Episode #3

             

                           

     

About this episode

Have you had moments that defined your scientific tastes? For Dr. J Silvio Gutkind, a class on oncogenes and his interests for GPCRs helped shape his scientific interests. These took him from the University of Buenos Aires in Argentina to UC San Diego and through the National Institutes of Health in Bethesda, Maryland. In this episode, Silvio discusses G protein signaling in the context of cancer, immunotherapies, and combination therapies that could help improve patients’ lives. 

 

Dr. J Silvio Gutkind on the web

• Dr. J Silvio Gutkind on LinkedIn

• Gutkind Lab – UC San Diego Moores Cancer Center

• Gutkind Lab publications

• Gutkind Lab on Pubmed

• Gutkind Lab on Twitter

• UCSD Moores Cancer Center

• Dr. GPCR Ecosystem

 

Episode transcript

   

Dr. Yamina Berchiche  0:00  

Hello everyone. Today we are speaking with Dr. J. Silvio Gutkind is currently a professor of Pharmacology and Associate Director of basic science at the University of California, San Diego. And thank you for being here today with us. How are you?

Dr. J Silvio Gutkind  0:16  

Doing Excellent. Thank you Yamina for the opportunity.

Dr. Yamina Berchiche  0:20  

Thank you for being here. Can you talk to us a little bit about your career path and how you got to where you are today?

Dr. J Silvio Gutkind  0:26  

Hmm. It's a long career. So, so, initially, I will say, I did my PhD in Argentina at the University of Buenos Aires doing extremely, extremely basic classical GPCR biology, working on alpha two and adrenergic receptors in hypertension, modulating so, in Argentina is where initially, the first description of the alpha two presynaptic adrenergic receptors were made. So there was a big school for them in pharmacology. So I got my PhD there. And then I moved to NIH as postdoc in when I moved there I went up one step. In the sense I focused mostly in the CNS working neurobiology, but again GPCRs was focus mostly angiotensin in remaining working on cardiovascular in the sense of angiotensin receptors in the brain in neurobiology, but in concept of regulation of blood pressure control, focusing on the brain, neural beta receptors. And from there I I take a very very unusual change. So I was taking a class on so at NIH a class on hormones, receptors and modulation etc. So it was very, very exciting appropriate for my training. And it was one class on oncogenes and it was very cold winters in washington dc in Bethesda in the USA, what the heck am I doing called in listening to oncogenes. In the end of the class, I fell in love with oncogenes. And then I switch, and I was I had an opportunity to move to the Cancer Institute at NIH, NCI, where it was a time of the discovery of most of the oncogenes. So in the context of viral oncogene etc. in that it was very interesting switch, molecular biology became my technique, etc. And from there that became absolutely focused on cancer. And so, initially working on non receptor tyrosine kinases, and then I was given the opportunity to basically as a successful postdoc, you negotiate, and I was given the opportunity to do something by by myself, if you will, in addition to working on on the main topics of the lab, instead of working on GPCR, oncogenes, so the time it was very fueled only one paper on the ras oncogene nobody really followed that and I was given that opportunity. So like, like a side project while complying or working on exciting projects within the lab and it worked. So, basically, I was given the opportunity then to move to the dental Institute where I helped establish another cancer program in the opportunity to start beginning to develop a program on that so and then GPCR in concept became what I have been working since ever since. And I I went through the ranks and got tenure when I was very young, and very lucky and then become eventually Branch Chief, which is equivalent to like department chair. And I was Branch Chief for many years working on GPCRs in cancer and oral cancer that was part of our mission. And very recently in 2015, I left NIH and moved to UCSD following by my students in our team so 12 people come with me from NIH. So there was a recall. So it was, it was a big move in. So but essentially, we establish our lab here, the cancer center, but I'm associated to focusing science in the same time working with my colleagues in pharmacology. You name all of them in, in working on GPCR in cancer. So that's how I ended up. We ended up as a team here.

Dr. Yamina Berchiche  4:27  

That's fantastic. So every 12 people moved with you cross country. How much do you think the weather versus the love for GPCR has led them to move with you?

Dr. J Silvio Gutkind  4:37  

Well, first we call Operation Exodus. Just to give you an idea, because it was a big move. Many people came driving in and I came flying so there was easier. It was so it was already neither was was probably the passion for  everything in our team. It was very, very active people like each other a lot and everybody was over the beginning to be successful, some very successful. Few people stay one of them stay with a tenure track position at NIH, in at NCI and several others, so we were able to identify positions for everybody. Those that did not come here. But the idea was to return the team in to be honest with you, we'll have other opportunities in everybody persuaded me the amount of opportunities that were there probably encourage everybody to select UCSD as compared to other locations. But everybody came with us. Yeah.

Dr. Yamina Berchiche  5:35  

That's great. So you had mentioned just a minute ago that when when you moved and you started working on oncogenes, you you started working on this very tricky project that involves GPCRs and in cancer. At that time, were you the only one who was thinking about GPCRs and their role in cancer?

Dr. J Silvio Gutkind  5:53  

Probably does is always I know many people were thinking about it, but but the reality was only one at a time. The ras oncogene showing so from the Wigler lab, it was pretty nice Cell paper, there were days in which you can grind it to more transfecting two cells get the focus or a few foci in the new sequence, and then you end up identifying an oncogene and you you were able to publish a paper in Cell. But But, but but the reality is that besides that, the technical aspect, very few people really were really focusing on that and the main reason was because the ras oncogene did not have any mutations. And so that, at that time, they were wonderful publications in some that you can say were, were questionable in the sense that relevance to cancer was questionable in this particular case, three people did not embrace it became like a well happens so be it, in so many people explore it, that was basically how we started. And the very simple concept is that GPCR do not need to be mutated to be oncogenic all you need have is expression, abberant expression usually expression in the wrong place at the wrong time in concomitant excess of ligand either produce locally, like an autocrine mechanism of production, we call this oncocrine in the stage is new term oncocrine signaling, or or just circulating, so if you have, you don't need So, the most pressing issue was when when doing this focus formation assets everybody was doing at that time to identify oncogenes is that when you do compare transforming efficiency of I use the muscarinic receptors especially. And the reason I use muscarinic receptors is because the carbachol was so cheap and so stable it is if you leave with any other receptor, it will take forever and would be so expensive. So carbachol you can boil it's still active.  And so that was the reason I selected muscarinic receptors. But the bottom line is if you looked into the number of foci you get which was our way to measure the transforming potential was amazing similar to Ras or many other oncogenes. So the shape of the foci was different. It was very clear you can distinguish them, but the number of just phenomenal so that was really shocking, not one or two here and there and you go from there, it was just so you need to dilute a lot of the DNA. So it really prompted us to work. I mean, at that time, we were very few it was one or two, my team to like what the heck it was a our reaction and that was why we that's it was probably know, most people were not working on that. In even today. So probably is the limit under explorer which is, which is something being under explore or under exploited means it's an opportunity. So that's exactly the way we see it.

Dr. Yamina Berchiche  8:51  

So it gives you an opportunity to  make a point and explore GPCR is in the context of cancer. That was actually one of my questions is how does that The cancer community who's mostly focused on mutated proteins, think about GPCRs that are overexpressed in these cancers?

Dr. J Silvio Gutkind  9:10  

So I think it's changing. So they so so what is the turning point? In certainly the turning point is a cancer genomics. So with a revolution of few years ago, the ability to sequence cancer genomes in more recent is in availability from TCGA and other international you can say, operations and in this area, in combined with the use of bioinformatics tools, that that changed completely because for the first time A) we do see that there are many copy number variations in specific GPCR in specific very specific cancers. There's not just widespread it's very specific cancer types that we can see copy number variations increases with expression levels and we are beginning to see the rationale behind that. But also, we can see many mutations in GPCR 20% of the cancers have mutations in GPCRs is much more than we ever expected. So that was initially we published in in Nature Reviews Cancer in 2015. Together with Tracy Handle here and Sekar in Genentech so they helped with a lot of bioinformatics that was eye opening. In the part, that became even more No, I won't call eye opening like da. So instead we start seeing patterns have mutations in G proteins now, they're very they're very hotspot, they are similar to Ras. So it's like in a sense is single nucleotide is always mutated in Gs in GQ, primarily in Qq 11. So we see very hot spot mutations in that there are some concepts now we know in everybody knows it's not what we know about the community accepts they're driven by G protein. So the concept that there are cancers there are three of them by G proteins, change the land, the landscape in terms of understanding appreciation, in the other part they align with that is the fact that many G proteins, some GPCRs are part of the current sequencing panels. So most cancer patients, at least in UCSD, when when they have the tumors, usually they will be sequenced. And so but they don't do whole genome sequence in general, they do a panel of 400 or 500 genes. So there are many companies do that in in house as well. But But these very frequent most big cancer centers and they will sequence they are several G protein, so Gas is it 13 Q and 11 that are part of the panel. In that means there are many, many new concepts. People have no idea. They don't have a drive, you don't go to you know, saying wow, they have Gq , Gs mutations, etc. And so that people are beginning to appreciate they don't know what that means. But we're beginning to understand in in, but but but people now the cancer genomics again, raise to the bar in the sense that mutations they are there in their course also they do contribute in or initiate cancer. And so that's why it change a lot.

Dr. Yamina Berchiche  12:18  

And that's, that's new and we're learning more and more about G proteins about the fact that they go to the nucleus and they do things there. So I think it's, it's a good, it's a, it's good to be able to identify these mutations, but there is a lot of work that needs to be done in order to understand how these mutations drive cancer. So then, that gets me to my next question, do you have a favorite G protein?

Dr. J Silvio Gutkind  12:41  

So it's almost like let me say very simple. I have two daughters. It's like asking which one you like more. I love both of them are the same, the same way. So so so they asking what is your favorite is you may be neglecting another one. So this one so I will say that we have our favorite G proteins as precisely as I was telling, saying, so that because they do have mutations driving mutations, the ones that we love is a G alpha Q, or they, most of the medical community don't call it G alpha Q. They call based on the name is called GNAQ. Also, if you ask medical students don't even know what a GNAQ is they call it G-NAQ. Well, you need to learn we need to learn the language so to communicate so when because I was given a talk recently on in G alpha Q, and then said GNAQ , ah that's G-NAQ. Yes, that's G-NAQ. So G alpha Q /11 and the other ones, G alpha S is mutated in many cancers happy to discuss. And then in terms of the GPCR it has been usually we have been focused on model GPCRs. So we've never really focused on one GPCR. Now we're changing a little bit based based on our interest, shall I say new interest or renewed interest or just new interest in in chemokine receptors in immuno-oncology. So, in that case probably we are falling in love in with CXCR3 and XCR1. So, these are the two ones that we are beginning to really focus on in the context of cancer immunology. This was so I will say this for will be very different from each other and we embrace them, all of them, we love them.

Dr. Yamina Berchiche  14:30  

That's great. You can have more than one GPCR  or G protein love. I particularly am fond of CXCR3. I've worked on deciphering,  dissecting the signaling of CXCR3 splice variants, which I find also very interesting about you know why a cell would decide to make three variants of a chemokine receptor.

Dr. J Silvio Gutkind  14:49  

Absolutely indeed it is absolutely for cancer immunology you The world may not be even aware that CXCR3 is central to cancer immunology for example, because of happy to talk to talk more about it. So, absolutely,

Dr. Yamina Berchiche  15:03  

Of course, so since we're talking about CXCR3 what is the current status of research about on this receptor in the context of cancer immunology?

Dr. J Silvio Gutkind  15:13  

If I tell you what, CXCR3 most people will know, I mean most. shall I say most scientists or physicians working in cancer will not will not resonate with CXCR3 but they see if I tell you CXCL10, 11 and 9 or IP 10, it was called CXCL10. So it's interferon-alpha 10 everybody will know that these three genes are central to the response of immune therapies. Everybody's perfectly aware that if you if you can measure CXCL10, 11 and 9 in pre existing levels prior to immune therapy is part of the most robust signature predicting that a cancer will be responding say, melanoma or many other cancers, lung cancer, that's it. And most of them is part of even the panel. There will be used by some pharmaceutical companies as a predictive marker. So it's part of the predictive marker, but it's one of the best predictive markers of response. And so in then the reason is, I mean, you say da, because these are the ligands for CXCR3. So because these three lines are the lines CXCR3 and CXCR3 is expressed primarily in NK cells in T cells, CD8 positive T cells, so it's expressing other but it's usually the good, the good immune so so do you have the good ones and the bad ones CXCR3 see the good ones. So it's an express in the in the NK cells, they will be the first responders if you will, in cytotoxic CD8  positive T cells is very highly expressed in the reason is very simple. You have the ligand being produced in response to interferon or usually is part of the STING  pathway that this a lot of research being done. The STING distinct pathway will activate these expressions through interferon response of these chemokines in that will recruit the the T cells and NK cells to fight the cancer. So that's why that may be the main reason. So in there are many opportunities to two dimensional knowledge are still under exploited. So this is a very recent paper in using CXCR3 knockout mice. And basically they found that there's no response to an anti-PD1 or anti-CTLA4, for example. So that's a primary I would say this raises the bar in terms of the need to target that particular receptor.

Dr. Yamina Berchiche  17:42  

Definitely, definitely. It's one of my favorites. Absolutely.

Dr. J Silvio Gutkind  17:47  

I checked your papers.

Dr. Yamina Berchiche  17:49  

I had a lot of fun working on that paper and it's one of my favorites. And you know, it's important, this receptor is also important in IBD, and then all of these immune diseases and I always wondered why people aren't working on it. And I'm glad that it became more and more important now.

Dr. J Silvio Gutkind  18:06  

So it's beginning to be part of our our lab work. But again, so one of the reasons sometimes you go with the, with the flow, sometimes you lead the flow, lead the pack. In some times you have opportunities in this case, the opportunities that based on our interest in oral concept, which we still very heavily invested into. In new in my model is in clinical trials that we're conducting for kidney cancer in the context of cancer immunology. Now we have the opportunity to investigate specifically that and so that is why we will be focusing on those receptors.

Dr. Yamina Berchiche  18:42  

That's fantastic. And tell me more about one or two of your G proteins that are your favorite.

Dr. J Silvio Gutkind  18:47  

so the most exciting right now. So of course, the GalphaS. Is is very exciting, you will see much more coming is the most widely mutated G protein in cancer, almost many many cancers that are extraction. The most basic one is appendix cancer is almost 70%. Pancreatic cancer is around 12%. Colon cancer is around four to 5%. But there are so many colon cancer cases. That is very, very important. But the one where we're making a big push for now, G alpha Q, so it's mutated. So the light dynamic, so it's mutated preventing UVA melanoma, so is the driver of melanoma of the eye, which is the most prevalent cancer adults in the eye. 50% will develop metastases in the liver in his fulminant. So once metastasize the 50%, they their life expectancies around six months to one year max, so it's really fulminant in so the there's a lot to be done with G alpha Q. Number one, number two, also four to around 4% of cutaneous a melanoma have mutations in G alpha Q which are absolutely under under investigated, underappreciated in both of them, but mostly in uveal melanoma. They do not respond to immune therapies. And in particular uveal melanoma, melanoma of the eye, so the number of mutations in total, so usually in most cancers you will have four or 500 mutations 600, 1000, you name it. In the, in the melanoma of the eye, there are only four or five mutations. So it's almost like a childhood type of cancer. The driver is G alpha Q. 93% or G 11. You have 93% of mutations in G alpha Q as a single amino acid  209, when if you have 183, but the vast majority almost 90% of 209, very precise hotspot mutation, the mess that makes the G protein constitutively active because prevents the GTP-ase activity. So this is canonical activating mutation. So in the other mutations are in the RNA splicing molecules that regulate RNA splicing or modifiers of epigenetic. So, is the driver in in that context, there are no therapies currently available. People have continued dying there are several treatments, several clinical trials are conducted in perhaps a living this is where the science can come in is when you need to be open minded. Another canonical G alpha Q signaling or or scientists or student you will always put PLC in PKC and then you go from there ERK and whatever is your choice. We went a little bit more open minded we run it in  siRNA screen or dsRNA screen in Drosophila cells to try to investigate what's really important in terms of we knew AP-1 one was important, a transcription factor and what we found is that there is another component, completely different. It's called the molecure is called Trio. So the idea is the GQ will activate PLC that's very important but every attempt to target that has failed in the clinic in its sense of not having any impact. I'm not saying it's not important, I'm saying that this target in that alone is not sufficient. And  so in the most recent publications, etc. so we will we keep up with people who are brilliant doing bioinformatics. And so, we did some synthetic lethality analysis using all in silico not even running a single CRISPR analysis and we came up with some new ideas, but the take home message is that in addition to the canonical pathway, there is a very poorly investigated non canonical pathway through the activation of this exchange factor called Trio that activates Rho in to some extent Rac but mostly its Rho and then Rho will activate many things. The one that whether with other team here or independently, and in our lab, we show the YAP was central in addition to AP-1. In more recent our most recent studies For druggable because YAP is wonderful, but it's not druggable in the part of the HIPPO pathway, which did not expect before. So we did this  bioinformatic approach to synthetic lethal interactions, we found a product called FAK (Focal Adhesion Kinase) was center activation of, of YAP and downstream from GQ and Rho. So that led to a clinical trial that was just opened by a by a team and our own clinical trial will be open relatively soon both a based on our observations so again, so you need to be open minded. If you go to the textbook you will see PLC stay with that fine, but for the immediate effects, but for cancer in probably for many things they involve cell growth promotion can be hyper proliferation can be many other diseases. The Trio-Rho pathway that is very fully investigated is also central.

Dr. Yamina Berchiche  24:04  

Well and what is the so in uveal melanoma if I remember correctly, there have been reports of the Cys-LTR2 receptor being mutated and being considered actively active. How does that the receptor fit with the GQ mutation?

Dr. J Silvio Gutkind  24:21  

Yeah, I was so so 93% have a G alpha Q. The few ones that don't have a G alpha Q, they have a mutation is Cys-LTR2  which is a typical canonical GQ activator and so it's perfectly so it's a cannot it's a single nucleotide mutation. All of the patients have in one place where it's mutated that a receptor is now being sequenced as well as part of the panels is a GQ. So it's a very small number but fits perfectly well with a GQ.

Dr. Yamina Berchiche  24:51  

And can they be mutated at the same time or it's usually one or the other?

Dr. J Silvio Gutkind  24:56  

Mutually exclusive. So G alpha Q and G alpha 11 they're almost identical, there are 40 something percent each, they are mutually exclusive. 

Dr. Yamina Berchiche  25:05  

Okay.

Dr. J Silvio Gutkind  25:05  

They're also mutually exclusive with Cys-LTR2. So there is one or the other. But it's not that it cannot be mutated. Instead there is no gain to be mutated. So in cancer, we often use mutual exclusivity as an indication that there is no gain in this enough with one of them in saying this there are complimentary

Dr. Yamina Berchiche  25:29  

Definitely that that it seemed like the mutation and Cys-LTR2  are two very similar driving elements on that matches the G alpha Q. I'd love to hear more about the clinical trials that you're conducting, looking at the alpha, the mutated G alpha Q, can you tell us more about that?

Dr. J Silvio Gutkind  25:45  

Yes. So sort of the trial one trial was just open based on these findings for from so one has to understand so that they you can call it again the day the cancer patient is that is really what we need to help, who does it when when who benefits financially who takes the credit who these are details they will be in few few months few years will be less less relevant. So the important issue again so that several trials were conducted blocking primarily blocking MEK as part of the ERK pathway, all of them fail in terms of progression free survival in in, in survival of the patients, three independent trials were conducted. Two PKC trials were conducted- one initially there were some toxicities in a recent trial, there are less toxicities and yet very limited activity some activity, but limited. So, so basically saturating if you will, the the canonical pathway seemed not to be sufficient, that's the message. So with based on our study with FAK, so the there are several companies that have FAK inhibitors in their pipeline. We're working with one of them ourselves. But basically there are many for different indications. So for fibrosis, to enhance response to immune therapies because the anti fibrotic effects so they're already in the safety is very high. They have very limited toxicity. You know, it's not it's not aspirin, aspirin it also has toxicities, but it's not yet. But but they're very limited toxicity when compared to other conventional cancer therapies. And so based on our publication, so they even before the publication based on abstracts presented, one company started over the clinical trial of developing a clinical trial there was just open in three centers  and focus on FAK in they plan in the future to combine with a typical MEK inhibitors, in our case, our trial, we are developing so the trial is ready to be submitted very soon the company is sponsoring the trial. So it would be a FAK inhibitor or a different effect inhibitor for some reasons we believe we will be better. The only reason is because blocks and other kinds called PTK2B, PTK2 is the gene name for FAK, PTK2B which is very typical also has been reported a long time ago by Joseph Schlessinger to be downstream from GQ couple receptors just in India, which is fully expressed in uveal melanoma but we think that this dual activity will be better because it's a chance that you block FAK, PTK2b maybe be a make rescue. So we feel the blocking both will be important. In recent studies are unpublished so we are ready to submit soon. So we did a crispers claim but then what can kick what can you do to enhance the response to FAK, the paperwork has been submitted, I should say in so what we found is that targeting the canonical pathway was the best way to synergize. So we'll be using a different MEK inhibitor is experimental is not approved by the advantage of these second MEK inhibitors is we think is more potent because blocks the MEK-RAF interaction preventing the over activation of the pathway which usually is what have a lead in melanoma can be rough mutations can lead to resistance. So, we think this would be almost like a perfect match in there is also from this model where they some preliminary data using this combination that shows some activity I cannot disclose more than that in some cancers, some interesting activity or shall I say, but but also limited toxicities. So this way so we feel this may be the winner combination. Hopefully we can conduct the trial and we already have the preliminary information about toxicities, dosing, so there are many in terms of drug development in advance into the clinic. So these are very important components that they will facilitate the process of the trial. That's our perspective.

Dr. Yamina Berchiche  30:15  

That's great. So it's a combination therapy. 

Dr. J Silvio Gutkind  30:18  

Yeah. 

Dr. Yamina Berchiche  30:18  

And of potentially repurposed drugs at this point.

Dr. J Silvio Gutkind  30:23  

Yeah. 

Dr. Yamina Berchiche  30:24  

That's, that's great. Because you're, you're, you're, you know, jumping hoops and stepping over the whole toxicity, toxicity issues. What do you think, at this moment, are the biggest hurdles in treating these cancers and advancing research to understand the function of these proteins of  G proteins or GPCRs in cancer?

Dr. J Silvio Gutkind  30:44  

So  I think there are no no roadblocks is basically they're not they're not real roadblocks. It's a it's a mindset. I think it's a mindset so that they, so it's a mindset in the sense that the often often not always, it most people were doing GPCR don't work on cancer, they use cancer cells  293 of cancer cells, okay. But most people work on GPCR. They don't fully work on cancer, these cancer cells as a test case, but the cancer has a lot of details that sometimes escaped to someone who focuses on the biochemistry or function of GPCRs. So many details are very important that you need to embrace. And on the other hand, again, most people will in GPCR, don't work on cancer. Most people in cancer don't work on GPCR. So I think that the now is is a perfect perfect match to start developing together in the community to develop this this understanding in exploiting GPCRs in cancer and G proteins. Perhaps one approach so if you ask me what is missing, A- is the acceptance in the community in the cancer community of embracing the prospect of GPCRs and G protein bieng important in cancer, which is slowly advancing based on genetics, genomics, etc. So, as I was telling you, but the other is to have a real so the problem so G protein are easy. So, targeting is difficult, but you brought in studies because there are a few, four classes of G proteins beta gamma's, of course, beautiful. You can embrace, you give it the name, GNAQ, GNAQ, G alpha S. In perfect you can embrace anyone can embrace. GPCRs because the number of GPCRs themselves is different classes. And  because that, with the exception of Cys-LTR2, there are very few hotspot mutations in cancer, it's very difficult for the community to embrace them. So this why a we tried to go back to say Back to the Future, trying to re-emphasize the issue of these, these the issue that maybe mutations may not be the only thing that activates GPCR in focus specifically on the on the possibility of these oncocrine networks where ligands secreted locally call it chemokine, call it neurotransmitters, beta2-adrenergic receptors, it is important for some cancers and prostaglandin receptors we feel are really important etc. So, the idea is to embrace this concept of the oncocrine or autocrine, paracrine signaling that may facilitate cancer or even in some cases may even initiate And so, in that is bring into the concept of cancer immunology is even better, because again so, now, it makes a lot of sense that the for the immune cells to be recruited, they don't go by chance to the tumor they're recruited in they're recruited by chemokines like this. Back to the Future again. So the concepts are very simple. Even if the inactivated even a CAR-T is ready to go to kill a cancer, unless it's recruited nothing's going to happen. So That's why so I think embracing this in I know that sometimes it's difficult because the name settled with a funny, nobody knew what PD-1 before nobody knew about CTLA-4, maybe having a number or having another name. But all for example going to CXCR3, if you explain about the the CXCL11. So CXCL10, 11 , 9 being so centric, which everybody embraces already, that's perfect. And then you explain that CXCR3 is central to that response, then people start beginning to embrace. So that's what we think is missing. So communication in in, in embracing number, you know, that would be one of the issues. The way we try to overcome that is by cataloging and cataloging is very important developing data sets, or databases. So we recently published a paper in JBC in the main reasons of some of my colleagues as to why you publish that in JBC is fairly simple. When we published the other paper in nature reducing cancer, my colleagues working on GPCRs usually don't read it. So it is cancer centric. So say okay, let's go to hard core what we all read, a make it absolutely available. So, it's all freely available all of the data sets out there or wherever, wherever express we are doing, continue working on that in in the context of cancer immunology. But by cataloging, showing, making available these data sets, people can can can can really see whether they express which cells express the cells whenever they express, whether in responder versus non responders. So you can put some flavor they are clinically relevant in this what we want to achieve. So make it available, we focus ourselves some in and happy to share the field so it's not that we won't really it's a competition is really moving the feet forward.

Dr. Yamina Berchiche  35:56  

I agree. I think communication is key. And I think the biggest thing issue in science and working on GPCR, as in in cancer is the availability of that information, and how fast you can get the information and how fast you can, you know, form partnerships with the experts, like yourself, for example, if you had to wish for a drug, would you want to drug the targets of GPCR? Or would you prefer having a drug that targets a G protein and what are the difficulties of having a good drug?

Dr. J Silvio Gutkind  36:25  

So regarding the the G proteins, the proteins of GQ is the G alpha Q when we're studying. So, certainly, this is a signal transduction driven cancer, uveal melanoma. So if you if you will, it will be almost like the best example of a signal transduction driven cancers tend to have mutations in terms of any other - this is a driver. So then intracellular signaling is what you want to accomplish. You want to kill the cancer cell, but not the not the normal cells. In this is the concept of precision therapy or precision medicine. So again, uveal melanoma is the best example of precision medicine, what you need to target only the primitive pathways in the, in the specifically in these cancer cells, which I think is doable. If you want to GPCRs I think that the big explosion right now would be with the use of two things. One is antibodies, targeting antibodoies, this so the first example was CCR4. Mogamulizumab was the first antibody was FDA approved, targeting GPCRs. Right now he's very focused on one particular subset of of the cancers very, very small subset, if you will. But But the reality is that that that opens the use of antibodies blocking GPCR or stimulating GPCRs.  Antibodies, I think in the long term that will be probably the future given me the huge specificity. And impossibilities of long term use it there are so many many reasons why antibody will be the long term. The long term and the short term probably more than canonical blockers or or agonist /antagonist the possibility of of molecules that will be modulating them. So, the data will be allosteric modulators of GPCRs will be probably the way to go in the sense that you you don't block every GPCR in in the body, you block only when you modulate switch you know turn on and off a little bit modulate rather than block for simple for chemokine receptor we think is the way to go. So, you can turn it on a little bit and so, there will be more responses to the locally produced ligands in I mean not turning, tuning that will be what you want to do to tune up/ tune down probably that is the best way to go so in in the future. So, basically allosteric modulators would be one one area for GPCRs, which is a exploding right now. In the other one would be eventually antibodies, targeting antibodies.

Dr. Yamina Berchiche  39:08  

It's Yeah, it's that sounds like a good path forward. When it comes to allosteric modulators to tune the function of the receptors. I mean, I guess you need a structure to try and find an allosteric modulator site.  How much do we know about potential sites? And how much do we know and how much we want to tune? For example, receptor functions in the case of CXCR3, for example.

Dr. J Silvio Gutkind  39.34

So, no, that would not be me. I don't work on structural biology of GPCR. But then is when partnerships or collaborations are fantastic. And so so we're working here with Tracy Handle. She's the expert in chemokine receptors in the structure, and she can close her eyes and tell me precisely where to mutate and we are doing that so I will not be able to do so this is the typical case where you would like to you it's not you'd like to you have to collaborate with like minded colleagues, scientists in quite often wonderful friends, which is always helps. But at the end of the day, I think there is enough you want to be general but for different receptor subtypes for which there is already many, many structures, I think there is already opportunity to start predicting this. So, this also based on our work in terms of tackling for example, so, it's a paper together with my colleagues in Japan in Germany, which we published in  Cell. So, we use you can call artificial intelligence or are other approaches so to to identify structural determinants of coupling for example, that gives you a very excellent example of that this is doable. So if you have a lot of this structura; information you can use is called machine learning so you can use machine learning approaches combined with a with the wet lab experiments guided by these and then reformulating the the equations that you use, I think this is a combination that will be used as a winner. So, having, people who are doing a lot of crystal structures, they have the expertise then they know in field every amino acid what they will do, you can ask my colleagues here I will not give you all of the names but they really know what they do combined with a more experimental approach targeting that and probably machine learning approaches to come up with overriding a you can say trance or perspective how can you modulate.

Dr. Yamina Berchiche  41:45 

Using machine learning reduces the number of experiments that you can you have to do in the lab and at the same time, the processing information processing capability of a computer very much exceeds our ability to do pipetting and then dnd then screening but for that as you had mentioned, you need a good crystal structure

Dr. J Silvio Gutkind  42:04  

 And you need the team.

Dr. Yamina Berchiche  42:05  

Obviously,

Dr. J Silvio Gutkind  42:07  

we have  computational people in Germany now moving to Italy and in our colleagues in Japan doing high throughput screens in, in our emphasis in terms of biology, so it was perfect and each one contributed to that. So it's in from different perspective. So it was you needed different expertise and team therefore.

Dr. Yamina Berchiche  42:28  

Definitely it goes back to communication and being / wanting to form partnerships to advance drug discovery and understanding the role of these proteins in these signaling cascade in diseases like cancer. You had mentioned pancreatic cancer is basically a death sentence, getting getting a diagnosis of that. You had mentioned that while at NIH, you went to this class in the middle of winter and you became enamored with oncognes. That to me was kind of an aha moment that you had mentioned and that shaped your career. Were there any moments in your career that you could qualify as aha moments?

Dr. J Silvio Gutkind  43:12  

So it's not easy that almost everything in science is called but but but probably the one that was. So, old fashion if you will using COS cells transfecting with there was the idea of the ERK, how on ERK or MAPK is regulated by G proteins in so, you know, few papers get in there and it was very confusing. In his focus on Gi coupled receptors, everybody know, you take Gi coupled receptor, you stimulate with ligand, you get ERK activation or MAPK at the time, and then you block that with pertussis toxin. And it's very easy. Gi is involved, nobody will argue. And so then doing a working on something else. So we started expressing G alpha i mutant and wild type in COS cells so we didn't see any activation of ERK in it no matter how you do Western blot here Western blot there, DNA, new preps, whatever mutant here mutant there, we couldn't see any activation. So this is what the heck. And then once you start reading how pertussis toxin works, that basically ADP rebosylates, they C-terminus preventing G protein coupling to the receptor and say what the heck. So maybe it's not it's not the alpha, maybe it's the beta / gamma, and at that time it was very controversial with beta gamma did or did not do it very, very interesting arguments in the context of meetings so but what they were not doing. And I said let's do it. And so one of my colleagues at NIDDK so he got all of the beta gammas already expressing for different reasons. Let's give it a shot. And we express the betas and gammas. No, really, and then we combine wow for the first time, we saw ERK activation. And only betas not beta 5  but mostly beta 1 with the 2 anyone but beta 5, which makes sense. And normally with a gamma that was not a gamma 1 there's only in the in the eye in only myristoylation. So how to be a modulated so they would have a lot of controls afterwards but the first was that beta gamma alone, but not alphas were able to activate ERK that was happened to be our first Nature papers. So there was it was not easy to publish because we were not known in the community. So that was probably one in aha moment. Probably the second similar to what the heck aha a moment was when when working on JNK kinase. So when ERK was the first kinase to identify the MAP kinase pathway, the second one was JNK kinase. And it was part of the JNK kinase  was activated by Ras in Cell papers etc was very, very compelling. And so we tested in GPCR the activation of JNK kinase is extremely well. Perfect, but much better than PDGF or EGF that activates very poorly, but very well ERK. So there was a disconnect between the two. And so when they say okay, let's do again our analysis and we put Ras in we saw, a two fold increase in ERK and JNK kinase activation. If you remove the background, multiply by the number of times you do and select the right example you can find that there was an activation but I would suggest to four to three fold max. And in GPCRs, will activate Gq coupled receptors, specifically and G 12/13 will activate 10 times so we're going to justify and so then what the heck so we collected we have a number of oncogenes in the lab, from the DBA path etc. We have many oncogenes because I work at the cancer center. So we have many of these in our fridge. And in most of them do not although published in very well known journals you name it, some of them inducing ERK because it was very popular at that time everything will go through ERK we didn't see much activation. Said well these oncognes do not do not activate ERK but they do activate beautifully JNK kinase something else should be there. And so in we collect the Rho GTP-ases was emerging. The Rho GTP-ases may be acting downstream from these oncogens, not Ras in in that was fantastic. So we should basically Rho, Rac, CDC42, but mostly Rac and CDC42  whatever to activate extremely highly JNK kinase, almost no activation of ERK. And so that was that was our first Cell paper. Still our most cited paper so it was all based on what the heck this doesn't make sense in that basically is just being open minded, not trying to push anything in order to publish. It's really getting deeper in really question yourself. Maybe we are doing something wrong, number one of course it's possible. Maybe we're not doing something wrong, maybe the concepts we are basing our experiments on may not be correct. And that is when you have huge opportunities to be breakthrough discoveries. Both of them are very recognized and highly cited, but the bottom line, those were probably very exciting moments.

Dr. Yamina Berchiche  48:15 

 

That's great. So speaking of aha moments, and looking back through to all these papers, if you had a young scientist right now in front of you, what would be your advice to them so that they can you know, jump on the train and start contributing to the field.

Dr. J Silvio Gutkind  48:31  

Be open minded in following your heart and soul into it, don't get me wrong, read certain you have to read a lot. But you need to make slowly try to distinguish what's published and what's I will call it real, in the sense of get the sense of because many, many times many findings don't don't don't pay attention to the impact factor of the journals. Pay attention to the impact. How many you know who reproduce who did not etc or which context sometimes is nothing wrong, but it's a very context dependent so try to be extremely open minded read but mostly think so that would be the most important in in try to follow your heart and find something that doesn't doesn't match the expectation based on the publication's based on all of these, but you think is relevant relevant in my case would be in our case, but it mostly based on cancer, and cell proliferation. Some other case would be neurotransmission. Some other case would be I mean, so it's not only cancer, of course, something so the biological relevance in in translational, potentially, I will argue, maybe the major drivers of deciding where to focus, but the how to focus and how to have fun. Do your experiments, plan it,  keep it simple, keep it simple, though. You don't need to do knockout to prove a point. Prove it anywhere and then be ready but mostly be open minded, open minded and be absolutely free and  enjoy challenging pre existing dogmas they may have been established for in a very specific area and very specific cells, very specific context.

Dr. Yamina Berchiche  50:15  

All right. Thank you so much for that. And if you have job openings in your lab to join your team of 12 that moved cross country with you where can people find you?

Dr. J Silvio Gutkind  50:26  

That is an interesting question. So happens that so often we do so now in the old days at the NIH, we have a fixed number of positions. Now, It changes based on reviews of course, which is the way intramural scientists are being review and resources are located. In the extramural is based on on opportunities, mostly on grants, or or opportunity but mostly grants. So we have been very fortunate. We have several students in there very extremely passionate. So they they local students based on communication. We do have openings from time to time or based on the resources in it to some extent I'm not advertising anywhere. So people receive emails from colleagues, former students that are now a directors of Institute's. So they have many of my students are doing amazing everywhere. They're really wonderful people and they continue doing science, so I am not posting it almost anywhere. So that is maybe a mistake, because there is a pool of people, they don't even think about the potential for applying that I'm missing it myself. And the same is true maybe from my colleagues. So it's basically if you if I can give an advice to a more junior person in training, don't be afraid. So you like a paper you like this subject you think the person may be okay. So, digging in the web, try to, you know, all of the parameters you can you can see in the websites when it's a fun place or is boring place both of them can be wonderful for you, up to you. But then  contact, send an email. Don't be afraid to send emails.

Dr. Yamina Berchiche  52:09  

Yes, because what's the worst thing that can happen? People can say no, but if you don't send the email, you won't know. 

Dr.J Silvio Gutkind 52:15

Exactly So after I finished talking to you actually, I have an interview with a one one student potential candidate, they send me an email a few days ago and I immediately contacted everybody around that person, supervisor, etc. Even he was very positive. I mean, we'll see. And then for me, it's very important that the team interviews the person will be by zoom not physically but initially discussed with the person and then the team will will interview because it's important the team. So the structure of the team is central to making progress. Mostly the opportunity for the midterm day. Here, if you don't move, this is California. Move the light switches off. You need to do need to continue being active. So even stretching so the team is central. So we are very lucky with a our our students there, again, they're fantastic but the same time, there personalities, and team efforts and I prefer always to during the  students and postdocs play a very, very important central role in really analyzing in identifying who they would like to work with for the next 2, 3, 4, 5 years because they will be interviewed. So, you know, as a junior scientist, don't be afraid, send an email, contact the person, contact the students. At the end of the day if something is more important contact the students get to know and move from there.

Dr. Yamina Berchiche  53:47

I agree. I agree. It's very important to interview people but also let the give the opportunity to the interviewee