. It will be combined with sodium glucose CO transporter inhibitor.

Oh, I didn't even realize I'm bringing up all this stuff. I'm teeing it up for you here. Right?

Yeah. That trial will be starting any day now.

What type of cancers, Lou, are you looking for? Enrollment.

So in phase one here, it's going to be a combination with sodium glucose cotranporter. Inhibitors inhibitor/PI3 kinase alpha inhibitor. Initially we'll be looking at solid tumors, breast endometrial, where you see high rates of mutations, but we won't.

So breast endometrial, in what stage of disease is this? Stage four.

Yes, in stage four disease.

So have you fully enrolled or.

No, no, we're just. The trial just hasn't enrolled.

So in other words, if someone's listening to this that either has or knows someone with breast or endometrial cancer who is stage four, how can they best find out information on this trial?

So, Petra Pharmaceuticals. So of course this will be registered@clinicaltrials.gov where you can find every single trial. So if you just go to ClinicalTrials.gov and type in PETRA P E T R A that will.

We'll link to that for sure. How many patients will you take in the phase one?

I've forgotten the exact number, but it depends on how many responses we see and so forth.

But the dose escalation, the dose escalation.

Is going to be pretty quick because we already have. We know what the dose is without a key.

Each of these drugs, you sort of know what they do.

We know we don't anticipate any toxins.

So the hope is to get to phase two quickly.

Yeah, we should get to phase two very quickly. And the patients on phase one could transition into the phase two.

This is really exciting, Lou.

So in that case, we will recommend that they have for breakfast because all these drugs, most of these drugs require you eat breakfast before you take the pill because that limits some of the toxicities you get from and increases the absorbance. But I think it makes a hell of a lot of difference what you have for breakfast.

So you'll recommend scrambled eggs and avocado.

And whole milk yogurt and not yogurt.

With sugar or a high fat yogurt.

And high fat yogurt with some crunched.

Almonds in it or something like that.

Yeah, right.

Now, Lou, is there any way to pair.

Absolutely no juice, no ensure in that time of the morning. Now, by the evening, the drug will have already be below effective dose. So one might have some elasticity for what you had for your evening meal.

Although I will tell you something, Lou, you probably see this thing on my arm, right? That's a glucose meter. I know what a nighttime high glucose meal. I know what a dessert at dinner can do to my glucose in the morning and my insulin and it's still high. So for people listening to this who want to enroll in this trial, or even for you, Lou, I would just say the more stringent people can be based on what you're saying, if the hypothesis that's being tested is more insulin is worse, I think you're almost better off just biting the bullet and saying we're going to do ketogenic diets. In fact, is there any probability in this trial of actually providing the meals to the subjects to make it easier?

You know, so I have another conflict here. I've also started a company that is making meals for dietary intervention in cancers in general. And this company also, Sid Mukherjee is a co founder of this company as well, called Fayess Pharmaceuticals.

How do you spell that?

Fayess? F A E T H, which is a Welsh name for health. F A E T H. So this my co founders are Karen Valsden and Oliver Maddox from her laboratory, Ben Hopkins and Marcus Goncalves from my laboratory.

These are a lot of the folks that were on that paper last summer.

Right.

Ben was the first author. Right?

Right. Sid Mukherjee, also founder of this, also Scott Lowe and Greg Hannon. I think I got everybody.

And the idea here is for patients who have cancers which the KD will be potentially a viable adjunct to what they're doing. The idea is let's pharmaceuticalize it, basically. So is Faith already selling product?

No, it's just now getting started. We've gotten it funded and we're looking now for where we're going to be making the meals and how and whether we contract some of the meals out or we do them internally. This isn't going to be purely ketogenic, although we will focus on preparing the meals, make sure they have exactly the composition that we expect, rather than just giving advice and hoping people find the right foods. But it's not purely ketogenic in some cases. There's evidence coming out of Karen's lab and also my lab that reducing serine levels can make some drugs more effective. Producing methionine levels can make other drugs more effective in different mutational backgrounds.

Say more about those two. I mean, those methionine and leucine might be two of the more potent activators of the target of rapamycin. Tor, tell me about serine. That one's a bit. We don't hear about that as much.

So serine and some tumors rely on synthesizing, can either use serine in the serum or they can synthesize their own serine. So the enzyme phosphoglycerate dehydrogenase PhDH is the first step in converting intermediates of glycolysis into serine and glycine synthesis. You need serine and glycine in order to make nucleotides as well as glutathione. So for redox potential and for combating Ross rather, and also for building nucleotides, you need serine and glycine.

So you're trying to selectively deprive a cancer cell from nucleotide precursor.

That's right.

How difficult is it to restrict methionine and serine in food?

So this has been done both in humans and in mice. Diets have been tried and you can get this serine level down about tenfold and still have quite viable human.

And what about methionine?

Similar. You can also.

Without just restricting protein.

Right. We've been using diets for metabolic disease intervention for years. Children lack an enzyme in particular.

Sure. Ketogenic diets for epilepsy and things like.

That, but even ketoneuria.

Pku.

Yeah, pku. You need to reduce the fennel phenylalanine in the diet. So this idea that you could get better clinical outcomes by changing amino acids, for example, in their diet has been proven clinically in born areas of metabolism. So the idea that the same kinds of interventions, in fact ketogenic diets, as you say, were developed not for cancer, but for epileptic seizure syndromes.

Yeah. Have you seen the data that have suggested this is a little changing gears. I don't want to let you leave without at least some discussion of this, that in certain cancers, when you restrict glucose, which would then imply potentially a ketogenic diet, you actually make the cancers more robust. And in particular in pancreatic cancer. Have you seen some of these data?

Actually, I've seen the opposite in pancreatic cancer. If you use a ketogenic diet in combination with a drug. So in our hands, putting a mouse that has a KRAS P53 mutant, pancreatic cancer, which is majority of human cancers, have those mutational events. So in that context, ketogenic diet doesn't really do anything. It doesn't accelerate, doesn't slow it down. But if we combine that with a PI3 kinase and PI3 kinase inhibitor doesn't do anything to those tumors either. But we give the two together, the tumors completely disappear. So you have to combine the diet with the right drug or it doesn't really work.

And it's really this diet drug pairing that is really on the cusp of innovation right now. We're scratching the surface of that. Right. And will these two companies faith and what Was the other one Petra? Petra, is the goal that these companies collaborate and start to pair diet with drug?

Yes, we could certainly do that at this point. As I say, for Petra, we focused on on the sodium glucose co transporter inhibitor because we think compliance will be easier to monitor. And at this stage, we aren't yet revved up to produce the diets that would allow those trials to continue. But yes, we could move on to a ketogenic diet and even possibly on top of a sodium glucose cotransporter inhibitor. But we'd have to do a phase one study.

Yeah, you still have to go back to phase one.

Phase 1B.

How many PI3K inhibitors are currently approved by the FDA?

So there's the alpalisib Novartis drug just approved in May. That hits specifically only the alpha isoform. Now, that's the isoform that insulin activates that mediates everything insulin does. And that's the one that's caused all the problems with hyperglycemia. But there is also a Delta isoform of pi3 kinase that's seldom mutated, but it's actually required for B cell growth. And we discovered that back when we started knocking out pi3kinase in hematopoietic lineage cells in mice back in the late 1990s. David Fruman, a postdoc in my laboratory, was doing those mouse knockouts and found that B cell lineage cells failed to thrive if you knocked out pi3 kinase. T cells, on the other hand, were fine if you knocked out pi3kinase. And ultimately it turned out to be the delta isoform that was critical for the growth of B cells. So when delta inhibitors were developed, they went into B cell lymphomas as the first possible place to work, since that enzyme was already known to be critical in B cells, and they turned out to be effective. Adelalisib was approved about five years ago. It's a delta specific PI3 kinase inhibitor.

Who made that?

That was made by Gilead.

Oh, Gilead. Okay.

Yeah. And then more recently, a drug called Kapan lisab was approved about three years ago by Bayer. This is a drug developed by Bayer.

And which isoform is that again?

It also hits delta, but it also hits alpha.

So the alpha one is the only one that causes the hyperglycemia. Yeah, that's the one that my friend was on, by the way. Right, but that was approved in May. And that seems to have the most ubiquitous application, doesn't it?

It's certainly where you see the most mutations. And of course, breast cancer is far More numerous than lymphoma.

Yeah, but that's the one that's going to need the most management of the hyperglycemia and the hyperinsulinemia. Is that message reaching the oncologists?

Slowly. I think they're beginning to understand what we're saying, why it's important to keep the insulin down. The problem, as you know better than I, that we're so specialized today in medicine that people commit to go into either oncology or endocrinology. And from there on, their studies are completely. They're studying completely different things. And if you have an endocrine problem as an oncologist, you don't try to solve it yourself. You call in an endocrine consult. So your brain isn't really even focusing on how to solve a problem because you have an expert who can do it for you. And the problem is the endocrinologists are not really that alert about what's going on with cancer. Their job is to make sure the glucose is in line.

Yeah, sort of. I think it's time that we either. I mean, there's really two ways around this. I see. One is you sort of create a new specialty, which is metabolic oncology. So just today, if someone wants to study oncology, they choose between surgical oncology, medical oncology, radiation oncology. Of course, the challenge is you have to differentiate very early. You don't do oncology first, then surgery, then medicine, then radiology. It's you do radiology or medicine or surgery and then subspecialize. So therein lies a problem. How would you become a metabolic oncologist? So the other option is basically oncologists learning more about the metabolism of cancer, so that when it comes to managing understood and predictable complex applications of these medications, which are actually desired features, as you point out, if you're using this drug correctly, you should see hyperinsulinemia and hyperglycemia, and you should treat it accordingly. Which reminds me, by the way, how often with the alpha isoform does that drug need to be administered? Or in your trial decision of how.

Frequently to dose is based on pkpd fast the drug level goes into the bloodstream and how long it stays high and following how quickly it declines. And so most of these drugs that make it in the clinic can stay at their therapeutic dose for three or four hours. So a lot of the companies want to do buy two drugs a day, one in the morning, one in the evening.

And what's the total cycle? How long are you being given drug? How long are you given A rest from the drug.

And so, for example, the Novartis drug is daily. It's once a day, daily after breakfast, on a full meal.

And why does it need a full stomach?

Because otherwise, absorption problems. You don't get as good a PKPD if you eat it on an empty stomach. And you get more side effects, diarrhea or nausea or whatever if you try to do it on an empty stomach. But there's no recommendation in the approval trial as to what you're supposed to eat for breakfast before you take the drug or whether you take it with inshore or apple juice or water.

Hence the importance of a company like Faith that could start to standardize that. Because, look, everybody wants to do the right thing here. And wouldn't it be great if, as a doctor, you could write a prescription that's not just covering the drug but covering the food?

Yeah, so we have. That's what we're anticipating at Faith. At some point, if you can prove in a clinical trial setting that you need this particular food for the drug.

To be attacked, to maximize absorption, minimize symptom side effects, and most importantly, maximize.

Efficacy, you should be able to get that food paid for. The delivery of the exact food that matches what your need is.

And think about it, what a trivial cost it is compared to the drug. I mean, you could send people caviar spread all day long at 1/100 the cost of a drug.

Exactly. But you have to prove it. You've got to prove it in a phase three.

Well, don't get me started on that. I mean, think of how many drugs have been approved that haven't proved Jack. I mean, it's Ambien. Should have been approved anyway. Don't get me started on the drug approval process process here, but I'm completely with you on that, Lou. I want to be kind of mindful of your time. There's kind of one last theme I want to explore. So you're going to be 70 this year. Right. Which is kind of hard to believe because any.

I already passed it.

You already passed it. So you don't look a day over, I don't know, 56. You've got this ageless phenotype to you. So I don't want to for a moment suggest your career is anything other than continuing to go up in terms of the work work you have to do. So as you think about the next chapter of your career, what is the question that you are most obsessed with? What is the question you want to spend as much time as you can Trying to answer that currently we really.

Don'T have an answer to that's hard for me because I'm the most unfocused in some ways I'm compulsive about getting the answer of every question that I get curious about. And as you can tell from this interview, I have an opinion on how everything works. And having never taken any biology courses, I have no prejudice about how it should work. I just. So for me, everything that I run into biology is here's a mystery. I don't bother reading the textbooks because I know they will prejudice me to the wrong answer. And so I'd like to get to the bottom of what's really going on. But the bottom line is that I get absolutely brilliant. Postdocs and graduate students come to my laboratory. I allow them to do anything they want to do and if it obviously give them advice and help interpret the experiments. But this has kept me in so many different fields for 40 some years as I've run my own laboratory. It keeps my mind continually alert about every possible thing. So yes, epileptic seizures. Turns out probably most of the people have epileptic Seizure fits have PIK3CA mutations as a mosaicism in neurons in the brain. Ketogenic diet was developed to minimize that. It makes perfect sense keep insulin down. If a PIK CA mutant neuronal cell you're going to get less firing of that neuron. That's going to solve the problem. But that wasn't how the ketogenic diet was developed. So my curiosity if just studying everything that's which pi3 kinase is involved at some level could keep me busy for several years. But that's not the only thing I'm interested in. I'm interested in whatever curious observation that comes up from experiments in my lab that can't be easily explained by current knowledge. To me that means there's an opportunity to break open some new explanation for some disease. So I'm willing to work in almost any disease that's not yet solved and there's an opportunity to figure it out. I'm a chemist, so to me, unless I understand something at the molecular level, I don't really understand it. But I think with pi3kinase I really understand at the molecular level.

I think that's an understatement.

Very few, there's still some mysteries there. I should say that we're about to publish a couple of papers hopefully over the next six to nine months that are going to completely change how Everybody thinks about pi3 kinase regulation. So there's still some major things to learn about this enzyme.

Well, there's so many other things I'd love to speak with you about, Lou, but I know that some of them are still work that you're doing, and therefore, I know you would prefer to wait until things are a little further along to talk about them. But we'll have to come back in a year or so and do this again because there's so much other stuff that I know we've talked about informally about cancer. That is, I want to believe it's where the field is going to start to go. I want to believe that because I get so many emails from people who say, you know, my mother has cancer or my husband has cancer. And they're being offered this therapy, and it's a very standard therapy. The patient is progressing through it. What metabolic options are there? And people like you are. People like Sid, people like all your colleagues in Matt and. And Ben and all these guys. I mean, you guys are the guys that are kind of driving that forward. And you've made a lot of progress in a decade. It doesn't feel like it sometimes. Sometimes I feel like it's not going fast enough. But I think having a discussion like we did today makes me realize how much has happened in a decade, and it's actually quite a bit. I have no doubt that you're going to be here a decade from now working just as hard.

Well, my real motivation, of course, is to convert every observation we make in the laboratory to actually change in practice, for whether it's diabetes or cancer or what other disease that we begin to understand, the molecular mechanism. If we don't convert those observations into actual change in behavior or change in practice or change in drug metabolism and diet, then in the end we failed. And so that's my goal. And as being a cancer center director, people sometimes listen to me now when I suggest this is the way, this is the trial we should do, and this is the way it should be designed to test the idea. For me, it's spectacular being here in New York City at Weill Cornell, New York Presbyterian, where we really can translate these breakthroughs into new therapies. That's what my goal will be over the next ten.

I hope you enjoyed that discussion with Lou Cantley, and I hope you found it half as interesting. As you can probably tell, I did. As you may recall, at the outset of this podcast, I said that the first 45 minutes, which was a much more detailed technical description of Lou's initial work in the 70s and 80s that led to the discovery of PI3 kinase. That content would be bumped as almost an appendix to the end of the podcast. And that's what you're about to hear now. So I hope you enjoy what was originally the first part of my discussion, but ultimately the appendix to this fun discussion with Luke Hanley. There are a lot of things I want to talk about today, but I do want to start with the discovery that you led that probably most people would associate with you. If they go to your Wikipedia page, that's probably, probably the first thing they're going to see. Although I haven't been to your Wikipedia page in a while, so I don't recall. But let's talk about pi3kinase. How do we define it, by the way? Tell people what.

So pi3kinase stands for phospho inositide 3kinase. And the 3 means that it phosphorylates the 3 position on the inositol ring, which is the head group of this lipid. And so in the middle early mid-1980s, phosphatidylinositol phosphorylation, which was known to occur from 1949, the purpose of it was totally ambiguous. Why did this lipid get phosphorylated? And in early 1982, 83, there was a breakthrough with the discovery that the phosphorylated form phosphatidyl inositol with phosphate at the four and the five positions of the inositol ring. By the way, inositol is hexahydroxycyclohexane. So there's six hydroxies. One of them connects to the glycerol backbone, and then the others are potentially available to be phosphorylated. Anyway, the 4 position and the 4 plus 5 position were both identified the year I was born, 1949. And the purpose of that was not known. It was called the futile cycle. Maybe a way of just getting rid.

Of getting rid of phosphates or something.

Or getting rid of ATP. Yeah, yeah, Just phosphorylate it and dephosphorylate.

It, sort of like an ATP sync.

And then the idea, the observation that this phosphorylation could be stimulated by various GPCR pathways and growth factor pathways got people thinking about what it might be doing. And it tended to correlate with calcium elevation. And so the breakthrough paper showed that in fact, when cells are stimulated with certain growth factors like EGF or certain subset of GPCR activators, and those stand.

For G protein coupled receptors and EGF Just for folks.

Epidermal growth factor.

Okay, so all growth.

So these signaling pathways would activate the hydrolysis of that lipid that the inositol 1,4 5 trisphosphate that comes off when you hydrolyze it away from the lipid turned out to regulate calcium release from cells. And that was a huge breakthrough because no one knew how calcium got elevated, and that explained it. So I. In the meantime, it was not working on that particular pathway.

And let's pause for a moment and remind everybody you trained, you were interested in science from a young age, but you were sort of deciding, I think, a little bit, if I recall, between chemistry and biology. You were very interested in both, correct?

I was a pure chemist. The last biology course I took was in 1964, when I was a sophomore in high school.

So you very quickly declared chemistry your obsession.

I hated biology because at that time, at least in the backwoods of West Virginia where I was growing up, biology was just a bunch of descriptive memorization. And so I was bored with that and decided I would never take another biology course. And I stuck to that. I was pure chemistry, mainly organic chemistry, and then I switched into physical chemistry, but was totally uninterested in biology at all until I took one semester of biochemistry and realized maybe there's something interesting going on in biology. So I went to Cornell in Ithaca to get my PhD, and there I focused on biophysical chemistry. It was the chance to apply chemistry to questions like how do you get molecules across membranes? And that's what I worked on. How do you synthesize ATP in the mitochondria or chloroplasts? So that kind of the slippery slope of going into biological questions that could be addressed by simple chemical questions or physics questions.

Now, what got you interested in the mitochondria so early?

I read a paper when I was an undergraduate that was an idea that Peter Mitchell had proposed that the way you make ATP in the mitochondria and or chloroplasts was using a proton gradient. And his idea was that that gradient would allow you to pull the protons in one direction, the hydroxy ions in the other direction, and remove water from phosphate plus ADP and condense that into ATP. That was just a very simple physical property that if you could move those two ions in different directions, it would produce ATP. In effect, the idea was not correct. It was correct that ATP was made from the proton gradient, but the actual mechanism turned out to be different. There are actually three Nobel Prizes given for how ATP is synthesized in the mitochondria.

I wasn't aware that there were three separate Nobel Prizes. Do you recall who won all of them?

So Mitchell. Mitchell, obviously Peter Mitchell got one. Then there was a second one by Boyer on figuring out the actual mechanism by which this occurs. And then a third one from Peter Walker, who figured out structural basis for how this all worked.

Oh, I didn't know the Walker one. Okay.

He was a crystal structure, did crystal structures. And I met all of them when I was a young man because before becoming a professor. And so I was very invested in that. That's what I worked on.

For the person who's listening to this, whose world is not necessarily wrapped up in this, which is most people, it's worth pausing for a moment on how much is involved. For the Nobel committee to recognize one body of work that says something that three times they would recognize. A different angle of the same body of work probably speaks to why there is so much, to this day, complete interest and fascination with the mitochondria beyond just the obvious. The energetics, which is what was initially how we would think about it.

Well, it was a fascinating question. In the 1960s, this was the biggest mystery because we knew how you made ATP from glycolysis, but how you made it into mitochondria, and how could the mitochondria do it so much more efficiently than glycolysis? That was the big question when I was a graduate student.

I think this podcast may represent the point at which mitochondrial topics exceed all others. It also speaks to that nature. So Mitchell the Great. Tell me about how you interacted with Mitchell. How did you come across him?

So he came to give a talk at Cornell, and I should say F. Racker, Ephraim Racker, who was chaired at the biochemistry department at Cornell and who I got to know as first year graduate student because I was getting mitochondria from his laboratory in order to purify the enzyme that synthesized ATP. Every time I would be waiting for the centrifuges to stop running, if he saw me, he would grab me and pull me into his office and start throwing out ideas of how he thought ATP was synthesized. And the truth was, at that time, he did not believe Mitchell's chemiosmotic hypothesis. Peter Mitchell was proposing. And so as a consequence, I got all of these ideas that he was throwing at me. I personally believed that Kimi Osmotypoth was correct. Between those discussions, as I continued to. I was working in physical chemistry. I was in the chemistry department, not the biochemistry department, but the Fact that he would spend so much time with me, even though I wasn't even in his department, to me was quite flattering and really exciting that this world quality person had done it, was willing to talk to me. So he went on because he was trying to prove Peter Mitchell wrong. He completely purified every component, generated a synthetic membrane, reconstituted the proton pump into that membrane, generated a proton gradient and was able to quantitatively synthesize ATP. And it was the year after, after he published that paper saying that the chemiosmotic theory is actually correct. But he did the definitive experiment that the prize went to Peter Mitchell. Today I almost guarantee they would have shared the prize. Mitchell had the idea, Racker proved that it was correct even when he was trying to prove that it was incorrect. So that was an interesting story that Mitchell was invited to come and give a talk at Cornell just at that time. But my last year as a graduate student and so he met with me and my advisor Gordon Hammis and I went through my entire PhD thesis with him and F. Racker, showing them the various experiments I did about the mechanism by which this enzyme worked. It was really quite interesting because this was before Rackert actually published the definitive proof paper. So he had still some questions and every slide I would show, Mitchell would say that's consistent with the chemoesmotic theory. And Racker would say no, but this is why it's not right. And so I hardly got to say a word because the two of them argued every piece of data I showed.

But you provided the substrate for the argument?

Yeah, it's the data from my PhD thesis that was fun experience.

So this is early 70s.

It was 1974 when I met Peter Mitchell at Vinnievent. The next year I decided finishing my PhD that I would look for a place that was asking questions that were a little more biological. I didn't want to dive too deeply into biology. So I was looking for someone who was working on like red cells or something that was pretty simple study. And ended up going to Guido Guidadi's laboratory who focused on membrane biochemistry. He was an MD, PhD, but he understood medicine quite well. But his real focus was in biochemistry, was not a practicing clinician. And he was in the Department of Biochemistry and Molecular Biology at Harvard, recruited there by Jim Watson. He was absolutely is still a brilliant biochemist. I learned a lot from him. And he was working on Sodium Potassium ATPase. It had first been purified in his laboratory, the enzyme that pumps sodium out of cells and potassium into cells. And since I'd already worked on how you pump protons into cells with an ATP molecule. I thought this would be easy for me to understand. I knew how to do those kinds of assays, so that's why I started working on it. But it turns out that Guido was also very interested in how insulin worked, because whenever you add insulin to cells, within seconds to minutes, they take up a massive amount of glucose, but they also turn up the sodium, potassium, atps. The sodium pump use that sodium gradient to move amino acids into cells. And so there was a lot of membrane transport being regulated by insulin.

Was it understood at that time how anabolic insulin was? What you've described are anabolic attributes, but was it understood clinically how anabolic the hormone was?

Yes, it was, because, just by observation, that type 1 diabetics failed to thrive. They were very small, they did not put on weight, their cells didn't grow. And so during development, insulin is an anabolic molecule. In a mature adult, it no longer, except under certain situations, drives growth, it will continue to drive growth of fat cells. It can also facilitate growth of muscle. But most tissues do not grow further, other than those two major tissues. So, yes, it was well known, it was an anabolic process.

But now you were seeing the steps by which it could carry out that anabolic property.

Yeah, and it was generally known. What I just said was generally known that amino acids uptake accelerated immediately. Within a minute or so, sodium export of the cell went up, and glucose flux, of course, went in, particularly in muscle and fat cells, and glycogen was stored. And then, of course, there was a whole lot of change in transcriptional regulation of all kinds of things. So we knew all of that, but the actual mechanism was totally unknown. In the mid-1970s, no one had even purified the insulin receptor, had no idea what the receptor was. People could quantify it because if you added radioactive insulin to fat cells, it would stick to the surface and with very high affinity, and that correlated with the responses that you see. So we knew it existed. We knew roughly how many copies there were per cell. We could quantify by radioactivity, but no one had quite purified the enzyme to homogeneity, and so numerous labs were trying to purify it. So eventually the enzyme receptor was purified. And it was about the same time that src, the src tyrosine kinase, the SRC oncogene for Rous sarcoma virus, was shown by Harold Varmus lab to be protein that had homology to pka, a protein kinase, and that Also was an endogenous protein that had been picked up by the virus and altered. So that really was a major breakthrough. It got Nobel Prize for Harold Varmus.

He shared that one with Michael, right?

Michael.

Yeah. Michael Bishop.

Michael Bishop.

89. They shared it.

79, I think.

Oh, that. They won the prize in 89. Maybe. Maybe I'm.

Oh, yeah, the prize was later.

The prize was award at the end.

Yeah. The opposite Discovery was 78. 78, yeah. Yeah. So that was about the time the insulin receptor was being purified as well. And then Ray Erickson's labs showed that SRC had a kinase activity. They thought it was a threonine kinase. They looked for that possibility. Joan Brugge was his lab at that time, given the sequence homology to protein kinase A, that it's probably also a protein kinase. And then Tony Hunter showed that actually by thin layer, the molecule that was thought to be phosphotynine was actually phosphotyrosine. And phosphotyrosine had never been seen as the product of a protein kinase before. It had been picked up as an intermediate in DNA unwinding. And so he could get a marker for that from Jim Wang's lab that showed that he could use on the thin layer and showed that what SRC was actually producing was actually phosphotyrosine. So that opened up a whole field. And then comparing the sequence of the insulin receptor to PKA versus src, it was clearly most highly related to src. And so multiple labs at that point showed that, yes, the insulin receptor was like SARC tyrosine kinase, but in this case, a transmembrane protein had an extracellular insulin binding component and then the intracellular kinase activity.

So putting that in English for folks, insulin hits this transmembrane tyrosine kinase. And when the molecule of insulin hits it inside the cell, this kinase pathway kicks up. It moves the GLUT 4 transporter. Well, there's another step in there, but oversimplifying a little bit, that chemical reaction is necessary to translocate this transporter across the cell to bring glucose in. And that's so the leap there. The new insight was exactly how insulin got glucose in the cell chemically and mechanically. Right?

So the initial observation was then insulin binds, and about the same time, EGF receptor was purified and FGF receptor and PDGF receptor. And they all sort of were like insulin receptor. They had an extracellular.

And this was back. I mean, today a young student who's in a lab Doing this now is scratching their head going, what do you mean? Why didn't you just use PCR or something like that? But I mean, you guys were literally, what, crystallizing a protein or how were they actually figuring out what the structure was?

So these proteins were all being purified to homogeneity by just plain fractionation. There was no way to knock out genes and knock them down. So you just had to purify the protein based on its ability to bind insulin. You just run over column after column after column and eventually you've got to a single band on a gel and they said, that's your protein.

And that only gives you the primary structure of the protein. It doesn't tell you how it's folded, does it?

No, that only tells you.

It tells you the molecular weight.

Basically the only thing you get is the molecular weight. Yeah, you see, it's.

Here it is on the band. Yeah.

Insulin receptor is.

It gets cleaved, but it's a heterodimer, isn't it?

It's a single polypeptide that gets clipped during processing into two proteins, but are held together by disulfide bonds. So it's dimer of that composition. So that we knew late 1970s and EGF receptor and all these other receptors are all look a lot like the insulin receptor. And they all had tyrosine kinase activity that was triggered by binding the growth factor insulin. IGF1 receptor, also very similar to insulin receptor. One by one by one, different labs purified them. They all found the same thing. You had the growth factor of the cell and you get the tyrosine kinase activity activated on the inside of the cell. You know, that was a huge breakthrough. But still, if you ask when you triggered the cell with insulin or EGF or pdgf, and you ask what was the major thing phosphorylated? It was the receptor itself. So you just ask, where's the radioactivity going? Phosphate. Radioactive phosphate. It goes mainly to the receptors isolate domain. It's autophosphorylated. So now we are left with this several years of. We have a tyrosine kinase that explains how all these growth factors work, including insulin and how SRC works. But we can't find anything other than autophosphorylation. How does that help? So the breakthrough ultimately then came with the observation that these autophosphorylations or phosphorylation of adapter proteins were recruiting a host of other proteins to the membrane that were actually doing the work that was required to drive the cell Growth. And so that was again early 1980s. These were all being figured out. So I got interested as soon as I saw that there was a different kinase activity, that's tyrosine kinase. I got very excited about it and realized there's an opportunity now, if I can figure out what that tyrosine kinase does, to figure out what might be the downstream signal. So we got one step into the cell. We didn't know how many steps we needed to explain glucose uptake or amino acid uptake, et cetera, et cetera, or glycogen synthesis. So I saw a paper by Ray Erickson's laboratory in which he found an activity associated with SRC that would phosphorylate glycerol. And he published it in jbc. It co purified with the protein through numerous steps of purification. And so in addition to having the ability to phosphorylate tyrosine antibodies that got phosphorylated on tyrosine, on the purified protein, there was also an activity there that phosphorylated glycerol. And I looked at that and thought, glycerol looks like half of an inositol. So inositol is 6 carbons, glycerol is 3 in both cases.

But in the pure glycerol case, it has. Oh on each of the carbons.

Yeah. Three carbons with an oh on it.

Yeah.

Inositol has six carbons with an oh on all. So if you took two.

Yeah, if you took the two rings and shoved them together. Yeah.

Glued them together. You would get inositol. And what the paper showed was the KM. There was a 50% concentration of glycerol you needed for it to be phosphorylated was something like 100 millimolar.

Enormous concentration.

Huge. Like 100 to 1000 fold higher unit ever found in a cell.

Yeah.

So they weren't claiming this was a physiologically relevant.

I see. They were just saying, here's a chemical reaction that can take place.

And they just noticed it because they saw this molecule running on their thin layer that ran faster than proteins when they isolated it turned out to be phosphorylated glycerol. It was sort of out there. We saw this other activity. What might it be? And so I went to Ray Erickson and said, well, that looks a lot like inositol, so why don't we.

Was Ray at Harvard at the time?

Yeah, one floor below me at Harvard. So my graduate student, Malcolm Whitman, who knew how to do kinase assays on small molecules collaborated with Ray Erickson's postdoc to see whether phosphatidylinositol might be a better substrate than glycerol. And he tried it and sure enough, he got this km now of like 5 micromolar.

Wait, wait, wait. How is this even possible? Let me make sure I understand what you just said. You take glycerol and you need a km of 100 millimolar to get it phosphorylated. You simply take the same structure, but now it's basically two of them stuck together in a ring and you get down to 5 micromolar and it's also.

On a membrane component. So the inositol is in a membrane bilayer. So you sonicate the lipid and instead of free molecule floating around.

Oh, so it's not a free inositol, it's to the inositol and the phospholipid or in the lipid.

Yeah.

Tell me from a chemistry standpoint, and I apologize. I know that for some of you listening right now you're thinking, wow, you guys are really in the weeds. I promise we're going to get out of the weeds in a minute. But I also think this is just an interesting example of the specificity of biology too. What is it about that lipid holding that? Is it the position with which it holds the ring in place that enables that phosphorylation?

Well, now that we know the structure of pi3kinase and how it works and its mechanism, it's easy to explain in retrospect. But at that time we didn't know that there was anything there other than src. So it was hard to explain why the SRC tyrosine kinase itself was carrying out this reaction.

What did the editors even say of that? I mean, that's one of those things where people are like, is there a mistake here? Because that's like a five log difference.

Physicists had no problem understanding what I just said. If you confine things to two dimensions, they can come together much more readily than if you do it in three dimensions.

And that would account for the difference.

Yeah, easily. Try closing your eyes, bringing your two fingers together in space, in three dimensional space, and then do it again on the table.

When they're on the table.

Yeah, you'll find, you'll find each other. So combining things to two dimensions is the way nature continually uses membranes and.

The lipid's basically keeping it in a two dimensional plane.

Yep. Yeah. So any event, we're really getting into weeds Here.

Now, just personally, I. I mean, all of that is intuitive to me. What's not intuitive to me is five log difference. I would take a one log difference for what you said. That's what's amazing to me.

I didn't. Well, there are other reasons that will come clear as we move on. So the bottom line was that that SRC preparation, which we assumed was completely pure, wasn't completely pure. It was a certain amount of the SRC harvested out of the cell brought along this second enzyme, which ultimately turned out to be pi3k phosphoenositide 3 kinase. But we didn't know that at that time. And so we suggested in the paper that the src very same enzymatic pocket could both accommodate tyrosine as a substrate and also accommodate the head group of phosphatidyl inositol. That in the end turned out to be incorrect in that even though by all criteria that we could characterize at that point, we couldn't prove that there was a separate function. If you used a kinase dead src or a mutant of SRC that no longer had activity, then the phosphatidyl andositol kinase also went away. That's why we thought it was the same pocket that was doing both. Retrospect, it turns out that the enzyme has to be active in order to bind to pi3kinase, and that's why that activity was coming along. So we published that. We're very, very clear to say that we used sonicated membranes to do the assay which was necessary to get this mini log preference for that substrate. In the meantime, after publishing that, multiple other labs tried to reproduce it, but they didn't have the machine that would sonify the lipids to make the membrane bilayers, so they just added detergent. So even though we showed in our paper that if you added detergent, the enzyme activity completely disappeared, wouldn't the detergent.

Break the glycerol off the lipid?

The phosphatidylinositol would now get embedded in the detergent rather than being a bilayer.

Yeah, it wouldn't stick to it at all. Right, right. Yeah.

So you're going to lose that advantage of having a membrane.

And that wasn't known at the time, I guess.

Well, we knew it because I'd worked on membranes for the previous 10 years.

But the other labs didn't. I guess at the time they didn't know.

They were not membrane biochemists, they were all molecular biologists. So they tried to reproduce the result. But they didn't have a sonifier to make membrane lipids, and they assumed all that phosphorylates, they had group phosphatidyl inositol, it shouldn't make any difference how you present it. And so they presented a detergent micelle and they could not reproduce the results. So there were three prominent papers coming out a year or so later saying.

Nope, this is incorrect, this is incorrect.

This is incorrect from major laboratories. So at that point had already started a collaboration with Tom Roberts.

But at that point, Lou, did you have confidence that you had done this correctly or were you questioning yourself?

I had no doubt.

So you knew this was a methodologic error on the part of these other three labs, and despite the fact that at this point the scientific community would look at you and say, lou, you're probably wrong, you were confident in your methodology?

Yeah, there was no doubt. All my research had been on membrane enzymes and membrane reconstitution, et cetera. So I knew what I was doing made sense and I knew our results were reproducible because not only could multiple people in my laboratory do it, but multiple people in Tom Roberts laboratory, who we started a collaboration with, could do it, and Brian Schaffhausen's lab, which was a third collaborator, could also reproduce it. So multiple people in multiple independent labs all got the same result. If you sonified the lipid and if you added MP40, a detergent to Solubilize the lipid, then the activity went away. So there was no doubt about that. And so I had no lack of confidence that we were right. But the problem was how to convince these other laboratories that they needed to do the assay. Right. They didn't want to buy a sonifier. They were like, I'm done with that when they're there and it doesn't work. So we actually sent David Kaplan, a graduate student in Tom Roberts lab, and Malcolm Hoytman, the graduate student at my laboratory, to these other laboratories with the sonifier. They went there, they sonified the lipid form, gave them to their graduate students, who then did the assay again, and now they got the same result we got.

Are you pushing this hard to get the other labs to see it? Because you know how important this is going to be, or you have sort of premonition about the role that pi3k is going to play in growth. I mean, you couldn't have possibly seen in the early 80s what, you know today. So how much of this was just scratching an intellectual itch versus A biologic intuition about the importance of this to overall growth, which is obviously where we're going. I'm amazed we are this far in and we haven't used the C word yet.

So the reason I was completely convinced by 76, 77 that this was driving growth of cancer cells is the collaboration we started with Tom Roberts lab. And Tom Roberts had been working on polyoma middle T. So polyomavirus is a DNA virus, while SARCA is an RNA virus. So it causes, as the name implies, the formation of multiple tumors. So you infect a mouse with this virus and all kinds of tumors show up everywhere. And so Tom had begun mutating various regions of polyoma middle T and polyma. Reason we looked at polyma middle T is because his lab and Sarah Kortnitz's lab and others in Schaffhausen had shown that SRC co purifies with polyomy T. We already knew by the mid-70s that src was somehow implicated in how polyamidal T transforms cells and forms all these tumors. That's why I went to collaborate with Tom, because he had additional tools that we could collaborate with to understand. And so he had made all kinds of mutations. He'd also found that SRC phosphorylated a site in polymer metal T, a tyrosine site that was highly phosphorylated. And even more importantly, that if you mutated that tyrosine residue to phenylalanine, polymemidal T completely lost its ability to transform cells with a single point mutation. Even though the Src protein was still bound, there was no longer tyrosine phosphorylation 315. And that eliminated the ability to transform cells. So we said, well, if pi3kinase is not SRC itself, maybe it binds to middle T independently. But we found that if you prevented SRC from binding to middle T, then you didn't get that tyrosine phosphorylation, and now there was no pi3kinase activity bound to middle t. So you needed src for middle t to bind to pi3kinase. At this time, we didn't know it was phosphorylating the 3 position, which we'll come back to. However, the shocking result was that if you eliminated that tyrosine residue, now pi3 kinase would no longer bind and middle T would no longer transform cells, even though SRC was still there and was still activated. So that said that activating SRC is not sufficient to transform a cell unless you also activate pi3 kinase. And we published that in nature in 76, I think. 76, 77. In nature.

Were you collaborating with Bishop and Varmus at this point in time?

86.

Okay, okay.

And then 86, 87. So 84 was when we published the paper with Ray Erickson and then a couple years later with Tom Roberts and a couple of papers we had. And we showed that PDGF receptor also, when activated, brought down COPROs of the PI3 kinase activity, et cetera, et cetera. So that's where we were in 1986. 87 or so. And at least some of the people who had gotten sonicators also believed us that this was really uniquely associated with tyrosine kinases after they were activated by growth factors. We saw the insulin receptor would also bring down pi3kinase. Only if you stimulate with insulin first, then you could also bring down this PI kinase activity. But at that time, the only monophospholated form of phosphatidylinositol known was phosphatidylinostitol. For that was discovered in 1949. I mentioned earlier, there's a 4 phosphorylation and there's a 4,5 phosphorylation, and those are the only two species of phosphorylated phosphatidyl inositol. So we assumed that this was phosphorylation at the 4 position. But as we began to characterize the ability of proteins from cells to phosphorylate phosphatidylinositol, we found that there were two activities. There was one that required that you sonify the membranes for it to bind and get for it to have activity, and another that would work perfectly well, in fact, even better if the lipid was dissolved in a detergent. So we called those two enzymes type 1 and type 2 PI kinase. And it was only the type 1, the one that required sonified lipids that co precipitated with all these tyrosine kinases. The type 2 had completely different enzymatic characters, so it was inhibited by adenosine. The type one wasn't. So we had a whole lot of profiles that said these were two different enzymes. So Malcolm Whitman registered in my lab, decided to separate them. So he ran column fractionations and activities with or without detergent, with or without adenosine. And he characterized the two enzymes, separated them completely. And we had a lab meeting which every other spot on the thin layer, which is how we characterized the lipid phosphorylation, we ran it out to separate the molecules based on migration in a solvent on A silica plate. And we noticed in the lab meeting that every other spot migrated about 1 millimeter different from the previous spot. And the way that Malcolm had spotted them on the thin layer was it was type 1, then type 2, then type 1, then type 2, type 1, type 2, all the way across. If you looked at where the radioactivity ran, it went up, down, up, down, up, down, up, down, up, down. And the bottom line was, to me as a chemist, the fact that they migrate differently means they have to be chemically different. So that meant that one of those molecules was being phosphorylated at a different position on the inositol ring from the other. And so we began the process then of chemically characterizing the product of the type 1 and the type 2 enzyme.

And how subtle was this, by the way? Was this obvious in looking at it, or was it something that could have easily been dismissed?

I guarantee almost everybody would have dismissed this one millimeter.

Yeah, I was about to say that just doesn't sound like thought was 10.

At least 10 millimeters diameter in the center of the spots were 1 millimeter different.

Do you naturally have an eye that gravitates towards symmetry?

I have an eye that gravitates towards unexpected results. And to me, there's no way to explain it. If you had only two spots beside each other, you could say, well, that side of the thin layer migrated a little faster because of the solvent front, was not completely horizontal. But this, you couldn't explain the. Up, down, up, down, up, down, all the way across.

Do you still have a photo of that?

It's in Figure 1 in the Nature.

Paper that we published from 87. 88.

88 published in 88.

We'll link to that figure.

But the observation was made in 1987. I bumped into Peter Downs, who's one of the best lipid chemists in the world, and at a meeting at Cold Spring harbor, and I told him this result, and I said, I know you must have standards for inositol degradation, so can we work together? And so we knew it had to be some site other than the four position. But it could have been the five. It could have been the three.

Yeah. Was there a chance it could have been? You were looking at 4 versus 4 plus 5, 4 versus 4 plus 5.

No, because we knew at 4 plus 5, it would migrate about 3 inches different.

So that would look totally different.

Yeah.

So the null hypothesis is you're just looking at four and your assay is a little dirty, but you're Thinking, no, it's something else.

Yep. Of course, we repeated it multiple times. And we also did HPLC separation, reverse phase. Every way you could look at it, and in every way we did it. They were chemically different, they migrated differently and numerous. The Nature paper, we only put that one slide, that one figure of the slightly different migration, because from there on we did the chemistry to prove that it was a three position.

Why do you think the three position had such a similar look to the four versus the five?

Well, keep in mind there were only two species known to exist at the time we made this discovery. Phosphatidyl inositol 4, phosphate, oh, and 4, 5 and 4, 5.

There was not a 5 by itself.

It was not a 5 by itself.

Got it.

So this told us there's a 3 by itself. And then we went on to show there's also 3, 4 and also 3, 4, 5. Pi3 kinase could phosphorylate the 3 position whether or not the 4 or 5 were already phosphorylated. So we now that generated three new species, pi3p, pi3, 4 and pi3, 4, 5. But I have to say the three hundred and forty five. This is sort of another sidebar that we could drop out of this. But it's so cool. Never published it because it was embarrassing, but, you know, at this age you may as well get embarrassed. So we knew that there were species that had been claimed to be 4, 5 that were probably 3, 4 for the same reason that the 3p was thought to be 4p, because separating the 3, 4 from the 4, 5 was also hard. So we looked for that carefully and found that in fact there was a 3, 4 being made and proved that the same enzyme was making 3P, and if you gave it PI 4P to the substrate, it would make 3, 4P 2. Leslie Sarounian, a postdoc in the laboratory, was doing the experiments to really verify that those two species really were being made from the same enzyme. And in the course of that work, she came into my office one day and said, I no longer get the same results that I've been getting for the last six months. Something has changed. And so we went over all of her thin layers again. This is how we separated all the species. And sure enough, after getting numerous results in which she could give pi4p and get pi34p2, suddenly over the past week, she no longer got that doubly phosphorylated lipid when she added purified pi3 kinase to that lipid. So the question was, what had changed? And she said, well, the only thing that changed is we ran out of the Pi4P and I had to buy a new jar of Pi4P from Barringer Mannheim. And I said, oh, that's interesting. As I looked at the thin layer more carefully, I noticed that just when the experiments were no longer working, there was a new spot on the thin layer that was just off the origin where we assumed it would probably just be ATP. There was always a little bit of contaminating ATP that got left over when you separate the lipid out. And we assumed that that was ATP ATP. But I noticed it ran just a little bit faster than ATP and that it only appeared in experiments where she could not find the 3.4p2. And I said, okay, stain this with iodine. It'll tell you where the lipid that you used from Behringer Manheim runs. And I'll bet that it's actually 4.5p2. And that turned out to be the case that they mislabeled the bottle. What was commercially sold as phosphatidylinositol 4 phosphate was actually phosphatidylinositol4.5 bisphosphate. And so, by accident, I almost think that God looked down on us and said, look, they're missing the Most important lipid PI345P3. It never occurred to us to look for it because we were. No one had ever claimed there was something like that that ran on a thin layer. That was because it runs so close to the ATP that was always missed.

Because it's got three phosphates.

Yeah. It's very highly charged and therefore runs.

I never once thought of that until you just said that, Lou. I never. I never would have had that thought, actually. And then, of course, when you say it now, I mean, that's. Think about how dominant those three phosphates are. Right.

In fact, a year after we made this obstacle, the paper came out. In fact, I got called by Nature saying there's this paper claiming they found something that looks like triply phosphorylated phosphatidylinositol. And they asked, do you think that's possible? And I said, yeah, in fact, I know it's possible. We already have results showing that that can happen with purified enzyme.

Lou, thank you so much for the generosity with your time and your insights, and most importantly, for the work you're doing. You and your colleagues are helping a lot of people.

Thank you.

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