The Nobel winners, 2019: Physician-scientists who discovered how cells adapt to changing oxygen levels

The Nobel Prize in Physiology or Medicine, 2019 was awarded to Gregg L. Semenza, William G. Kaelin Jr. and Peter J. Ratcliffe and for their work on how cells sense and adapt to oxygen availability. Their research elucidated the genetic mechanisms through which cells respond to changes in oxygen levels. The findings have implications in treating many diseases, including cancer, anaemia, heart attacks and strokes. These scientists had also shared the Albert Lasker Basic Medical Research Award in 2016. 

Left to Right: William G. Kaelin Jr., Sir Peter J. Ratcliffe, Gregg L. Semenza

If Semenza, Kaelin and Ratcliffe had one thing in common, besides their area of research, it is that they chose to publish houses of brick rather than mansions of straw. It sure took a while for the world to notice them, but it did.

About the papers that earned him the Lasker, Kaelin wrote, “Most would be considered quaint, preliminary and barely publishable today.” These papers would get him the Nobel Prize three years later.

Semenza and Kaelin woke up to the call from the Nobel media that day. While Semenza’s first reaction was to hug his wife, Kaelin told Adam Smith – the Chief Scientific Officer of Nobel media – how he missed his late wife on that occasion. Ratcliffe, on the other hand, was working on a grant when Smith called him. “I was writing and will continue to write an EU Synergy grant for collaborative work with friends and colleagues in Finland, and also my good friend and colleague Christopher Schofield, so of course the EU’s on our minds at the moment, and we’re writing a Synergy grant. And despite this good news, I guess I’ll continue doing that and meet the deadline,” Ratcliffe said.

What made these scientists embark on their journey in research is worth knowing. In an interview with The Journal of Clinical Investigation (JCI) in 2016, Semenza said that it was his high school biology teacher, Rose Nelson, who inspired him to pursue biology. “I had her as a freshman for biology and then as a senior for AP biology when it was genetics within biology that I got really excited about. With her help, I was able to enrol in an NSF-sponsored summer program at the Boyce Thompson Institute for plant research. It was really my first exposure to research and experiments,” he said. His interest in genetics and his meeting with a family friend’s child, who had Down syndrome, motivated him to pursue MD-PhD to study genetics and to care for those with genetic disorders.

It wasn’t easy for him at this stage either. Being the first graduate student of the lab with a very ambitious project in hand, he had reached the stage where he felt it wasn’t working out. He then switched to another lab at Children’s Hospital of Philadelphia in the second year, where he studied β-thalassemia.

“I was tasked with studying an unusual family, where one of the alleles was a silent carrier,” Semenza told JCI. “Normally, you can tell from looking at the blood cells whether someone is a carrier. In this case, it was the father who was an obligate carrier, as he had two affected children, but you couldn’t tell he was a carrier. It was suggested that the mutation was in some way different; this was going to be my project. The idea was that you take the blood, isolate the DNA, make a library, pull out the β-globin gene, and sequence it. I did all that and at the end of one year, I got the sequence, and the mutation was the exact same mutation as the previous study, which meant that somehow the clone had become contaminated, and the whole thing had been for nothing. So now I was going to have to start over the third time. I went back, started over again; things went smoothly because technically I’d already done it, and we went through the project and got an answer.”

He then moved to Johns Hopkins for his post-doctoral education, where he worked with Haig Kazazian and Stylianos Antonarakis – leaders in finding the molecular basis of β-thalassemia. He started working on this gene called Erythropoietin (EPO), which Chuck Shoemaker from Genetics Institute in Cambridge had asked Kazazian and Antonarakis to consider. Semenza tried to identify the DNA sequences in the gene responsible for its expression in different tissues. They put different fragments of human DNA spanning the EPO gene into mice and checked if and where the gene was expressed.

This was the turning point for Semenza. He figured that the gene was regulated by oxygen. Now, he had to understand where these DNA sequences regulated by oxygen were located. On investigating further, he realized that this sequence was in the DNA sequence that came after the gene (3’ flanking sequence). This was unusual as such regulating regions usually lie before the gene (5’ flanking sequence). But the data said that it was the 3’ flanking sequence. So, they took this sequence and put them in a plasmid which had a reporter. They then put this plasmid inside human cells grown in the dish and deprived them of oxygen. At low oxygen levels, the EPO gene would be expressed because of the sequence present in the plasmid. They then made smaller fragments of that sequence and repeated the process until they got down to 33 base pairs. On mutating individual nucleotides in this region, they found that the EPO gene expression was stopped.

“We suspected that there was a very important factor, which was binding to that sequence and was responsible for turning on the gene. And so, we tried to find the protein that was the key factor for turning on the EPO gene,” Semenza told JCI.

At this point, he was a young faculty with a post-doctoral fellow, Guang Wang. Semenza and Wang started looking for a protein that would bind to this DNA sequence (Hypoxia Responsive Element). The challenge was to figure out the conditions that allowed binding. They were also hoping to find something that would be present in the nucleus of cells with low oxygen (hypoxic) but absent in those with normal oxygen levels (normoxic). “Guang would do several of these experiments a day and had this stack of blots with negative results on his desk. I was thumbing through them one day, and I came across this one gel where there seemed to be a faint band in the hypoxic lane. I got all excited and said, ‘Did you see this? Did you see this?’ After optimizing the assay in a very short period of time, he generated really strong definitive results of a binding activity that we called hypoxia-inducible factor 1,” Semenza said.

For Ratcliffe, the start was serendipitous. In his Lasker acceptance speech, Ratcliffe reflects on an incident from his days at the Lancaster Royal Grammar School for the boys. “I was a terrible schoolboy chemist and following the career of some distant relative, I was keen to study industrial chemistry. One day the ethereal but formidable headmaster appeared in the chemistry laboratory, summoned me to one side, and said, ‘Ratcliffe, I think you should study medicine.’ I said ‘Yes, Sir’ decisively. Without further thought, the university application papers were changed accordingly,” he said. “To this day, I don’t really know whether he felt I would be a good doctor or a bad chemist. But the moment sticks with me as a reminder of the importance of serendipity in a scientific career, at least in mine,” he added.

He trained in Medicine as a Kidney specialist and started his research career fascinated by the extraordinary sensitivity with which the kidneys regulate the hormone Erythropoietin (EPO) to regulate red blood cell production. “I felt that the problem was interesting, conceivably attractable and there was a new opportunity for study with the cloning of the erythropoietin gene. But some people felt, with the emerging success of recombinant Erythropoietin, that understanding how the hormone was regulated was a niche area unlikely to be of very general importance. They advised me accordingly to study something else,” he said.

In the telephone interview with Nobel Media, Kaelin shared what drew him to science. “You know, I’m a big believer of curiosity-driven, hypothesis-driven research,” he said. “I know that’s complementary to other ways of generating knowledge but I think in the end what drew me to science and what draws a lot of scientists to science is that we like interesting puzzles, like clinical features of patients who had mutations in the VHL gene, were a curious constellation of findings but one way to unify them was there was some abnormality in the way the tumours they were developing were sensing and responding to oxygen, and we thought if we could understand that we could understand more globally how cells and tissues sense and respond to changes in oxygen.”

Little did these physician-scientists know while addressing their research problems, that they were reaching for the Nobel with their findings. But that’s the beauty of Science. Think about these Nobel laureates. As someone who made a serendipitous entry into medicine later pursuing an area of research, which many thought had no scope beyond the niche area of kidney research, Ratcliffe won the Nobel Prize for the findings that have immense physiological importance. Sharing the award with him are Semenza, who struggled in the early days of PhD and found his way through, and Kaelin, whose pre-medical school mentor remarked on his college transcript, “Mr. Kaelin appears to be a bright young man, whose future lies outside of the laboratory.” Their life and work are a testament to the fact that houses of brick stand the test of time.

Sources:

Kaelin Jr., William G. (2017). Publish houses of brick, not mansions of straw. Nature. Retrieved from https://www.nature.com/news/publish-houses-of-brick-not-mansions-of-straw-1.22029

Neill, Ushma S. (2016). A conversation with Gregg Semenza. The Journal of Clinical Investigation.  Retrieved from https://www.jci.org/articles/view/90960

Albert and Mary Lasker Foundation. Peter Ratcliffe, Acceptance Speech, 2016 Lasker Awards. Retrieved from https://www.youtube.com/watch?v=wB5gzwZMvTM

Albert and Mary Lasker Foundation. William Kaelin Jr, Acceptance Speech, 2016 Lasker Awards. Retrieved from https://www.youtube.com/watch?v=K2Ds_S48IWg&t=54s.

Sir Peter J. Ratcliffe Interview. Retrieved from https://www.nobelprize.org/prizes/medicine/2019/ratcliffe/interview/

William G. Kaelin Jr. Interview. Retrieved from https://www.nobelprize.org/prizes/medicine/2019/kaelin/interview/

Gregg L. Semenza Interview. Retrieved from https://www.nobelprize.org/prizes/medicine/2019/semenza/interview/