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

00:00:00.000 Hey everyone, welcome to the drive podcast. I'm your host, Peter Atiyah. This podcast,

00:00:15.480 my website and my weekly newsletter all focus on the goal of translating the science of longevity

00:00:19.800 into something accessible for everyone. Our goal is to provide the best content in health

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00:00:28.880 If you enjoy this podcast, we've created a membership program that brings you far more

00:00:33.280 in-depth content. If you want to take your knowledge of this space to the next level at

00:00:37.320 the end of this episode, I'll explain what those benefits are. Or if you want to learn more now,

00:00:41.740 head over to peteratiyahmd.com forward slash subscribe. Now, without further delay, here's

00:00:48.080 today's episode. I guess this week is Lou Cantley. Lou is the Meyer director and professor of cancer

00:00:54.680 biology at the Sandra and Edward Meyer Cancer Center at Weill Cornell Medical College in

00:00:58.800 New York City. Lou's research has made significant advances in cancer research stemming primarily

00:01:03.880 from his discovery of the signaling pathway for PI3 kinase or PI3K in the mid 80s. And his results

00:01:12.020 here have really touched multiple fields, including cancer first and foremost, but also diabetes

00:01:17.300 and autoimmune disease. Lou is a very decorated scientist to put it mildly. It would take me too

00:01:25.080 long to rattle off all of the awards that he's won, but I'll leave it to the four most prestigious,

00:01:29.620 the Gairdner Award, the Breakthrough Prize in Science, the Wolf Prize, and most recently the Horowitz

00:01:34.980 Prize. Arguably the only two prizes that are not on that list would be the Lasker Prize and the Nobel

00:01:40.360 Prize. And I think that many people suspect that Lou will ultimately be awarded at least the Nobel

00:01:45.400 Prize, if not both. This is a podcast that at times gets very technical, but most of the technical

00:01:51.440 stuff falls at the end of the podcast. It's actually what we started with, but much like we did in a

00:01:57.460 previous podcast, we're going to move that to the back end of the podcast so that we can jump right

00:02:02.540 into the more concrete stuff that I think people want to hear about with respect to science. And

00:02:07.580 we'll treat what was the first 45 minutes of this interview as a technical tour de force of the

00:02:13.220 discovery of PI3K. And we'll move that as basically an appendix. So if you're listening to this

00:02:18.640 and you really want to understand science and you really want to understand how discovery is done,

00:02:23.360 stuff that I really like to hear about, by all means, you'll just listen to this as it's laid out.

00:02:28.040 And if you really just want to hear about the implications of Lou's work as it pertains to

00:02:32.680 cancer, then you will probably just listen to the first hour and 45 minutes or so of this and not the

00:02:39.180 remaining 45 minutes. Before we jump into this, there are just two things I want to say. The first is

00:02:44.520 I want to note Lou's disclosures. Lou is the co-founder of two pharmaceutical companies,

00:02:50.740 Agios and Petra. And he is also the co-founder of a company that is just getting off the ground

00:02:56.320 called Fayeth. That's F-A-E-T-H. Fayeth is a company that hopes to pair nutrition with pharmaceuticals. And

00:03:03.560 that will all make sense during the podcast. These are all really interesting companies,

00:03:07.960 but I think it's important obviously to list those disclosures at the outset. Secondly, I want to be

00:03:12.580 sure that everyone understands that we have a discussion here that is very detailed at times

00:03:16.860 and very tactical at times with respect to cancer treatment. And certainly every time I talk with

00:03:22.220 Lou, I come away with a new way of thinking about the care of patients with cancer. Please do not

00:03:28.200 mistake what we are talking about here as medical advice. It is impossible to provide medical advice

00:03:33.520 to someone with whom you do not have a doctor-patient relationship. And so unfortunately, I hate saying that,

00:03:38.460 but it is an obligatory disclaimer that I must provide. And whatever we talk about here,

00:03:44.280 the combinations of therapies with respect to drugs for diabetes, drugs for cancer, and of course,

00:03:49.340 nutritional therapies really needs to be had in discussion with your physician. Actually,

00:03:54.520 I lied and I said two things. There's one other thing I want to talk about, which is there is a

00:03:59.080 cancer trial that is about to take off that is a really interesting and exciting prospect through one

00:04:07.000 of Lou's companies. The enrollment for that trial is just kicking off now. And we will list to that

00:04:12.420 should anybody listening to this podcast either have the type of cancer that's being studied or know

00:04:17.560 somebody who does. And it would be great to know that people are able to more readily get enrolled in

00:04:22.100 that clinical trial. So without further delay, please enjoy my conversation with Lou Cannon.

00:04:32.040 Lou, thank you so much for coming over.

00:04:33.820 Oh, it's great to be here, Peter.

00:04:35.400 You live like 10 feet from me here. And yet this is the first time you've been here, I think, right?

00:04:39.900 Yeah. It took me five minutes to walk here.

00:04:42.300 It's great to see you always. You're definitely one of the most generous people with his insights

00:04:47.860 and with the work you've done. And I consider you an absolute mentor in how I think about these

00:04:53.340 problems in biology. So it's, we've been trying to get on this podcast together for a year,

00:04:56.900 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.

00:05:01.120 I'm busy, but you are even busier than me.

00:05:03.700 I do keep pretty busy.

00:05:06.660 Well, there are a lot of things I want to talk about today. I think to set my own expectations,

00:05:12.640 we probably won't be able to get through them all, which speaks in some ways to the breadth of

00:05:16.680 your work. We're going to talk so much about cancer and metabolism and things here, but there's

00:05:21.880 what we know today about PI3K, which I think would be a nice thing to spend a moment on. But I also love

00:05:28.080 this idea. Like I have this idea, Lou, which is we should be teaching kids science, not the way you

00:05:34.220 were taught biology as a 16 year old, which was rote memorization. We should simply teach science

00:05:40.700 through the lens of discovery. So in other words, every biology class, even at the high school level

00:05:46.760 and even younger should be less about memorizing what it is that you see. Look under this mic up and

00:05:53.620 memorize all the organelles or something like that. It's more of let's tell the stories of science

00:05:58.680 because one, if our goal is to train scientists, well, they're going to go and become experts in

00:06:05.500 whatever they're going to become expert in. We don't have to worry that in high school,

00:06:08.420 they're not going to learn enough content. But if we're trying to screen for people who are

00:06:12.640 interested in science, what you have to be screening for is the process, is the discovery,

00:06:16.500 is the game, is the thinking that went into the experiment. It's the blind luck. It's the bad

00:06:23.700 luck. And that comes out in the story of everything. I've yet to hear a single world-class scientist,

00:06:31.160 and I have the privilege of sitting down with so many of them, tell their story without some element

00:06:36.500 of good luck and bad luck combined.

00:06:38.760 Serendipity.

00:06:39.580 Yes.

00:06:40.220 Serendipity in science is absolutely critical that when you get a result that's unexpected,

00:06:45.660 you should be laser-focused on understanding at the biochemistry, at the chemical level,

00:06:51.860 why you got the different result than you expected to get.

00:06:54.840 And following these journeys is, to me, a better way to weave the narrative arc of science. I mean,

00:07:00.420 I think, frankly, it's not that there's no value in knowing that DNA is a helical structure and it

00:07:06.460 has these base pairs and it has this backbone, but that shouldn't be the focus. To me, actually,

00:07:11.500 walking through the fumbling of how those guys got there, that's a very interesting story.

00:07:18.440 And that probably will serve a non-scientist better when they're done with science class,

00:07:24.640 because they'll have an appreciation for the process. And now I'm getting on my soapbox about

00:07:29.820 it would be better if policymakers understood that that's how science worked, as opposed to

00:07:33.980 trying to remember what they learned in a biochemistry class 30 years earlier.

00:07:38.420 And of course, for the people who are going to go into science, I still think they're going to go

00:07:42.560 back and learn the basics anyway. So we don't have to, through rote memory, get that stuff into them.

00:07:47.420 But it's this process of thinking and collaborating and walking down the hall

00:07:50.820 and seeing the result, as you said, that doesn't quite make sense and saying, well, wait a minute,

00:07:57.420 there's something in there. There's a story. I mean, science is basically just one big mystery.

00:08:01.020 It's really about curiosity. That's what I'm hearing in your story is it's less about your

00:08:05.760 innate brilliance and more about your obsessiveness, your ability to make an observation

00:08:11.060 and not let it go. Yeah. If I get a result that suddenly doesn't make sense, to me, that's more

00:08:16.840 exciting. It means there's something more complicated going on than the simplest explanation

00:08:21.820 for what I'm seeing. If the simple explanation works, then fine, you publish it in some second

00:08:27.240 rate journal. But if you've broken up a whole new field because your simple explanation doesn't

00:08:32.660 work and you figured out why it didn't work and what was wrong, then that's where most breakthroughs

00:08:38.040 come from. When did cancer become such a high focus? So we met, we met in 2011, two years after

00:08:46.380 you, Matthew Vander Heiden and Craig Thompson wrote what I thought was sort of the one of the most

00:08:52.140 interesting papers I'd read on cancer in the journal science. It's hard to believe we're 10 years away

00:08:56.540 from that paper. What was the lead up to your collaboration in that paper and less about the

00:09:02.700 paper, but more about thinking about cancer metabolism? Because today, when I think of

00:09:07.320 Lou Cantley, I think of cancer metabolism. When did that transition occur? It was a postdoc during my

00:09:13.380 laboratory, Matt Vander Heiden, who had trained with Craig Thompson. Matt's an MD, PhD, now on the faculty

00:09:19.880 at MIT. But he had been obsessed about glucose metabolism as a graduate student with Craig.

00:09:29.100 And in fact, I'd gotten one of his papers to review, which I was very impressed by.

00:09:34.040 And so he suddenly showed up at my office one day saying that he wanted, he was interested in doing

00:09:40.500 his postdoctoral work as he finished his clinical training. And at that time, a graduate student,

00:09:46.620 Heather Kristoff, had been working in my laboratory. And she'd made the observation that if she used

00:09:55.620 tyrosine phosphorylated peptides to pull down a degenerate sequence context, it wasn't a single

00:10:03.360 peptide, it was a whole mixture of billions of peptides, all the same length, biotinylated so you

00:10:09.620 could pull down anything they bound to, and then use mass spec to identify all the things that came down.

00:10:15.220 First of all, she rediscovered all the proteins that were known to bind to, that have SH2 domains.

00:10:22.420 This is a domain that many proteins, including PI3 kinase, have that allow them to bind to tyrosine

00:10:29.200 phosphorylated proteins. And so in that one experiment, she rediscovered everything that had

00:10:35.600 been published of all the things that bind to tyrosine phosphorylated proteins. But she discovered

00:10:39.960 the thing that bound better than anything else in the cell, or that was most massively pulled down,

00:10:46.120 was an enzyme in metabolism, pyruvate kinase. And that was shocking. And I remember her first

00:10:52.980 response to that was, well, the only novel thing I found here that wasn't already known

00:10:57.660 was this...

00:10:59.160 This affinity for pyruvate kinase.

00:11:01.020 Pyruvate kinase. And she said, well, that's a metabolic enzyme, and they're all boring,

00:11:06.020 so I don't think I really want to work on that. And I said, no, no, that's whenever you get the result.

00:11:10.100 You pulled the loo.

00:11:10.900 The totally unexpected result. That's the thing you should focus on. And so she did.

00:11:16.680 And at the time that Matt was interviewing with me, I told him about her results. And he got very

00:11:23.280 excited about that. And the two of them worked together to show how that was working.

00:11:28.520 So this is early 2000s, right?

00:11:31.240 This was 2000 and...

00:11:33.800 Mid 2000?

00:11:34.800 Like 2005?

00:11:35.340 2005, maybe.

00:11:37.380 Matthew Vanderheiden was a classmate, I believe. I've obviously met Matt since, but I believe he was

00:11:42.860 a medical school classmate of one of my close friends, Ted Schaefer, who actually I had on the

00:11:46.740 podcast. And Ted also was an MD-PhD, did his PhD in Harold Varmus' lab. So it all kind of comes full

00:11:52.180 circle. A lot of smart folks came out of the University of Chicago.

00:11:55.100 So anyway, so he, Matt and Heather ended up publishing two back-to-back papers in Nature

00:12:01.420 that both explained structurally how phosphotyrosine peptides or proteins binding to pyruvate kinase

00:12:09.480 could turn off its activity. And then also explained why turning off the activity of pyruvate

00:12:15.840 kinase was important to increase anabolic growth. And that is kind of the basis for

00:12:21.660 why we decided to start Agios, that Craig was interested in how metabolism was regulated in

00:12:28.100 cancer cells. And we had made this observation that pyruvate kinase regulation was important in

00:12:33.580 oncogenic regulation, anabolic processes in cells. And so we proposed pyruvate kinase

00:12:40.420 activators rather than inhibitors as a way to reduce tumor growth as one of the targets to go

00:12:46.840 after at Agios as we were starting the company. So that review, which I should say that Matt played

00:12:54.080 a much bigger role in writing that review than Craig or I did.

00:12:56.760 He was the first author. So yeah, that was pretty clear.

00:12:59.560 Yeah.

00:13:00.060 When did the Warburg effect, when did you start paying attention to it? I mean,

00:13:04.200 obviously it was around before you were born.

00:13:06.020 Yeah. So actually I mentioned F. Racker earlier. F. Racker was, knew Warburg quite well and they

00:13:15.180 overlapped in Germany. He was obsessed by understanding the Warburg effect. The Warburg

00:13:20.520 effect being a result published by Warburg back in the 1921 or so, almost a hundred years ago,

00:13:27.580 that if you chemically induce cancer in rats, the tumor that evolves takes up glucose and metabolizes

00:13:35.180 in an anabolic way much, much faster than the tissue of origin prior to transformation. This

00:13:41.940 was chemical induced cancer formations.

00:13:43.980 And this also, this observation was that even when there was sufficient cellular oxygen to generate

00:13:50.660 ATP much more efficiently through the mitochondria, it was as though the cancer cell elected to go this

00:13:57.120 faster, less efficient route.

00:13:58.960 That's right. The surprising result was, well, there's plenty of oxygen. We've taken the tumor out.

00:14:02.580 We were in an oxygen environment. Why doesn't it make ATP through mitochondrial oxidative phosphorylation

00:14:08.120 instead of through glycolysis?

00:14:10.620 Why do you think the Warburg effect was largely forgotten in the forties and fifties and sixties?

00:14:15.380 I mean, it wasn't, it fell out of favor really not until late sixties, early seventies. And what was

00:14:21.980 taking over at that time was evidence for virally induced cancers. V. Sark, Harold Varmus and Mike

00:14:28.480 Bishop. So once it was shown that viruses could turn on cancers, the idea that it was just an altered

00:14:36.220 metabolism fell out of favor. Of course, the discovery of oncogenes of which V. Sark was the

00:14:41.860 first clear oncogene because it told us that we've met the enemy and it is us. It is our own genes that

00:14:48.440 are getting altered in cancer. And so proto-oncogenes were being picked up by retroviruses.

00:14:53.580 Did anyone at the time think these two mechanisms aren't mutually exclusive? The expression of,

00:15:00.360 or the amplification of, or the mechanism by which this viral injury to the genome can perpetrate

00:15:09.280 and can perpetuate could be through this defective metabolism?

00:15:13.580 I think people who thought about it deeply and who were trained as biochemists who were doing

00:15:18.440 metabolism, which F. Racker was one of the prime examples, continued to realize you have to alter

00:15:25.560 metabolism to do cancer. You need to switch a cell from a quiescence, not only from a quiescence state

00:15:31.480 to a dividing state, but in order to divide and make a bigger cluster of cells, you have to drive

00:15:38.100 glucose and amino acid uptake and anabolic processes, converting those to proteins and lipids

00:15:44.240 in DNA and RNA. So there was no doubt you had to turn up metabolism. The question was, at what level

00:15:51.680 did metabolism play the role? Was it just a consequence? Yes, exactly. Of the viral transformation

00:15:59.260 or the oncogenic transformation? Or did they work cooperatively in some more complicated way?

00:16:05.860 So our people in pyrovate kinase suggested, yes, there is some clear cooperation where the active

00:16:10.940 tyrosine phosphorylating proteins was directly regulating the activity of a metabolic enzyme

00:16:15.720 rather than just turning up the expression, for example, through a transcriptional cascade.

00:16:20.120 So that's why it shifted the emphasis back to there may be targets in metabolism that could give you

00:16:27.020 an efficacy toxicity ratio for treating cancer. The truth is chemotherapy, as you know better than I,

00:16:33.860 is really targeting metabolism. You're blocking DNA synthesis or other steps in metabolism as a way

00:16:41.720 of killing cancer cells. So that's, in a way, targeting metabolism is our first approach to

00:16:47.940 curing cancer. And now we're back to doing it in a more serious way today.

00:16:51.580 What was the point that you guys made that was so novel in that 2009 paper? Because it's a subtle

00:16:57.340 point that I think you can easily miss. You could easily look at that paper and say, oh, this is just

00:17:01.560 another paper discussing the Warburg effect. But you came up with a very clear explanation for why

00:17:06.540 the Warburg effect might exist that was different from the explanation that I think I had looked at

00:17:13.560 before, which was the mitochondria are defective. So I think the easy explanation for the Warburg

00:17:18.680 effect is the mitochondria don't work. Therefore, the cancer cell has no choice but to undergo

00:17:24.740 anaerobic glycolysis. You guys propose something different.

00:17:28.540 Yeah, we propose that you could, even with totally functional mitochondria,

00:17:33.700 you could divert intermediates in glycolysis into anabolic processes by regulating steps in glycolysis.

00:17:42.740 And so the biggest question, we know that if you have high ATP in the cell, it will shut off glycolysis.

00:17:51.440 And that's a feedback that there's high citrate and high ATP, either one of those directly bind

00:17:56.900 the phosphofructokinase, shut off its activity, and the remaining glucose that comes in that gets

00:18:02.840 phosphorylated either goes to the pentose phosphate shunt or it goes to glycogen storage or it just goes

00:18:08.660 back out of the cell again. So there's a break right there. And so F-Racker was obsessed by how

00:18:15.360 you relieve that break, because how do you get a 10 to 50 fold increase in glycolysis if you're making

00:18:22.440 so much ATP, it should shut it back off again. So the idea that it was defective mitochondria was a

00:18:29.220 simple explanation. There's no other way to make ATP, so you have to make it through glycolysis.

00:18:33.960 But the idea that there was something more complicated than that was really what we were

00:18:39.780 discussing in that review, that there was a way to regulate steps in glycolysis that would divert

00:18:46.020 intermediates in glycolysis from making ATP to the carbon atoms of glycolysis that go into lipid

00:18:52.160 synthesis, serine synthesis, glycine synthesis. So it was sort of like a mass balance argument.

00:18:57.880 It was an energetics for construction argument. It was basically saying, look, the way to really

00:19:04.120 think about this is cancer's growing like crazy. It's easy to talk about the energy requirement,

00:19:09.780 but just think about the carbon requirement of growth.

00:19:12.660 Right. So one analogy I like to make is, imagine you have a river flowing through a valley

00:19:18.240 and you want to flood some fields in order to get enough water to grow plants. So if you put a dam

00:19:25.160 here, then those other little canals that you've generated, the water level will go up to flood into

00:19:30.500 those other canals and allow you to grow plants. That's sort of what happens if you turn off pyruvate

00:19:36.280 kinase. Then you slow down ATP production. That means there's less ATP made. That means there's

00:19:42.640 means glycolysis, part of glycolysis to continue, but instead of going to make more ATP, it goes off

00:19:49.320 to make serine and glycine and ribose and lipids. And that gives you all the molecules you need to

00:19:56.060 grow.

00:19:56.840 Without shutting the system off because you don't let it go all the way to ATP.

00:20:00.720 Right. Yeah.

00:20:01.620 And that allows the mitochondria to make whatever ATP you need. And glycolysis is now used to do

00:20:07.280 anabolic processes and sort of make ATP. That still makes some ATP, but you can now rebalance

00:20:13.120 that whole equation so that you can grow better rather than just make ATP.

00:20:18.580 Now, this had a profound impact on the way you thought about your life. You're one of the,

00:20:23.760 I don't want to say the few, but there are a lot of people who, a lot of great scientists, frankly,

00:20:29.760 who their professional work and their personal, the way they go about conducting themselves and their

00:20:35.900 own choices around food or whatever. There seems to be a disconnect there. Not the case with you.

00:20:41.780 I remember the first time we had a meal in probably 2012, you were quite particular about

00:20:47.260 what you ate and what you didn't eat. And the thing that you were most careful about was fructose.

00:20:52.780 So what is it about the work that you were doing in the 2000s, the early 2000s into this

00:20:58.120 decade we're in today, the beginning of the decade we're in today, that had you start to think

00:21:02.740 about nutrition in that way? So I have to say that I made my decision about what to eat and not to eat

00:21:08.560 before I knew anything about metabolism. And it came from a simple empirical observation. So I grew

00:21:15.200 up, I mentioned earlier, I grew up in Blackwoods of West Virginia. In retrospect, I realized that

00:21:20.400 as I was growing up in the 50s, we were eating incredibly healthy meals. In those days, the most

00:21:27.020 expensive thing to buy at the grocery store was sugar. And my grandparents, who I spent a lot of

00:21:32.440 time with working on their farm, raised everything they ate. They were sustenance farmers. They had

00:21:38.020 essentially no income. They sold some strawberries in the spring. My grandfather would plow fields for

00:21:43.380 people to make additional money, but they had very minor income. But they lived off of everything

00:21:48.300 they grew. The one thing they couldn't grow, for a while they grew some sugar beets, but it was hard to

00:21:53.280 get enough sugar. And so when my grandmother went shopping, essentially the only thing she would buy 0.89

00:21:59.060 was flour and sugar. And one five-pound bag of sugar had to last several years. And so when she would 0.54

00:22:06.620 make what she called a cake, we would call it a biscuit today. It had essentially no sugar in it

00:22:12.600 because it had to be sprinkled in very small amounts. And so that, as a consequence, we were all very

00:22:18.820 healthy. Nobody in my family. In fact, I didn't know anyone who was overweight in West Virginia

00:22:23.080 because most people lived like my grandparents. And that was in the 50s. What happened in the 60s

00:22:29.540 was really what was tragic. It was all triggered by Castro, as you know the story. And Castro's 0.91

00:22:37.960 taking over Cuba and us boycotting sugar from Cuba and the anticipation the cost of sugar would go up

00:22:44.400 even higher. A German scientist had worked out a way to convert

00:22:48.820 cornstarch into high fructose corn syrup, which could be made much, much more cheaply than raising

00:22:55.540 cane. It's funny. I always thought it was a Japanese scientist. I didn't realize it was a

00:22:59.500 German scientist. I could be wrong on that. I would take your word over mine. We'll blame 1.00

00:23:03.500 somebody from World War II. The irony of it is it's always someone that got hurt in World War II. And

00:23:09.060 the joke is this was their payback. Exactly. And Castro's payback. So Castro did this to us.

00:23:14.840 We did it to ourselves, is the truth. And we still are doing it. And as long as I was

00:23:19.860 where the primaries are held, first primaries, and where nobody's going to cut off taxpayer support for

00:23:27.580 cornstarch or growing corn. But you had this profound belief. I mean, it's,

00:23:32.140 I guess you got to experience something very few people do, which is a natural experiment.

00:23:37.300 Yeah. So I watched West Virginia go from what I would argue in the 50s was the skinniest state in

00:23:42.640 America. I never saw anyone overweight. None of the kids in my school, none of my relatives,

00:23:49.460 none of them were overweight in the 50s. Most of them were very thin. And then along came high

00:23:55.420 fructose corn syrup. And suddenly the cheapest thing you could buy were sodas that normally at a

00:24:02.140 six ounce Coke would be my total allowance for a week to buy one six ounce Coke. And now suddenly

00:24:08.820 you five, 10 times as much for that cost. And so people advent of very sugary drinks,

00:24:15.780 people would start consuming 10, 20, 50 times as much sugar as they were consuming before.

00:24:22.180 And over from the mid sixties to mid seventies, I watched when I went back, I was at that time at

00:24:30.080 Cornell getting my graduate degree. And they didn't go back to West Virginia that often. But when I did go

00:24:36.220 back, I every time noticed a dramatic change in that all my friends and relatives were suddenly

00:24:42.780 becoming obese. And I just watched what they ate and I realized they were consuming massive amounts

00:24:48.600 of sugar. Many of them were consuming diet drinks, but then at the end of the day, because of their

00:24:54.900 addiction, the sweets would eat like a gallon of ice cream per person. When I asked them, how can you

00:24:59.480 eat a gallon of ice cream? And they would tell me if I don't eat a gallon of ice cream, I'll wake up two

00:25:04.000 hours later and go read the refrigerator. So I can't sleep through the night without eating a

00:25:08.560 massive amount of sweets. So at that point, I realized this is a real addiction. And I cut out

00:25:15.220 drinking anything that was sweet. And I cut out eating dessert. So that was 1975. Didn't know anything

00:25:22.380 about the metabolism of fructose or glucose. Just empirically, I saw the change in diet and the

00:25:28.800 consequent massive change in obesity. So you basically have these two sort of separate

00:25:34.260 stories, which is this is this observational journey of your life. And then you have the work

00:25:40.200 of your career that is bringing you closer and closer to cancer metabolism. When did you finally

00:25:46.080 realize in the laboratory that this observation of yours had mechanisms to it that could go far beyond

00:25:53.860 obesity, but also play a role in metabolic diseases, inclusive of cancer? What really brought it all

00:26:00.100 together, and this is why I keep emphasizing insulin, as a postdoc and ultimately starting my own lab, I

00:26:05.900 kept coming back to how does insulin work. And whenever we managed to see this PI kinase activity

00:26:13.420 co-precipitating with SARC and insulin receptor, I realized that the same enzyme was mediating the effects of

00:26:22.640 the oncogenes and also effective normal insulin signaling. And so that by 1990, it was very clear

00:26:31.240 in my mind that insulin was triggering cell growth in exactly the same way that oncogenes were.

00:26:38.740 They were both activating PI3 kinase. And that suggested then a correlation between the two.

00:26:44.900 And particularly as we began to notice that the mutations, so many years later, mutations in PI3 kinase

00:26:52.080 were identified. It was more than 15 years after we discovered the enzyme, the mutations were picked up

00:26:57.040 from Bert Vogelstein's laboratory. And as we began to explore what those mutations did, we realized they

00:27:03.680 increased the ability of insulin to activate the enzyme. So this whole connection between everything

00:27:09.840 that insulin does goes through PI3 kinase, and most oncogenes manage to activate PI3 kinase by either

00:27:18.680 directly binding or indirectly activating it. Not necessarily through insulin, but they were bypassing

00:27:23.700 the need for insulin. We're going to come back to that very important observation in a moment, right?

00:27:29.440 Yes. You know exactly where I'm going with the next level of questioning. Okay.

00:27:33.440 Right. But it also raised the possibility that being insulin resistant, which results in elevated serum

00:27:42.320 insulin because the pancreas has to generate more insulin. If your liver and muscle and fat cells are

00:27:48.620 failing to respond to insulin, then more insulin has to be made in order to bring glucose back down.

00:27:54.640 So insulin resistance is pretty much a silent disease because you don't really know it if you

00:27:59.080 go for an overnight fasting glucose measurement in the urine, your glucose is okay. But if you do a

00:28:05.800 glucose tolerance test, which very few people do, you realize that the glucose goes up much higher,

00:28:10.780 takes longer to come back down, and the insulin level is much, much higher than normal during that

00:28:16.720 process of adapting. So that's how insulin resistance is defined. But I realized that high

00:28:22.940 level of insulin, if we add it to our cells and culture, cancer cells and culture, it makes them grow

00:28:29.080 better. And we've known that since the 1960s and 70s. We use fetal calf serum to make our cancer cells

00:28:36.920 grow in culture. And if fetal calf serum doesn't work, we either order another batch, you know,

00:28:44.000 another lot from the company, or we just add insulin. And adding insulin always makes it work again.

00:28:50.200 And so we've known for a long time that if you have high levels of insulin, you can make almost any

00:28:54.700 cancer cell grow better than just pure fetal calf serum, which already has some insulin, and it's variable

00:28:59.680 from batch to batch. So in a way, it's been staring us in the face for years that insulin will drive the

00:29:07.760 growth of cancer cells. And cancer cells tend to have more insulin receptor than the tissue from

00:29:13.280 which they emerged. So in the process of tumors growing out, evolving, they turn up the expression

00:29:19.860 of the insulin receptor. And that allows them to respond to insulin better. So as I kept seeing more and

00:29:25.420 more data on this over the last 15 years or so, I became convinced that the act of being insulin

00:29:33.300 resistant or the state of being insulin resistant in humans sets them up to accelerate tumor growth.

00:29:40.940 When I interviewed Sid, gosh, I don't know how long it's been, probably six, eight, nine months ago,

00:29:46.320 he shared something with the listeners that I think for many people is still hard to believe, which is

00:29:50.100 smoking is the leading environmental cause of cancer. Obesity is second. In fact, I think he even

00:29:57.800 mentioned that in the documentary that was made after his book. You were in that documentary as

00:30:03.420 well. Great documentary. It was not named after the book, was it? Was the documentary also called

00:30:08.620 The Emperor of All Maladies? Yes, it was. Okay, okay. Phenomenal. We'll link to it in the show notes

00:30:12.820 because it's a real gem. I mean, it's worth every penny you've got to pay to download on Apple and

00:30:17.760 watch that PBS special. Ken Burns, of course, is a master. But Sid made the point there as well.

00:30:23.280 And hearing you say what you're saying makes you think it's really less the obesity and more the

00:30:29.000 hyperinsulinemia that accompanies obesity in 80% of cases, which is high enough that you could easily,

00:30:36.240 just from an epidemiologic standpoint, identify obesity per se as the trigger, meaning excess adiposity.

00:30:42.540 But my belief, and I think yours, is that no, it's probably the hyperinsulinemia, which

00:30:46.840 means if you're lean and hyperinsulinemic, you're worse off. And I think those data are

00:30:51.840 becoming abundantly clear. There was a paper published a few years ago out of Montessori,

00:30:57.780 Albert Einstein, showing that in fact, if you take breast cancer patients and separate them into

00:31:03.260 overweight slash obese, insulin sensitive, because a lot of people can, if you put on peripheral fat,

00:31:10.700 but don't have visceral fat, you don't necessarily become insulin resistant with overweight. So body mass

00:31:15.720 index is not. Yeah, it's not enough. It's a first order term.

00:31:18.720 That subset of overweight women who were insulin sensitive did not have an increased risk of breast 0.64

00:31:24.340 cancer, but the ones that were insulin resistant did. So that says a lot. And that paper also had

00:31:31.020 data on lean women who got breast cancer. And that correlated with insulin resistant lean women.

00:31:37.740 And in many nationalities, particularly Asians, they may look very lean, but still have a lot of visceral

00:31:43.960 fat. And so that I think we, our current mechanism of using body mass indexes, our correlation is not

00:31:51.120 a good one. Yeah, it's just not good enough. And Mitch Lazar in 2013, I believe, published a paper

00:31:55.920 showing basically the two by two of lean and not lean, metabolically healthy and not as a proxy for

00:32:02.980 hyperinsulinemic. And it turned out that it wasn't the body mass that was the thing that tracked with

00:32:08.980 health. Now, a moment ago, you gave this very eloquent explanation of the role of insulin in

00:32:14.460 cancer. Now, four months ago in science, you published a really interesting paper. What did that

00:32:20.540 paper show? So this is a paper in which was inspired by Mayor Bloomberg's attempt to ban the 40 ounce Coke.

00:32:26.820 So G.A. Yun, a postdoc in my laboratory who migrated with me from Harvard to Ball Cornell

00:32:33.120 2012-13, decided that she was inspired by Mayor Bloomberg and she would try to test the possibility

00:32:41.520 that high sugar consumption would increase colorectal cancer. She'd worked with Burt Fogelstein as a

00:32:48.060 graduate student. So she knew a lot about colorectal cancer and was working on mouse models of colorectal

00:32:53.540 cancer as she migrated into my lab. And so we decided that we would design an experiment to test

00:33:00.160 whether there was higher risk for colorectal cancer with high sugar consumption. And so it was really

00:33:07.080 two questions there. And my hypothesis going into this was the insulin level, that if you go on a high

00:33:13.380 sugar diet, that you'll eventually become insulin resistant and that will drive high levels of insulin,

00:33:20.540 which may accelerate the growth of colorectal cancer. And in fact, we had data that said that

00:33:26.960 what I just said was true. That in fact, yes, if we allowed the mice to have sugar in their drinking

00:33:34.600 water so they could ad lib feed as much sugar as they wanted on top of their normal chow diet.

00:33:41.240 How much sugar was in the chow?

00:33:43.140 So the normal chow diet, I don't remember exactly.

00:33:45.420 Normal chow might be 10%, but you can buy these really high sugar standard American chows that

00:33:50.300 are... Yeah. So we just gave them the normal

00:33:52.120 chow diet. And so because they were consuming massive amounts, they could drink as much of

00:33:57.920 the sugary water as they want. They drank three to four times as much water, sugary water, as if

00:34:03.440 they just had the water without the sugar. And a consequence, they became massively obese and

00:34:09.260 they had accelerated polyp formation in the context of having knockout of the APC gene. So APC is the

00:34:18.260 first gene in human cancer that gets lost to initiate the process of colorectal cancer development as

00:34:24.480 Bert Vogelstein is.

00:34:25.520 So the controls drinking normal water versus the sugar drinking waters both had the APC knockout?

00:34:32.760 They both had the APC knockout.

00:34:34.080 And what was the difference in polyp formation?

00:34:35.900 So in that three or more fold size of polyps, they were much more aggressive.

00:34:41.220 And what about the cancerous transformation?

00:34:43.940 Well, these mice, the polyps became so big that we had to sacrifice the mice because they

00:34:48.720 literally could not digest food anymore. They didn't go into metastatic disease. So we couldn't

00:34:53.880 call it a progressive other than the fact that they were massive and it was beginning to occlude

00:34:58.440 polyps.

00:35:00.040 Now, in this experiment, you also tried to figure out what was playing the role in this. And you

00:35:05.220 looked at glucose. So you could do this experiment with glucose water and fructose water, correct?

00:35:09.620 That's right. But let me get to the next step first.

00:35:12.360 Okay. Okay.

00:35:13.080 So that was nice and fine. It was consistent with my idea. But GA wanted to challenge me on the idea

00:35:19.820 that maybe the sugar was directly feeding the growth rather than just making the mouse insulin

00:35:26.700 resistant and obese.

00:35:28.040 Oh, I'm sorry. Because at this point, your hypothesis was all still through the insulin. This was all being

00:35:32.720 mediated through insulin.

00:35:33.940 Yeah. That was my hypothesis. So in order to try to always challenge people in my lab to prove me

00:35:40.500 wrong rather than prove me right. And because it's always learned more if my simplest idea is wrong.

00:35:47.180 Another great tenant of science.

00:35:48.760 So she challenged me on this. And Marcus Goncalves is the other postdoc in my lab that worked with her.

00:35:54.320 And the two of them worked together. And they designed a diet that would not allow the mice to

00:35:59.520 become obese. In other words, they were taking the total amount of sugar they were consuming. It was

00:36:03.480 the equivalent of a 12-ounce cola sugary drink. And so on that diet, at least over the six months or so

00:36:12.300 that they were on the diet, they really didn't gain weight. Maybe 5% increase in total calories consumed.

00:36:18.360 So they did not gain weight. They did not become insulin resistant. We didn't see elevation in C

00:36:22.980 peptide, glycosylated hemoglobin. So by all characteristics, those mice were normal. They

00:36:28.140 were not insulin resistant. And yet they still had increased polyps by two to threefold size. So at

00:36:35.240 that point, that raised the possibility that the sugar might be directly feeding the growth of the

00:36:41.360 polyp. And so we characterized that by using either radioactive glucose or radioactive fructose

00:36:48.440 or carbon-13 labeled, a non-radioactive but traceable form of carbon atoms in fructose or in

00:36:55.580 glucose. And we also tried feeding them only glucose, the same number of calories of glucose

00:37:01.600 or the same number of calories of fructose versus the mixture of glucose plus fructose. And they had to

00:37:09.300 have both sugars, fructose and glucose before the polyps would grow faster. So that was really

00:37:15.240 surprising.

00:37:16.940 And do you think that that could be because the glucose provides the insulin and the fructose is,

00:37:22.300 what do you think the fructose is doing in there? Because based on everything you said earlier,

00:37:26.100 a glucose water alone, just a pure dextrose solution should have been sufficient, right?

00:37:31.460 That's right. So that turned out not to be the case. And the way to figure it out is again,

00:37:36.240 using carbon trace, either by radioactivity or heavy atom. So we did both. And first of all,

00:37:42.620 does the fructose or glucose get all the way to the colon? Question number one.

00:37:47.440 It would seem highly improbable.

00:37:49.380 But it turns out if you add, if you have glucose plus fructose, the glucose competes for the fructose

00:37:55.780 for entry into the small intestine. And as a consequence, the glucose is not as efficiently taken up.

00:38:01.840 And it makes its way all the way to the colon if you give it in a sugary drink.

00:38:06.440 I was just about to say, Lou, does this have something to do with it being in a liquid so

00:38:10.340 the transit speed is quicker?

00:38:11.760 That's right. So if you gave the same amount in a solid food, it would never make it to the colon.

00:38:18.720 But because it was in a watery mixture, it would transit the intestine fast enough that there was

00:38:24.280 still fructose left.

00:38:25.500 Your experiment was elegant because you didn't have to do what my next experiment would have been,

00:38:29.700 which is why I'm not a postdoc in your lab, is I would have done colonic lavage. I would have gone

00:38:34.360 retrograde and seen if I could bathe the polyps and not even deal with the absorption. But I would

00:38:40.320 have missed this detail.

00:38:42.220 We wanted to replicate what-

00:38:43.900 What's physiologic, of course, yeah.

00:38:45.680 The condition in which humans do it. And other labs had already shown that, again,

00:38:50.460 giving a fructose-glucose mixture as a liquid at a certain volume, the fructose would make it all

00:38:56.720 the way to the colon. So what we found, if the fructose made it in the colon, the glucose got

00:39:00.100 absorbed in the small intestine, but there was some local increase in glucose level in the bloodstream

00:39:07.100 that's full, that is-

00:39:08.720 This is the first time I've ever seen this elegant demonstration of how you could miss in blood sugar

00:39:17.340 a problem. Because by definition, the faster it's going through you, the faster the glucose and

00:39:26.480 fructose are getting through and bypassing the small intestine, the more likely you'll get actual

00:39:33.140 colonic content of them. And the less likely you'll see glucose in the blood sugar, which is why the

00:39:39.380 group that in the second experiment, who were not being given ad libitum access to it, didn't get obese

00:39:45.580 and didn't develop hyperinsulinemia, but still developed the polyp phenotype.

00:39:50.840 Yeah. As a side issue, along the lines you're saying, we actually found in the case where we had the

00:39:55.320 water fed, rather than a fixed amount, the 12-ounce soda equivalent, that the subset of mice that had

00:40:03.160 the APC mutation actually were protected from insulin resistance. Those polyps were eating so much of

00:40:10.800 the sugar that it was protecting the mouse. That is unbelievable. And at some point,

00:40:16.900 you'll blow through that and they'll become quite insulin resistant and get hypertrophic polyps.

00:40:21.340 Yeah. Yeah. But in this case, we weren't allowing that to happen. It was a very small amount of sugar,

00:40:26.480 equivalent of a single drink a day, 12-ounce sweetened drink. The real surprise was when we got

00:40:33.200 to the molecular mechanism. And that was that you had to have fructose and glucose because the actual

00:40:42.220 carbon atoms that were being used to grow the tumor were coming from the glucose and not from

00:40:49.000 the fructose. And what the fructose was doing is when it went into the polyp, it was converted to

00:40:56.100 fructose 1-phosphate by an enzyme called ketohexokinase, also called fructokinase.

00:41:02.440 That enzyme is only found in three tissues in any significant concentration, the liver, the kidney,

00:41:08.800 and the gut. And so these polyps, like the normal gut cells, have that enzyme. That has been something

00:41:16.740 of a mystery of why that enzyme exists at that location. But in any event it does, what happens

00:41:23.220 is the fructose goes in, it gets phosphorylated by that enzyme, and it happens very rapidly. It's a very

00:41:28.280 active enzyme. And that drops the ATP level in the cell because you're consuming ATP to phosphorylate

00:41:35.820 that fructose that's coming in. Now, I mentioned earlier that the thing with the Warburg hypothesis

00:41:41.740 is that in order to get glucose to flux at a high rate into a cell, you have to drop the ATP level.

00:41:48.460 Well, this is a way to drop the ATP level. Instead of consuming it in some other way,

00:41:53.680 you're consuming it by phosphorylating the fructose. There's an additional complication,

00:41:58.320 or not complication, but intervention that is also important, which is once you start doing

00:42:03.880 glycolysis at a higher rate, the process of doing glycolysis incorporates an inorganic phosphate

00:42:09.620 to make the doubly phosphorylated glycerol and 1,6-bisphosphate. And so that additional consumption

00:42:17.600 of inorganic phosphate drops a negative regulator of inosine deaminase, and that drops the ability

00:42:25.060 to keep ATP synthesis going on in the cell. So the combination, too, drops the ATP level dramatically.

00:42:32.580 And now the glucose that's coming in is flooding through glycolysis, but it's going into all these

00:42:38.520 anabolic processes. It's being used to make lipids. We see all the label from glucose going into fatty

00:42:44.940 acid synthesis and serine synthesis and nucleotide synthesis going up five or tenfold. It's really

00:42:51.480 quite dramatic what happens. But if you leave the fructose out, even though the glucose gets

00:42:56.500 in the cell, it can't go through glycolysis at a higher rate, and so you don't get growth.

00:43:00.800 You have to have both molecules. The carbon atoms are coming from the glucose.

00:43:05.500 The fructose is basically driving the kinetics.

00:43:08.060 Exactly.

00:43:08.940 When you describe it this way, you sound crazy. I mean, you're the guy that nobody wants to talk

00:43:14.560 to at the party. Your wife must be annoyed senseless by her crazy husband, who's got this 1.00

00:43:20.260 hypothesis that now has so much emerging data behind it that says, if you wanted to create a molecule 0.99

00:43:27.080 to kill people, it's not just glucose. It's not even just fructose. It's put the two together.

00:43:33.440 And guess what? Nature did that. Nature came up with a 50-50 mixture of this thing.

00:43:39.680 And if that weren't enough, she made it taste so good.

00:43:44.600 Right.

00:43:44.900 How do we reconcile this?

00:43:46.720 Yeah. So that's the part I like best because I think about everything in regard to evolution.

00:43:51.120 Why do things evolve? Why do we evolve to be addicted?

00:43:53.600 Because you're ruining the party right now.

00:43:55.400 Exactly. So I think this allows, if you think about humans 100,000 years ago,

00:44:02.760 our metabolism hasn't changed in the last 100,000 years. So 100,000 years ago, how often

00:44:08.740 during a typical year in a temperate climate would you have available high amounts of fructose and

00:44:17.680 glucose?

00:44:18.700 I mean, maybe in the fall.

00:44:20.560 A month, a month or so. So you would get berries ripen, apples ripen. Keep in mind,

00:44:25.720 these are very small berries, very small apples before domestication of plants. And so as a

00:44:31.440 consequence, what that means is at the end of the growing season is when most of the sugary

00:44:37.480 producing fruits are being produced. If you could eat enough of those and you could keep your appetite

00:44:43.660 up enough to just keep consuming anything in place, anything available, you could put on weight.

00:44:49.760 As you go into the cold season.

00:44:50.840 As you go into the cold season. If you put on enough weight, then you might actually survive

00:44:56.480 to the next spring period when there's actually some roots to dig up to eat and keep going. And

00:45:02.560 anyone who probably, almost anyone 50,000 years ago who didn't put on 50 pounds or so in the fall

00:45:10.420 would not be alive for the next spring. It's a very strong requirement to gain weight in order to

00:45:17.820 survive a period of time when no food is available.

00:45:21.440 Yeah. Rick Johnson has written so eloquently about this and I'm blanking on the name of his

00:45:25.720 collaborator. Who's an anthropologist. And they even trace it back to which primates probably

00:45:31.620 developed this first. And they were primates that had left Africa, gone to Northern Europe,

00:45:36.120 and most of them die off. It was only the primates that could develop this mutation. And I believe it

00:45:42.420 was a mutation in both uricase and fructokinase. I could be wrong on that. And I've interviewed Rick

00:45:47.400 and so we can go back and listen to that, but you had to develop this mutation or else you wouldn't

00:45:52.640 survive the winter. And then what happened was tens of thousands, if not longer of years

00:45:57.600 of strengthening that is what allowed those primates to then come back to Africa to basically

00:46:03.680 become our descendants. So we as humans, as a species have these mutations that would have served

00:46:10.060 us well when glucose and fructose combined were provided just at the right level.

00:46:15.800 Yeah. So if you didn't anticipate that you needed to gain weight, your body would tell

00:46:21.420 you to do it, basically how it came about. And so it makes perfect sense. The extreme example

00:46:26.720 is the hibernating bear. We call it the honey bear often, right? Because in the fall, not

00:46:33.520 only do they eat every berry they can find in the woods, but they also climb up the trees

00:46:38.640 and get to the honey bees. And they put on 150, maybe even 200 pounds within a period of two 0.95

00:46:45.760 or three months. And then they go into extreme insulin resistance and they hibernate, they fall

00:46:52.340 asleep. But because of being insulin resistant and having metabolic syndrome, their ability to

00:46:59.500 break down fats is impaired. And that keeps them from quickly burning up.

00:47:05.640 Their metabolism slows greatly. They preserve the little bit of glucose they have for their

00:47:09.480 brain and they feed their body off ketones and fats, I assume. 0.61

00:47:14.300 Very long period of time. And then they survive. So we don't hibernate, but we probably became

00:47:19.940 insulin resistant every fall, a hundred thousand years ago in order to survive these periods of

00:47:26.200 starvation.

00:47:27.220 I mean, you've touched on this briefly from a public health perspective. I know you're not a

00:47:32.400 policymaker, you're a scientist. Many would say it's impossible. Like we're just never going to

00:47:38.780 see the day when sugary beverages go away, but the data are becoming harder and harder to ignore

00:47:45.780 that there is something uniquely toxic, chronically toxic. People don't like the word toxic because they

00:47:51.660 think it only has an acute implication. Like ethanol can be acutely toxic, but I'm talking about the

00:47:57.520 chronic toxicity. But the chronic toxicity associated with sugar-sweetened beverages,

00:48:03.020 inclusive of juices and things like that, right? It's not just a Coke. These data are becoming

00:48:08.300 almost impossible to ignore. Yet they still proliferate. I believe their consumption is down.

00:48:14.620 I don't believe people consume nearly the amount of sugar-sweetened beverages that they

00:48:18.420 consumed 20 years ago. I believe we peaked at around the turn of the century. But we're nowhere near

00:48:25.040 what you consumed growing up in West Virginia. Is it possible to get back to that? Is there a

00:48:30.640 solution or is it simply something that each person must be accountable for? Do you have a view on that

00:48:35.260 even? How you reverse the policy? I think changing cigarette smoking, there's a lesson to be learned

00:48:41.700 there. What was the incentive? How did the government make this happen? Reducing advertisement is one way

00:48:47.480 to do it. I mean, if you look at TV commercials, almost every commercial about food is about sugary food.

00:48:52.880 And there are many of them tailored to young children. To get them to buy the sugary cereals,

00:48:58.880 to get them addicted to young children, you've got them for life. Just like cigarette smokers,

00:49:02.240 you get them addicted at 15, 14, they're addicted for life. And there's a market for you. So I think

00:49:07.840 that's the appeal we have to make. And the question is, how do you do this? One way taxing also helps.

00:49:14.800 If sugar suddenly becomes, like it was in the 50s, the most expensive thing that you bought at the

00:49:20.340 grocery store, then maybe you would quit buying it. But of course, most people don't buy sugar

00:49:25.500 anymore. They buy processed foods that have sugar already added to them. By the way, looking at your

00:49:29.840 experiments, was there any distinction between if you used sugar as the substrate versus high fructose

00:49:36.200 corn syrup? No. Sugar, sucrose, by sugar we typically mean sucrose from cane sugar, which can be

00:49:43.100 crystallized because it's a pure molecule. While high fructose corn syrup is a mixture of fructose

00:49:48.140 and glucose, that's 60-40 rather than 50-50. But in our experiments, the difference between those two

00:49:55.140 doesn't make a whole lot. The fact that you need to, if it's sucrose, you have to hydrolyze the bond

00:49:59.680 is irrelevant to the kinetics that you described. Yeah, that happens very quickly.

00:50:04.500 So you don't buy the argument that some, I don't buy it either, by the way, but that you should eat

00:50:09.260 your sugar in natural form, which is, it should say cane sugar or beet sugar or something on the

00:50:14.540 ingredient. And there's this whole group of folks who believe that high fructose corn syrup is

00:50:18.940 horrible, but quote-unquote naturally occurring sugar is not. There's so much confusion around this,

00:50:23.820 but I just want to know if experimentally you see any difference. No, I think whether sucrose,

00:50:29.040 what shows the difference is whether the sugar is embedded in a fibrous fruit as opposed to being

00:50:35.280 pure in a water. There's something about that liquid that is devastating.

00:50:39.620 There are two things. One, for colorectal cancer, it is, as I said before, the fact that having it in

00:50:45.700 a sugary water drink gets it all the way to the colon. If you had the exact same amount of sugar,

00:50:52.660 but it was embedded in a fibrous fruit like an apple, then none of that fructose would make it to

00:50:57.900 the colon. The transits to this intestine would be so slow that the sugar would get absorbed.

00:51:04.080 But does that increase the risk of cancer in other organs?

00:51:08.400 No, I think this is absolutely unique to colorectal cancer. But I would argue, on the other hand,

00:51:14.280 the difference between eating an apple and eating apple juice not only is whether the fructose makes

00:51:19.680 it all the way to the colon, but whether or not your glucose levels spike after consuming an apple,

00:51:27.720 a whole apple eating it, as opposed to apple juice. And the answer is, with apple juice,

00:51:33.420 you're going to get a glucose spike for sure. Eating an apple, you may get hardly any change

00:51:39.040 in your glucose because it takes so long to break it down that the absorbance is slower.

00:51:44.400 Well, and you might, you know, what I've observed, I don't drink apple juice. I don't drink beverages

00:51:48.120 that have sugar in them. But what I notice is nothing has ever spiked my blood sugar more than a raisin

00:51:54.940 on a per mass basis. You took a fixed mass of raisins and you contrast it with a fixed mass of

00:52:01.540 anything else. It's pretty stark because it doesn't have the water, doesn't have the fiber,

00:52:05.960 doesn't have anything else. And even if you ate, say, call it, compare apple to apple juice. If you did

00:52:12.680 equal grams of glucose, you might even get a similar area under the curve. But where it's going to look

00:52:19.360 different is the apple juice, which will have, you'll get a big spike. And the apple, you'll get

00:52:25.540 a gradual. You're going to have different insulins in response to those. So the glucose AUC could be

00:52:30.840 the same, but the insulin response could be quite different. That's the point. And it's, in my opinion,

00:52:35.020 it's all about insulin. Most endocrinologists worry about the glucose. I worry about the insulin.

00:52:40.960 So really there's three things you're worried about. You're worried about glucose, fructose,

00:52:44.400 and insulin, but in how they coexist. Right. So colorectal cancer is unique and can feed

00:52:50.220 directly off the fructose if you get it all the way to the colon through. But it has to be in

00:52:55.920 liquid form. In a liquid form. But the difference between liquid form, apple and apple juice is,

00:53:00.920 as you say, the area in the curve is the same, but the fact that the glucose isn't going up as high

00:53:05.940 means you don't get as much insulin release. And of course, if digested very slowly, the rate at which

00:53:11.940 you're ambiently burning glucose in your brain and muscle may be almost equal to the rate at which

00:53:18.060 it gets absorbed in the bloodstream, in which case you hardly see any increase at all. And that means

00:53:22.720 no spike in insulin whatsoever. So that's the ideal situation is to never allow that insulin to get

00:53:29.880 high. Now, before we go into the why, I want to go back to some experiments, some drugs, and

00:53:38.420 something you said earlier. Let's go back to PI3 kinase. Everything you said about PI3 kinase

00:53:45.080 would make someone listening to this think, if you could block it with a drug, could you block

00:53:51.180 cancer? Well, obviously you've thought of that. So tell us about drugs that block PI3K inhibitors when

00:54:00.100 given to cancer patients. Okay. So by 1990, I was quite sure that everything insulin did went through

00:54:08.180 PI3 kinase. And it was also quite clear to me that many cancers have emerged because of activation of

00:54:14.020 PI3 kinase. Some pharmaceutical companies came to me and said, should we develop the PI3 kinase

00:54:20.860 inhibitor? And I said, well, I can't imagine how you would be able to thread that needle of being able

00:54:28.600 to inhibit PI3 kinase. Without causing diabetes. Without causing severe diabetes. And unless insulin

00:54:36.460 went up high enough to offset the inhibition of PI3 kinase, you would have to go off the drug because

00:54:41.840 of the hyperglycemia. If you raise the insulin level, it could override the PI3 kinase. It's also

00:54:47.300 going to activate the tumor to grow more. So it could be worse. Yeah. So I was skeptical and never,

00:54:54.540 in spite of all of our data saying that PI3 kinase was driving a lot of cancers, I never pulled the

00:55:00.840 trigger in starting a company or even encouraging anyone else to start a company to make inhibitors

00:55:06.180 of PI3 kinase. The fact that mutations in PI3 kinase were picked up in Bert Vogelstein's laboratory

00:55:12.700 in colorectal cancer and ultimately shown to be in many types of cancers led the charge to actually

00:55:18.500 develop an inhibitor. Because by then it was clear that other mutant tyrosine kinases, when they were

00:55:25.080 hyper mutated or hyper produced in cells, could be drugged and those would be effective ways to block

00:55:31.500 cancer growth. So with those observations, numerous companies, probably 15 companies went after projects

00:55:38.600 to develop in PI3 kinase inhibitors. I was still skeptical that you would be able to manage the

00:55:44.160 insulin problem, but agreed, in fact, applied for and got funding from Stand Up to Cancer, American

00:55:50.380 Association of Cancer Research, to put a team together to try to figure out how to most effectively use

00:55:55.560 those inhibitors as they went into the clinic. And I remember after we received that money, several

00:56:03.260 pharmaceutical companies contacted me and said, will you work with us? We have a PI3 kinase inhibitor

00:56:09.780 in human phase one trials. And they said, what's more, it doesn't cause hyperglycemia, so it's going

00:56:14.940 to be a safe drug. And I said, well, if it doesn't- How does that work? If it doesn't cause hyperglycemia,

00:56:19.060 it's not hitting PI3 kinase. So no, I'm not interested in working with a drug that doesn't cause hyperglycemia.

00:56:27.040 Ultimately, we decided to work primarily with Novartis that had a drug that we, several drugs actually,

00:56:33.040 that clearly caused hyperglycemia. So I was convinced they were hitting PI3 kinase and that

00:56:38.500 that was an on-target effect. And the question then is, can you thread that needle? Can you

00:56:43.820 manage the glucose level without getting insulin levels so high that they would reactivate PI3

00:56:51.240 kinase in the tumor? And that had been the challenge. So we suspected, I should say that in

00:56:57.620 working with Novartis, we got them to agree that if, when the patients got hyperglycemia,

00:57:03.620 that the endocrinologist would judge was not tolerable, that they would ensure that if the

00:57:11.140 patient could not be managed on metformin, that they would have to go off to trial. In other words,

00:57:15.560 I didn't want- You didn't want them being given insulin.

00:57:17.820 I didn't want them to be given insulin or an insulin secretagogue, which also raises serum insulin.

00:57:23.980 And I should say that the endocrinologists who were called in and consult for these patients

00:57:29.640 who were hyperglycemic on the trial disagreed with me. They said, insulin is totally safe.

00:57:35.540 They should be able to get insulin and we should do it that way. But I was quite adamant. I said,

00:57:40.480 well, I won't work with you on these trials unless you exclude patients who have to be managed on

00:57:46.520 insulin or insulin secretagogue. So Novartis kind of reluctantly agreed to do that and went

00:57:50.980 all the way through their approval trial with that as their requirement. Anyone who couldn't

00:57:56.800 be managed, who would need an insulin or insulin secretagogue would be off the trial. And their

00:58:02.820 drug got approved just in May this year. It took a long time because roughly half the patients who

00:58:08.640 tried to enroll could not be managed on metformin. Could you also use SGLT2 inhibitors?

00:58:14.120 So at that time, the SGLT2 inhibitors had not yet been approved. So we're talking about 10 years ago

00:58:21.220 when the phase 1b trials that were the lead-in to the approval trial were being where we worked

00:58:27.900 with Novartis on that. But Novartis stuck with the initial requirement through their approval trial

00:58:33.700 that everything had to be managed on metformin. Otherwise, they'd go up the trial. And that's why

00:58:38.740 it took them a long time to enroll enough patients to complete the trial. Now the SGLT2 inhibitors are

00:58:44.680 approved sodium glucose co-transporter inhibitors that reabsorb glucose from the ultrafiltrate in

00:58:51.040 the kidney. If you block that, then the glucose ends up in the urine. And so that's a good way to

00:58:57.180 lower glucose and thereby lower insulin. So that tool wasn't available at that time. It's now available

00:59:03.840 today. So we did a study over the last four years. It took a long time to do this. Lots and lots of

00:59:09.640 mice and lots of experiments to test whether in a mouse model for cancers, a variety of cancers,

00:59:16.620 12 different types of cancers, if you give a PI3 kinase inhibitor and use either a sodium glucose

00:59:23.500 co-transporter inhibitor or put the patients on a ketogenic diet, which means that they only have

00:59:30.480 about 8% of their total diet as carbohydrate. And even that is slow release carbohydrate.

00:59:37.700 The rest, 80% is fat and 12% is protein. And we ask now comparing metformin versus insulin versus

00:59:46.680 sodium glucose co-transporter inhibitor versus a ketogenic diet, what works best in getting the PI3

00:59:53.480 kinase inhibitors to shrink the tumor? And I want to just pause for a moment. This is something you

00:59:58.180 alluded to in passing 30 minutes ago, which was very important, which was insulin can go around

01:00:05.040 the PI3K inhibitor when you block it. And that's why just blocking PI3K alone wasn't going to be

01:00:12.480 enough. Is that correct? More accurately, we know that if you take a tumor that has PI3 kinase

01:00:21.160 mutations, for example, ex vivo. So we did a lot of this work in organoids from our human patients at

01:00:27.400 Weill Cornell, and we would take an organoid, an endometrial tumor, for example, and give it a PI3

01:00:33.560 kinase inhibitor at the therapeutic dose, and we could kill every cell in that organoid. But if we

01:00:40.340 now added back the level of insulin that would be in the bloodstream... At the level of glucose that

01:00:46.080 they would have experienced. And the level of insulin that we actually measure in the blood of the

01:00:50.320 patient 30 minutes after giving them the inhibitor. How high is an insulin level typically in that

01:00:55.120 patient? So 10 nanograms per mil is roughly what we see within 15 to 30 minutes. And by 90 minutes,

01:01:02.200 it's up to twice that, 10 to 20 to 30 nanograms per mil of insulin. And by two hours, the insulin

01:01:09.060 level is high enough that the glucose now in the bloodstream starts to drop. So that tells us that

01:01:16.180 that massive amount of insulin is enough to reactivate PI3 kinase in the liver and muscle.

01:01:21.920 And so it's not going around PI3 kinase. It's just reactivating it in spite of the presence of the

01:01:27.400 drug. Because you can't develop a complete inhibitor for physiologic reasons. That's right.

01:01:31.800 The dose that you can tolerate, fighting this battle, if you completely turn off PI3 kinase...

01:01:36.760 So insulin still has to go through PI3 kinase no matter what. You're putting up a porous dam

01:01:42.120 because a complete dam would kill the patient. Exactly. And insulin is tougher, and in the end,

01:01:48.120 it's going to win. So let's suppose you're giving an estrogen receptor antagonist

01:01:52.700 and you start getting toxic effects of losing estrogen. Would you decide that you would now

01:02:00.420 give estrogen to correct that problem? And so that's what the endocrinologists say. We want

01:02:08.300 to give them insulin to bring their glucose down. You're like, that's the whole thing we're trying

01:02:12.080 to stop here. That's what we're trying to stop is insulin because insulin is what's driving

01:02:15.460 the activation. Those mutations in PI3 kinase will not activate PI3 kinase unless insulin is added to

01:02:22.520 the tumor. And I recall, I think I even told this story. I may have told the story when I was talking

01:02:27.260 with Sid on the podcast, but we were having dinner one night and I talked about a friend of mine with

01:02:34.040 breast cancer who was in a trial and she was in an arm in one of the Boston hospitals and she was on a

01:02:40.400 PI3 K inhibitor. And I think it was a phase two. She was the only woman who survived. She's still

01:02:48.020 alive today. The only woman with metastatic breast cancer that survived who on her own went on a

01:02:53.660 ketogenic diet. And I remember telling you this out of curiosity, like Lou, what do you think of this?

01:02:58.880 Do you think this is just an odd coincidence? And that was the time that you and Sid were starting

01:03:04.840 these experiments. As Sid described it, that was sort of maybe the final clinical, the little pearl

01:03:12.260 that sort of made you guys go off and do this. And that paper was published a year ago, right?

01:03:16.720 That was published was last summer, last July? Last July, yep. So what was the efficacy of a ketogenic

01:03:21.680 diet versus an SGLT2 inhibitor and metformin? Were they equal? So if you did a run-in to the ketogenic

01:03:29.240 diet, so they have to have an entire week on the diet and then they're given the inhibitor,

01:03:33.900 it's incredibly effective because by then you've depleted all the glycogen, all the relevant

01:03:39.520 tissues. If you just start on a ketogenic diet a day before you give the first PI3-cannase

01:03:45.580 inhibitor, it's still pretty effective, but not as effective as the whole week run-in to deplete

01:03:52.720 glycogen. Is there any reason not to combine these ketogenic diet, metformin, SGLT2 inhibitor,

01:03:57.680 PI3K inhibitor as a FU to cancer? For a cancer is dependent on this pathway, of course.

01:04:03.220 Let me address the sodium glucose co-transporter inhibitor first, because that is almost as

01:04:08.800 effective as the ketogenic diet in our mouse models. It keeps the glucose level not quite

01:04:14.100 as low as the ketogenic diet, but pretty damn low. And the importance, of course, is in both

01:04:19.740 cases it brings the insulin, ambient insulin in the serum drops dramatically. But the important

01:04:25.200 thing is that every single 12 different types of cancers, some of them had PI3-cannase mutations,

01:04:32.060 others didn't. Some were RAS mutant tumors. Didn't matter. Some were, one was AML in Sid's

01:04:38.500 study.

01:04:39.680 That's right. I remember talking about that that night.

01:04:41.380 And I was skeptical that AML would have, because you never see a PI3-cannase mutation in AML.

01:04:46.700 But the bottom line was, there was a benefit in every single cancer we looked at. The reason we did 12

01:04:52.940 different types, we were trying to find one example of a cancer.

01:04:55.740 That would violate this principle.

01:04:56.940 That would violate the principle. The bottom line is, I think all cancers require some amount

01:05:01.080 of PI3-cannase to survive. And so if we can keep the insulin level low, and insulin, of course,

01:05:07.800 is the best way to activate PI3-cannase. If you can keep the insulin level low and now turn off PI3-cannase,

01:05:13.060 you don't get that rebound of insulin and the tumors just go away. So ketogenic diet, if you can stay on

01:05:19.640 it, would be my recommendation if you have cancer and you're going on a PI3-K inhibitor.

01:05:24.900 Sodium glucose co-transporter inhibitor, however, is easier for most people to take. It's a pill.

01:05:30.020 And that's, I would say, the second best.

01:05:32.600 And of course, again, it's hard, because we're sitting here, we can't give medical advice to

01:05:36.100 people. I get asked this question all the time. It's a very difficult question to answer,

01:05:39.960 because I'm not managing the given patient. But cancer patients are among the most motivated

01:05:44.920 patients you'll ever meet. I mean, you know this, you've been involved in these trials.

01:05:48.520 They will do anything they believe has some modicum of science behind it that can increase

01:05:53.020 their odds of survival. Tragically, we're still kind of living in a world where

01:05:58.460 the mainstream approach to this is a little bit convoluted. Obviously, one of the most

01:06:04.660 significant concerns that many oncologists have, well-meaning, is that patients are going to lose

01:06:09.840 weight. I don't know how bad it is today, but certainly back when I was on the wards,

01:06:14.320 we would force-feed cancer patients with Ensure. I don't know if you've ever had Ensure,

01:06:19.360 Lou, but let me ensure you, it is full of sugar. So now we're back to force-feeding patients liquid

01:06:24.940 sugar when they have cancer, because we're afraid of them losing weight.

01:06:28.060 Exactly. So one caveat, or one concern, which came out of the SIDS part of our trial,

01:06:35.840 is that, at least in the mouse models of AML, ketogenic diet alone...

01:06:40.800 It was actually negative, wasn't it?

01:06:43.280 Ketogenic diet alone in most of the tumors doesn't have too much. So just switching to a ketogenic diet,

01:06:48.300 I think it's rare that that alone is going to work, maybe in some cases. But in the case of

01:06:54.040 the AML, there was some evidence of some toxicity that came in the context, not, you know, normal

01:07:00.960 mice, ketogenic diet, they're perfectly healthy. They live a normal lifespan, in fact, probably live

01:07:05.440 a little longer. But in the context of this AML model that SIDS lab was using, there could be some

01:07:11.580 adverse effects. So we're cautioning people, don't automatically go on a ketogenic diet if you have

01:07:17.540 cancer unless you couldn't have somebody paying attention to potential side effects.

01:07:21.420 Well, I had the luxury of sending... I mean, you've been so generous, and I've sent many tissue

01:07:27.180 specimens to you from patients or friends, relatives of patients, and you've got your own CLIA-developed

01:07:33.420 assay to look for insulin receptors. And we've even made clinical decisions based on that, which is,

01:07:38.820 this is a patient who has lots of insulin receptor on their tumor. This is a favorable approach

01:07:43.960 to cancer. This is a patient who doesn't, might not make that sense. How far is that type of an

01:07:49.660 assay away from being a mainstream assay that any physician can use as part of their toolkit to start

01:07:57.240 to customize cancer therapy? To reveal my conflicts here, I do have a company I've started,

01:08:03.660 Petro Pharmaceuticals, that is beginning a PI3 kinase inhibitor trial. It will be combined with

01:08:09.460 sodium glucose co-transporter inhibitor.

01:08:11.300 Oh, I didn't even realize that. I'm bringing up all this stuff. I'm teeing it up for you here,

01:08:14.640 right? Yeah. That trial will be starting any day now.

01:08:18.240 What type of cancers, Lou? Are you looking for enrollment?

01:08:20.780 So in phase one here, it's going to be a combination with sodium glucose co-transporter

01:08:24.740 inhibitor plus PI3 kinase alpha inhibitor. Initially, we'll be looking at solid tumors,

01:08:30.320 breast endometrial, where you see high rates of mutations, but we won't... 0.79

01:08:33.700 So breast endometrial in what stage of disease? Is this stage four?

01:08:37.180 Yes, in stage four disease.

01:08:38.380 So have you fully enrolled or...?

01:08:40.900 No, no. We're just... The trial is just... Hasn't enrolled.

01:08:43.140 So in other words, if someone's listening to this that either has or knows someone with breast or

01:08:47.160 endometrial cancer who is stage four, how can they best find out information on this trial?

01:08:52.660 So Petro Pharmaceuticals. So of course, this will be registered at clinicaltrials.gov,

01:08:57.000 where you can find every single trial. So if you just go to clinicaltrials.gov and

01:09:01.320 type in Petra, P-E-T-R-A, that will...

01:09:05.040 We'll link to that for sure. How many patients will you take in the phase one?

01:09:09.060 I've forgotten the exact number, but it depends on how many responses we see and so forth.

01:09:14.180 But the dose escalation...

01:09:15.480 The dose escalation is going to be pretty quick because we already have... We know what the dose is

01:09:20.800 without a condition.

01:09:21.700 Each of these drugs, you sort of know what they do.

01:09:23.660 We know. We don't anticipate any toxicity.

01:09:25.380 So the hope is to get to phase two quickly.

01:09:27.260 Yeah. We should get to phase two very quickly. And the person patients on phase one could

01:09:31.240 transition into the phase two.

01:09:32.900 This is really exciting, Lou.

01:09:34.660 So in that case, we will recommend that they have for breakfast because all these drugs,

01:09:39.660 most of these drugs require you to eat breakfast before you take the pill because that limits

01:09:43.280 some of the toxicities you get and increases the absorbance. But I think it makes a hell of a

01:09:48.520 lot of difference what you have for breakfast.

01:09:51.240 So you'll recommend scrambled eggs and avocado and...

01:09:55.360 Whole milk yogurt and not yogurt with sugar.

01:09:58.040 Yep. Yep.

01:09:58.980 Or a high-fat yogurt and...

01:10:00.580 High-fat yogurt.

01:10:01.460 With some crunched almonds in it or something like that. Yeah.

01:10:04.080 Right.

01:10:04.580 Now, Lou, is there any way to pair...

01:10:06.200 Absolutely no juice, no insure...

01:10:08.640 Yeah.

01:10:09.100 ...in that time of the morning. Now, by the evening, the drug will have already be below

01:10:13.960 effective dose. So one might have some elasticity for what you had for your evening meal.

01:10:19.000 Although I will tell you something, Lou, you probably see this thing on my arm, right?

01:10:23.220 Mm-hmm.

01:10:23.480 That's a glucose meter.

01:10:24.900 Yeah.

01:10:25.060 I know what a nighttime high-glucose meal, I know what a dessert at dinner can do to my

01:10:30.460 glucose in the morning and my insulin, and it's still high. So for people listening to

01:10:35.620 this who want to enroll in this trial, or even for you, Lou, I would just say the more stringent

01:10:40.040 people can be, based on what you're saying, if the hypothesis that's being tested is more

01:10:45.660 insulin is worse, I think you're almost better off just biting the bullet and saying, we're 0.93

01:10:50.740 going to do ketogenic diets. In fact, is there any probability in this trial of actually providing

01:10:57.580 the meals to the subjects to make it easier, you know?

01:11:00.960 So I have another conflict here. I've also started a company that is making meals for dietary

01:11:06.800 intervention in cancers in general. And this company, also Sid Mukherjee is a co-founder of

01:11:12.860 this company as well, called Faeth Pharmaceuticals.

01:11:16.420 How do you spell that?

01:11:17.360 Faeth, F-A-E-T-H, which is a Welsh name for health, F-A-E-T-H. So this, my co-founders are Karen

01:11:26.900 Valsden and Oliver Maddox from her laboratory, Ben Hopkins and Marcus Goncalves from my laboratory.

01:11:34.020 These are a lot of the folks that were on that paper last summer.

01:11:36.580 Right.

01:11:36.900 Ben was the first author, right?

01:11:38.060 Right. Sid Mukherjee, also founder of this, also Scott Lowe and Greg Hannon. I think I got everybody.

01:11:44.660 And the idea here is for patients who have cancers, which the KD will be potentially a viable

01:11:53.680 adjunct to what they're doing. The idea is let's pharmaceuticalize it basically. So is

01:11:59.040 Faeth already selling product?

01:12:01.000 No, it's just now getting started. We've gotten it funded and we're looking now for

01:12:05.680 where we're going to be making the meals and how and whether we contract some of the meals out or

01:12:10.420 we do them internally. This isn't going to be purely ketogenic, although we will focus on

01:12:15.920 preparing the meals, make sure they have exactly the composition that we expect rather than

01:12:20.320 just giving advice and hoping people find the right foods. But it's not purely ketogenic. In

01:12:27.260 some cases, there's evidence coming out of Karen's lab and also my lab that reducing serine levels

01:12:33.380 can make some drugs more effective. Reducing methionine levels can make other drugs more

01:12:39.720 effective in different mutational backgrounds.

01:12:42.460 Say more about those two. I mean, those methionine and leucine might be two of the more potent

01:12:47.140 activators of the target of rapamycin, TOR. Tell me about serine. That one's a bit,

01:12:52.120 we don't hear about that as much.

01:12:53.200 So serine, some tumors rely on synthesizing, can either use serine in the serum or they can

01:12:59.820 synthesize their own serine. So the enzyme phosphoglycerate dehydrogenase, PHEDH, is the first

01:13:07.820 step in converting intermediates of glycolysis into serine and glycine synthesis. You need serine and

01:13:14.020 glycine in order to make nucleotides as well as glutathione. So for redox potential and for

01:13:20.760 combating ROS rather, and also for building nucleotides, you need serine and glycine.

01:13:28.600 So you're trying to selectively deprive a cancer cell from nucleotide precursor.

01:13:32.580 That's right.

01:13:33.500 How difficult is it to restrict methionine and serine in food?

01:13:38.480 So this has been done both in humans and in mice. Diets have been tried to, and you can get this

01:13:45.120 serine level down about tenfold and still have quite viable human.

01:13:50.740 And what about methionine?

01:13:51.940 Similar. You can also-

01:13:53.220 Without just restricting protein.

01:13:55.480 Right. We've been using diets for metabolic disease intervention for years. Children lack an

01:14:01.400 enzyme in particular.

01:14:02.540 Sure. Ketogenic diets for epilepsy and things like that.

01:14:05.960 But even kinonuria, PKU, you need to reduce the phenylalanine in the diet. So this idea that

01:14:14.680 you could get better clinical outcomes by changing amino acids, for example, in your diet has been

01:14:22.240 proven clinically in born areas of metabolism. So the idea that the same kinds of interventions,

01:14:28.640 in fact, ketogenic diets, as you say, were developed not for cancer, but for epileptic seizure

01:14:33.420 syndromes. Yeah. Have you seen the data that have suggested, this is a little changing gears.

01:14:39.280 I don't want to let you leave without at least some discussion of this, that in certain cancers,

01:14:43.660 when you restrict glucose, which would then imply potentially a ketogenic diet, you actually make

01:14:49.160 the cancers more robust. And in particular, in pancreatic cancer. Have you seen some of these data?

01:14:54.980 Actually, I've seen the opposite in pancreatic cancer. If you use a ketogenic diet in combination

01:15:00.320 with a drug. So in our hands, putting a mouse that has a KRAS, P53 mutant pancreatic cancer,

01:15:08.400 which is the majority of human cancers have those mutational events. So in that context,

01:15:14.200 ketogenic diet doesn't really do anything. It doesn't accelerate, it doesn't slow it down.

01:15:19.000 But if we combine that with a PI3 kinase, and a PI3 kinase inhibitor doesn't do anything to those

01:15:23.540 tumors either. But we give the two together, the tumors completely disappear. So you have to

01:15:28.540 combine the diet with the right drug, or it doesn't really work.

01:15:33.820 And it's really this diet-drug pairing that is really on the cusp of innovation right now.

01:15:40.300 We're scratching the surface of that, right? And will these two companies,

01:15:44.300 Fayeth and what was the other one?

01:15:45.760 Petra.

01:15:46.320 Petra. Is the goal that these companies collaborate and start to pair diet with drug?

01:15:51.000 Yes, we could certainly do that at this point. As I say, for Petra, we focused

01:15:55.760 on the sodium glucose co-transporter inhibitor because we think compliance will be easier to

01:16:00.600 monitor. And at this stage, we aren't yet revved up to produce the diets that would allow those

01:16:07.060 trials to continue. But yes, we could move on to a ketogenic diet, and even possibly on top of

01:16:13.160 sodium glucose co-transporter inhibitor. But we'd have to do a phase one study.

01:16:17.840 Yeah, you still have to go back to phase one.

01:16:19.640 Phase one B.

01:16:20.480 How many PI3K inhibitors are currently approved by the FDA?

01:16:24.740 So there's the alpalisib, the Vardis drug just approved in May that hits specifically only

01:16:31.080 the alpha isoform. Now that's the isoform that insulin activates and mediates everything insulin

01:16:36.120 does. And that's the one that's caused all the problems with hyperglycemia. But there is also a

01:16:42.420 delta isoform of PI3 kinase that's seldom mutated, but it's actually required for B-cell growth.

01:16:50.300 And we discovered that back when we started knocking out PI3 kinase in hematopoietic

01:16:55.940 lineage cells in mice back in the late 1990s. David Fruman, a postdoc in my laboratory, was doing those

01:17:05.140 mouse knockouts and found that B-cell lineage cells failed to thrive if you knocked out PI3 kinase.

01:17:13.840 T-cells, on the other hand, were fine if you knocked out PI3 kinase. And ultimately, it turned

01:17:18.280 out to be the delta isoform that was critical for the growth of B-cells. So when delta inhibitors

01:17:25.580 were developed, they went into B-cell lymphomas as the first possible place to work since that

01:17:32.160 enzyme was already known to be critical in B-cells. And they turned out to be effective.

01:17:37.140 Adel-elisib was approved about five years ago. It's a delta-specific PI3 kinase inhibitor.

01:17:43.680 Who made that?

01:17:44.880 That was made by Gilead.

01:17:47.380 Oh, Gilead. Okay. Yeah.

01:17:49.340 And then more recently, a drug called capanelisib was approved about three years ago by Bayer.

01:17:58.360 This is a drug developed by Bayer.

01:18:00.420 And which isoform is that?

01:18:02.160 Again, it also hits delta, but it also hits alpha.

01:18:05.620 So the alpha one is the only one that causes the hyperglycemia. Yeah. That's the one that

01:18:09.420 my friend was on, by the way.

01:18:10.780 Right.

01:18:11.220 But that was approved in May. And that seems to have the most ubiquitous application, doesn't it?

01:18:16.700 It's certainly where you see the most mutations. And of course, breast cancer is far more numerous

01:18:22.520 than lymphoma.

01:18:23.820 Yeah. But that's the one that's going to need the most management of the hyperglycemia and the

01:18:28.120 hyperinsulinemia. Is that message reaching the oncologists?

01:18:31.420 Slowly, I think they're beginning to understand what we're saying, why it's important to keep

01:18:35.960 the insulin down. The problem, as you know better than I, that we are so specialized today in medicine

01:18:41.900 that people commit to go into either oncology or endocrinology. And from there on, their studies

01:18:47.960 are completely, they're studying completely different things. And if you have an endocrine

01:18:52.180 problem as an oncologist, you don't try to solve it yourself. You call an endocrine consult.

01:18:57.500 So your brain isn't really even focusing on how to solve a problem because you have an expert

01:19:03.700 who can do it for you. And the problem is the endocrinologists are not really that alert about

01:19:10.780 what's going on with cancer. Their job is to make sure the glucose is in line.

01:19:15.800 Yeah. Sort of, I think it's time that we either, I mean, there's really two ways around this. I see

01:19:22.040 one is you sort of create a new specialty, which is metabolic oncology. So just today, if someone

01:19:28.420 wants to study oncology, they choose between surgical oncology, medical oncology, radiation

01:19:32.840 oncology. Of course, the challenge is you have to differentiate very early. You don't do oncology

01:19:38.160 first, then surgery, then medicine, then radiology. It's you do radiology or medicine or surgery and

01:19:44.040 then subspecialize. So therein lies a problem. How would you become a metabolic oncologist?

01:19:48.300 So the other option is basically oncologists learning more about the metabolism of cancer

01:19:55.320 so that when it comes to managing understood and predictable complications of these medications,

01:20:01.160 which are actually desired features, as you point out, if you're using this drug correctly,

01:20:06.160 you should see hyperinsulinemia and hyperglycemia and you should treat it accordingly.

01:20:10.040 Which reminds me, by the way, how often with the alpha isoform does that drug need to be

01:20:14.760 administered or in your trial? The decision of how frequently the dose is based on PKPD,

01:20:20.560 fast as the drug level goes into the bloodstream and how long it stays high and following how quickly

01:20:26.540 it declines. And so most of these drugs that make it in the clinic can stay at their therapeutic dose

01:20:33.380 for three or four hours. So a lot of the companies want to do buy two drugs a day, one in the morning,

01:20:39.360 one in the evening. And what's the total cycle? How long are you being given drug? How long are

01:20:43.740 you given a rest from the drug? And so, for example, the Novartis drug is daily. It's once

01:20:48.640 a day daily after breakfast on a full meal. And why does it need a full stomach? Because otherwise

01:20:53.900 absorption problems, you don't get as good a PKPD if you eat it on an empty stomach and you get more

01:20:58.860 side effects, diarrhea or nausea or whatever, if you try to do it on an empty stomach. But there's no

01:21:06.040 recommendation in the approval trial as to what you're supposed to eat for breakfast before you

01:21:10.600 take the drug or whether you take it with insure or apple juice or water. So hence the importance of

01:21:17.000 a company like Fayeth that could start to standardize that. Because look, everybody wants to do the right

01:21:21.980 thing here. And wouldn't it be great if as a doctor you could write a prescription that's not just

01:21:25.960 covering the drug but covering the food? Yeah. So that's what we're anticipating at Fayeth at some

01:21:31.200 point if you can prove in a clinical trial setting that you need this particular food for the drug to

01:21:36.340 be effective. To maximize absorption, minimize symptoms, side effects, and most importantly,

01:21:39.880 maximize efficacy. You should be able to get that food paid for, the delivery of the exact food that

01:21:46.240 matches what your need is. And think about it. What a trivial cost it is compared to the drug. I mean,

01:21:50.980 you could send people caviar spread all day long at one one hundredth the cost of a drug.

01:21:58.300 Exactly. But you have to prove it. You've got to prove it in a phase three clinical trial.

01:22:02.460 Well, don't get me started on that. I mean, think of how many drugs have been approved that haven't

01:22:06.120 proved jack. I mean, it's Ambien should have been approved. Anyway, don't get me started on the

01:22:10.800 drug approval process here, but I'm completely with you on that.

01:22:14.760 Lou, I want to be kind of mindful of your time. There's kind of one last theme I want to explore.

01:22:18.980 So you're going to be 70 this year, right? Which is kind of hard to believe because anybody-

01:22:22.760 I already passed it.

01:22:23.340 You already passed it. So you don't look a day over, I don't know, 56.

01:22:27.640 You've got this ageless phenotype to you. So I don't want to, for a moment, suggest your career 1.00

01:22:34.460 is anything other than continuing to go up in terms of the work you have to do.

01:22:39.600 So as you think about the next chapter of your career, what is the question that you are most

01:22:46.080 obsessed with? What is the question you want to spend as much time as you can trying to answer

01:22:51.460 that currently we really don't have an answer to?

01:22:53.480 Yeah. That's hard for me because I'm the most unfocused. In some ways, I'm compulsive about

01:22:59.260 getting the answer of every question that I get curious about. And as you can tell from this

01:23:04.740 interview, I have an opinion on how everything works. And having never taken any biology course,

01:23:09.900 I have no prejudice about how it should work. I just, so for me, everything that I run into biology

01:23:16.220 is, here's a mystery. I don't bother reading the textbooks because I know they will prejudice me

01:23:21.320 to the wrong answer. And so I'd like to get to the bottom of what's really going on. But the bottom

01:23:26.020 line is that I get absolutely brilliant postdocs and graduate students come to my laboratory.

01:23:32.200 I allow them to do anything they want to do. And if it obviously gives them advice and help

01:23:39.280 interpret the experiments. But this has kept me in so many different fields for 40 some years as I've

01:23:47.540 run my own laboratory. It keeps my mind continually alert about every possible thing. So yes, epileptic

01:23:55.040 seizures. It turns out probably most of the people have epileptic seizure fits, have PIC3CA mutations as a

01:24:01.160 mosaicism in neurons in the brain. Ketogenic diet was developed to minimize that. It makes perfect

01:24:08.180 sense. Keep insulin down. If a PIC3CA mutant neuron was so, you're going to get less firing of that neuron

01:24:14.740 that's going to solve the problem. But that wasn't how the ketogenic diet was developed. So my curiosity,

01:24:22.240 if just studying everything that's, which PI3 kinase is involved at some level, could keep me busy for

01:24:27.520 several years. But that's not the only thing I'm interested in. I'm interested in whatever curious

01:24:33.640 observation that comes up from experiments in my lab that can't be easily explained by current

01:24:39.980 knowledge. To me, that means there's an opportunity to break open some new explanation for some disease.

01:24:46.500 So I'm willing to work in almost any disease that's not yet solved. And there's an opportunity to figure it

01:24:51.940 out. I'm a chemist. So to me, unless I understand something at the molecular level, I don't really

01:24:57.540 understand it. But I think with PI3 kinase, I really understand it at the molecular level.

01:25:02.820 I think that's an understatement.

01:25:04.260 Very few. There's still some mysteries there. I should say that we're about to publish a couple of

01:25:10.340 papers, hopefully over the next six to nine months, that are going to completely change how everybody

01:25:15.740 thinks about PI3 kinase regulation. So there's still some major things to learn about this enzyme.

01:25:20.940 Well, there's so many other things I'd love to speak with you about, Lou. But I know that some

01:25:24.540 of them are still work that you're doing. And therefore, I know you would prefer to wait until

01:25:30.820 things are a little further along to talk about them. But we'll have to come back in a year or so

01:25:35.680 and do this again, because there's so much other stuff that I know we've talked about informally about

01:25:40.420 cancer that is, I want to believe it's where the field is going to start to go. I want to believe

01:25:46.580 that because I get so many emails from people who say, you know, my mother has cancer or my

01:25:53.180 husband has cancer and they're being offered this therapy. And it's a very standard therapy.

01:25:58.880 The patient is progressing through it. What metabolic options are there? And people like

01:26:05.800 you are people like said, people like all your colleagues and Matt and Ben and all these guys.

01:26:10.780 I mean, you guys are the guys that are kind of driving that forward. And you've made a lot of

01:26:16.400 progress in a decade. It doesn't feel like it sometimes. Sometimes I feel like it's not going

01:26:20.040 fast enough. But I think having a discussion like we did today makes me realize how much has happened

01:26:24.700 in a decade. And it's actually quite a bit. I have no doubt that you're going to be here a decade from

01:26:29.780 now working just as hard. Well, my real motivation, of course, is to convert every observation we make

01:26:35.660 in a laboratory to actually change in practice for whether it's diabetes or cancer or what other

01:26:41.720 disease that we begin to understand a molecular mechanism. If we don't convert those observations

01:26:48.340 into actual change in behavior or change in practice or change in drug metabolism and diet,

01:26:55.800 then in the end we've failed. And so that's my goal. And as being a cancer center director,

01:27:01.780 people sometimes listen to me now when I suggest this is the way, this is the trial we should do

01:27:08.500 and this is the way it should be designed to test the idea. For me, it's spectacular being here

01:27:15.100 in New York City at Weill Cornell, New York Presbyterian, where we really can translate these

01:27:20.620 breakthroughs into new therapies. That's what my goal will be over the next 10 years.

01:27:31.780 I hope you enjoyed that discussion with Lou Cantley, and I hope you found it half as interesting

01:27:36.200 as you can probably tell I did. As you may recall, at the outset of this podcast, I said that the first

01:27:41.960 45 minutes, which was a much more detailed technical description of Lou's initial work in the 70s and

01:27:49.420 80s that led to the discovery of PI3 kinase, that content would be bumped as almost an appendix to the

01:27:55.760 end of the podcast. And that's what you're about to hear now. So I hope you enjoy what was originally

01:28:00.040 the first part of my discussion, but ultimately the appendix to this fun discussion with Lou Cantley.

01:28:10.740 There are a lot of things I want to talk about today, but I do want to start with the discovery

01:28:17.080 that you led that probably most people would associate with you. If they go to your Wikipedia

01:28:22.500 page, that's probably the first thing they're going to see. Although I haven't been to your

01:28:25.480 Wikipedia page in a while, so I don't recall. But let's talk about PI3 kinase. How do we define

01:28:31.760 it, by the way? Tell people what it was. So PI3 kinase stands for phosphoinositide 3 kinase.

01:28:37.860 And the 3 means that it phosphorylates the 3 position on the inositol ring, which is the head group of this

01:28:44.820 lipid. And so in the mid, early mid-1980s, phosphatidyl inositol phosphorylation, which was known to occur

01:28:55.480 from 1949, the purpose of it was totally ambiguous. Why did this lipid get phosphorylated? And in early

01:29:04.780 1982-83, there was a breakthrough with the discovery that the phosphorylated form, phosphatidyl inositol

01:29:14.180 with phosphate at the four and the five positions of the inositol ring. By the way, inositol is

01:29:20.640 hexahydroxycyclohexane. So there's six hydroxys. One of them connects to the glycerol backbone,

01:29:27.860 and then the others are potentially available to be phosphorylated. Anyway, the four position and the

01:29:34.320 four plus five position were both identified the year I was born, 1949. And the purpose of that was

01:29:42.620 not known. It was called the futile cycle, maybe a way of just getting rid of-

01:29:46.300 Getting rid of phosphates or something.

01:29:47.620 Or getting rid of ATP.

01:29:48.740 Yeah, yeah.

01:29:49.080 Just phosphorylated and dephosphorylated and-

01:29:51.700 Sort of like an ATP sink.

01:29:53.080 And then the idea, the observation that this phosphorylation could be stimulated by

01:29:58.380 various GPCR pathways and growth factor pathways got people thinking about what it might be doing.

01:30:05.200 And it tended to correlate with calcium elevation. And so the breakthrough paper showed that, in fact,

01:30:12.180 when cells are stimulated with certain growth factors, like EGF, or a certain subset of GPCR

01:30:19.020 activators-

01:30:19.760 And those stand for-

01:30:20.940 G-protein coupled receptors.

01:30:22.740 Mm-hmm. And EGF just for folks.

01:30:24.740 Epidermal growth factor.

01:30:26.200 Okay. So all growth.

01:30:27.500 And so these signaling pathways would activate the hydrolis of that lipid, that the inositol

01:30:35.680 1,4-5-trisphosphate that comes off when you hydrolyze it away from the lipid turned out to

01:30:42.980 regulate calcium release from cells. And that was a huge breakthrough because no one knew how

01:30:47.540 calcium got elevated, and that explained it. So I, in the meantime, was not working on that

01:30:53.400 particular pathway.

01:30:54.760 And let's pause for a moment and remind everybody, you trained, you were interested in science from a

01:31:00.060 young age, but you were sort of deciding, I think, a little bit, if I recall, between chemistry and

01:31:05.000 biology. You were very interested in both, correct?

01:31:07.440 I was a pure chemist. The last biology course I took was in 1964 when I was a sophomore in high school.

01:31:14.420 So you very quickly declared chemistry your obsession.

01:31:16.920 I hated biology because at that time, at least in the backwoods of West Virginia where I was growing up,

01:31:22.000 biology was just a bunch of descriptive memorization. And so I was bored with that and decided I would

01:31:29.760 never take another biology course, and I stuck to that. I was pure chemistry, mainly organic chemistry,

01:31:36.260 and then I switched into physical chemistry, but was totally uninterested in biology at all until I took

01:31:43.060 one semester of biochemistry. So I took one semester of biochemistry and realized maybe there's something

01:31:48.000 interesting going on in biology. So I went to Cornell in Ithaca to get my PhD, and there I focused on

01:31:56.200 biophysical chemistry. It was a chance to apply chemistry to questions like, how do you get molecules across

01:32:04.180 membranes? And that's what I worked on. How do you synthesize ATP in the mitochondria or chloroplasts?

01:32:11.560 So that kind of the slippery slope of going into biological questions that could be addressed by

01:32:17.040 simple chemical questions or physics questions.

01:32:19.940 Now what got you interested in the mitochondria so early?

01:32:22.280 I read a paper when I was an undergraduate that was an idea that Peter Mitchell had proposed,

01:32:28.080 that the way you make ATP in the mitochondria and or chloroplasts was using a proton gradient.

01:32:36.000 And his idea was that that gradient would allow you to pull the protons in one direction,

01:32:41.620 the hydroxy ions in the other direction, and remove water from phosphate plus ADP and condense that into

01:32:50.040 ATP. That was just very simple physical property that if you could move those two ions in different

01:32:56.140 directions, it would produce ATP. In effect, the idea was not correct. It was correct that ATP was made

01:33:03.440 from the proton gradient, but the actual mechanism turned out to be different. There are actually

01:33:08.900 three Nobel Prizes given for how ATP is synthesized in the mitochondria.

01:33:13.380 I wasn't aware that there were three separate Nobel Prizes. Do you recall who won all of them?

01:33:17.640 So Mitchell.

01:33:18.180 Mitchell, obviously.

01:33:19.160 Peter Mitchell got one. Then there was a second one by Boyer,

01:33:21.320 only figuring out the actual mechanism by which this occurs. And then a third one from Peter Walker,

01:33:27.500 who figured out structural basis for how this all worked.

01:33:31.580 Oh, I didn't know the Walker one. Okay.

01:33:32.880 He was a crystal structure, did crystal structures. And I met all of them when I was a young man before

01:33:38.880 becoming a professor. And so it's, I was very invested in that. That's what I worked on.

01:33:44.840 For the person who's listening to this, whose world is not necessarily wrapped up in this,

01:33:48.240 which is most people. It's worth pausing for a moment on how much is involved for the Nobel

01:33:54.720 committee to recognize one body of work that says something that three times they would recognize

01:34:01.260 a different angle of the same body of work probably speaks to why there is so much to this day,

01:34:08.880 complete interest and fascination with the mitochondria beyond just the obvious, the energetics,

01:34:14.440 which is what was initially how we would think about it.

01:34:16.720 Well, it was a fascinating question. Uh, in the 1960s, this was the biggest mystery

01:34:21.140 because you, we knew how you made ATP from glycolysis, but how you made it in the mitochondria

01:34:26.420 and how could the mitochondria do it so much more efficiently than glycolysis? That was the big

01:34:31.640 question when I was a graduate student. I think this podcast may represent the point at which

01:34:37.480 mitochondrial topics exceed all others. It also speaks to that nature. So Mitchell, the great,

01:34:44.040 tell me about how you interacted with Mitchell. How did you come across him?

01:34:46.960 So he came to give a talk at Cornell. Okay. And I should say F. Racker, Ephraim Racker, who was

01:34:53.080 chaired the biochemistry department at Cornell and who I got to know as first year graduate student

01:34:58.460 because I was getting mitochondria from his laboratory in order to purify the enzyme that's

01:35:05.620 synthesized ATP. Every time I would be waiting for the centrifuges to stop running, if he saw me,

01:35:11.260 he would grab me and pull me into his office and start throwing out ideas of how he thought ATP was

01:35:17.100 synthesized. And the truth was at that time, he did not believe Mitchell's chemiosmotic hypothesis

01:35:23.740 Peter Mitchell was proposing. And so as a consequence, I got all of these ideas that he was throwing at me.

01:35:31.340 I personally believed that chemiosmotic hypothesis was correct. Between those discussions,

01:35:37.300 as I continued to, I was working in physical chemistry, I was in the chemistry department,

01:35:41.020 not the biochemistry department. But the fact that he would spend so much time with me, even though I

01:35:45.220 wasn't even in his department, to me, was quite flattering and really exciting that this world

01:35:51.160 quality person had done it, was willing to talk to me. So he went on because he was trying to prove

01:35:58.320 Peter Mitchell wrong. He completely purified every component, generated a synthetic membrane,

01:36:05.360 reconstituted the proton pump into that membrane, generated a proton gradient, and was able to

01:36:11.720 quantitatively synthesize ATP. And it was the year after he published that paper saying that the

01:36:18.300 chemiosmotic theory is actually correct, but he did the definitive experiment that the prize went to

01:36:24.340 Peter Mitchell. Today, I would almost guarantee they would have shared the prize. Mitchell had the

01:36:29.620 idea, Racker proved that it was correct, even when he was trying to prove that it was incorrect.

01:36:34.980 So that was an interesting story that Mitchell was invited to come and give a talk at Cornell just at

01:36:40.440 that time, my last year as a graduate student. And so he met with me and my advisor, Gordon Hammes,

01:36:48.120 and I went through my entire PhD thesis with him and F. Racker, showing them the various experiments I did

01:36:56.040 about the mechanism by which this enzyme worked. It was really quite interesting because this was before

01:37:02.940 Racker had actually published the definitive proof paper, so he had still some questions. And every slide I

01:37:09.620 would show, Mitchell would say, that's consistent with the chemiosmotic theory. And Racker would say, no, but this

01:37:16.760 is why it's not right. And so I hardly got to say a word because the two of them argued every piece of

01:37:22.880 data I showed. But you provided the substrate for the argument. Yeah, it was the data from my PhD thesis.

01:37:28.960 That was a fun experience. So this is early 70s? It was 1974 when I met Peter Mitchell. At any event,

01:37:37.620 next year I decided, finishing my PhD, that I would look for a place that was asking questions that were a

01:37:46.740 completely into biology. So I was looking for someone who was working on, like, red cells or

01:37:50.440 something that was a pretty simple study and ended up going to Guido Guadagni's laboratory,

01:37:56.000 who had focused on membrane biochemistry. He was an MD-PhD, but he understood medicine quite well.

01:38:03.720 But his real focus was in biochemistry. He was not a practicing clinician. And he was in the Department

01:38:08.860 of Biochemistry and Molecular Biology at Harvard, recruited there by Jim Watson.

01:38:12.760 And he was absolutely, is still a brilliant biochemist. I learned a lot from him. And he was

01:38:20.560 working on sodium-potassium ATPase. It had first been purified in his laboratory, the enzyme that

01:38:27.220 pumps sodium out of cells and potassium into cells. And since I'd already worked on how you pump protons

01:38:34.740 into cells with an ATP molecule, I thought this would be easy for me to understand. I knew how to do

01:38:41.400 those kinds of assays. So that's why I started working on it. But it turns out that Guido was

01:38:47.580 also very interested in how insulin worked. Because whenever you add insulin to cells,

01:38:53.300 within seconds to minutes, they take up a massive amount of glucose, but they also turn up the sodium

01:38:59.680 potassium ATPase, the sodium pump, use that sodium gradient to move amino acids into cells.

01:39:05.920 And so there was a lot of membrane transport being regulated by insulin.

01:39:11.060 Was it understood at that time how anabolic insulin was? What you've described are anabolic

01:39:16.400 attributes, but was it understood clinically how anabolic the hormone was?

01:39:22.100 Yes, it was because just by observation that type 1 diabetics failed to thrive. They were very small. 0.54

01:39:29.460 They did not put on weight. Their cells didn't grow. And so during development, insulin is an anabolic

01:39:36.860 molecule. In a mature adult, it no longer, except under certain situations, drives growth. It will

01:39:44.320 continue to drive growth of fat cells. It can also facilitate growth of muscle. But most tissues do not

01:39:51.460 grow further other than those two major tissues. So yes, it was well known. It was an anabolic process.

01:39:59.500 But now you were seeing the steps by which it could carry out that anabolic property.

01:40:03.980 Yeah. And it was generally known. What I just said was generally known that amino acids uptake

01:40:08.720 accelerated immediately within a minute or so. Sodium export of the cell went up and glucose flux,

01:40:16.460 of course, went in, particularly in muscle and fat cells. And glycogen was stored. And then,

01:40:21.560 of course, there was a whole lot of change in transcriptional regulation of all kinds of things.

01:40:25.920 So we knew all of that. But the actual mechanism was totally unknown. In the mid-1970s, no one had

01:40:33.920 even purified the insulin receptor. They had no idea what the receptor was. People could quantify it

01:40:39.840 because if you added radioactive insulin to fat cells, it would stick to the surface and with very

01:40:45.720 high affinity. And that correlated with the responses that you see. So we knew it existed. We knew roughly

01:40:52.600 how many copies there were per cell. We could quantify the rate of activity. But no one had

01:40:57.180 quite purified the enzyme to homogeneity. And so numerous labs were trying to purify it.

01:41:01.800 So eventually, the insulin receptor was purified. And it was about the same time that SARC,

01:41:07.520 the SARC tyrosine kinase, the SARC oncogene for Rals sarcoma virus was shown by Harold Varmus's lab

01:41:16.900 to be protein that had homology to PKA, a protein kinase. And that also was an endogenous protein

01:41:27.980 that had been picked up by the virus and altered. So that really was a major breakthrough. It got Nobel

01:41:34.380 prize for Harold Varmus. And he shared that one with Michael, right? Michael. Yeah. Michael

01:41:39.460 Bishop. Michael Bishop in 89. They shared it. 79, I think. Oh, but they won the prize in 89. Maybe,

01:41:44.920 maybe I'm. Oh yeah. The prize was. The prize was awarded. Yeah. The discovery was 78, 78. Yeah. Yeah.

01:41:51.220 So that was about the time the insulin receptor was being purified as well. And then Ray Erickson's

01:41:55.780 labs showed that SARC had a kinase activity. They thought it was a threonine kinase. They looked

01:42:01.160 for that possibility. Joan Brugge was his lab at that time.

01:42:04.380 Given the sequence homology to protein kinase A, that it was probably also a protein kinase.

01:42:10.840 And then Tony Hunter showed that actually by thin layer, the molecule that was thought

01:42:15.520 to be phosphatrhenine was actually phosphatyrusine. And phosphatyrusine had never been seen as the

01:42:22.640 product of a protein kinase before. It had been picked up as an intermediate in DNA unwinding.

01:42:29.100 And so he could get a marker for that from Jim Weying's lab that showed that he could use on a thin

01:42:35.800 layer and showed that what SARC was actually producing was actually phosphatyrusine. So that

01:42:41.100 opened up a whole field. And then comparing the sequence of the insulin receptor to PKA versus

01:42:47.900 SARC, it was clearly most highly related to SARC. And so multiple labs at that point showed that,

01:42:54.280 yes, the insulin receptor was like SARC, a tyrosine kinase. But in this case, a transmembrane protein

01:43:00.140 had an extracellular insulin binding component, and then the intracellular kinase activity.

01:43:06.240 So putting that in English for folks, insulin hits this transmembrane tyrosine kinase.

01:43:10.940 And when the molecule of insulin hits it, inside the cell, this kinase pathway kicks up. It moves the

01:43:17.900 GLUT4 transporter. Well, there's another step in there, but oversimplifying a little bit,

01:43:24.000 that chemical reaction is necessary to translocate this transporter across the cell to bring glucose

01:43:28.460 in. And that's, so the leap there, the new insight was exactly how insulin got glucose in the cell

01:43:35.000 chemically and mechanically, right?

01:43:37.280 Right. So the initial observation was then insulin binds and about the same time EGF receptor was

01:43:42.840 purified and FGF receptor and PDGF receptor. And they all sort of were like insulin receptor.

01:43:50.660 They had an extracellular.

01:43:52.160 And this was back, I mean, today, a young student who's in a lab doing this now is scratching their

01:43:57.680 head going, what do you mean? Why didn't you just use PCR or something like that? But I mean,

01:44:01.540 you guys were literally what, crystallizing a protein or how were they actually figuring out what the

01:44:06.200 structure was.

01:44:07.280 So these proteins were all being purified to homogeneity by just plain fractionation. There

01:44:11.720 was no way to knock out genes and knock them down. So you just had to purify the protein based on

01:44:17.860 its ability to bind insulin. You just run over column after column after column, and eventually

01:44:22.220 you've got to a single band on a gel. And they said, that was your protein.

01:44:26.140 And that only gives you the primary structure of the protein. It doesn't tell you how it's folded,

01:44:29.600 does it?

01:44:30.180 No, that only tells you.

01:44:31.240 It tells you the molecular weight, basically.

01:44:32.540 The only thing you get is the molecular weight.

01:44:34.320 Yeah.

01:44:34.560 You see it's...

01:44:35.320 Here it is on the band, yeah.

01:44:37.440 Insulin receptor is, it gets cleaved, but it's...

01:44:40.040 It's a heterodimer, isn't it?

01:44:40.880 It's a single polypeptide that gets clipped during processing into two proteins, but are

01:44:48.240 held together by disulfide bonds. So it's a mid-dimer of that composition. So that we knew

01:44:55.860 late 1970s and EGF receptor and all these other receptors are all look a lot like the insulin

01:45:02.920 receptor. And they all had tyrosine kinase activity that was triggered by binding the

01:45:08.900 growth factor insulin. IGF-1 receptor also, very similar insulin receptor. One by one by

01:45:15.620 one, different labs purified them. They all found the same thing. You had the growth factor

01:45:20.280 of the cell and you get the tyrosine kinase activity activated on the inside of the cell.

01:45:26.740 You know, that was a huge breakthrough. But still, if you ask, when you triggered the cell

01:45:31.900 with insulin or EGF or PDGF, and you ask what was the major thing phosphorylated, it was

01:45:39.400 the receptor itself. So if you just ask, where's the radioactivity going? Phosphate, radioactive

01:45:43.740 phosphate, it goes mainly to the receptor cytosolic domain. It's autophosphorylated. So now we're

01:45:49.960 left with this several years of, we have a tyrosine kinase. It explains how all these growth factors

01:45:55.480 work, including insulin and how SARC works, but we can't find anything other than autophosphorylation.

01:46:02.200 How does that help? So the breakthrough ultimately then came with the observation that

01:46:08.120 these autophosphorylation or phosphorylation of adapter proteins were recruiting a host of other

01:46:14.660 proteins to the membrane that were actually doing the work that was required to drive the cell growth.

01:46:20.440 And so that was, again, early 1980s. These were all being figured out. So I got interested. As soon as I

01:46:28.880 saw that there was a different kinase activity, this tyrosine kinase, I got very excited about it

01:46:33.580 and realized there's an opportunity now if I can figure out what that tyrosine kinase does

01:46:38.180 to figure out what might be the downstream signal. So we got one step into the cell. We didn't know

01:46:43.960 how many steps we needed to explain glucose uptake or amino acid uptake, et cetera, et cetera,

01:46:48.980 or glycogen synthesis. So I saw a paper by Ray Erickson's laboratory in which he found an activity

01:46:58.020 associated with SARC that would phosphorylate glycerol. And he published it in JBC. It co-purified

01:47:06.900 with the protein through numerous steps of purification. And so in addition to having the

01:47:12.220 ability to phosphorylate tyrosine, antibodies that got phosphorylated on tyrosine, on the purified

01:47:19.100 protein, there was also an activity there that phosphorylated glycerol.

01:47:23.180 And I looked at that and thought, no, glycerol looks like half of an inositol. So inositol

01:47:30.800 is six carbons, glycerol is three in both cases.

01:47:34.120 But in the pure glycerol case, it has OH on each of the carbons?

01:47:37.780 Yeah, three carbons with an OH on it. And inositol has six carbons with an OH on all.

01:47:42.560 So if you took two-

01:47:43.860 Yeah, if you took the two rings and shoved them together, yeah.

01:47:46.060 Glued them together, you would get inositol. And then what the paper showed was the KM,

01:47:50.720 in other words, a 50% concentration of glycerol you needed for it to be phosphorylated,

01:47:58.840 was something like a hundred millimolar.

01:48:01.960 Enormous concentration.

01:48:03.260 Huge. Like a hundred to a thousand fold higher unit ever found in a cell.

01:48:07.240 Yeah.

01:48:08.320 So they weren't claiming this was a physiologically relevant.

01:48:12.000 I see. They were just saying, here's a chemical reaction that can take place.

01:48:15.700 And they just noticed it because they saw this molecule running on their thin layer that ran

01:48:19.780 faster than proteins. When they isolated, it turned out to be phosphorylated glycerol.

01:48:24.740 It was sort of out there. Here's, we saw this other activity. What might it be?

01:48:29.120 And so I went to Ray Erickson and said, well, that looks a lot like inositol. So why don't we-

01:48:35.340 Was Ray at Harvard at the time?

01:48:36.580 Yeah. One floor below me at Harvard.

01:48:38.380 So my graduate student, Malcolm Whitman, who knew how to do kinase assays on small molecules,

01:48:45.340 collaborated with Ray Erickson's postdoc to see whether phosphatidyl inositol might be a better

01:48:51.340 substrate than glycerol. And he tried it and sure enough, he got this KM now of like five micromolar.

01:49:00.240 Wait, wait, wait. How is this even possible? Let me make sure I understand what you just said.

01:49:04.040 But you take glycerol and you need a KM of a hundred millimolar to get it phosphorylated.

01:49:12.660 You simply take the same structure, but now it's basically two of them stuck together in a ring

01:49:17.740 and you get down to five micromolar.

01:49:19.960 And it's also on a membrane component. So the inositol is in a membrane bilayer.

01:49:25.120 So you sonicate the lipid and instead of free molecule floating around-

01:49:28.760 Oh, so it's not a free inositol. It's the inositol in the phospholipid or in the lipid.

01:49:32.780 Tell me from a chemistry standpoint, and I apologize. I know that for some of you listening

01:49:37.280 right now, you're thinking, wow, you guys are really in the weeds. I promise we're going to

01:49:40.540 get out of the weeds in a minute. But I also think this is just an interesting example of

01:49:44.760 the specificity of biology too. What is it about that lipid holding that? Is it the position with

01:49:50.520 which it holds the ring in place that enables that phosphorylation?

01:49:53.620 Well, now that we know the structure of PI3 kinase and how it works in its mechanism,

01:49:57.880 it's easy to explain in retrospect. But at that time, we didn't know that there was anything

01:50:03.400 there other than SARC. So it was hard to explain why the SARC, tyrosine kinase itself,

01:50:10.160 was carrying out this reaction.

01:50:12.380 What did the editors even say of that? I mean, that's one of those things where people are like,

01:50:15.500 is there a mistake here? Because that's like a five log difference.

01:50:19.880 Physicists had no problem understanding what I just said. If you confine things to two dimensions,

01:50:24.420 they can come together much more readily than if you do it, do it in three dimensions.

01:50:28.820 And that would account for the difference?

01:50:30.220 Yeah, easily. Try closing your eyes, bringing your two fingers together in space,

01:50:35.620 in three-dimensional space, and then do it again on the table.

01:50:38.600 When they're on the table, yeah.

01:50:39.440 You'll find each other.

01:50:41.360 Yeah, yeah, yeah.

01:50:41.680 So combining things to two dimensions is the way nature continually uses membranes.

01:50:46.060 And the lipids basically keeping it in a two-dimensional plane.

01:50:49.080 Yep.

01:50:49.340 Yeah.

01:50:49.840 So in any event, we're really getting in the weeds here now.

01:50:51.900 Just personally, I mean, all of that is intuitive to me. What's not intuitive to me is five log

01:50:58.440 difference. I would take a one log difference for what you said. That's what's amazing to me. I

01:51:02.720 didn't...

01:51:02.980 Well, there are other reasons that will become clearer as we move on. So the bottom line was

01:51:07.920 that that SARC preparation, which we assumed was completely pure, wasn't completely pure. It was

01:51:15.280 a certain amount of the SARC harvested out of the cell brought along the second enzyme,

01:51:21.900 which ultimately turned out to be PI3K, phosphoinositide 3 kinase. But we didn't know that at that time. And so

01:51:29.040 we suggested in the paper that the SARC, very same enzymatic pocket, could both accommodate

01:51:35.360 tyrosine as a substrate and also accommodate the head group of phosphatidyl inositol.

01:51:41.640 That, in the end, turned out to be incorrect in that even though by all criteria that we could

01:51:48.540 characterize at that point, we couldn't prove that there was a separate function. If you used a

01:51:53.880 kinase dead SARC or a mutant of SARC that no longer had activity, then the phosphatidyl inositol

01:52:00.160 kinase also went away. That's why we thought it was the same pocket that was doing both. Retrospect,

01:52:05.700 it turns out that the enzyme has to be active in order to bind to PI3 kinase. And that's why

01:52:11.020 that activity was coming along. So we published that. We're very, very clear to say that we used

01:52:17.240 sonicated membranes to do the assay, which was necessary to get this mini log preference for

01:52:23.280 that substrate. In the meantime, after publishing that, multiple other labs tried to reproduce it.

01:52:28.620 But they didn't have a machine that would sonify the lipids to make the membrane bilayers. So they

01:52:35.340 just added detergent. So even though we showed in our paper that if you added detergent, the enzyme

01:52:41.420 activity completely disappeared. Wouldn't the detergent break the glycerol off the lipid?

01:52:47.580 The phosphatidyl inositol would now get embedded in the detergent rather than being a bilayer.

01:52:53.260 Yeah, it wouldn't stick to it at all, right?

01:52:54.980 Right. So you're going to lose that advantage of having a membrane.

01:52:57.860 And that wasn't known at the time, I guess?

01:53:00.520 Well, we knew it because I'd worked on membranes for the previous 10 years.

01:53:04.960 But the other labs didn't, I guess at the time they didn't know that?

01:53:07.400 They were not membrane biochemists. They were all molecular biologists. So they tried to reproduce

01:53:12.340 the result, but they didn't have a sonifier to make membrane lipids. And they assumed, oh,

01:53:17.520 it phosphorylates the head group. And phosphatidyl inositol shouldn't make any difference how you

01:53:21.940 present it. And so they presented a detergent micelle and they could not reproduce the

01:53:27.780 results. So there were three prominent papers coming out a year or so later.

01:53:32.380 Saying, nope, this is incorrect. This is incorrect. This is incorrect.

01:53:35.280 From major laboratories. So at that point, it already started a collaboration with Tom Roberts.

01:53:40.260 But at that point, Lou, did you have confidence that you had done this correctly or were you

01:53:44.220 questioning yourself?

01:53:45.520 I had no doubt.

01:53:46.640 So you knew this was a methodologic error on the part of these other three labs.

01:53:52.040 And despite the fact that at this point, the scientific community would look at you and

01:53:55.140 say, Lou, you're probably wrong. You were confident in your methodology.

01:53:58.720 Yeah, there was no doubt. All my research had been on membrane enzymes and membrane reconstitution,

01:54:04.600 et cetera. So I knew what I was doing made sense. And I knew our results were reproducible

01:54:09.020 because not only could multiple people in my laboratory do it, but multiple people in Tom Roberts'

01:54:14.320 laboratory who we started a collaboration with could do it. And Brian Shafelson's lab, which

01:54:20.620 was a third collaborator, could also reproduce it. So multiple people in multiple independent

01:54:25.740 labs all got the same result. If you sonified the lipid and if you added MP40, a detergent

01:54:31.680 to solubilize the lipid, then the activity went away. So there was no doubt about that.

01:54:36.800 And so I had no lack of confidence that we were right. But the problem was how to convince

01:54:42.640 these other laboratories that they needed to do the assay right. They didn't want to buy

01:54:47.740 a sonifier. They were like, I'm done with that, went there, and then it doesn't work.

01:54:52.740 So we actually sent David Kaplan, the graduate student in Tom Roberts' lab, and Malcolm Whitman,

01:54:58.120 the graduate student in my laboratory, to these other laboratories with the sonifier.

01:55:03.640 They went there, they sonified the lipid form, gave them to their graduate students who then

01:55:08.980 did the assay again. And now they got the same result we got.

01:55:12.660 Are you pushing this hard to get the other labs to see it because you know how important

01:55:17.680 this is going to be? Or you have sort of premonition about the role that PI3K is going

01:55:22.200 to play in growth? I mean, you couldn't have possibly seen in the early 80s what you know

01:55:27.480 today. So how much of this was just scratching an intellectual itch versus a biologic intuition

01:55:34.440 about the importance of this to overall growth, which is obviously where we're going. I'm amazed

01:55:42.380 we are this far in and we haven't used the C word yet.

01:55:44.840 So the reason I was completely convinced by 76, 77 that this was driving growth of cancer cells

01:55:53.400 is the collaboration we started with Tom Roberts' lab. And Tom Roberts had been working on

01:55:59.280 polyoma middle T. So polyoma virus is a DNA virus, while SARC is an RNA virus. So it causes,

01:56:09.080 as the name implies, the formation of multiple tumors in mice. So you infect a mouse with this virus

01:56:15.800 and all kinds of tumors show up everywhere. And so Tom had began mutating various regions of

01:56:24.340 polyoma middle T. And middle T, the reason we looked at polyoma middle T is because his lab and

01:56:31.120 Sarah Courtney's lab and others in Schaffhausen had shown that SARC co-purifies with polyoma middle

01:56:37.380 T. We already knew by the mid-70s that SARC was somehow implicated in how polyoma middle T

01:56:45.260 transforms cells and forms all these tumors. That's why I went to collaborate with Tom, because he had

01:56:50.040 additional tools that we could collaborate with to understand. And so he had made all kinds of

01:56:54.940 mutations. He'd also found that SARC phosphorylated a site in polyoma middle T, a tyrosine site that was

01:57:01.400 highly phosphorylated. And even more importantly, that if you mutated that tyrosine residue to

01:57:08.500 phenylalanine, polyoma middle T completely lost its ability to transform cells. It's a single point

01:57:14.700 mutation. Even though the SARC protein was still bound, there was no longer tyrosine phosphorylation

01:57:21.220 315, and that eliminated the ability to transform cells. So we said, well, if PI3 kinase is not SARC

01:57:29.180 itself, maybe it binds to middle T independently. But we found that if you prevented SARC from binding

01:57:36.140 to middle T, then you didn't get that tyrosine phosphorylation. And now there was no PI3 kinase

01:57:40.980 activity bound to middle T. So you needed SARC for middle T to bind to PI3 kinase. At this time,

01:57:48.180 we didn't know it was phosphorylated 3 position, which we'll come back to. However, the shocking

01:57:53.640 result was that if you eliminated that tyrosine residue, now PI3 kinase would no longer bind,

01:58:01.320 and middle T would no longer transform cells, even though SARC was still there and was still activated.

01:58:06.320 So that said, that activating SARC is not sufficient to transform a cell unless you also activate PI3

01:58:13.480 kinase. And we published that in Nature in 76, I think, 76, 77 in Nature.

01:58:19.560 Were you collaborating with Bishop and Varmus at this point in time?

01:58:22.000 I'm sorry, 86, 87.

01:58:22.520 Okay, okay, okay.

01:58:23.160 I meant 86, 87. So 84 was when we published the paper with Ray Erickson,

01:58:27.700 and then a couple of years later with Tom Roberts, and a couple of papers we had. And we showed that

01:58:32.160 PDGF receptor also, when activated, brought down co-perceptive PI3 kinase activity, et cetera,

01:58:39.040 et cetera. So that's where we were in 1986, 87 or so. And at least some of the people who had gotten

01:58:47.100 sonicators also believed us that this was really uniquely associated with tyrosine kinases after they

01:58:55.100 were activated by growth factors. We saw the insulin receptor would also bring down PI3 kinase,

01:59:01.080 only if you stimulated with insulin first. Then you could also bring down this PI kinase activity.

01:59:07.800 But at that time, the only monophosphorylated form of phosphatidyl inositol known was phosphatidyl

01:59:13.800 inositol 4-phosphate. That was discovered in 1949, I mentioned earlier. There's a 4-phosphorylation,

01:59:22.500 and there's a 4-plus-5-phosphorylation. And those were the only two species of phosphorylated

01:59:28.160 phosphatidyl inositol. So we assumed that this was phosphorylation at the 4-position.

01:59:34.960 But as we began to characterize the ability of proteins from cells to phosphorylate phosphatidyl

01:59:42.600 inositol, we found that there were two activities. There was one that required that you sonify the

01:59:48.660 membranes for it to bind and get, for it to have activity. And another that would work perfectly well,

01:59:55.060 in fact, even better, if the lipid was dissolved in a detergent. So we called those two enzymes type 1

02:00:02.080 and type 2 PI kinase. And it was only the type 1, the one that required sonified lipids that

02:00:09.800 co-precipitated with all these tyrosine kinases. The type 2 had completely different enzymatic

02:00:16.460 characters. So it was inhibited by adenosine, the type 1 wasn't. And so we had a whole lot of profiles

02:00:22.660 that said these were two different enzymes. So Malcolm Whitman, registered in my lab, decided to

02:00:27.500 separate them. So he ran column fractionations and meditative acid activities with or without

02:00:33.440 detergent, with or without adenosine. And he characterized the two enzymes, separated them

02:00:39.220 completely. And we had a lab meeting, which every other spot on the thin layer, which is how we

02:00:46.860 characterized the lipid phosphorylation, we ran it out to separate the molecules based on migration

02:00:52.340 in a solvent on a silica plate. And we noticed in the lab meeting that every other spot migrated about

02:00:59.920 one millimeter different from the previous spot. And the way that Malcolm had spotted them on the thin

02:01:06.500 layer, was it was type 1, then type 2, then type 1, then type 2, type 1, type 2, all the way across.

02:01:13.440 If you looked at where the radioactivity ran, it went up, down, up, down, up, down, up, down.

02:01:20.160 And the bottom line was, to me as a chemist, the fact that they migrate differently means they have

02:01:25.120 to be chemically different. So that meant that one of those molecules was being phosphorylated at a

02:01:32.120 different position on the inositol ring from the other. And so we began the process then of chemically

02:01:37.560 characterizing the product of the type 1 and the type 2 enzyme.

02:01:42.320 And how subtle was this, by the way? Was this obvious in looking at it? Or was it something that could have

02:01:47.160 easily been dismissed?

02:01:48.600 I guarantee almost everybody would have dismissed this. One millimeter.

02:01:52.340 Yeah, I was about to say, that just doesn't sound like...

02:01:54.780 Spot was 10, at least 10 millimeters diameter. And the center of the spots were one millimeter different.

02:02:00.760 Do you naturally have an eye that gravitates towards symmetry?

02:02:04.660 I have an eye that gravitates towards unexpected results.

02:02:09.940 And to me, there's no way to explain it. If you had only two spots beside each other,

02:02:14.600 you could say, well, that side of the thin layer migrated a little faster because of

02:02:18.420 the solvent front was not completely horizontal. But this, you couldn't explain the up, down,

02:02:25.080 up, down, up, down, all the way across.

02:02:26.760 Do you still have a photo of that?

02:02:27.980 It's in figure one in the nature paper that we published.

02:02:30.760 From 87?

02:02:32.440 88.

02:02:33.260 88.

02:02:33.720 Probably say 88.

02:02:34.660 We'll link to that figure.

02:02:36.300 But the observation was made in 1987. I bumped into Peter Downs, who's one of the best lipid

02:02:44.100 chemists in the world, and had a meeting at Cold Spring Harbor. And I told him this result. And I

02:02:50.200 said, I know you must have standards for inositol degradation. So can we work together? And so we

02:02:57.820 knew it had to be some site other than the four position, but it could have been the five,

02:03:01.680 it could have been the three.

02:03:02.500 Yeah. Was there a chance it could have been, you were looking at four versus four plus five,

02:03:07.280 four versus four plus five?

02:03:08.680 No, because we knew at four plus five, it would migrate about three inches.

02:03:13.280 So that would look totally different.

02:03:15.520 Yeah.

02:03:16.380 So the null hypothesis is, you're just looking at four, and your assay's a little dirty. But you're

02:03:21.840 thinking, no, it's something else.

02:03:24.580 Yep. Of course, we repeated it multiple times. And we also did HPLC separation, reverse phase,

02:03:30.280 every way you could look at it. And in every way we did it, they were chemically different. They

02:03:35.920 migrated differently in numerous. In the nature paper, we only put that one slide, that one figure

02:03:41.940 of the slightly different migration. And because from there on, we did the chemistry to prove that

02:03:48.800 it was a three position.

02:03:49.560 Why do you think the three position had such a similar look to the four versus the five?

02:03:56.400 Well, keep in mind, there were only two species known to exist at the time we made this discovery,

02:04:01.220 phosphatidyl andositol, four phosphate.

02:04:03.060 Mm-hmm. Oh, and four, five.

02:04:05.000 And four, five.

02:04:05.520 It was not a five by itself.

02:04:06.780 It was not a five by itself.

02:04:08.140 Got it.

02:04:09.280 So this told us there's a three by itself. And then we went on to show there's also three,

02:04:15.500 four, and also three, four, five. PI3 counties could phosphorylate the three position,

02:04:22.080 whether or not the four or five were already phosphorylated. So we now, that generated three

02:04:28.760 new species. PI3P, PI3-4, and PI3-4-5. But I have to say the 3-4-5, this is sort of another

02:04:37.760 sidebar that we could drop out of this, but it's so cool. Never published it because it

02:04:44.240 was embarrassing. But, you know, at this age, you may as well get embarrassed. So we knew

02:04:49.620 that there were species that had been claimed to be 4-5 that were probably 3-4 for the same

02:04:56.160 reason that the 3-P was thought to be 4-P, because separating the 3-4 from the 4-5 was

02:05:02.540 also hard. So we looked for that carefully and found that, in fact, there was a 3-4 being

02:05:10.020 made and proved that the same enzyme was making 3-P. And if you gave it PI4-P to the substrate,

02:05:17.120 it would make 3-4-P2. Leslie Cerunian, a postdoc in the laboratory, was doing the experiments

02:05:23.000 to really verify that those two species really were being made from the same enzyme. And in

02:05:28.920 the course of that work, she came into my office one day and said, I no longer get the same

02:05:34.520 results that I've been getting for the last six months. Something has changed. And so we

02:05:39.900 went over all of her thin layers. Again, this is how we separated all the species. And sure 0.99

02:05:45.320 enough, after getting numerous results in which she could give PI4-P and get PI3-4-P2, suddenly,

02:05:54.940 over the past week, she no longer got that doubly phosphorylated lipid when she added purified

02:06:01.020 PI3 kinase to that lipid. So the question was, what had changed? And she said, well,

02:06:09.000 the only thing that changed is we ran out of the PI4-P and I had to buy a new jar of PI4-P from

02:06:17.700 Beringer-Mannheim. And I said, oh, that's interesting. As I looked at the thin layer more

02:06:23.980 carefully, I noticed that just when the experiments were no longer working, there was a new spot on the

02:06:29.700 thin layer that was just off the origin where we assumed it would probably just be ATP. There was

02:06:35.440 always a little bit of contaminating ATP that got left over when you separate the lipid out.

02:06:42.220 And we assumed that that was ATP, but I noticed it ran just a little bit faster than ATP and that

02:06:48.920 it only appeared in experiments where she could not find the 3,4-P2. And I said, okay, stain this with

02:06:56.360 iodine. It'll tell you where the lipid that you used from Beringer-Mannheim runs. And I'll bet that

02:07:03.560 it's actually 4,5-P2. And that turned out to be the case, that they'd mislabeled the bottle.

02:07:09.480 What was commercially sold as phosphatidyl inositol 4-phosphate was actually phosphatidyl inositol

02:07:16.260 4,5-bisphosphate. And so by accident, I almost think that God looked down on us and said, look,

02:07:24.340 they're missing the most important lipid, PI3-4,5-P3. It never occurred to us to look for it

02:07:30.280 because we were, no one had ever claimed there was something like that that ran on a thin layer.

02:07:36.920 That was because it runs so close to the ATP that was always missed.

02:07:41.060 Because it's got three phosphates.

02:07:42.980 Yeah. It's very highly charged and therefore runs.

02:07:45.500 I never once thought of that until you just said that, Lou. I never, I never would have had that

02:07:53.440 thought actually. And then of course, when you say it now, I mean, that's, think about how dominant

02:07:59.360 those three phosphates are, right?

02:08:00.860 In fact, a year after we made this observation, the paper came out. In fact, I got called by nature

02:08:05.580 saying there's this paper claiming they found something that looks like triply phosphorylated

02:08:09.960 phosphatidyl inositol. And they asked, do you think that's possible? And I said, yeah. In fact,

02:08:15.580 I know it's possible. We already have results showing that that can happen with purified enzyme.

02:08:22.000 Lou, thank you so much for the generosity with your time and your insights. And most importantly,

02:08:25.800 for the work you're doing, you and your colleagues are helping a lot of people. Thank you, Peter.

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02:11:09.480 Oh, oh, oh.

02:11:26.760 Oh.

02:11:27.840 Oh.

02:11:28.900 Oh.

02:11:29.900 Oh.

02:11:30.940 Yo.

02:11:31.620 Oh.

02:11:32.520 Oh.

02:11:33.460 Oh.

02:11:33.580 Oh.

02:11:33.960 Oh.

02:11:34.300 Oh.

02:11:34.880 Oh.

02:11:35.100 Oh.

02:11:35.300 Oh.

The Peter Attia Drive - May 11, 2020

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

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