Special Feature Tony Hunter How an animal virus discovery more than 40 years ago led to one of today’s most successful cancer drugs

In 1979, Professor Tony Hunter had only recently started his lab at Salk when he saw something no one else had even thought to look for before—a cellular switch called tyrosine phosphorylation. Today, more than 60 cancer drugs work because they inhibit that switch.

To survive and replicate, viruses hijack their host’s cell machinery, turning them into mini virus-making factories. It’s in a virus’ best interest to force its host into making more cells. But it’s bad news for the host when those cells start dividing out of control, and eventually become tumors.

In the 1970s, excitement was growing about tumor viruses—viruses that cause cancer in animals. Hunter, then a junior faculty member at Salk, Karen Beemon, a member of his lab, and Bart Sefton, another junior faculty member, were studying two such viruses: polyomavirus and Rous sarcoma virus, which cause cancer in mice and chickens, respectively.

“Because these viruses are so simple—one has just four genes and the other has six—we hoped we could figure something out about how one or a few genes are able to convert normal cells into cancer,” says Hunter, who, turning 80 this year, is now Salk’s most senior faculty member, American Cancer Society Professor, and holder of the Renato Dulbecco Chair.

Not only would that information help them understand virally induced tumors in animals, Hunter thought, but it might also illuminate the ways human cells become cancerous.

Celebrating anniversaries of the discovery of tyrosine phosphorylation at the Post-Translational Regulation of Cell Signaling meeting, a conference of kinase experts co-organized by Tony Hunter and held at Salk, in 1990 (left) and 2022 (right).

Kinases as the cancer-causing culprit

A breakthrough came when a research group in Denver discovered that one Rous sarcoma virus gene encodes a kinase, and that it’s responsible for the virus’ ability to trigger tumors. Kinases are enzymes that add phosphate groups—chemical tags—to other proteins, altering their structure and function.

Phosphorylation by kinases is a common way cells make changes on the fly to meet the body’s ever-changing needs. And it’s easy to reverse the change, with phosphate-removing enzymes.

But the idea that a virus could use a kinase to remove the brake that keeps cells from constantly dividing was new and fascinating. Scientists around the world got to work, testing their favorite cancer-causing viral proteins to see if they had kinase activity.

Hunter’s team made the exciting discovery that a polyomavirus protein, known as Py middle T, has kinase activity that was needed for the virus to convert normal cells into cancer cells. This gave Hunter the idea that there might be a general process in which these viral kinases phosphorylate cell proteins and force host cells to replicate themselves even when they shouldn’t.

But two big questions remained: Which proteins were being phosphorylated by the Py middle T kinase? And what parts of those target proteins were being phosphorylated? At the time, the only known places that kinases tagged proteins with phosphate groups were on the amino acids threonine and serine. (There are 20 different types of amino acids, and all proteins are made up of some combination of them.)

Tony Hunter’s key June 1979 experiment: This is the X-ray film taken of a cellulose thin layer electrophoresis plate in which Hunter separated the amino acids that make up Py middle T protein. He saw an unexplained radio-labeled spot (which he initially labeled “X”) between two well known phosphorylated amino acids, serine and threonine. That mystery spot turned out to be the first evidence of phosphorylated tyrosine.

“X” marks the mystery spot

In June 1979, Hunter ran an experiment with Py middle T protein, which itself is phosphorylated by the viral kinase, expecting to find that it was phosphorylated at threonine or serine.

Using a technique called cellulose thin layer electrophoresis, Hunter first took a sample of phosphorylated Py middle T protein in which the phosphates were labeled with a radioactive marker for easy detection. He used a strong acid to break the protein up into individual amino acids, loaded the amino acids onto the plate, wet it with pH 1.9 buffer, and ran an electric current through it.

The phosphorylated amino acids migrated toward the positive end of the plate. Phosphorylated amino acids with the greatest negative charge-to-mass ratio move further through the plate’s cellulose pores, meaning phosphorylated serine moves further than phosphorylated threonine, which is bigger.

Hunter then dried the plate and laid an X-ray film on it. After developing it the next day, he ended up with a film with dark spots indicating where the radio-labeled, phosphorylated amino acids had run on the plate.

At that point in time, scientists knew where phosphorylated threonine and serine would run on these plates, based on their size and charge. Because Hunter had added unlabeled phosphorylated serine and phosphorylated threonine to the sample run on his plate, he saw where those should be on his film, but they were not labeled with radioactive phosphate.

Instead, he saw an unexplained radio-labeled spot between phosphorylated serine and phosphorylated threonine, which he initially labeled “X.” (See image above.)

As a biochemist, Hunter knew that, at least theoretically, another amino acid called tyrosine could also accept a phosphate group. But if this mystery spot was indeed phosphorylated tyrosine, why would it suddenly appear here, when no one had ever seen it before? This possibility intrigued Hunter, who made up some phosphorylated tyrosine to test if it was “X.” It was.

“The protocol had called for the separation buffer to be pH 1.9. But I kept reusing the old stuff instead of making it up fresh, and unbeknownst to me the pH had dropped to 1.7,” Hunter says.

The small pH drop was responsible for the separation of phosphorylated tyrosine from threonine, which normally move together at pH 1.9 and appear as one spot on a plate.

And that is how Hunter discovered the first kinase that phosphorylates the amino acid tyrosine.

Before Gleevec, only 22 percent of people with chronic myelogenous leukemia (CML) survived five years past diagnosis. Today, more than 70 percent of patients diagnosed with CML survive, and 90 percent of CML patients are still alive after five years.

Telling the world

Hunter and team published their revelation in the journal Cell in December 1979, followed by another landmark paper in Proceedings of the National Academy of Sciences in March 1980, where they showed that the Rous sarcoma virus’ cancer-causing kinase was also phosphorylating proteins at tyrosine.

“I spoke about our work at a phosphorylation meeting in Switzerland in December 1979. And then the world just exploded,” Hunter says. “Everyone who heard about tyrosine kinases wanted to get the phosphorylated tyrosine marker we had used to detect it.”

He went on to publish many additional papers about other tyrosine kinases, in viruses and humans, over the following decades. Human tyrosine kinases, it turns out, normally play important roles in embryonic development and cell proliferation. But they also become overactive in many cancers, where they help drive tumor development.

Turning the tide on cancer

Understanding what drives tumor growth gives scientists a potential way to stop it. Drugs that treat cancer by inhibiting tyrosine kinases began emerging in the early 2000s, and since then more than 60 have been approved for clinical use.

The most famous tyrosine kinase inhibitor, imatinib (brand name Gleevec), is now routinely used to treat chronic myelogenous leukemia (CML). Before Gleevec, the only treatment options available to patients with CML were bone marrow transplantation and daily infusions of interferon, a molecule that helps a patient’s immune system to fight their cancer. Both had serious side effects. And even with those therapies, in the 1970s only 22 percent of people with CML survived five years past diagnosis.

Now, more than 70 percent of patients diagnosed with CML survive. One large study found that 90 percent of CML patients treated with Gleevec were still alive five years later.

“A gratifying outcome of this set of experiments on a simple chicken tumor virus,” Hunter modestly wrote in a 2015 Proceedings of the National Academy of Sciences perspective recounting his discovery.

“When I’m teaching students, I say an awful lot has already been discovered, but an awful lot remains unknown. I’m sure that new biological principles will emerge that we can’t even imagine right now. I’m excited because there will always be new things to be discovered for as long as I’m alive.” – Tony Hunter

Tony Hunter

Creating community

June 2019 marked the 40th anniversary of Hunter’s discovery of the first tyrosine kinase. A small group of colleagues gathered in a Salk conference room that month to celebrate it. But the milestone wasn’t truly noted until June 2022, at the 21st Post-Translational Regulation of Cell Signaling meeting—a conference of kinase experts co-organized by Hunter and held at Salk. Many of the speakers at the recent gathering were involved in the field from the very beginning, including Harold Varmus, who won the 1989 Nobel Prize for his seminal work on tumor viruses.

Over the years, Hunter has co-organized more than 30 scientific meetings at Salk on different topics, including previous milestone celebrations of tyrosine phosphorylation’s discovery in 1990, 2000, and 2010.

“I felt it was important to bring great science and scientific meetings to the San Diego area, which wasn’t something that was happening regularly in the 1980s or ‘90s,” Hunter says. “Most meetings were being held only on the East Coast.”

The future of phosphorylation

Now well into his sixth decade at Salk, Hunter is at the front lines of what he calls “a new area of phosphorylation.” It’s emerging that some kinases also phosphorylate histidine, another of our 20 amino acids, and that this phosphate label may play roles in human health and disease much like phosphorylated tyrosine. Hunter’s lab generated the first antibodies recognizing phosphorylated histidine. Now they are engineering better antibodies that bind with higher affinity and are using them to probe cells to unravel phosphorylated histidine’s function in normal cells and cancer.

“When I’m teaching students, I say an awful lot has already been discovered, but an awful lot remains unknown. I’m sure that new biological principles will emerge that we can’t even imagine right now,” Hunter says.

“I’m excited because there will always be new things to be discovered for as long as I’m alive.”

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