Ewings U: Ramon Sun, PhD | Leveraging Next Generation Spatial Technologies to Identify Actionable Targets in Ewing's Sarcoma

EPISODE 5: Dr. Ramon Sun discusses how his lab uses cutting-edge analytical techniques to discover a unique metabolic dependency of Ewing's Sarcoma cells, and how this newfound knowledge has led to further preclinical research that exploits this metabolic vulnerability, significantly reducing the growth rate of Ewing Sarcoma tumor cells.

NOTE: THE FOLLOWING IS THE OUTPUT OF TRANSCRIBING FROM AN AUDIO RECORDING. ALTHOUGH THE TRANSCRIPTION IS LARGELY ACCURATE, IN SOME CASES IT IS INCOMPLETE OR INACCURATE DUE TO INAUDIBLE PASSAGES OR TRANSCRIPTION ERRORS. IT IS POSTED AS AN AID TO UNDERSTANDING THE WEBINAR, BUT SHOULD NOT BE TREATED AS AN AUTHORITATIVE RECORD.

Piero Spada: [00:00:03] Good afternoon everyone, and welcome to the fourth episode of Ewing's U. I am Piero Spada. I'm going to be your host today and I'm one of the co-founders of Little Warrior Foundation and dad to 12 year old Little Warrior Lucy. Today, we are thrilled to be hearing from Dr. Ramon's Sun, a bit about him. Dr. Sun is a newly appointed, endowed chair and associate professor of biochemistry and molecular biology and also happens to be the Director of Center of Spatial Biomolecule Research within the College of Medicine at the University of Florida. He recently made his transition from the University of Kentucky and prior to that received his PhD in cancer biology at the Australian National University, followed by postdoctoral training at Stanford University. Lastly, Dr. Sun is also a member of Little Warrior Foundation's medical advisory board. As we'll see here today Dr. Sun specializes in using cutting edge tools to quantify metabolites which are signaling molecules that can sometimes be used as cellular energy sources. Specifically, his characterization of glycogen stores across many cancer types has led to a unique and potential dependency by Ewing Sarcoma cells (more on that later). In his spare time, Dr. Sun is an avid dog lover who cares for four dogs ranging in size from a small Pomeranian all the way up to a Doberman Pinscher and Greyhound, and is also father to an active three and a half year old son.

Piero Spada: [00:01:33] But before we kick things off with Dr. Sun, I just want to let everybody know and remind you that all that Ewing's U is a webinar and podcast production of the Little Warrior Foundation. We are a National Childhood Cancer Foundation with a laser focus on finding a complete and permanent cure for Ewing Sarcoma. And we were found in April of 2020 and have granted out $1 million in high potential therapies for income, survival and facing toxicity. We very much so rely on the support of our communities of the big and little warriors who are joining us on this call and across the country. We really appreciate all of our community support to continue our mission so that we can bulldoze our way to a solution and continue this vital work. And with that, before I hand it off to Dr. Sun, I'm going to pass it off to Emily to introduce herself and a couple of housekeeping items.

Emily McFadden: [00:02:25] Thank you, Piero. I'm Emily McFadden, one of the other co-founders of the Little Warrior Foundation. And real quickly, I just wanted to take you through some housekeeping items before we begin. Dr. Sun is going to talk for about a half hour and then we'll leave time for questions at the end. Please use the Q&A function at the bottom of your Zoom chat to submit questions. And then Piero and I at the end will ask those questions for Dr. Sun. As always, we can't discuss specific medical cases or provide medical advices in the context of this webinar. And then I think we're going to cover some pretty dense material today, and I want you to know that this is going to be recorded and we will have the transcript in the recording available for you to re-listen to if you miss anything. So with that, I will kick it off to Dr. Sun.

Dr. Ramon Sun: [00:03:27] Well, thank you guys so much for the invite. And thanks, Piero, for that awesome introduction. I just wanted to say that, you know, I stumbled upon Ewing sarcoma research, and it is really like meeting meeting people like you and the rest of the little warrior families that keeps the fire going for me. Just continue to work on Ewing sarcoma and to continue pushing it along the line of research to try to eventually bring something from the bench to the bedside, hopefully in the near future. You know, having said that, my talk is going to be a little science heavy. I apologize up front if it wasn't directed to the right audience. Blame Piero, I'm just joking. But feel free to ask any kind of questions. If anything in the science is not fully understood, just let me know and or email me later. Happy to chat with anyone online or offline. So I'm just going to get started. Like Piero said, I just moved over from Kentucky to Florida. As you can see, I'm I'm still at my own house. My lab is not set up yet. And half of my people still in Kentucky doing work. So hopefully the transition will finish in about a month or so. But in the meantime, there's a lot of writing and a lot of like housekeeping stuff.

Dr. Ramon Sun: [00:04:54] So, so my lab has, as a research group really is interested in how do we connect genotype to phenotype. And what I meant by that is when you look at the genetic makeup of all of us, the DNA. So the genetic makeup humans have 20 to 25,000 genes, but when you look at other organisms, dogs have 19,000 genes. A mouse was frequently do experiments on, they also have 25,000 genes and a plant that's on the back in your backyard or in your front lawn, they have greater than 40,000 genes. So it's hard to tease apart organismal complexity just by looking at the genetic material alone. You know, human and dogs and mice have over 90% shared genetic sequences. It's really hard to basically just tell what sets us apart from dogs or mice, just based on looking at the genetic material alone. So when you when you expand from that DNA gets translated, gets transcribed (sorry) into RNA and RNA again, translate it into protein. And so and every single protein has a metabolic function. So these proteins, either enzymes that continue to do metabolism in our body or they become structural is where they form long chains of structural components that lines the outside of your cells, your bone, or any any kind of your body component. And this protein to metabolite interaction or metabolism, that's normally what give us what we call organismal complexity of human physiology. And and it is really the control of how protein interacts with metabolite that really gives us that interactome network. So for example, a single protein can have multiple different ways of getting regulated inside of a cell. So you have phosphorylation acetylation, glycosylation, miracylation (sp?), alkylation, you know, the, the endless possibilities of how we can regulate a single protein to do this function.

Dr. Ramon Sun: [00:07:13] But the other interesting thing is all of our proteins are ubiquitinated, which means it's getting destroyed and re-synthesized within our cell and 80% of all of our protein in our body, human body, it does a complete turnover in about seven every 72 hours or three days. So, you know, if you really think about it, we're really essentially brand new human being every three days, which is kind of cool when you when you think about it that way. So it is this complex interactome network that's beginning to break. And when this any protein that breaks it perturbs the metabolic network. And then that's what happens when you lead to metabolic diseases.

Dr. Ramon Sun: [00:07:55] So when I was a graduate student and most of my postdoc, you know, we're really cool that we're living in an age where rapid technology innovations are being applied to do academic research and having these, you know, amazing data being translated into the clinics. So when I was when I was just a trainee, we used to study transcriptome, proteome, and metabolome in a variety of different ways. But one thing is certain is we generally mix up our organs. So I've actually used a physical blender to blend a a cow liver before because it's so massive. So you mix up everything that you want to study inside of this organ, and then you kind of study the metabolic profiles and phenotypes and genotypes. So one of the major problems of doing this approach is the lack of - account for cellular or heterogeneous metabolism or responses. For example, if you look at a tumor, you have the cancer cells, you have the stroma and you have the necrotic regions. So if you put all of these regions together, for example, something is decreased in the tumor, but increase in stromal and necrotic regions, which doesn't mean it's not important. Not everything happens instead of a tumor, it's pro-tumorogenic has to happen inside of a tumor. It can happen the surrounding microenvironment. So if you pull everything together, this is actually a piece of Ewing sarcoma, human tumor actually. So it's kind of cool. So when you put all of these regions together, a lot of these exciting phenotypes that we should be going after or we should be investigating suddenly come out with a net zero. When you see that net zero as scientists, you kind of you generally kind of just let it go and you don't go down that route anymore. So it's kind of cool that we should be able to, tease apart all these cellular responses and rather than mixing them all together. And this this piece of tumor is no bigger than an a M&M. There's just that mean, like, it's a very small cancer. Not, not Ewings in general. But, you know, even from such a small piece of a part of a tumor, it's already have there's many regions. How are we really teasing apart what is really a tumorgenic or tumor-repressive?

Dr. Ramon Sun: [00:10:23] So in the last decade, I think for those of you who's really following the academic research and science, there's a lot of advances in terms of how we study a lot of these.

Dr. Ramon Sun: [00:10:33] So a lot of these things, you know, single cell RNA-seq and I believe single cell RNA-seq has been applied to Ewing sarcoma as well. What you see here is all of these different, unique sub cell types getting clustered by  single cell RNA-seq. But what they don't have is a spacial - where in the solid tumor these cell types are present. Then you come into the spatial proteomics analysis where you have all you can do single cell proteomics, and now you can look at cell types and how they are represented next to each other. And then you can do spatial metabolomics, which is what I'm really interested in and specialize in, where you can see where all the different cellular layers and where all the metabolism occurs in all the different cell types. And in the future, my lab is really focusing on - Sorry -  really focusing on integrating a lot of these different next generation techniques, moving them together and really using them to tease apart specific cellular and regional metabolism with unique diseases. And if you think if you think if you're like me, you think all of these technologies are cool. We're not the only we're not the only people who are doing it. You know, Nature and a lot of different articles are beginning to highlighting the innovative and the importance of doing single cell metabolomics analysis by multiple different organizations. If you just give me one second. Sorry about that.

Emily McFadden: [00:12:17] Dr. Sun.

Dr. Ramon Sun: [00:12:19] Yeah.

Emily McFadden: [00:12:20] This is Emily. I think this audience would benefit from just zooming out one level on what spatial metabolics is, kind of at a really kind of a high level and how it's different from other types of research.

Dr. Ramon Sun: [00:12:39] Yeah. So that's spacial metabolism. It's really. So if you as I progress is really the spatial metabolism is teasing out these differences versus looking at this part of the interrogation. So majority of the current work in the lab currently you're looking at this bar graph over here where it is no change because it's from a mix of material. Spacial metabolism is different is where you can look at each individual regions uniquely separate from all the different other regions where there's no responsive mixing or any other kind of responses. I think it will become more clear as I start doing - there's a lot more actual examples of this.

Emily McFadden: [00:13:27] Perfect. Thank you.

Dr. Ramon Sun: [00:13:30] In the later part of the slide, I just wanted to also like the way we do this is really just cut a piece of tissue coated with some ionized material and put in this laser ionized this this really cool machine. So you kind of coat your tissue was this this and like special chemical material that helps the ions ionize and this is where the laser ionization happens. So you put this in the machine, laser would hit the slide and you have the empty slide and you have the brain regions and you can see as the laser tracks through the different regions you have. And it's starting to collect a lot of metabolic information as it hits actual piece of tissue. And the end results is what I'm like mentioned earlier. It's all of these different metabolites that uniquely localize in all the different regions, which all of these information that you would be losing you that would be lost when you do a pooled analysis, when you put everything together. And you can represent it in a special spatial setting where you overlay all the different metabolites that's happening. So what I mean by this is you really need to tease apart what happens in each regions inside of a tumor before you can really come out with usable or effective therapeutics. If you just look at it, a bigger picture kind of a way, we're losing a lot of scientific information that just rushing into to do a therapeutic development, a lot of the times, a lot of drug fails is because we're we missing a lot of spatial information.

Dr. Ramon Sun: [00:15:11] So we can either do these by looking at single metabolites overlay. Again, these are all human tumors. So that's where the cancer is. You can see like a lot of the amount of metabolism actually does not happen inside of a tumor. So this is where a cancer is and these are stromal regions. And you can see this is a colon cancer. You have those little hairs coming out of it that's attached to your colon. You can see a lot of metabolism doesn't actually happens in the cancer. A lot of these these surrounding cells, they act as supporting structures that support cancer metabolism and facilitate cancer growth. So not everything that's pro-tumorigenic, or pro-cancer, are directly happening inside of a cancer. And you need to identify all of these combinations are kind of a point together before you really can design a very effective therapeutics, in our mind. Because if you say if you want to target as red, for example, that's very high in the special form of lipids. It does not happen in the tumor. So if you just you just hitting a lot of these cells around the tumor, you really not impacting how the tumor is growing. It may be helping the tumor, but you're not really directly hitting the tumor, which is what anti-cancer therapeutics aimed at doing.

Dr. Ramon Sun: [00:16:31] So based on this, we can profile a lot of different metabolites within within a cell. That goes from Glycogen, OxPhos, which is like ATP bio-energetics, lipids, as well as like a structural carbohydrates. So that's the outside and the extracellular matrix components that's really make up the different parts of a cancer cell. And today we're going to go a little bit more in depth into like our role in glycogen metabolism, what we've been doing with that.

Dr. Ramon Sun: [00:17:06] So. Just even my worst part of my undergrad experience is study biochemistry and glycogen metabolism, even as an undergrad. So hopefully this does not put all of you to sleep, which is really boring biochemistry. So glycogen is how our body stores glucose, which is mostly the stuff that we eat. So when you eat a big bowl of pasta, for example, during lunch, maybe a lot of we just had lunch. So a lot of glycogen synthesis happening right now in your liver. So our body uses the liver to store all of our circulating glucose and suck it up into the liver as stored as glycogen when you overeat. But during a period of starvation, for example, if you missed a meal, that's when glycogen breakdown occurs. And it's really just aimed at leveling your circulating glucose. And inside of a cell you can find glycogen kind of in multiple different set of locations in the nucleus, in the mitochondria, and the cytoplasm, and the ER.

Dr. Ramon Sun: [00:18:11] And glycogen is degraded by multiple different mechanisms inside of our cell. So we have lysosome as well as glycogen phosphorylated degradation.

Dr. Ramon Sun: [00:18:19] So different route leads to different roles for glycogen. And glycogen is also heavily phosphorylated again, decides which route a cell takes. This is important to know. Again, scientific background is important because it will come in handy later when we talk about Ewing sarcomas because we have to know how glycogen is utilized and different route and how it's controlled have been utilized before can actually design a therapeutic strategy because you can't we can't just hit one or the other one and ignore the other because one of the things we in research, we all come to the realization that a cell is very what they call metabolic fluid. It's almost like. A leaky pipe with multiple different points, and you block one, it just comes out the other end and you plug the other end just comes out another one. So the metabolism of a cell is very metabolically fluid. So you have to find out that the exact mechanism, how things is utilized before you actually hit it, because otherwise it would - all you're doing is redirecting the nutrient pathway and it really doesn't do anything to what happens inside of a cell.

Dr. Ramon Sun: [00:19:28] So to do this, we designed a brand new technology, something that hasn't changed in the last 80 years, really in the lab, how we measure glycogen. That's part of the reason why we're missing a lot of this Ewing sarcoma is because the technology hasn't changed in 80 years - much, very much similar to the treatment of Ewing sarcoma patients. It hasn't really changed. The technology hasn't changed. Treatment, Therapeutic treatment hasn't changed. So because of that reason, we developed this new MALDI imaging technique that we can actually starting to visualize where glycogen is a localized in a boosted sensitivity that is like, we're guessing, 10,000 fold more sensitive than what the previous technique is, which is the PAS stain. So many of you might have heard the PAS staining before because that is one of the clinical diagnostic features of Ewing sarcoma. So when you resect the tumor, you put it under a microscope. And if we do a PAs staining and if it's red, it's being that it's being shown that given the diagnosis of Ewing sarcoma.

Dr. Ramon Sun: [00:20:34] So with this we can suddenly starting to do a lot of different cool analysis in different tumors and all the different tissues. And we can starting to look at how big the glycogen are, how much phosphorylated they will be, and really starting to understand what is underlining the accumulation or the lack of glycogen when you look at a tissue specific set. With this, we're suddenly starting to see glycogen in many unexpected tissues. So you have glycogen in the lung, glycogen in the brain, you can beginning to see that all of the different structures are very different among different human organs.

Dr. Ramon Sun: [00:21:09] So our body -  not just liver stores glycogen, and many, many other features like your brain, the lung, kidney, they all kind of stored glycogen and they all use them for different purposes. And to this end, we don't really know what each organs' metabolic demand is for glycogen. That's some of the things that we're trying to actively work-on and discover in the future. But one of the cool things about having any technology is that we can profile a lot of different tumors and different subsets of different tumor types. And you can see here a prostate, two different lung cancers, and then Ewing sarcoma on the far right and looking at their glycogen accumulation - and glycogen - and Ewing sarcoma is included is because we're interested in this, this PAS positive phenotype in Ewing sarcoma. And you can you can kind of see that Ewing sarcoma accumulate glycogen by far the most amounts compared to all the other kind of human tumors. So you can see that it's the Ewing's sarcoma is probably you know - 10 to 50 fold higher than prostate. And this lung adenosar -  Lung squamous cell carcinoma and it's about 1.5 fold higher than the lung adenocarcinoma. Glycogen is actually important in this tumor as well, but I'm not going to go into it. But really, we're just going to try to figure out, is this a viable, therapeutic target inside of Ewing sarcoma? And again, you can kind of see because of spatial technology, you can see that this is where the tumor is and this is the stromal region, the fat region.

Dr. Ramon Sun: [00:22:46] There's a fat pad (?) here that's attached to this human tumor. And these are all muscle fibers. And you can, you can see that glycogen is very, very uniquely localized inside this tumor, and it's not there at the other regions, which suggests that it's a very specific Ewing sarcoma tumor feature.

Dr. Ramon Sun: [00:23:07] So I think usually I do a little introduction on you, but I really for you guys, you need no introduction. We all know how devastating this disease is, is it's extremely heartbreaking to see our kids have to go through surgery and chemotherapy, and it's even harder as a parent. I can't even imagine how parents would deal with losing a child from Ewing sarcoma. So, that's part of the reason that we decided to work on it and two really cool clinical phenotypes that you often see in, in at least children's Ewing sarcoma cases is one that everyone kind of know that is the EWS-FLI1 fusion oncogene and the two is the PAS positive histology, which which looks like that. So this is what clinicians, pathologists use to diagnose with the help to diagnose Ewing sarcoma. We know that they form these big giant red lumps inside the tumor. But no one - you know -  we know that clinicians has been knowing about this big red patches that you can see over here inside of Ewing sarcoma for decades.

Dr. Ramon Sun: [00:24:14] I mean, since since it was discovered, we knew that these big red patches are forming inside the tumor. Which is why I was super surprised about three years ago when I started working on Ewing sarcoma. And then I asked around. Literally no one knew why what those red patches are. They kind of guess as glycogen, but no one really know and to no one's really looked into it because this is such a striking phenotype inside of a Ewing's tumor. No other tumor, very few other tumor have this phenotype. So you're looking at hundreds and hundreds of different tumor types. You can count the number of tumors with these specific red patches on the on the number of your hand. So there's very few tumors that have these - this phenotype, but it's just really surprises me that no one really going after this phenotype figure out why and is why is accumulating and is it a therapeutic intervention or target? So, like I said, majority of the ES tumors has been focused on EWS-FLI1. And I think me talking to a few people alone, I know that there's a number of drugs that's trying to hit this. This, you know, we call it an oncofusion gene(s). And I think there's a lot of good preclinical efficacy that's been showing that it's very effective at targeting, showing a lot of tumor regression. So, you know, hopefully we're going to see a lot of advances in this in the next couple of years and help a lot of children. Ideally, if this drug is working, I would be gladly to be put out of a job for looking into glycogen if this drug works. But before this is proven to be a cure, there's no point of stopping Ewing sarcoma research. And it's better to have multiple shots on goal, what we call multiple shots on goal, so that just in case one fails, you have multiple different backup options to be able to follow up, if your primary option is failing. I'm not saying that [inhibiting] EWS-FLI1 is failing, I'm just saying we should have backup options and not put our eggs into the single oncogene basket. I wish with all my heart that this works and everyone else can be cured of Ewing sarcoma. This is honestly the best case scenario.

Dr. Ramon Sun: [00:26:35] So let's move on to the PAS staining. So this is what, when when your children have Ewing sarcoma and their cancer gets cut out, they get sent out for analysis. This is what the pathologists see. So the doctor look at the slide and you see all these little circular pink granules that's, that's pretty much everywhere inside of a Ewing sarcoma is these arrows are pointing this. And this is what I mean by PAS positive staining. This is being used to diagnose for the diagnosis of Ewing sarcoma in the last couple decades and to the fact that the doctors nor researchers know what they are or how we, what do we deal with it? Right. Is it a therapeutic target? So PAS, just as a background a sees all form of carbohydrates. And if you don't know what carbohydrates are, honey, maple syrup, they're all just kind of carbohydrates. Long chains of sugars joined together. And carbohydrate is, by nature is sticky. So if you want to know what carbohydrates do instead of a cell, just imagine, put a drop of honey between a finger and try to stretch it. That is exactly what carbohydrates are doing inside of a cell, just how it's doing. It's very sticky. It's gluing cells together. It helps support it in place. So. When we first worked on this project, we're really curious about just what exactly is PAS staining. It could be any of these carbohydrate related molecules, we guess is glycogen just because it's shape. But there's really no way to know until recently that we got our technology in place.

Dr. Ramon Sun: [00:28:10] So here we got a number of patient Ewing Sarcomas from shoulder, chest, ward, rib, abdominal and bladder. And you can kind of see that glycogen is heavily accumulated strictly inside the Ewing tumors.

Dr. Ramon Sun: [00:28:28] And what's more interesting is that when you look at how glycogen is accumulated so that the structure of glycogen, the tumor, actually looks very much look like, the muscle that is surrounding it. So now that you can kind of beginning to tease this apart and you can see this is a, this is one of the shoulder, Ewing sarcoma patients. Here is the tumor region. Here is the muscle that's, that's resected at the same time when you take the tumor out. And then you kind of know that, Hang on a second. And then when you compare it to tumor versus necrotic regions, you see how the tumor is exclusively in the tumor, but also the glycogen, (sorry), the glycogen exclusively in the tumor in this case. But the glycogen is also heavily accumulated in the muscle regions surrounding the tumor, which actually give out or give rise to that. The interesting hypothesis is like, look, Ewing's is probably somewhat similar to the muscle cells surrounding it than than other tumors of the similar subtype. They share very similar glycogen characteristics with the muscle cells surrounding surrounding the tumor.

Dr. Ramon Sun: [00:29:39] And then you can more or less confirm this so you can get them as a kind of stem cells, that is. Give rise to all the connective tissue such as the muscle, the bone, and they actually contain a lot of a lot of glycogen. When you compare it to like a Ewing's tumor. So this actually give us the hypothesis that tumor cells, Ewing's tumor cells probably arise from these these progenitor cells that give rise to all the bone and muscle that's that's surrounding a normal tissue.

Dr. Ramon Sun: [00:30:14] And therefore, you know, when, as a scientist, you always when you have two different phenotypes, EWS-FLI1 or glycogen, are they connected, right? So are they connected. So when we did that experiment, so we put EWS-FLI1 in a kidney cells. So if, for example, if this is the driver of glycogen accumulation, if, if this if this fusion oncogene drives glycogen accumulation, you should see that if I put this oncogene in a different cell types, it would drive the glycogen accumulation up - much, much higher. But what we what we don't see is that this oncogene is not a primary driver, specifically over here, you see that EWS-FLI1 is not a primary driver of glycogen, which tells you that they came from independent responses. So you probably have the high glycogen phenotype in the progenitor cells, such as the mesenchymal stem cells. And then you have a secondary hit, which is EWS-FLI1. So you have the combination of the two that give rise to Ewing sarcoma rather than the oncogene shows up and then it drives glycogen and then it drives tumor growth. So they kind of independent events, which means that you can kind of hit them as individual, individual events in terms of therapeutic targeting.

Dr. Ramon Sun: [00:31:39] And like I said, glycogen is a fuel in your liver, right? So when you overeat, glycogen accumulates. And then when, you when you're during a fasting glycogen gets broken down, it's really just the metabolic reserve. So you can kind of see it's doing very similar things in terms of Ewing sarcoma. So if you look at the number of cells when you take away glucose, so we grow these tumor cells inside of a dish and we give them a media full of glucose. But then when you take them away, you can kind of see that the Ewing sarcoma is very resilient when when during a nutrient deprivation. So after you take glucose out of this nutrient, Ewing Sarcoma very resilient for up to like three or four days without glucose because the glycogen is being utilized. But if you take a lung cancer, for example, that doesn't have a lot of glycogen, the moment you take glucose away from the cells, they all just kind of die. So you kind of know that when you look at lung cancers, glucose is absolutely important because you take it out, they die, they don't survive. But when you look at Ewing sarcoma, you take away glucose from them, you take away their nutrient choice. They can still survive because they have large amounts of glycogen that's built into the store. And you can see that it's slowly releasing this glycogen. The glycogen store goes down slowly, but in in other tumors it completely disappears after 2 hours.

Dr. Ramon Sun: [00:33:09] So in a lot of the cases where we're current thinking is that high glycogen is really just like how liver is functioning. High glycogen Ewing sarcomas are really acts as a metabolic fuel reserve to sustain tumor growth and in future cases to sustain metastases. So when, when a cell breaks down to go and move to a different location, the glycogen help facilitate that whole process by providing a lot of fuel or nutrients for the cell themselves and to, to continue to divide and metastasize.

Dr. Ramon Sun: [00:33:46] So with that in mind, we really wanted to know, is glycogen a therapeutic target, assuming it is a fuel for the cells? So we have two different modalities. One, this is a research tool and these are both research tools, right? So they're not therapeutic tools, but they give us a window into whether we can continue to develop therapeutic agents against glycogen. So, one, you have this CRISPR knockout. So CRISPR is a way to knock out glycogen synthase. So glycogen synthase, the single only enzyme in a cell, including Ewing sarcoma cells that makes glycogen. So you kind of either knock it out or we have a small molecule inhibitor called Guaiacol. So this is actually a compound that's heavily concentrated in whiskey, not not advocating for whiskey consumption, but it's, it's present in very low amount that gives whiskey the cool aromatic taste but in much higher concentrations it actually blocks glycogen synthase.

Dr. Ramon Sun: [00:34:47] So when you look at both of these approaches, so first of all, when you look at the CRISPR knockout, when you when you ablate a Ewing sarcoma to be able to to make glycogen and this is how it has the tumor growth we put them in in the mice on the flanks and we can measure how fast the tumor grows inside of a mouse. And you can see that's a normal Ewing sarcoma that's growing, grows in really fast, even on the flank of a mouse. But when you knock out this glycogen synthase, which is responsible to make glycogen, the tumor essentially completely dead, it blocks (?)new growth in the mice. And you can you can kind of see a very similar response is by us injecting this research grade compound that when you inject it into the mice, you can it has a very similar effect where it kind of reduced tumor growth growth or tumor burden by these mice quite a bit. So this gave us a lot of hope, like glycogen. We know that the PAS positive things are glycogen and we can pharmacologically manipulate glycogen synthesis to lower that amount of glycogen. And by doing so, we can significantly lower tumor growth in these mice. It does give us a lot of hope to continue to work on this drug or this enzyme as an inhibitor to really move this into the clinics in the, in the, in the near future - hopefully.

Dr. Ramon Sun: [00:36:20] Before I finish my talk, one of the things what I would like to highlight is this this enzyme called AMPK, which is an AM protein kinase. So AMPK is a master regulator that regulates a lot of these different cellular metabolisms. But what, what matters in the in the case of Ewing sarcoma is Metformin. So we we all know and I don't know if your child was part of the metformin trial that was a multisite trial. So there was a metformin trial for a lot of different refractory solid tumors. And Ewing Sarcoma was part of this and one of our collaborators, Lars Wagner, is actually one of the co-lead of Duke University. So there was a there's a number of children that was part of this trial. And one of the this is really want to highlight the importance of lab research, is to really we need to understand the molecular mechanisms or cellular mechanisms of why something works before we give them to our children. Because if we don't, you know, it might be potentially a waste of time that we can be trying a lot of things. So one of the things that what happens AMPK is glycogen is a potent inhibitor of AMPK and that's what your metformin is trying to activate. So when glycogen is present, it shuts down AMPK activity, like this. So it shuts down all the AMPK activity and I overrides any kind of activator, activating products of metformin. So metformin may be useful in a lot of different tumors that doesn't have glycogen, but at least from our lab based research, that based on our knowledge in how glycogen works inside of a cell. Metformin wouldn't be a very useful therapeutic agents for kids that have Ewing sarcoma because of the heavy amounts of glycogen that's present inside of that tumor. So in a lot of cases, I think getting rid of glycogen in Ewing sarcoma cases would actually benefit the metformin efficacy. So I don't know what the I think the the trial is ongoing, but it's coming to an end. And I think at least early reports based on just direct communication with the clinicians that metformin really didn't have a really synergistic or additive effect to Ewing Sarcoma patients which, you know, knowing what we know now could be a reason why glycogen could be part of the reason why our kids are not benefiting from metformin from a from a cancer regression perspective.

Dr. Ramon Sun: [00:39:22] With that, I think I like to stop my talk right here. I think it's about 35 minutes. I'm a little bit over, but I skip the next slide and would like to thank everyone in my in my lab. Lab is getting, Lab is rapidly expanding. We have a lot of different funding agencies. I wanted to highlight St. Baldrick's Foundation, V Foundation, and Rally Foundation, who support most of the Ewing sarcoma work. So they are very pediatric cancer focused foundations that fund a lot of laboratory research as well as junior faculty. And with that happy to take any questions.

Piero Spada: [00:40:10] Thanks for that, Dr. Sun. That was fascinating on many levels. Let me give Emily some time to delve through the questions in the Q&A box, but I had a couple of questions, and I was wondering if you could kind of allude or let's focus on the translational aspect of these therapeutics. Can you can you talk about the other diseases like Pompe diseases and others that are heavily reliant on glycogen stores and where those therapeutics are at in the clinical trials now, if you know whether you're inhibiting glycogen synthase or stopping its degradation, can you talk a little bit about that?

Dr. Ramon Sun: [00:40:46] Yeah, totally. So the most well known disease that is getting treated with a FDA approval approved therapeutics is the Pompe disease. So they're - they're form glycogen aggregates. So in those diseases, the glycogen physically cannot be broken down. So there's this no fuel perspective. So the glycogen is not being utilized. It just keeps getting bigger and bigger and bigger and it's blocking a lot of cellular processes. So Pompe disease is uses this they call it acid alpha-glucosidase. It comes in, it's an enzyme replacement therapy. So this enzyme comes in and clear the glycogen and actually show a pretty good improvement. Like the patient is not cured, but they end up living longer. Their their physical motor neural responses are much better. They can walk longer. They have a lot more energy from from a glycogen storage - glycogen storage perspective. And then among myself and colleagues, suddenly there's that the debate of is Ewing sarcoma a glycogen storage disease. And if so, how are we going to to treat it looking at a glycogen storage disease. And we do also work on I don't know if Maze Therapeutics actually works on a glycogen synthase inhibitor that actually is clinical grade. By clinical grade, I mean, that is actually very potent, low toxicity, and they've tested in humans. And if you guys Google Maze Therapeutics, they just got approval for orphan disease status, which means the door can be open very soon for a lot of different diseases using that drug. And they're going through 100 volunteer Phase one clinical trial to assess actual toxicity.

Dr. Ramon Sun: [00:42:45] So with that in mind, we think glycogen is a therapeutic target. How you're hitting it matters, because if you hit it with a(n) enzyme therapeutics, right. What it does is it breaks the parties glycogen to many, many smaller pieces. And in the case of glycogen storage, in the case of Pompe disease, that is good because once you break it apart, it can be just digested as the big glycogen that is the disease driver. The cell is holding this big glycogen in and because keep making bigger and bigger, it's not being utilized. The cell shuts down. In the case of Ewing sarcoma, we've got to realize that you can think glycogen is a rechargeable battery. If you consider glycogen and Ewing sarcoma as a rechargeable battery, what happens when you break that resource down? You're just giving the cells, more resources. It's a big ball of glucose sitting inside of a cell that's getting slowly broken down to support tumor growth. But if you come in too quickly. To break this cancer down - to cancer to break this glycogen down - all it does is you providing more glucose to the cell to continue to divide. What we really need to do, you know, this is, again, very - needs to be scientifically designed, is that we need to stop glycogen synthesis from the very beginning. And that is why we chose to hit glycogen synthase, because we have access to these enzyme therapeutics and we never tested those in Ewing sarcoma cases because we know that based on a battery model, if you actually hit it with an enzyme therapeutics glycogen is going to get released, the cells are actually going to be happier.

Dr. Ramon Sun: [00:44:38] So believe it or not, I know that cancer grows, but Ewing sarcoma is still a sarcoma. They're not in a very energy favorable environment. You could make them grow even faster by giving them more, more fuel because there's no vasculature They're surviving under odds because there's, there's low oxygen, low nutrients. If you have something that has big glycogen that you, you're helping the cells to break down the glycogen, they're just going to grow faster. So what we really need to do is just nip it at the source of glycogen synthesis, which is we really need to kill glycogen synthesis from the very beginning and don't just see a phenotype and treat - we don't want to treat the symptom, we want to treat the disease. We don't want to treat the symptom of having large glycogen inside of a cell because large glycogen have different disease drivers. In Pompe disease it's because it's just getting too big, it's blocking everything. In Ewing sarcomas' case, it's because it's acting as a battery to store. So if you if you cleave this, it's going to get released and continue to add on a lot of different cells to give it more energy store. So I think the opportunity is there but different. Different glycogen storage diseases also have different target entry points and therapeutic entry points. So we all kind of need to figure out what the actual disease driver is and rather than just hitting that glycogen that's inside of our cells.

Piero Spada: [00:46:10] Thank you for that.

Emily McFadden: [00:46:13] We have a question here asking if this is really popular topic in the Ewing space. Would patients benefit - does your research suggest that patients may benefit from extreme low carb diets like a keto diet to limit the amount of glycogen in the body?

Dr. Ramon Sun: [00:46:31] Yeah, OK. So, that's a really tough question because I was literally just having another conversation with another colleague the other day. How do I put it? So if you look at in the case of athletes, so if you look at the case of athletes who's actually doing ketogenic diet, ketogenic diet lowers liver glycogen. But it doesn't do much to muscle glycogen. Your muscle still has a lot of glycogen. And the reason is. But those of us who believes in evolution, we were evolved to run away from predators like a lion shows up around the corner, we need to be able to take off. And to be able to take off, you need energy. And what do you need that - how do you store that energy for that sudden burst of activity? You - You store it in your muscle. So to deplete muscle glycogen you don't go ketogenic, ketogenic depletes plays liver glycogen it doesn't touch muscle glycogen, so it really deplete muscle glycogen. And you see in my talk we saw that you and sarcomas very much similar to muscle glycogen it doesn't look like liver glycogen right. So you know, professional athletes, which does a lot of sprinting and running, they have lower muscle glycogen. That's one way of depleting it. But again, coming back to the ketogenic diet thing. There's a lot of benefit of eating a ketogenic diet just from a low carb diet for both adults and kids, from a health perspective, because you're no longer eating garbage, you pick and choose fresh food and you cook it yourself. So those alone should have benefits for families, for kids to try. But I don't know if it would benefit Ewing sarcoma from that perspective, from a glycogen perspective. I my my philosophy for my own kid is like, what's the worst that could happen? I'm giving him healthier, a healthier diet. I guess from that perspective, I think it's okay, but I can't tell you yes or no. The ketogenic diet would in any mean affects Ewing sarcoma glycogen.

Emily McFadden: [00:49:02] Great, so when a kid wakes up from surgery and wants a donut and nothing else, it's all right.

Dr. Ramon Sun: [00:49:11] I think so. I think so. I wouldn't. I wouldn't. That's only going to hit your liver glycogen, I think. I think we're looking at a much deeper problem here, just, just because it's accumulated in the muscle and it's a lot harder for your body to control from that perspective. Yeah, but so from that perspective, yeah. Like a donut. I don't I don't think it's making the cancer grow faster. It's got other underlying mechanisms.

Piero Spada: [00:49:36] Thanks for that, Dr. Sun is a tough question.

Dr. Ramon Sun: [00:49:39] Tough question Yeah.

Piero Spada: [00:49:40] I want to focus again more on the targeted therapy potential of this. It sounds like you're of the mindset that this is if it's going to make it to the clinic, it's going to have to be an almost like a 1-2 combo with another targeted therapy, right? Because you kind of highlighted that E.W., or Ewing Sarcoma, we know, is driven by this EWS-FLI1 oncogene, which is independent of glycogen stores. And then you've also highlighted that the PAS positive hallmark of this cancer is highly glycogen dependent. So given that, is there a theory as to when you would time that out? Like if you're - I guess to me, if you're blocking glycogen synthase in the mesenchymal cells, which are the precursors - does that happen - Is it almost like a maintenance therapy or how would you how do you see that moving forward?

Dr. Ramon Sun: [00:50:31] Yeah. So a lot of factors goes into that. We don't really know how toxic hitting inhibiting glycogen synthase is. The current data does suggest that it's, the only bad side effect is you, lose a lot of energy. It doesn't have the same effects as chemotherapies. Right. So if if that's the case, this is a big IF and we're hopeful if that's the case. It is possible as a maintenance therapy, because I really I don't have a lot of data to support this, so that's part of the ongoing research. And and that is actually a major barrier because there's not, there's not a lot of good metastatic Ewing models. So it's the metastatic disease that's killing our children. It's not the primary disease. You find it early use resected, chemotherapy. If it's early stage, the chance of survival is very high, it's the metastatic disease that's killing our kids. So what my honest-to-God thought is, once the primary tumor is breaking off into metastatic phases, it's utilizing its glycogen to become metastatic. That's the hypothesis, and I think that's where the therapeutic window would be. I don't want it to be replacing surgery or radiation or that first line of defense. I think it should be a maintenance therapy. I envision it if there's more data to support this as well, is like a maintenance therapy that prevents future metastatic events in Ewings.

Dr. Ramon Sun: [00:52:07] So that's that's one part of the question. The other part of the question is: Is glycogen, hitting Glycogen can augment and boost chemotherapy responses to the tumor? So chemotherapy actually really relies on a lot of oxygen tension and permeability of the actual tumors. So your solid sarcoma in general is just a solid sheet of tumors. There's not a lot of vasculature. It's highly hypoxic. Is - is remodeling the tumor by getting rid of glycogen can remodel the tumor itself to make it more susceptible to chemotherapy or radiation therapy, that's another question that we're also very interested in knowing. Hopefully once we get the lab settled, we can get on to that testing. I think to that by far would be the easiest way to bring glycogen synthase therapeutics into the clinics, is to be able to demonstrate that we can use this in combination with already established therapy and give its added bonus of killing the cancer cells and increase survival. Because, it's hard. I know a lot of these therapies is not super effective, but they do work. A lot of these chemo, surgery and radiation, they do work a lot of people. So we don't want to take those proven therapy away from Ewing sarcoma patients. The best case scenario is to add something to improve on the already known therapies to be able to boost that their cytotoxicity or efficacy really brings to better combat the cancer using the current conventional therapies. So yeah, two routes that we're really, we're hopeful is this research is one as a maintenance therapy to prevent future metastases and two is to maybe help augment current therapies to help that. And the second one, the current therapy, that's an easier route to bring it into the clinics. To do a combination therapy. I think clinicians would be more willing to do that kind of a test as well, because you're not having them to take away the already known therapies that they know that works. You're not having to stop doing that kind of thing.

Piero Spada: [00:54:37] Okay. Thank you.

Emily McFadden: [00:54:42] Great. And then one more question. Are there potent supplements or naturopathic options that reduce glycogen stores that you're aware of?

Dr. Ramon Sun: [00:54:55] Oo. I might have read something on PubMed a lot of the natural, some natural plant substrates does hit glycogen synthase. I can't tell you the exact names or where they come from right now, but I definitely remember reading those on PubMed. There is this this relationship between plant and humans because we eat them from evolution and the plant produces some kind of a chemical in them that affects our glycogen store because they're our food and modulating our store affects our ability to eat them and how often, how frequently we eat them, too. And because what you eat from plants is also glucose, you convert all of that starch back to glucose, back to glycogen. So there is I definitely read literature out there from natural compounds, but again, all of the natural compounds are not - Very - one low potency, but two, they hit other targets in your body, too. It's not a specific glycogen synthase inhibitor. I can't tell, I can't make a recommendation to take them. I really can't. But I know they're out there. If you just if you do a PubMed or Google scholar search, I think you can find them. But I as. I don't. I don't. I don't know if they're not going to work, but I also don't know if they are - I can't make recommendations.

Emily McFadden: [00:56:28] That's fair.

Dr. Ramon Sun: [00:56:29] I never personally tested them, I guess, if that makes any sense. Yeah.

Emily McFadden: [00:56:34] That's more than fair.

Dr. Ramon Sun: [00:56:35] Yeah.

Piero Spada: [00:56:37] Got a couple more minutes here, Dr. Sun. I guess I just want to ask one final question. What, is seems like you have a really clear path towards targeting this tumor type. And is, what is the rate limiting step for you guys moving forward as a lab and really taking this next step and moving it to the clinic.

Dr. Ramon Sun: [00:56:57] Rate limiting step. Let's see, number one, people, I need somebody to move down here to Florida. And same as every other lab is the, the everything in the lab is expensive. It runs $1,000,000 operation with a lab of seven or eight people. The annual cost is close to $1,000,000 just from paying everybody's salary. And every, you know, a nude mice, single nude mice, it's like $5-600. You buy them. And then you do a powered analysis where people accept the numbers. You can do like 20 per group, and then you inject the tumors. It's the resources are limited just because, you know, we can't do everything we want to do in the lab. Sometimes we have to pick and choose just because there are plenty of research labs that end up losing all of their funding from going down the rabbit hole and then just collapsed and the PI ended up teaching, right, become it become a full time teaching professor. That happens all the time in academia. In fact, half of my department is like that. Half of my department, they're supposed to do research, but they all kind of lost their, lost their funding and can no longer do research anymore. It's it's a sad kind of a thing in academic research is the resources and funding. The successful ones, I'm somewhere in between, but the real successful people, They know what to do, what to write, to go for, like, things that will get them funded. And then. You'll never hear me heard this. But we would. It's hard to get funding for Ewing sarcoma research, but we've managed to keep going through from resources from our other funding. It's easier to get lung cancer research. It's easier to get an Alzheimer's disease research. But then there's also institutional support. So you kind of mix it apart and just trying to keep pushing on this. Right. That's the reason that V-scholar and St. Baldrick's Scholar, those foundations exist as well is because there's not enough money out there for pediatric cancer research. There's just really not. So those foundations exist as well. So yeah, I'd say human, and human, just manpower and resources I think to really trying to push but we do have a really good relationship with Maze Therapeutics. In fact, we helped them establish their current drug that's going into the Phase 1 clinical trials right now. Just because they need a lot of validation from their glycogen models. So. We've mentioned Ewing sarcoma multiple, multiple times. So you guys are definitely on their, in their mind when they're continuing to push this through Orphan status, Orphan drug status.

Piero Spada: [00:59:57] Well, thanks for keeping us on the radar and for keeping us in the mix, especially with regards to all of those larger cancers. I think we're out of time, so I'm going to close this out here. I want to thank you, Dr. Sun, for presenting and giving us your time.

Dr. Ramon Sun: [01:00:09] Thanks for inviting Me.

Piero Spada: [01:00:10] And from the Greater Ewing Sarcoma community, just thank you for keeping this a priority in your lab, as you mentioned.

Dr. Ramon Sun: [01:00:17] And if anyone needs to have more questions, just feel free to email me.

Piero Spada: [01:00:21] Okay. And then just one last thing. Thank you to our viewers for all joining us today as well. We'll see you back here in October. Please be sure to subscribe to our Ewing's you email. You can do that by going to Little Warrior.org and navigate to Ewing U's tab on the website and also follow us on Instagram and or Facebook as we always post about our next speaker that way as well. Lastly and in closing, we have a sign off here at Little Warrior that we feel embodies our fight in honor and in memory of many little and big warriors who have faced this cancer. So, Doctor Sun, we're gonna ask you to join us is just simply a 1,2,3 swords up. So if you will, I know the rest of you are all muted, but the three of us, will sign off that way. So, one, two, three. Swords up, everyone. All right. Thanks again. Take care.

Dr. Ramon Sun: [01:01:07] See you.