HIF-independent synthetic lethality

Episode 6: HIF-independent synthetic lethality

TIL Frequency & Tumor Reactivity

Posted: Oct 13th, 2019

HIF-independent synthetic lethality

On Oct. 7th, 2019, the Nobel Assembly announced that William G. Kaelin, Jr., Dana-Farber Cancer Institute, shares the 2019 Nobel Prize in Physiology or Medicine with Sir Peter J. Ratcliffe of Oxford University and the Francis Crick Institute, and Gregg L. Semenza of Johns Hopkins University, for their discoveries of how cells sense and adapt to oxygen availability.

In this episode Tracy interviews Hilary Nicholson, Ph.D., a postdoc research fellow in Kaelin Lab at Dana-Farber Cancer Institute. Hilary talked about her perspectives of Dr. Kaelin receiving the Nobel Prize, how Dr. Kaelin mentors and inspires her, and how her work is built upon this textbook discovery.

Hilary is the first author of a recent publication on Science Signaling, "HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species". Kidney cancer is one of the top ten most common forms of cancer in developed countries, and the most common type of kidney cancer is clear cell renal cell carcinoma (ccRCC). The von Hippel-Lindau tumor suppressor gene (VHL) inactivation is associated with ccRCC development, and hypoxia-inducible factor 2 alpha (HIF-2 alpha) is accumulated.

Hilary talks about synthetic lethality between CDK4/6 inhibition and VHL loss in two species and across various human ccRCC cell lines in culture and xenografts; The study also shows that HIF-2 alpha was not required for the synthetic lethality. "These findings support testing CDK4/6 inhibitors as treatment for ccRCC, alone and in combination with HIF-2 alpha inhibitors".

In addition, Hilary introduced the Science Cheer leaders, a non-profit organization comprised of 300+ current and former NFL, NBA and college cheerleaders pursuing STEM careers.

Full Transcript »
Tracy:
Hi, everyone. This is Tracy Yin. Thanks for tuning in. Our guest today is Hilary Nicholson. Hilary is now a Research Fellow scientist in Medicine at the Dana-Farber Cancer Institute. She's currently working in Dr. Kaelin's lab. Dr. Kaelin shared this year's Nobel Prize in Physiology and Medicine, which was just announced two days ago. Welcome Hilary.
Hilary:
Thank you.
Tracy:
I imagine the entire lab is in great excitement this week, right? Could you please share with our audience how the group reacted and celebrate?
Hilary:
Yeah, everyone is just thrilled for Bill and for this recognition of the discovery that he and the Ratcliffe Lab and the Semenza Lab discovered together, and it's been a very surreal experience, because we all hoped that he would get this recognition. And Bill is always so generous in, whenever he wins a prize or is awarded some sort of recognition, he is the first one to tell you that it's a result of all of the people who have been in the lab and are in the lab, and to share the fruits of that labor and those awards with all of us.
Hilary:
So, it was fitting that on the day he won the Nobel Prize, he came in and wanted to share that with all of the alumni and all of us who are in the lab now. So, it was a really exciting time, and it is wonderful to see everyone recognizing the years of work and dedication that Bill and his colleagues has put into that discovery and making sure that it's true, and it's fundamental, and that it lasts and really has an impact on, not just the fundamentals of science, but also it's had a great impact on a lot of people's lives.
Tracy:
You sound a lot of the excitement through your voice. I'm very excited about that, and congratulations. And so, did any of you know that it might be coming, before the announcement? I'm not sure about the entire process, but do they contact you saying that it's possible that Dr. Kaelin might be selected for the Prize?
Hilary:
So, none of us knew that he had won until it was announced in the press, really. Even Bill didn't know. You get a call just before 5:00 in the morning and that's the first time you know for sure. But I think we all hoped that this would happen for him, and I think there are a number of other recognitions and prizes that Bill has acquired along the way that indicate that he has made this amazing discovery and he and Dr. Semenza and Dr. Ratcliffe have together been recognized a few times by the Massry Prize, or the Lasker Award, and those often precede a Nobel Prize, so we were all keeping our fingers crossed and we were all hoping, but I don't think any of us were as prepared for the reality when it actually happened.
Tracy:
Yeah, I actually listened to the audio recording when he received the phone call. It's amazing. And so, yes, I see peoples talking about this discovery as textbook discovery, and there was three physicians, scientists, sharing the prize. As an insider, can you elaborate for our audience, who contributed to which part of this oxygen sensing process, and in general the discovery?
Hilary:
Yeah, so the discovery of how cells are sensing oxygen, the fact that HIF transcription factors are hydroxylated, the EGLNs that contribute, and VHL as a recognizing part of that E3 ligase to degrade HIF. I think all of that was certainly a team effort to figure out the whole process and in parts of it, Dr. Semenza, Dr. Ratcliffe and Dr. Kaelin were working in parallel and then there are parts of it where they would meet up at a conference and say, "Oh hey, that looks real familiar," or, "Gosh, we're really on this, different parts of the same project here." But I think it is sort of emblematic of Dr. Kaelin's approach to science that it was a joint effort, a collaborative effort to really get all the pieces in line.
Hilary:
And he continues to be that way now, he's a very collaborative scientist. He encourages us all within the lab to collaborate with each other and also to be very open to collaboration outside. But I have to be clear that I was certainly not part of the initial discovery. I was in kindergarten when he was publishing those papers. So I think it is for all of us working on sort of the fruits of that labor and the next steps that have resulted from their seminal discoveries has really clarified how important things really were and how important their contribution was to those pathways and all of the things that have followed from it.
Tracy:
Okay. So Hilary, can you talk a little bit more about that switch that was mentioned that during the Prize announcement?
Hilary:
Yes. So, the way that your body regulates how much oxygen, and senses how much oxygen it has available, is based on the use of elemental oxygen in a particular pathway. So when there is lots of oxygen around a particular transcription factor called HIF gets that oxygen added to it as a hydroxyl group by a series of HIF prolyl hydroxylase enzymes called EGLNs. And the discovery, that all of them contributed to, was the fact that this elemental oxygen was used as a marker on HIF, that the EGLNs participated and then that hydroxylated HIF, which is tagged with the available oxygen, is recognized by VHL, which is part of the E3 ligase complex. And VHL's recognition of hydroxylated HIF induces the degradation of HIF.
Hilary:
So HIF is degraded when oxygen is present, when there's no oxygen, HIF doesn't get that oxygen mark on it, that hydroxyl group, and so VHL doesn't recognize it, and so it can't be degraded. So HIF is stabilized.
Tracy:
Thank you. So what do you think is the most important application of this discovery?
Hilary:
I think there are a lot of applications that can come from it. Some of them already have been realized, some of them are still nascent ideas, but if you think about oxygen sensing and how those processes are involved in a lot of pathology, we work a lot on kidney cancer and one of the reasons for that is that VHL is often mutated and inactivated in kidney cancer. So the application of this discovery to how can we work on kidney cancer, what pathways are involved, how can we treat it? A lot of that is coming to reality right around now.
Hilary:
There are some clinical trials going, some drug that has recently been approved in Asia and hopefully it's coming to the US soon. And all of these are based on using this understanding of VHL, HIF and oxygen sensing. And I think those are some that are current, but there are also some that are down the line that we can still think about like stroke and ischemia, which are processes where you need to think about oxygen availability and keeping tissue alive when there may not be available oxygen. So our understanding of these pathways that has just exploded after the Nobel prize discovery has really allowed us to start thinking about those pathways and manipulating them and helping people that are suffering from issues of oxygen deprivation.
Tracy:
Yeah, I heard that there is already treatment approved in China for anemia using this process.
Hilary:
Yes. And that's built off of this, theseVHL, EGLN, HIF pathway. Exactly.
Tracy:
Okay, great. Congratulations again to Dr. Kaelin and the lab. And then back to you, actually, you published on Science Signaling on October 1st, which is like 10 days ago as the first author. Very exciting too, right?
Hilary:
Yes, very.
Tracy:
And Dr. Kaelin as the correspondence author. So congratulations. And before we dive in, can you tell us more about you? What's the path that led you here? Where did you go for school and what's your interest, research interest?
Hilary:
Yes, absolutely. I did my undergraduate degree at Colgate University in upstate New York. I majored in biochemistry there, and that's where I got my first longer term lab work experience. I worked in Roger Rowlett's lab, where we studied carbonic anhydrase and that's where I got an appreciation for understanding substrates of enzymes, how those enzymes work, some protein chemistry. And I found myself really interested in lab work and the only thing that I wanted more out of that was some sort of applicability to human health.
Hilary:
And so I decided to apply for graduate school for programs in pharmacology and studying how drugs affected the body, how the body responded to drugs, and how those interactions work. So I went to Brown and joined the Molecular Pharmacology and Physiology program there, and I studied under Wayne Bowen, who was the chair of the department, and we worked on sigma receptors. So again, staying in that sort of protein biology world, but sigma receptors are implicated in a lot of cancers and that's where I first got into cancer biology and I found myself just fascinated by what goes wrong in cancer, what is different between cancer and normal cells, and how can we possibly use that to understand what has happened to cause the cancer, and also what is available to us for intervention.
Hilary:
So I started using the protein biology background I had from my undergraduate work to apply to my graduate studies where I was starting to get into the cancer field. And then after graduate work I really wanted to do something very translational and I knew I wanted to stay involved in cancer. I knew I wanted to work on pathways and Bill's lab was a perfect fit. I loved the way that he approached science and I loved that a lot of his work seemed to be translating into the clinic. I knew that he was a brilliant mind and I knew that if I wanted to really see what my skill set could become and how good I could be as a scientist, I felt that Bill could get me there and push me hard enough and give me all of the mentorship and encouragement and training that I needed in order to really round out my skillset and learn to take research from an idea through what you always hope will be a clinical trial.
Tracy:
As you mentioned, when we were talking about the Nobel price, kidney cancer is one of the 10 most common forms of cancer in the developed word. And you were, in this study, you were working on ccRCC, which is the most common type, Clear Cell Renal Cell Carcinoma. So can you tell us what's your hypothesis in this study, and why you started to look into the VHL tumor suppressor gene, and how did you get interested in looking into CDK4/6?
Hilary:
So as you mentioned, Clear Cell Renal Cell Carcinoma is the most common type of kidney cancer, and over 70%, some studies say up to 90%, of Clear Cell Renal Cell Carcinomas have inactivating mutations in the VHL tumor suppressor. And because of that, a lot of these ccRCCs have very high levels of HIF, which VHL is normally responsible for degrading under normal oxygen conditions.
Hilary:
And so, we wanted to look at ways to target VHL inactivated ccRCCs, but it is a challenge to target something that is lost. You can't put a drug on to a tumor suppressor that doesn't exist. And so we decided to look at approaching kidney cancer through the lens of synthetic lethality, which means we look for situations in which in normal cell, that has VHL, may be able to tolerate loss of some protein, whereas a cancer cell that has lost VHL is hyper dependent on that other protein.
Hilary:
So we look for those differences where a healthy cell doesn't care, but a cancer cell very much cares, to find those context specific opportunities for intervention where we can harm the cancer cells without any harm to the positive VHL cells around it. So our idea with looking for synthetic lethality was we need to find something very robust. We need to find something so fundamental that by the time we get to looking at different cell lines and looking at different people, the variability between cell lines and people won't matter because this relationship between loss of VHL and loss of this other target that we're hoping to find will just be so robust that it will kill the cancer cells across all the cell lines, across all the patients, despite their variability.
Hilary:
So the way we thought to go about that was to use Drosophila cells, which have an analogous VHL HIF pathway, just like in humans. And we decided if we knocked out VHL in fly cells and we looked for something synthetic lethal in that setting, and we did the same, knock it out or use these ccRCCs where it's already knocked out, in human cells and we find something that's synthetic lethal there. Anything that overlaps between a Drosophila cell and a human cell, which are so different, that relationship that we might find should be something that is fundamental and really robust. And if it's true in a human, and it's true in a fruit fly, we expect it will hold true across small variabilities between cell lines and between human patients.
Tracy:
And that's, you said the Drosophila and the human, they are so different. So is there any homology gene that you work during the process?
Hilary:
Yes. So thankfully in Drosophila cells, the axis where you have VHL recognizing a hydroxylated HIF ortholog is conserved between humans and flies. So we were able to look at the same pathway in those two different settings.
Tracy:
Okay. And for your study, you already mentioned several times this synthetic lethality. Can you tell us more about this, and why we want to use this approach in cancer treatment?
Hilary:
Absolutely. So synthetic lethality is the concept that you have a different set of dependencies, a different set of things that matter when you are a normal cell as compared to a cancer cell. That's how we think about it in the context of cancer research. So the idea is that if you are a normal cell and you have intact VHL, we might be able to inhibit some factor, we'll call it X. And the normal cells don't really care if you inhibit X, because they have VHL and they're fine. But if you take a cancer cell that has lost VHL, those cells might be hyper dependent on X. They might have a much greater requirement for X because they've lost VHL.
Hilary:
So an analogy that we often use is to think about a car. And if you're driving down the road and you hit a pothole and you pop your tire, as long as you have a spare tire in your trunk, you change the tire, you move on and you're fine. But if you don't have that spare tire, then if you pop a tire in a pothole, you're in trouble.
Hilary:
So if you think of that spare tire sort of like VHL, when you inhibit some factor X, which might be a wheel on your car, as long as you've got VHL, or a spare tire in your trunk, you're okay. But if you don't have VHL, or you don't have a spare tire, then when you go and pop your wheel on your car, you're in real trouble because you don't have any backup plan because you're hyper dependent. So that's how we like to think about synthetic lethality and cancer.
Tracy:
I see. And in your study you basically worked on HIF-2 Alpha. What is the difference between HIF-2 Alpha and HIF-1 Alpha that was talked about during our conversation about the Nobel Prize?
Hilary:
So HIF-2 Alpha and HIF-1 Alpha are related. They both stand for the hypoxia inducible factor and then either one or two. And the difference between them depends a lot on the context. In some settings, HIF-1 is considered a tumor suppressor, in other settings it's considered oncogenic, so tumor promoting, and sometimes HIF-1 and HIF-2 have different roles in the cell. So when we talk about HIF in terms of kidney cancer, HIF-2 in the setting of kidney cancer is oncogenic, in fact it's an oncogenic driver in Clear Cell Renal Cell Carcinoma. Whereas in the setting of ccRCC, HIF-1 is sometimes thought of as a tumor suppressor or in some ccRCC cell lines, HIF-1 doesn't exist, it's simply isn't expressed. Whereas HIF-2 is always expressed in ccRCC cell lines.
Tracy:
Okay, thank you. And so in your study it's HIF independent. What exactly does this mean to the future application in clinical?
Hilary:
So the reason that it was important for us to understand the HIF dependence of this drug, and this drug combination, that we talk about in the paper, is because we don't yet have a great biomarker to determine whether or not a particular patient is going to have a HIF dependent or a HIF independent ccRCC. And part of that is just because there's a heterogeneous population of ccRCCs among patients. But when we were looking at inhibiting CDK4/6, which we talk about in our paper, we wanted to know is this true regardless of how these cells respond to and care about HIF, is this really synthetic lethal only with VHL or is this synthetic lethal because VHL acts upon HIF?
Hilary:
And the reason it's really wonderful that we found that this is a HIF independent effect is because we expect, based on our cell line data and our in-vivo data, that patients who might or might not respond to HIF inhibition or HIF modulation in the clinic, regardless of that, they should all respond to CDK4/6 inhibition, because it doesn't matter whether or not they are HIF dependent or HIF independent in their cancer. They should respond to this Palbociclib or Abemaciclib or any of these CDK4/6 inhibitors regardless of their HIF status.
Tracy:
Okay, and you tested both genetic CDK4/6 inhibitors and the pharmacological CDK4/6 inhibitors. Why was that?
Hilary:
One of the reasons that we looked at both pharmacological and genetic inactivation of CDK4/6 was, first, to be thorough, we wanted to make sure that our effects were on target. And so you can use genetics, you can use rescues of genetic phenotypes, and you can use pharmacology, you can use drugs and you can rescue those pharmacologic phenotypes. But for CDK4/6 in particular, this was extra important.
Hilary:
And one of the benefits of doing this study in the first place using both fly cells and human cells, is that fly cells have far less redundancy in their genome. So in humans we have a CDK4 and a CDK6 and those are two separate kinases. In fly cells, there's just one protein that works like both CDK4 and CDK6, and so in fly cells, when I genetically inactivated the orthologue of CDK4 and CDK6 which is just one gene, I saw synthetic lethality with VHL.
Hilary:
When I moved into the human and I tried to genetically inactivate CDK4 I saw no effect. And that is presumably because CDK6 was able to come in and compensate for the loss of CDK4, and when I did an activation of CDK6 alone, I similarly saw no phenotype in human, presumably because CDK4 could do that job. So when we started using the inhibitors, all of the drugs that are available to target CDK4 also target CDK6, and vice versa. So using those inhibitors, we could block the activity of both CDK4 and CDK6 at the same time. Whereas when I genetically tried to inactivate CDK4 and CDK6 at the same time, it proved to be lethal. I couldn't actually knock both of those out and keep the cells alive.
Hilary:
So using the inhibitors I was able to block most of the activity but keep the cells alive. Presumably due to the fact that there is some CDK4 and CDK6 that are not inhibited. So we were inhibiting most of the activity, but not all of it. Not enough that the cells would die, the way they did when I tried to knock out 4 and 6 genetically.
Tracy:
So can these mechanism be the target of treatment for CCRCC in the future?
Hilary:
I certainly hope so. I think our data shows very compelling evidence that this is worth trying in ccRCC patients, and that that clinical trial, hopefully, will show the same. We showed that this relationship between VHL and CDK4/6 is really fundamental. It's true in flies, it's true in cells. It was true in our in-vivo studies as well. And I think that showing that it was HIF independent, showing that it would work in a diverse array of ccRCC cell lines, both in-vitro and in-vivo, suggests that we should be looking at this for selective targeting of ccRCC cancers.
Tracy:
So in order to be applied in the future what do you see that needs to be done, and do you see any opportunities, any challenges there?
Hilary:
I think there are always going to be challenges moving into humans that you can't foresee and I think that no matter how careful you are and no matter how excellent your preclinical data looks, humans are not mice, and humans are not cells in a Petri dish, and there are always going to be interactions that you have to be careful about. I think that one of the promising things is that CDK4/6 inhibitors are already in use in the clinic for other applications. So we know that there are doses we can use that engage the target and are safe for humans. So that is definitely a good sign.
Hilary:
And I think that the other thing that we have to keep in mind is that the more carefully you do your preclinical work, the more likely it is to succeed. But even if you've done perfect preclinical science, there's always a challenge going into the clinic. So all you can do is be really careful about your preclinical work, and making sure that you take a lot of different approaches to show your effect is real and on target, and cross your fingers from there.
Tracy:
Yes. So in one or two sentence, can you summarize what is the most important part that Dr. Kaelin's work has the impact on your study?
Hilary:
So I think without Dr. Kaelin's work, we wouldn't know how to use the VHL loss that we see in ccRCC effectively. Part of this study we published shows that you can combine a HIF-2 Alpha inhibitor with a CDK4/6 inhibitor, and had we not known that VHL was responsible for HIF degradation, I don't think that combination would have been something we naturally would have thought of. But here we were able to look at CDK4/6 inhibition, ask whether or not HIF is involved and what we were able to show was a synergistic effect, which means that using CDK4/6 inhibitor with a HIF-2 inhibitor actually works better than either one alone, and it actually works better than the sum of either one alone. And so this is a pathway we wouldn't have known to look at if not for the discovery that VHL and HIF are connected.
Tracy:
Great. I actually wanted to ask a question about your daily interaction with Dr. Kaelin. What's the most inspiring part that you learn from him and also, what's the most part that you appreciate, as a postdoc fellow working in his group?
Hilary:
I think one of the most important things that I have learned in the last three years in the lab is that your experiment is only as strong as your controls, and every result is assumed to be off target until you prove otherwise. And because of looking through that lens of really questioning, "Did your assay work?"
Hilary:
Before you even bother to look at your results, before you look at the samples that you care about, did the assay itself work? Did it show what you expect before you even look at your treatment groups? Do your controls show that you can really trust what this assay looks like, and to just make sure that when you read a paper or when you look at your own work, what is the least interesting explanation? That's something that Bill always asks us to consider. What is the least interesting explanation for this data? And then start crossing off those possibilities. Show that it couldn't be that the cells are dying because you've put bleach on them.
Hilary:
You have to show that it's not just any killing drug, it's a specific killing drug. It's working this way. And I know that because I can rescue based on this experiment. So I think it's taught me to be a lot more careful when I plan experiments and also to question the experiments that I'm using to set my foundation. If I'm looking at a paper and I want to repeat that work, you have to hope that you can repeat it before you can even start trying to build on it.
Tracy:
Wow. He sounds like a great mentor. Thank you for sharing this with us. And on a side note, I learned that you are National Director at Science Cheerleader. Can you tell us more about that?
Hilary:
Absolutely. So that is one of the outside the lab activities that I spend most of my time on. Science Cheerleader is a nonprofit organization that is made up of current and former professional and collegiate cheerleaders who also have careers and advanced degrees in STEM fields. And we go out into the community to try to encourage everyone to become interested and excited about science and also to break down stereotypes about who can participate in science. It doesn't have to be all men in lab coats by themselves in a room. And you don't have to change anything about yourself in order to fit any mold of a scientist because there is no one mold. And the stereotypes about who can participate in science and what you have to look like and what you have to know and who you have to be. None of that is valid. Anyone can be a scientist, anyone can participate in science that really matters, as long as you are passionate about it. And you shouldn't let anything about what you have in your head as an image of science or scientists hold you back.
Tracy:
This sounds wonderful. Thanks for being such a role model and it was nice talking to you and congratulations again. So I guess we will end here and hope you have a great day.
Hilary:
Thank you very much. Same to you.