Impure Genius

To find out which enzyme in the v-Src preparation was really phosphorylating PI, Cantley and his colleagues ran the mixture through various protein-purification columns. And they discovered that the prep actually contained two different PI kinases: one that was inhibited by various pharmacological agents, another that wasn’t. Then, in one fateful experiment, they ran both enzymes through a series of reactions and compared their lipid products in alternating lanes on a single thin-layer chromatogram. “And we noticed that the migration positions of the products went up, down, up, down, up, down, like an elevator,” says Cantley. Although the spots only differed by a millimeter, that meant that the products of these two enzymes were not exactly the same. At the time, the only PI that was known to harbor a single phosphate group was PI(4)P, with its phosphate at the 4 position on the inositol ring. What Cantley and his team had discovered was an entirely new lipid—PI(3)P—and the enzyme that makes it: PI 3-kinase.

“We sent the paper to Nature,” says Cantley. “And I got a response saying they weren’t interested in another phosphorylated form of inositol. So I wrote back and said, ‘Well, maybe you might want to reconsider, because the last new form of phosphoinositide was discovered 30 years ago.’ ” The journal sent the article for review, says Cantley, “and then I got a letter back from the editor, extremely apologetic, saying, ‘We almost missed the discovery of the decade. Glad you got us to reconsider.’ ”

That finding—published in 1988—was the first of many game-changing discoveries Cantley would churn out. In addition to uncovering yet another new form of PI—the triply phosphorylated PI(3,4,5)P₃—Cantley has continued to explore the key roles that the PI 3-kinase pathway plays in cancer and diabetes. Here he talks about the chemical impurities that have fueled his success, the beauty of basketball, and the importance of proving yourself wrong.

CANTLEY AT WORK

Have sonicator, will travel. Cantley’s discovery of a novel lipid—and its corresponding kinase—was not welcomed with open arms. “A luminary in the field said if our results were true he would eat his hat,” says Cantley. One problem was that the new kinase did not care for lipids that had been prepared using detergents. Cantley’s team had been feeding the finicky enzyme sonicated lipids—a technique that was not widely used. “Eventually my student, Malcolm Whitman, and a postdoc from our [Harvard] collaborator Tom Roberts’ lab, David Kaplan, traveled to these other labs that had trouble reproducing our results—and they took a sonicator with them,” says Cantley. “They would sonicate the lipids, do the experiment, and show that it actually worked. So eventually, one by one, the people in the field began to see we were actually right. Of course, none of this was supported by any grant because nobody believed it. But I’ve always thought that when you get money to do research, you should use it to do what you know is right.”

“If you’re not often wrong, you’re not at the frontier. If you’re not willing to go out on a limb and throw out ideas, then you’re not pushing the envelope.”

Professor of impurity. “Impurities in commercial reagents have fueled my research from the early days,” says Cantley. “It was a chemical impurity that likely got me my first faculty appointment at Harvard.” This time the culprit was a bottle of ATP. Then a Harvard postdoc, Cantley was working on the Na,K-ATPase. “I was doing an assay using Sigma Grade ATP, the ‘purest ATP in the world.’ I noticed that the enzyme would take off really fast—zoom—and then, within five minutes would be dead. And it only died when I added ATP. So I wrote a paper, very speculative, suggesting that maybe the enzyme was designed to have an auto-shutoff mechanism. Just as the paper was coming out, I ran out of Sigma Grade ATP. So I scrounged around the lab and found some ATP from Boehringer Mannheim. And the enzyme didn’t die at all. So I had this paper coming out that was completely wrong, due to an impurity in Sigma Grade ATP.” And Cantley was determined to find out what that impurity might be. “By chance I ran the Sigma ATP over a little tiny Sephadex column, which was not designed to separate ATP from anything,” he says. Somehow the ATP came out clean—and the inhibitory activity followed five columns later. Cantley then ran the inhibitor through a battery of chemical tests and ultimately determined that it was a transition metal called vanadium. “It turns out that vanadate is a really potent inhibitor of any enzyme that goes through a phosphorylated intermediate,” he says. That discovery, published in the Journal of Biological Chemistry in 1977, Cantley says, “may be the most referenced paper I’ve ever published because people use vanadate in almost every study of kinases and phosphotyrosine phosphatases. And it’s all because I was embarrassed to publish a paper that said something stupid. Within a month of our paper coming out, the Sigma catalog read, ‘Sigma Grade ATP, vanadate free!’”

Boehringer’s bad. If the discovery of PI(3)P was a big surprise, the identification of PI(3,4,5)P₃ (aka PIP₃) was an even bigger one. After finding that their kinase could produce PI(3)P, Cantley and his lab discovered that if they fed the enzyme PI(4)P as a substrate, it would produce a doubly phosphorylated lipid. The only doubly phosphorylated PI known at the time was PI(4,5)P₂. But if the enzyme maintained its preference for the 3 position of the inositol ring, the doubly phosphorylated product should be PI(3,4)P₂—a lipid that had never been seen before. Cantley’s postdoc, Leslie Serunian, was exploring that possibility when, all of a sudden, the kinase stopped making any PIP₂. “I asked her what had changed,” says Cantley. “She said, ‘I’m doing everything the same way. The only difference is I just bought a new bottle of Boehringer Mannheim PI(4)P.’” Looking at the thin-layer chromatograms, Cantley noticed that after the switch to the new bottle of substrate, a new spot had appeared near where the samples had been loaded. When they analyzed this slowly migrating material, they discovered it was not a doubly phosphorylated lipid—but a triply phosphorylated one. “That stimulated us to ask whether we could see anything like that in cells,” he says. “If you take a quiescent 3T3 cell, there’s nothing at all that runs at that position. But when you stimulate with a growth factor for one minute, bang, out comes this lipid. That’s how we discovered PIP₃. And it was all because Boehringer Mannheim had mislabeled the bottle.” Instead of PI(4)P, the container was filled with PI(4,5)P₂. “They never refunded my money,” laughs Cantley. “Or sent me the right bottle.”

Hitting the clinic. “A lot of the work in my lab is becoming more clinically oriented. PI 3-kinase has become a target for pharmaceutical agents that are now entering Phase II clinical trials. The whole goal is to figure out who’s going to respond to PI 3-kinase inhibitors based on what we know about the pathway. This is a completely new way of doing clinical trials: looking at DNA sequences from tumors in a patient and trying to make a logical decision about whether that set of mutations is likely to respond to a single agent PI 3-kinase inhibitor. You need to be able to anticipate the consequences of putting the brakes on one step in the network. Based on experiments done in cells and in mouse models, we have some feeling for what’s going on. But until we get into humans, we’re not really going to be able to prove what’s going on. So we have to design clinical trials that really test these ideas—clinical trials where we learn as much from the patient who fails to respond as we do from the patient who benefits. It’s a huge challenge, but I’m finding it incredibly fulfilling.”

CANTLEY CONTEMPLATES

The road less traveled. “When I get an unexpected result that’s really reproducible, I always feel compulsive about figuring out why. Sometimes scientists are so obsessed about getting to the goal that they put on blinders and charge ahead. ‘My grant says I have to do this. So I shouldn’t get diverted by these side issues.’ Of course, you can also get so diverted by side issues that you never get anything done. So it does require making a real effort to make a decision whether or not you should chase something. I think in most cases when we decided to chase something, it’s turned out to be much more interesting than the original thing we were working on.”

Self-examination. “I think we need to train scientists to be more comfortable with proving themselves wrong. If you have a brilliant idea, you should look for the weak point. Go for the jugular. Not think ‘What’s the best set of experiments I can do to make people think it’s right?’ But rather, ‘Is there a killer experiment I can do where, if it comes out a particular way, it will tell me convincingly that that model is wrong?’ Because if your hypothesis is wrong, it’s best if you prove it wrong before somebody else does.”

Far out. “If you’re not often wrong, you’re not at the frontier. If you’re not willing to go out on a limb and throw out ideas, then you’re not pushing the envelope.”

Two steps forward, one to the side. “Once you get a grant to do a project, if you have a result that suggests there’s something more interesting in a completely different direction from what you’d planned, you should pursue it. Take the rest of that money and go after the exciting thing. Then you’ll have a much more interesting hypothesis to propose for your next grant. I’ve always said that, at the end of a five-year granting period, if I look back and see that I’ve accomplished every aim in the grant, I’ll retire. Because that would mean that six years earlier I knew exactly where the field was going. And that would mean the field was going nowhere.”

CANTLEY ‘IN THE ZONE’

Idea machine. “To me a wonderful way to spend a weekend is chasing down sequences of oncogenes, going through databases and looking at crystal structures to try to get some new insight into how a certain mutation contributes to cancer. My students will tell you that I throw them e-mails all the time, day and night, saying, ‘I just had this brilliant idea that would explain everything!’ The problem is, some students think that because I came up with the idea, it’s got to be right. So they jump in and try to test it. Then they come back and tell me, ‘Remember that idea you had?’ And I say, ‘What idea?’ But very quickly they learn that I just like to toss out ideas. So they ignore most of my e-mails.’

Big books. “Neal Stephenson is one of my favorite authors right now. I love his sense of humor and how he works science and personalities into his plots. It’s never possible to anticipate what will happen next in his books because they’re always off-the-wall, but in retrospect there’s not a single thing that goes on that’s not totally relevant to how the story ramps up. I especially love the Baroque Cycle, which is about 3000 pages. I probably would have never read it had I not gotten a Kindle and an iPad, because the books would be too heavy to carry on an airplane.”

Decadence and discovery. “I try to make it to Anguilla and Venice every year. Anguilla is just a perfect place to relax. No cruise ships land there, there’s nothing to buy, there’s no golf course. It has a dozen fantastic restaurants with good wine lists and spectacular beaches with nobody on them except me and my family. We read books all day and fight over which restaurant we’ll go to for lunch and which for dinner. Venice I enjoy because I feel like I’m stepping back into the fourteenth or fifteenth century. Things there haven’t changed for 500 years. Yet every time I go back, I always find something new.”

Hoop musings. “Basketball is a subconscious sport. If you stop to think about what you’re doing, you’re lost, because it happens faster than you can calculate. You’re 20 or 30 feet away from the basket, the basket is slightly bigger than the ball, you have to get the ball at exactly the right height, exactly the right angle, for it to go through. It has to do with how your hands release the ball as you’re jumping up in the air and moving sideways. I mean, if you tried to write a program to predict that, there’s no way you could do it. Yet the brain does it spontaneously. That’s always mystified me. At one point I was playing in three basketball leagues simultaneously. I just loved to get in that zone where I’d feel completely confident that every shot would go in. When I wasn’t ‘in the zone’ I could still end up winning by playing really hard defense, which meant I got a lot of exercise. So whether I was playing well or poorly, I felt like I was benefiting.”