Interview with Dr. Gregg Semenza

📷 Alexander Mahmoud/Nobel Media

Journalist: Anna Yang, Kian Kousha

Kian: Hi! I’m Kian.

Anna: I’m Anna.

Kian: And you’re listening to SciSection on 93.3 CFMU.

Anna: We’re beyond excited today to have Dr. Gregg Semenza on our show! Dr. Semenza won the 2019 Nobel Prize in Physiology or Medicine for discovering how the body’s cells sense and react to low oxygen levels. Thank you for joining us on our show, Dr. Semenza.

Dr. Semenza: You’re welcome, it’s my pleasure.

Kian: We’re super excited today! Now before we begin, we want to start off by some fun questions so our audience can get to know you a bit better. Our first question: who is your hero?

Dr. Semenza: Well, I guess I would say I had two: one classical and one contemporary. And that was Leonardo da Vinci and Francois Jacob.

Anna: That’s awesome! I love da Vinci. And so, our next question: what makes you laugh the most?

Dr. Semenza: I have a soft part for really dumb comedies. The ones that are so bad that they’re good. And they usually star a Canadian actor named Leslie Nielsen.

Kian: Ah, that’s nice! And what do you think has been one of the most important scientific discoveries of all time?

Dr. Semenza: I guess I would rank three: the first would be the discovery of oxygen, by Scheele and Priestly in the 1770s, the second would be Einstein’s theory of relativity, and the third would be the discovery of the structure of DNA by Watson and Crick.

Anna: Yeah, that’s awesome, those are three great discoveries. Now, could you tell us a bit about your personal journey and your professional journey to where you are today?

Dr. Semenza: Well, you know, it’s been an interesting journey, that’s for sure. I had a high school biology teacher named Rose Nelson who really inspired me in terms of my interests in biology, and I was interested in genetics, and then became interested in human genetics, and because I was interested in both research and potentially taking care of patients with genetic disorders, I got MD/PhD training and did a residency in pediatrics, and went to Johns Hopkins for my postdoctoral training in medical genetics, and I’ve been there ever since.

Anna: That sounds awesome. And so you won the 2019 Nobel Prize in Physiology or Medicine, could you tell us a bit about the discovery that won that award and the process leading up to that?

Dr. Semenza: Sure, so when I arrived at Hopkins for my postdoc, I was trying to figure out what I’d like to study for a research project, and I decided I wanted to study the expression of a gene in transgenic mice. And I originally thought I would study the factor H gene, because this was the gene that was being studied in the lab to look for mutations that caused hemophilia A. And I called up the person at the Genetics Institute named Chuck Shoemaker who had cloned the factor H gene, and he said “well, I can give you those clones, but there’s this other gene you might want to consider called EPO.” And so I did a little reading into the EPO gene, I saw that it was expressed in the liver in the fetus and in the kidney in the adult. And its expression seemed to be induced by low oxygen, because EPO is the hormone that controls red blood cell production, and of course red blood cells carry oxygen to all of the cells in the body. And so the body doesn’t count red blood cells, it just measures oxygen. And when oxygen levels get low, the cells in the kidney that make EPO make more of it. So we started out by making transgenic mice, to look at the regulation of the gene, and we found that sequences right around the gene allowed it to be expressed in the liver. But the sequences required for kidney production turned out to be quite far away from the gene in the 5’ flanking region. So after we had worked out the tissue-specific regulation, then we became interested in the regulation by oxygen, and we went into a tissue culture model, a cell line, where EPO induction by hypoxia could be used as a model system, and we first identified a sequence in the EPO gene that controlled the response, which we called the hypoxia response element, because you could take this very small DNA sequence of 33 base pairs and put it into a heterologous reporter gene, and the expression of that gene would be induced by hypoxia. And then we used that sequence to purify a protein that was induced by hypoxia and bound to the hypoxia response element. And we called that protein hypoxia inducible factor one, or HIF-1. And we purified the protein as I said by DNA affinity chromatography, based on its ability to bind to the hypoxia response element, and we found that it was composed of two subunits that we called HIF-1α and HIF-1β, and HIF-1β was constitutively expressed but the levels of HIF-1α were dramatically regulated by oxygen levels. So when oxygen levels were high, HIF-1α levels were low, and when oxygen levels went down, HIF-1α levels went up. And then the big question became: how was the level of HIF-1α being regulated by the oxygen concentration? And several labs, including Peter Radcliffe’s lab at Oxford and Will Kaelin’s lab at Harvard, found that HIF-1α was subject to a post-translational modification called hydroxylation. And they identified a series of proteins called prolyl hydroxylase domain proteins or PHDs that insert an oxygen atom into a proline residue in HIF-1α. And when that happens, HIF-1α can be recognized by a protein called VHL. And when VHL binds to the hydroxylated form of HIF-1α, it recruits a ubiquitin protein ligase that ubiquitinates HIF-1α, and that’s a signal for its degradation in the proteosome. So when oxygen’s available the protein gets hydroxylated and degraded. The enzymes use oxygen as a substrate. And so under hypoxic conditions, the activity of the hydroxylases is inhibited, and as a result VHL cannot bind to HIF-1α and the protein rapidly accumulates in the cells. In addition to this hydroxylation that regulates the HIF-1α stability, we had identified a protein that bound to HIF-1α and interfered with its transcriptional activity that we called FIH-1 for factor inhibiting HIF-1, and it turned out that FIH-1 was also a hydroxylase. And it hydroxylated not a proline residue but an asparagine residue in the trans-activation domain, again, when oxygen is available. And this hydroxylation prevents the binding of two proteins that are called co-activators, that are required for HIF-1 to activate gene transcription. And again, under hypoxic conditions, the hydroxylation doesn’t occur and the co-activators combined and HIF-1 is competent to activate transcription. So you can see, this is a really beautiful system, where changes in the amount of oxygen are transduced to the nucleus as changes in HIF-1 activity through these oxygen-dependent modifications.

Anna: Yeah, that’s a beautiful system, just as you said. And so could you tell us a bit about some of the potential applications or implications of this discovery of yours?

Dr. Semenza: Right, so, again, going back to our starting point with EPO, EPO of course is a recombinant protein that can be administered to patients, particularly patients who have kidney failure, who no longer can make EPO, but whose bone marrow is perfectly capable of making red blood cells, if it’s appropriately stimulated by EPO. So patients who are on dialysis for kidney failure are given recombinant human EPO, and this has been a great benefit for them. The hydroxylases, in addition to requiring oxygen, require alpha-ketoglutarate, which is a glucose metabolite. And because they require this small molecule, they are inherently druggable. Because one can design an analog of alpha-ketoglutarate that can bind to the enzyme but not allow catalytic activity. And these prolyl hydroxylase inhibitors induce HIF-1α levels, right, because if you inhibit the hydroxylases then you can build up the HIF-1α levels. And so there are four of these drugs, now, that are in phase three trials for the treatment of anemia in patients with chronic kidney disease. And one of them has been approved. So this is really the first translation of these discoveries to the clinic. And I think several more of these drugs will be approved. And the beauty of them is that because they’re small molecules, they can be given as a pill by mouth. And so this is obviously much more convenient than having an injection of a recombinant protein. And in addition, of course they don’t just turn on EPO, they turn on other genes. And what we know, is that the HIFs don’t just control EPO production, but they also control the production of proteins that are required for the absorption of iron from the GI tract and its transfer to the bone marrow, where it’s incorporated into hemoglobin, which is of course the protein within the red blood cells that carries oxygen. And this is typical of the activity of the hypoxia inducible factors, is that they coordinate these kinds of physiological responses that require the expression of multiple genes in multiple tissues in order to carry out a physiological response to hypoxia. And so there are some advantages for using these HIF inducers, and so that’s one major application. Another one of course is that, you know, we had shown that the HIFs play a major role in stimulating angiogenesis. And we had shown that in some models of ischemic cardiovascular disease that HIF-1α gene therapy could be beneficial in animal models. And so it’s possible that these HIF inducers will also be useful in that area. Then on the other hand, you have situations where you’d like to inhibit HIF activity, and that’s principally in cancer. Where the cancers have areas of tremendously severe hypoxia, because of the very rapid division of the cells and the formation of blood vessels that are not structurally or functionally normal. And so many cancers have very high levels of HIF-1, and in the case of breast cancer we’ve been able to show that HIF-1 activity plays a major role in stimulating invasion and metastasis. So we’d like to inhibit HIFs in cancer and the sort of hallmark cancer with regard to the HIFs is clear cell renal carcinoma because in that cancer, the hallmark genetic lesion is mutation of the HL. So the HL is mutated and inactivated and as a result the HIFs, both HIF-1α and sort of its sibling HIF-2α — the levels of those proteins, particularly HIF-2α, are very high and it’s believed that HIF-2α is driving the pathogenesis of kidney cancer. And so there’s a drug that’s now in clinical trials that binds to HIF-2α and prevents it dimerizing with HIF-1β and can inhibit kidney cancer growth in animal models. And the phase one trial of that drug showed that it was safe and also showed some remarkable responses in the patients who all had advanced kidney cancer and had failed all existing therapies. So that drug is now in advanced trials, and has provided proof of principle — or it will, I hope — that inhibiting HIFs will be useful for the treatment of some cancers.

Kian: That sounds awesome. It’s amazing to see all the applications of this research and what people can eventually get from it. Something which is purely scientific until it comes into the lives of a lot of people and helping them with cancer and a lot of other issues. Now moving on to your experiences as a Nobel laureate, can you tell us about the experience of winning the Nobel, and what was the first thing that you did after finding out that you’d won the Nobel prize?

Dr. Semenza: Well, you know, the phone rang in the middle of the night, and so I wasn’t quite awake, and by the time I got to the phone it had stopped ringing, so I went back to bed, and then some time later — it wasn’t right away — some time later, the phone rang again and I thought “I’d better be a little quicker this time” and so I managed to get to the phone and receive the good news that the folks at Stockholm were awarding us the Nobel prize. And, you know, the combination of the news and being not quite awake, I was kind of just totally flabbergasted, and my jaw dropped, and by that time I had the phone back in the bedroom and my wife could hear the call, and her jaw dropped, and I was just kind of speechless, so it was definitely a one-sided phone call. I’m sure they were wondering how I was gonna possibly give an address given my ability to articulate anything.

Kian: In one of your interviews you mentioned that many of the best discoveries are made by young scientists, and as students a lot of us usually get fascinated by success without noticing all of the challenges that went along the way. I’m sure being at your position right now did not happen easily, so can you tell us about some of the challenges you faced as a student?

Dr. Semenza: Oh, sure. So when I was a graduate student at Penn, I went into a lab and settled on a project, and let’s just say it was an overly ambitious project, and it didn’t work out. And then I tried another project in that lab which was equally overly ambitious, and that didn’t work out. And so after a year, I was kind of not feeling like I was gonna be able to be successful in that environment, and I went to the director of the program and I asked if I could find a different lab, and he said okay. And I managed to find a lab that was willing to take me, despite my record of failure, and this was a lab at a children’s hospital in Philadelphia led by Saul Surrey and Elias Schwartz that was studying the molecular basis of a disease called thalassemia, which is due to mutations in the beta globin gene. And so the plan was to study a family from an isolated area that had an unusual clinical phenotype, thinking that they might have an unusual mutation. And so we drew blood from several of the family members and isolated the DNA and made a phage library and screened the library and sequenced the beta globin gene. And when I sequenced the beta globin gene after a year — because at this time things were much different than now. So sequencing 4000 base pairs of DNA, that was a year’s worth of work. Now it’s, you know, a blink of an eye. So that’s another thing that students don’t appreciate, is what’s happened in terms of the power of the techniques. Anyway, so when I determined the sequence, I found the gene had the same sequence as the last beta globin gene that had been studied in the lab. Suggesting that somehow my DNA had become contaminated with DNA from this previous study. So that basically meant a whole year’s worth of work down the drain. And so I was very depressed. And in my med school class, there were a pair of twins and their father was the psychiatrist at Penn. So I went to see him and I told him what had happened, and then he looked at me and he said “well Gregg, it sounds like you have reason to be depressed.” And so when he said that I felt much better. So I went back to the lab and started again, and was able to sequence the gene and finish the project within a year, and manage to graduate on time. And my first mentor when I did research as an undergraduate at Harvard, he would say “search and re-search.” And so if you’re not able to overcome obstacles, you know, and don’t take failure well, research may not be a good area for you. Because it’s just a part and parcel of the enterprise.

Kian: Thank you for sharing that, that was really inspiring and I’m sure students will definitely appreciate hearing that from you.

Anna: Yeah, definitely, and so now that you have overcome these barriers, and you’ve won the most prestigious award in science, what kinds of goals are you working towards now, in your research?

Dr. Semenza: We’ve continued to study the molecular mechanisms by which the HIFs promote cancer progression. And we’ve focused on — particularly in breast cancer — we’ve focused on mechanisms of invasion and metastasis, then we spent some time identifying different pathways by which the HIFs are able to increase the specification of the breast cancer stem cell phenotype, which is really critical. The cancer stem cells are the cells that are really responsible for metastasis and for disease recurrence after therapy. And most recently we’ve been studying the mechanisms by which HIF activity enables the cancer cells to evade the immune system. And we’ve been able to show that in a number of these systems, if we add a HIF inhibitor to existing therapies, we have a better outcome in mouse models. And the inhibitors that we’ve identified work very well in the animal models but they’re not suitable for use in patients for various reasons. And so our goal has been to try to identify a HIF inhibitor that would be useful for treatment in cancer. And we have a compound now that we’ve been studying that is active in liver cancer, and several other cancers, and unlike the drugs that we had been using, does not seem to be very toxic, at least in mice. So we’re hopeful that we’ll be able to bring this drug to the clinic for the treatment of cancer. Unlike the drug I mentioned, this drug seems to inhibit both HIF-1 and HIF-2, not just HIF-2, so we’re working very hard on that, both testing the efficacy of this inhibitor, both in cancer models and in models of ocular neovascularization. Diseases like diabetic retinopathy, which is responsible for blindness in individuals with diabetes. And we find in mouse models of ocular neovascularization that the inhibitor is also active. So we’re hopeful that it may be useful for treatment of diseases like that as well.

Kian: And you already talked about the applications of your research, which sometimes connects people to science. So what do you think our scientific community needs the most right now?

Dr. Semenza: I think that science often benefits from collaboration. And there are some forces at work that sort of make collaboration more difficult: some political forces, some financial forces, but at least for me, one of the things that makes biomedical research so much fun is that you do get to meet people all over the world who share your passion for research. And that’s, for me, a great part of the fun of research. It is to be able to collaborate with people at Hopkins, elsewhere in the US, and all over the world.

Kian: Our final question for today: what advice do you have for students who are listening to our show?

Dr. Semenza: I’m concerned that a lot of times, that the students only hear the bad parts of research. They hear the mentors moaning about how difficult it is to get grants, and get your papers published, and they don’t hear the really good parts of the job-

Dr. Semenza: -which are the tremendous freedom that you have as someone doing biomedical research. And you can follow your ideas wherever they lead, and as long as you’re productive nobody’s going to bother you and tell you to do it one way or the other. And then as we mentioned, you have the opportunity to make friends all over the world. And then of course the icing on the cake is that there’s always the possibility that you may make a discovery that will have an impact on public health, which is ultimately why we do the research. So I think it’s a tremendous career, and I do worry that the students only hear the negative part of it.

Anna: Yeah, that’s a very positive outlook to have, so thank you for highlighting all of the positive aspects of a career in science. So that’s it for this week of SciSection, make sure to check out our podcast for our latest interviews, events, and episodes.

Kian: Thank you again, Dr. Semenza, for coming to our show.

Dr. Semenza: Thank you very much, good luck!

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