Transcript for:
Exploring Stem Cells and Brain Diseases

This program contains graphic images and discussion of medical procedures. Viewer discretion is advised. Welcome to a Closer Look, I'm Robert Signer, Deputy Director of the Sanford Stem Cell Institute Discovery Center, and associate professor of Medicine at UC San Diego. Today, we are here to take a closer look at stem cells and the human brain. Now, the brain controls everything: it controls our movement, our touch, our breathing, our temperature, our hunger, our vision, our thoughts, our memories, and our emotions. But the human brain does even more. It defines us as both a species and as individuals. Our intelligence, our cognition, and our capacity for reasoning are the essence of the human condition. The uniqueness of our brains distinguishes each and every one of us to make you uniquely you. As Emily Dickinson said, The brain is wider than the sky. Yet, there's probably no greater biological mystery than the human brain. Together, this is what makes diseases of the brain so terrifying and so difficult to treat. They are an attack on our essence and on our everything, and they challenge the limits of science and medicine, but today you'll hear from two people that are changing that. These are two of the world's leading experts that are completely transforming the way we see the human brain, as an organ, as an interconnected network, and even deep within our cells and molecules. Even better, they've teamed up to bring their brains together, converging on stem cells as a key for keeping our brains healthy. Our first guest today is Dr. Alexander Khalessi. Dr. Khalessi is Chair of the Department of Neurological Surgery and professor of Neurological Surgery, Radiology, and Neurosciences at UC San Diego. He's the president of the Congress of Neurological Surgeons and sits on the board of governors for the American College of Surgeons. He serves as a key advisor to numerous federal agencies, has published more than 150 peer-reviewed papers, and has spearheaded some of the greatest modern advances in neurosurgery. He earned his medical degree at Johns Hopkins University and completed his neurosurgical residency at the University of Southern California. He obtained his bachelor's and master's degrees from Stanford University and also holds an MBA from MIT Sloan School of Management. He also launched a new brain tumor and neural restoration program here at UC San Diego with our second guest, Dr. Frank Furnari. Dr. Furnari in addition to being co-director of the aforementioned program is a professor of medicine and head of the tumor biology laboratory at UC San Diego. Dr. Furnari has made seminal discoveries on the genetic and molecular mechanisms that drive the emergence of glioblastoma. His current research is utilizing state-of-the-art stem cell models to uncover why brain tumors resist treatment and is working to develop breakthrough therapies to treat both pediatric and adult brain cancers. Dr. Furnari earned his bachelor's degree from Hofstra University, his PhD from the University of North Carolina on Chapel Hill, and completed postdoctoral training at the Ludwig Institute for Cancer Research. He has been recognized by prestigious Scholar Awards from the V Foundation, the Kimmel Foundation, and the Goldhirsh Foundation, as well as major research awards from the Society for Neuro-Oncology. Join us as we take a closer look at stem cells and the human brain. Dr. Khalessi? Well, thank you, Rob, for that really generous introduction. I think you really beautifully framed, from a philosophical standpoint, the scale of the opportunity that we have when we actually think about the human brain. You've inspired me, actually, at least, to diverge for just a moment from my prepared slides because when we think about the human brain and the opportunity to innovate, I think you really captured for me, at least, why the future in neurorestoration specifically is so exciting because we've lived as neurosurgeons for a long time limited by our understanding of the structure of the brain, but as you'll see in some of what I'll share, we're moving into an era where advances in computational power and material science are really allowing us to deconvolute the electrophysiology of the brain in new and special ways. The reason that's important is because there are whole families of neurodevelopmental, neurodegenerative, and psychiatric conditions where are likely a perturbation of the function of the brain. As we understand those things better, they actually become opportunities and targets for a surgical intervention or the delivery of a biological payload like neurorestorative or stem cell therapies. I'd also say that in sharing some of my own personal background if we've had any measure of success here in San Diego, it's been precisely because I recognize that our progress can't be limited by my personal imagination or efforts. I've been deeply fortunate to have amazing partners that have really allowed us to answer questions in ways that other people's can't that are new and special and different. The ecosystem that's been developed by the Sanford Around Disease Teams is one interface and one opportunity. I'll share some of the structure and the work that we've actually done as a department that really position us to actually understand some of those opportunities a little bit better and a little bit differently. The reason people tend to focus and start with cancer just to take a half step back is because, obviously, cancer is a situation where the biology of the body has run astray. There are a lot of insights that come that actually provide immediate benefit to patients in terms of therapeutic targets, but as we actually understand the biology of cancer better, there's an opportunity then to think a little bit more broadly about those neurorestorative options. I share that framing just to provide some context for some of what I'm going to share. As I touched on earlier, I want to actually innovate, it really requires an entire team of people. I'm quite fortunate to be part of a much larger department that has outstanding expertise across the breadth and depth of neurosurgery, and some of my department partners are here today. We've developed an ecosystem here in San Diego that actually really allows for meeting the standard of care at a very high level. A lot of what I'm going to show you in later slides actually relies on quite a bit in terms of infrastructure. That once we actually know the right thing to do in the care of an individual patient, how do we make sure that we hit that mark every time? That's actually been reflected in the broader partnerships we've developed with the Sanford, with the School of Engineering, with our sister Department of Neurosciences to really advance that understanding. Unlike in other areas of science, if you can imagine, there's a phenomenology that's unique to the human brain that I think Rob really touched on beautifully. Even to advance a scientific question, what's really required is a commercial-grade product. That's required us to be quite intentional about engaging with industry partners that are unique to the ecosystem that allow us to deliver on some of the therapies that we'll talk about. We've been at the leading edge of three stem cell trials that requires a proprietary cell line, the ability to reliably deliver that vector in a way that's atraumatic, and to be able to exert a biological result. When you think about all the steps that are required for any form of neurorestorative therapy, there's a lot of essential elements that we're fortunate to have here in San Diego that allow us to do things that other people just frankly can't. That's required really leveraging and tapping into this much broader biotech VC ecosystem. Obviously, having support at the level of the chances been quite important. In a group of individuals who are really outstanding leading scientists within their area, I've always been measured in the contributions I could make actually as a practicing surgeon and some unique opportunities that actually come from the surgical moment, or we really are frontline observers to the physiology and natural history of diseases, and actually, particularly the biological implications of our interventions. I have this inset picture of Halsted because I think there's a belief among surgeons today, myself among them, that we're somehow much slicker and technically have a much greater command of anatomy than those who came before us, but the reality is, is the converse is likely true. The advances that we've made in surgery in the last 50 years and in the last 10 years, specifically in neurosurgery, has really been driven by advances in device, imaging and implants, and radiology. We need to engineer our procedures from artisanship to highly reliable segmented tasks, and so you're going to see that approach in some of the later work that I show. We've actually quite intentionally tried to develop in this environment, a lot of the essential infrastructure that's required to move the insights that I hope that in this research partnership that we'll develop as rapidly as possible into patients. It's quite important if you're dealing with a rare condition that there's an immediate actionable result that comes from the insights that we get from actually developing, for example, personalized avatars around your cancer. We are much farther along because of having done this intentional work than other institutions in furthering that goal and developing that network that's required. The best metaphor I can draw for people who actually don't think about neurologic disease every day is if we actually think about the progression that we've seen in astronomy in the last 10 years alone. The composition analysis of the James Webb Space Telescope has absolutely revolutionized our understanding of an object here, the Orion Nebula, actually hasn't changed over the time horizon of observation. What's allowed us to actually really make progress is what we're able to see and distinguish in the operating room. This is the courtesy of one of my partners, Dr. Baumont, who's in the audience, that you can actually see here now in the operating room where prior to maybe five or 10 years ago, it was very difficult to distinguish between normal and abnormal tissue when you're dealing with intrinsic tumors of the brain. We now have navigation and aids to actually outline in a heads up display normal structures and actually use fluorescen agents to distinguish normal from abnormal. We are also able because as Dr. Furnari will touch on, we're dealing with tumors that are quite heterogeneous in their composition, we're able to actually select cell populations quite specifically from different regions of the tumor that we can surveil and follow over time. This foundational work is going to be quite important as a baseline for the progress that we actually hope to make in our understanding of these diseases. We also have the benefit of infrastructure here, like interoperative MRI that really allows us to apply energy and laser ablation procedures, as you see in the inset left, and again, the use of highly reliable connectomics to actually map the insulated tracts of a brain to actually allow us to pick a very precise trajectory over the time. The reason that's important is because we used to be a leading center and still are in functional MRI mapping with Magneto and cephalography that's now cited at the Qualcomm Institute, but this used to be the level of our resolution. We would actually stimulate someone in an MRI, and we'd be able to map very basic things that are specific to the patient, like arm and hand function. You can see quite dramatically how we now are in a very different, both in mapping these insulated tracks in a way that's special and different, being able to map those structural findings to very specific biological insights that you'll see at the cellular level in some of what Dr. Furnari show, and we're actually really leading the national conversation in terms of actually creating much richer tempor spatial images of the function or the music of the brain. The reason that's important is it's really actually garnered much broader national attention that when people actually think about what's next in brain computer interface, the reason I'm personally excited about brain computer interface just to be clear is so that we can actually identify, as I said earlier, where are the structural and electrophysiologic abnormalities where there's a process that needs to be interrupted. There's going to be a subset of those conditions that the best way to interrupt that process is going to be the delivery of a biological payload. The best way to ensure in the long term that our neuro restorative therapies are successful is that we select the right patients and know the right target and have a clear, a traumatic path to get there. I hope you're taking away from what I'm showing to date is here in San Diego, we are in a very strong position to do that, and that's precisely why we've had the early opportunities to lead some of these stem cell trials because those are sources of failure, sources of variance. Those are things that we've actually eliminated or reduced to the point that now we can focus on the fun questions of what's the right stem cell line and how do we actually match that to the right clinical situation where the patient's natural history is such that it could justify that risk and that early insight in a way that really will over time, create real path to neurologic recovery in those patients. That also requires, obviously having the infrastructure to try all these approaches, and so we're quite fortunate to be among very few places in the world that have these high fidelity clinical environments on campus that allow us to de risk these procedures and do the required animal work to actually validate some of these approaches. I'd be remiss to also not emphasize that our advances are not just in the operating room and that it may be that a subset of these payloads are actually best delivered in an intravascular route. We've done a lot of intentional work to actually identify the subset of tumors that actually would benefit from that approach, and we're actually quite thoughtful in our current treatment plans of combined endovascular and open surgical techniques in the appropriate setting. I know we're here to actually celebrate what to me is actually a quite exciting research partnership for our entire department. But I do believe because so much of this work actually needs to be defined at the level of the human brain, and the only place you can access the living human brain is in the operating room right across the street that we're really working hard as a group of surgeons and with the right research partners to make sure that we're making the most of every single one of those opportunities, but I also think there's a responsibility for all of us to think about when we think about tumor cell biology or intrinsic brain cancer, what precipitates a metastatic event when so much of metastatic cancer is driven by a terminal or debilitating or defining CNS event, it's important for us to actually understand how our efforts fit into a much broader paradigm of care. When we think about what healthcare is going to look like over the 20 or 30 or 50 years in which we all together are going to make progress in these areas, I actually think it's quite important for us to contemplate the different settings of care. Obviously, a lot of what we're trying to do is an outpatient health maintenance as we develop longitudinal data collection on patients. There's going to be an opportunity to really optimize some of these things in a truly preventive way. Then, obviously, as we have longitudinal baseline data on patients, we're going to see that someone's going to potentially, for example, start to drift in their cognitive scores, and that may imply that there's an early change in their baseline and an opportunity to intervene. Obviously, there's going to absolutely be the need for acute care in the future, and will become more seamless in concentrating critical care and imaging and procedural services in a way, that really organizes in a high fidelity way around the patient. Then, we're going to actually have much better longitudinal data on patients and the use of wearable sensors and other things to make sure that we're avoiding complications that we actually achieved our functional goal of getting patients back to their lives. Then, most importantly, in the context of cancer, if you're intervening in the acute way, what actually really is required in the intermediate term getting a handle on the biology of that disease. Surgery and radiation are by definition, local therapies, immunotherapy, chemotherapy or systemic therapies, putting that all together in a way that provides a durable result for the patient is obviously the goal. If we actually think about that in terms of conditions, where you actually see inset here in gold are the five most common causes of death and disability in the United States. You can actually see neurologic disease and cancer feature prominently in two of the top five. If we actually think about, for example, individual susceptibility to disease, you can imagine a young woman who has a BRCA mutation, she's wearing accelerometers, she has a subtle asymmetry, she doesn't know that she actually has the diagnosis, and you'd be shocked to know how many patients with an occult diagnosis of breast cancer present for the first time with a solitary metastasis to their brain. Let's say she has an acute seizure event, the ambulance responds, they come to the hospital in the midst of an electrical storm. You can imagine getting that high resolution imaging to deconstruct for their family, what's actually taking place. You're able to get the required imaging to actually plan a trajectory for the removal of that mass. You take the mass out for that patient, and then in the acute phase, you actually get a lot of information to make sure that you've actually safely gotten that patient back to their lives. Maybe you actually have them, climb the tallest building in San Diego for breast cancer and brain cancer. Then, ultimately, this is the key part of what the center is going to do, you actually get very specific epigenomic data on that particular tumor, so that you can actually provide tailored therapies in the intermediate term that will make sure that solitary metasis that was resected gives them a durable functional life going forward. I really believe the opportunities for this center are quite broad, they really, in my view, touch all of the major aspects of human disease and disability that we're going to be facing together over the next 50 years. The reason I'm particularly excited about this research partnership is because it bears on a very devastating form of brain cancer, obviously, intrinsic brain tumors, which I know Frank expanded on. There's going to be implications for metastatic cancer. As I mentioned, CNS events in all forms of cancer are often defining in terms of the ultimate prognosis for that disease. Then, I know we're all concerned about neurodevelopmental psychiatric and neurodegenerative disease. I see some of the neuro restorative therapies we're going to be thinking about as we develop a more sophisticated map of the function of the brain, there's going to be real opportunities that are going to allow us to reverse disease in ways that we can only imagine today. I couldn't be prouder and more excited to be part of this ecosystem, I can tell you that it's really fun to surround yourself with people who are much better, smarter and faster and stronger than you. I'm just really excited to see what we're going to do together and really looking forward to the conversation and appreciate the opportunity. With that, I actually think it's left to me to introduce my partner in crime, and this, is that right, Rob? Let's bring up the good Dr. Furnari to show us some of the science that's going to lead to these advances. Thanks [APPLAUSE] Great. Thank you so much. Alex, that was just terrific. It's wonderful to be a partner with you. I'm truly excited about what the future holds for this program. I'm Frank Furnari. I'm a professor in Department of Medicine within the Division of Regenerative Medicine, and I'm co-director with Alex for the Sanford Stem Cell Institute Brain Tumor Program. What I want to tell you about today, in addition to stem cells in the human brain, I'm going to really focus on how we could use stem cells to model brain cancer and what lessons we can learn from this modeling system for therapeutic opportunity. This slide here shows the incidence of a variety of cancer types, notably brain, and other nervous systems circle there with a red circle. Cancers of the brains and the nervous system locations are rare as indicated here. But as indicated by the number of deaths, they are very difficult to cure. Even in cancers that are more clinically treatable when diagnosed early like breast, lung, and colon, such as has pointed out there, when they do metastasize to the brain, they do tend to be deadly. Whether it's a tumor that originates in the brain or metastasizes to the brain, as Alex mentioned, there's a lot we need to do to achieve durable cures. The tumor I'd like to tell you about, which is the most deadly for primary brain tumors is glioblastoma. This accounts for greater than 50% of all brain tumors in adults. There's about 13,000 cases of GBM per year in the US. If you include the European Union, that's about 25,000 cases. The five-year survival for GBM patients is less than 10% and we believe these tumors arise from glial cells, which are the cells that hold your brain together. The risk factors for GBM are very similar to other cancer types. Age is the number 1 risk factor. Males are slightly higher than females in terms of incidence, and Caucasians and Asians slightly higher than other populations. Overall, the prognosis is very poor, and what this shows here, this is the standard of care therapy which is surgery followed by radiation and chemotherapy. The chemotherapy here is temozolomide and prior to temozolomide, the overall survival was about 13 months. The inclusion in temozolomide in 2005, you can see we had an addition of two months of overall survival, so that's not good at all. More recently, we have the inclusion of tumor-treating fields as shown here. The overall survival has really increased by about an additional five months to 20.5 months, so we need to do better. But why are these cancers so difficult to treat? I think this can be boiled down to five main reasons. Number 1, they have limited response to traditional and targeted therapy, as I alluded to on the previous slide. They're highly invasive. You can see that in this slide. In this section, there's a tumor mass in one hemisphere of the brain, and there's another mass on the other side of the brain, so these cells have actually crossed the corpus callosum. They're highly invasive, making surgery not capable of curing these tumors. They're highly immunosuppressed or they're cold tumors, which means that cancer immunotherapies that have worked for other cancer types such as melanoma, don't work for GBM, and there's no early detection, as we see for other cancers such as breast, colon, and prostate. There's no detection that I'm aware of yet that would say you have the formation of a brain tumor. Above all these things, as Alex mentioned, these are very heterogeneous tumors. What is heterogeneity? Heterogeneity, in a word, is diversity. We have two types of heterogeneity that we think of when it comes to brain cancer. We have intertumoral heterogeneity. That's the difference in tumors between patients, and we have intratumoral heterogeneity. That is the heterogeneity you see within an individual tumor. That could present itself by different gene expression across the tumor landscape, different compositions of microenvironment cells. A good example of this was done by one of my students, Bret Taylor, where he used a technique called Murfish to look at gene expression across the landscape of a tumor that we had engrafted into a mouse and you can see there the image that says GBM, that mosaic part of that, those are actually different genes being expressed in the tumor, so there's no homogeneity within this tumor, so that is itself the problem. Although we would like the tumors to be like this, homogeneous, they're actually very much like this. How do we address this issue? I think of cancer like an evolutionary tree. How would I try to chop down this tree? Well, I would take a saw to the trunk and cut down the tree. However, we're faced with drugs that don't really tackle all the mutations in the tumor. For example, if you have a drug that targets mutation D shown in yellow, that would eliminate part of the tree. If you have another drug that targets mutation C, likewise, you'd only eliminate part of the tree, and the same for a drug that targets mutation B. We really haven't gotten to the root of the problem or the trunk of the problem, which I call truncal mutations in these cancers. That is where we need to go to try to eradicate these tumors. There's been a number of groups that have addressed this question to try to sequence tumors and figure out what are the early mutations in the cancer that are conserved throughout the landscape of the cancer. One very good paper that came out early this year, in which they took a whole series of GBM patients and took about 10 different tumor cores from each patient and did deep sequencing of those tumor pieces. They found that, by a large, there were many regions of the tumor that shared mutations. Some had very unique mutations, but what other regions shared. But they had one mutation that came out to be shared among all the sections of the tumor, and we'll call that mutation A. I'll come back to that in a minute. But before I talk about mutation A, I want to take a brief departure and talk about aging and immortality, aspects of which have been discussed by several of my colleagues at these closer-look presentations. What I really want you to focus on are these structures at the end of the chromosome. This is a chromosome here on the left. It looks like a pretzel, but it's actually a chromosome and on the very tip there, that's a telomere. With each cell division, these telomeres become shorter and shorter because the enzyme that controls the maintenance of these telomerase which is called telomerase, slowly turns off in most of our cells in the body and this correlates with genetic aging. Manifestation of aging is wrinkled skin, graying hair, some of which you see looking right at me right now, so what happens in our stem cells is that telomerase is actually kept on, and this maintains the ends of the telomerase so they do not sinus, and these cells maintain their proliferative capability throughout our lifespan. Cancers try to find ways to hijack telomerase. They acquire mutations that activate telomerase and therefore keep the cells or keep the tumors immortal. They have a full tank of gas all the time. Telomerase mutations are very frequent in cancer. Now, getting back to what that gene A was that I showed you on the previous slide, gene A actually turns out to be telomerase transcriptase or TERT. This is very highly mutated in GBM. It's an activating mutation. It keeps the expression of TERT on, and this mutation is present in 85% of GBM samples not only in GBM, but you can see here on this chart that it's also present in many other cancer types, so by mutation of TERT, we keep telomerase on, and GBM achieves immortality. Not only is TERT a vital mutation in a number of cancers, but it's the third most mutated gene in all of cancer. First is P53, and the second is KRAS. We have an hypothesis. Since TERT mutations are early events in glioblastoma formation that lead to tumor cell immortality, we can develop drugs that target this mutation, this would affect a rather a significant eradication of these tumors. How should we do this? My lab has developed a new program called Brain tumor Avatars. Hopefully, some of you have seen the movie many years ago. Avatars are actually, they're stand ins or icons or figures that represent a particular person or a particular thing. You can see here the image of the Avatar from the movie, and on the right, is an Avatar of yours truly. What we developed is a platform where we can make brain tumors in a dish, as well as in the animals. We start with stem cells, induced pluripotent stem cells into which we introduce gene mutations by CRISPR-Cas9 editing, and these mutations are specific for GBM. We then differentiate these cells to a cell of origin, such as a neural progenitor cell, which is a glial cell, and these will form tumors in a dish. We could also engraft these into mice, and then these will form tumors in the mice somewhere between 2-4 months. Then when we take these tumors out and we look at them histologically, as shown here, they're actually indistinguishable from human tumors. I've actually taken some sections of our models to our neuropathologist here on campus, and they actually thought these came out of an individual, but they're actually from our modeling system, our avatar system. When you compare the avatar to the patient GBM shown below, histologically, they're indistinguishable. When we do genetic analysis, mutational analysis, the mutations that are acquired in the avatar are also indistinguishable from the patient samples. This is what we refer to as Phase 1 of our program. Phase 2 is actually to leverage these avatars to look for therapeutic targets or therapeutic vulnerabilities. To date, my lab has generated a rather extensive avatar catalog. Each avatar is designed to explore tumor specific characteristics. For example, we've generated a model that allows us to look at gene amplification in the form of extra chromosomal DNA as shown here, which has never been done in other animal systems such as mouse. We've generated pediatric avatars, such as atypical teratoid rhabdoid tumor. It's a highly aggressive tumor that occurs in children under the age of one, typically. This particular model showed us that there is a defect in the maturation of a stem cell in the brain of the animals. These cells actually get stuck in a certain phase of differentiation, and we think that we can leverage this model to find therapies that force the cells to differentiate into mature neurons. We've also recently described or developed a model of leptomeningeal spread. This is driven by a bee raft mutation, and this is a very interesting disease because it's reflective of metastatic disease. We also see leptomeningeal spread into the spinal cord in this particular animal. Then, lastly, I will tell you a little bit more about our model to try to address the issue of heterogeneity and this once again has to do with the mutation in the TERT gene. As proof of concept, we made this model with the TERT mutation. We grew the cells in culture. These are cells growing in petri dish and you can see short term culture, looking at these cells, there's absolutely no difference in how well they proliferate. They proliferate identically. However, if we age these cells in culture for about three months, you'll note that the cells with no TERT promoter mutation actually begin to senescence. They stop dividing when compared to the cells with the TERT mutation, those cells have achieved immortality. This shows very clearly that the presence at that mutation is utilized by the tumor cells to overcome this cellular senescence in phenotype, to be a successful tumor, if you will. What happens when we engraft these cells into mice? These are primary tumors, so these are the original cells injected into mice, and we looked at survival and what you see here is that both the mutant and the wildtype TERT genes do not have any difference in the survival of these animals. However, when you take these tumors out and you re engraft them into secondary host, we see a very big difference. Those tumors with the wildtype TERT take much longer to kill the animals, whereas the mutant TERT still has that capability and you can see on the right, when we look histologically, the tumors with the wild type TERT are actually much smaller in these animals. With all of this, we're now at a phase where we can look for genetic vulnerabilities. In other words, the Achilles heal of these cancers, and so what we really want to do is to find genes which are specifically needed in a case with TERT promoter mutation. We did a genetic screen. I did this with our colleague Gene Yeo in this building, and we found genes that are specifically needed for the growth of tumors with TERT mutation, and they're shown there in the red circle. We have about 30 genes which are absolutely required if you have a TERT promoter mutation. I'll show you just the data of one of those. This is called METTL14. If we eliminate this expression in a GBM cell line that has a wild type TERT, there's absolutely no effect on cell growth. However, if we eliminate it in these two different GBM lines which have mutated TERT, it's very clear that the cell are beginning to senescence or slow down in proliferation. With this approach, I'm fairly confident we can find genes which are entirely approachable for drug therapy intervention. With that, I'm just going to stop and thank my lab, who over the years, I've had fantastic lab members who have really developed this platform. Alison, Tomo, Shun, and Daisuke are the originators of the Avatar platform along with colleagues in Gene Yeo's lab and currently Clark, Brett, Yohei, and Chris are taking over the mantle pursuing these models for different types of vulnerabilities that we hope to report back to you in not too distant future. With that, thank you all for listening. [APPLAUSE Thank you so much, Dr. Khalessi and Furnari, we're happy to take some questions. If you raise your hand, they'll bring a mic around to you, but maybe I'll start with the first one while people gear up. Dr. Khalessi, you showed us that image of the difference in seeing a nebula. From you as a surgeon in your experience, what's been the biggest change in terms of what you can see in the operating room maybe going back five or 10 years to now. What do you think is the thing that will change the most in the next five or 10 years? Sure. Thank you for the question. I think it varies a little bit on what you're intervening on. We obviously operate for a lot of different indications. If I had to pick one thing, I would say having the opportunity to map quite precisely structurally, the deep tracks of the brain has been enormously helpful because it used to be, and you can imagine, if you had a lesion in the substance of your brain, you'd look at a structural atlas and you'd say, in general terms for 90 out of 100 people, this lives here, but that doesn't really help you if you're one of the 10 people where it lives somewhere else. Being able to with high fidelity, pick a safe trajectory with the use of navigation, I think, has been the largest interoperative advance. The fluorescence I showed, as I mentioned, from Dr. Baumont's case, has been a huge advance in the treatment of intrinsic brain tumors specifically. If you have a glioma, having the availability of that fluorescent agent to aid your surgeon to maximize your resection when we know volumetrically achieving a higher resection while maintaining functional outcome is really important. I think that's been a quite important advance in glioma surgery specifically. When you think about the next 5 or 10 years, it's one thing to actually understand where the highways are for lack of a better term in the brain, what is even more helpful when you think about implications beyond cancer is actually understanding how those different networks communicate functionally. That's why I showed some of those very high density electrophysiologic maps of the cortical activity in the brain. Because I think once we actually understand that better, that's really the first best opportunity to modulate the nervous system in new ways, either with stimulation, with the application of laser energy or focused ultrasound or with a neurostorative stem cell therapies. That, to me, is what the next decade going to be about. Once you know how to safely get somewhere, now you need to figure out where you want to go. I think we're figuring out where we want to go for a whole host of diseases and beyond cancer. Thanks. While we get the mic up here, maybe Dr. Furnari you talked about how GBM cells hijack telomeras. Are there other stem cell pathways that GBM cells will hijack or how else are stem cells specifically involved in these glioblastomas? There's other ways that GBM could actually hijack the stem cell components. Another way that they can maintain their telomeres is through ATRX mutations. That's another telomere specific mechanism. They also can self renew. We can look at the lineages of these tumors and we see that there's a trajectory that represents very similar aspects of development of the brain. A GBM, although it is a tumor cell, is a tumor, if you look closely, it has aspects of glial differentiation, neuronal differentiation. It utilizes the same pathways to maintain this ability to self renew. If I could just add to that really quickly. I think the real power of Frank's work from my perspective as someone who has the opportunity to treat people with brain tumors every day is, you showed that beautiful slide of different colored M&Ms. Gosh, it'd be really helpful for me to know which color M&Ms I really need to worry about. That's helpful in terms of informing surveillance because when you think about all the different treatment decisions that are made over the course of a cancer patient's journey, you're always weighing competing risk and benefit. We think about it as surgeons in very simple terms as volumetric residual, but not all volumes are equal. It may be that you are more targeted in your radiation therapy. You have closer follow-up surveillance. You can seed a modest deficit to actually achieve a greater resection. All of those decisions are more informed as we actually better understand the specific biology of different components of tumors outside of actually being able to follow them over time. I think it's really fascinating how the advances in science aren't just informing the science and drug development, but also the surgical interventions as well and something that I think is a real breakthrough that you guys are making. If I can follow up on that, what we're also learning is that these multi-colored bowls of M&Ms can shape-shift. A cell that may be green or red or yellow one day can be a different color or next day. We're quickly learning which cells within the tumor are actually sensitive to radiation and other types of therapy, but they don't stay in that state. We need to figure out how do we make the tumor cells more susceptible to our standard of care therapy? How do we push them into a corner where they're synchronous in terms of sensitivity to radiation, let's say. That's something that we're working on. We have drugs that look like they may actually cause a tumor to be more of a pro-neural state, which we know is more sensitive to radiation. Take a question from the audience? I have a question for Frank and then a question for Alex. For the Metal 14 that places N6 methyladenosine marks on RNA, is there any indication that that's affecting turt expression in any way? It does. When we eliminate Metal 14 from the cells, there's a transcription factor which regulates the mutation in the turt promoter. That transcription factor, expression level goes down, which then leads to a decrease in turt. We found the link to the transcription factor which controls the mutated turt. Are there inhibitors for Metal 14? There's an inhibitor for Metal 14, which is not very good. There's one from Metal 3, which we've tried and that seems to work somewhat, but I think this is a great opportunity to actually develop inhibitors for Metal 14 that are much superior to what's out there. They work as a Complex 3 and 14, yeah. Dr. Khalessi, this was amazing what you conduct as yours and Frank, you're always amazing. I was actually really glad you asked that question of Frank, the last one. But weirdly, they're interrelated and now I'm going to make you nervous. Now, when you show the fluorescence image, my background, my PhD was on photodynamic therapy using these photoactive molecules with a specific wavelength and energy of light to activate signal and oxygen production. It looks like you could do that in the brain more readily than I thought you could, considering the new tools you have to image. Is that something that people have looked at resurrecting? Yeah. There's actually been a lot of work done in our neuroscience department here around optico genetics and the idea of actually providing a sensitizing agent and then exposing them to light as a way to deliver a therapeutic payload. There's been fewer applications to my knowledge for primary brain cancer. It's been largely used with five ALA to distinguish between normal and abnormal, but that absolutely would be a potential opportunity. Most of the local therapies that have been focused on has been around the polymer based delivery of known agents, as you know, but I think that field is wide open. Five amino leveulinic acid that five ALA is a very rudimentary version. There are huge amounts of things you can do, but the other thing I thought when you were showing the fluorescent imaging is we use fluorescent reporters of stem cell activity and we always thought they were amminogenic and we couldn't use them. Maybe you could actually use them. You could use a turt reporter with Frank. Well we have an ADR reporter. We've got a proteostasis reporter. There's so many things we could do now that we know you can image in such a fine. Obviously, as I mentioned, the center for future surgery would be a great opportunity to try a lot of those agents in animal models. You could deliver that vector and then we can actually see. The key thing is for those things is to actually figure out how specific they would be. I think that would be the main because obviously, you always have this trade off, because a lot of what we try to do is protect normal, obviously and we'd want to make sure we're in a position to do that. I think we've got another question in the audience. Thank you for all the great talks. I have a question for Dr. Furnari. Firstly, I would like to see that really a lot of fantastic model, really great models that you generated in your lab. I'm wondering when you show the two the mutation, you show that the first round injection transplantation, you don't really see the prognosis difference, but then the second round, you do see the difference. The patients, they don't really get transplantation so I'm wondering what will be the implication of this for the patients? Also, do you see association between the mutation and patient prognosis? Your second question first? TERT promoter mutation has definitely overall worse prognosis. Now, in terms of is the mutation really needed for these tumors to form? There has been some really good evidence of late, that TERT promoter mutation are actually existing in normal brain, so in autopsies, they've done some deep sequencing, and these mutations do exist, but they don't go on to become tumorigenic. Because they don't have the other mutations are associated with GBM. I do think it's definitely needed as to overcome the bottle neck of cellular senescence. But unless you have the other mutations that occur with it, it's going to be inert in our brain. Just like P53 mutations on our skin don't all develop into tumors. It's only the rare occasion where you have the acquisition of additional mutations that we get full blown cancers. Can I follow up with that and you say that the mutations are inert, is there any chance that those mutations in normal brain have a positive effect at all? Well, that's an interesting question. I would love to explore that if we can maybe make a mouse where we introduce this mutation and look and see if there's anything that might extend the pool of stem cells within the subventricular zone. That's an excellent question. These mutations are heterozygote. You never see a homozygote mutation in TERT. They keep the wild type gene intact to operate under normal situations. But in a tumor situation, it's the mutated gene which gets activated by your unique transcription factor. There's some interesting evidence in other tissues now that mutations that are associated with cancer may start coming originally on to help with regeneration or maintenance of the tissue and whether there's a positive side to that before it turns to the dark side, essentially, I think it is intriguing. Incredibly fascinating. It's something we can probably do in the mouse. Our next question from the audience? Thank you. You mentioned early on that the glioblastomas, and maybe this is a question for you, Frank, are heterogeneous. But you also mentioned that they can be metastatic from a breast cancer patient. Was the example given early on. Just wondering what the ratio of metastatic derived glioblastoma, versus primary glioblastoma, I guess, in the brain. Just fundamentally how different are those? Well I think this is a question maybe you can better address. Yeah, just to clarify. The example I was given was a patient with primary breast cancer and metastasized to the brain. But just to speak to it more directly, the reason you're hearing a broader conversation around metastatic cancer to the brain, is this reflects a more seismic shift in what neurosurgeons treat because of our success in treating systemic cancer. Ten years ago, 2/3 of the craniotomies done in the United States were actually for primary brain cancer, and the reason that was is because if you add metastatic cancer to your brain, that was thought to be a disabling or terminal event. In other words, there wasn't a meaningful benefit to try and achieve technical intracranial control because the systemic burn of disease was too high. Now that ratio is flipped, so 2/3 of the craniotomies I do for tumor are for metastatic lesions, as opposed to primary cancer. The reason that is is because our systemic treatment of cancer has improved so dramatically that if you can actually get someone good CNS control, that can buy them many years of functional life. That is a credit to the success and progress we've made in other forms of cancer, that we've been able to focus so much more on the biology of CNS metastases. Because there's this real value that comes with that. To me, and to speak to the earlier question that was asked about the application of some of the really exciting work that Frank's doing, is if you actually think about a GBM and it's right there in the name multiform, that there's all this distinct intertumoral heterogeneity. What becomes important is actually coming up with meaningful subcategories. To me, the most powerful insight in some of the avatar work that he showed, is we are starting to see those reproducible lines, that actually are a first step to then look at, okay, within this subset of tumors, what are the right targets for further work? Where are the potential therapeutic vulnerabilities? Because we used to, when we thought about the biology of GBM, let's say, ten or 15 years ago, we were totally focused on much higher order physiologic processes. We were worried about tumor angiogenesis. A lot of the effort was focused on reducing actually the tumor's ability to recruit blood supply. Then we were saying, Well, the local tumor environment's immunosuppressive, so how do we actually sensitize the immune system to the tumor? I think for the first time, as opposed to thinking about, the conventional way the body either supports or responds to tissue, we are now actually saying. Well, what's the biology of the disease we're trying to interrupt? Which is a fundamentally different question because we now actually have the power to characterize that in a way that's new and different, not just at the genetic level, but as Cat asked in how those genetics manifest at different time courses in the tumor's progression in an epigenomic way. That, to me is what's so exciting because the journey I take with cancer patients is a longitudinal one. Having the right information at the right moment. I think to me, is going to be the real power that comes out of this partnership in this work. We have a question here. Yes, question for Frank. Great talk as always. Two things: one, you showed in your avatar you differentiate stem cells along the glial lineage. I guess one question on that is, what is the cell of origin? How far have we come or the cell, as identifying the cell origin of glioblastoma, because as opposed to DIPG and medulla where we have, fairly good idea, which one it is. I guess for glioblastoma is much more difficult. How do you address that in your model? The other thing is you showed that you did genome sequencing of your avatar tumors. Have you also done methylome illuminate 150_K? Have we done the methylome, you said? No, we haven't. We've done whole genome sequencing, We've done obviously RNA Seq, single cell RNA seq, and those all perfectly aligned with what we see in patient samples. The cell of origin question? We've routinely pushed these towards a neuroprogenitor population to initiate our tumorogenesis. We've also pushed them towards becoming astrocytes, and they're less tumorogenic. I think, because they have more differentiation, such terminal differentiation associated with it. At this present time, I mean, neural progenitor cells seem to work for the GBM system, but that's not the same for the ATRT models that we've built. That's completely different. The cell of origin there, is more likely a progenitor cell on the way to becoming a neuron. We're learning a lot, and we can map onto the human disease to try to figure out trajectories and really determine the cell of origin for probably all of our models. We've got time for one final question. Great. Thank you very much. Those are really fantastic talks, and I have a question for Doctor Furnari. In your figure where you showed the example of the heterogeneity, in the xenographs you had four colors, and they were in patches rather than a mix of cells. Then you mentioned that even within that, the cells can also have plasticity to change and shift. Now I was wondering, how does the plasticity and the heterogeneity of your avatars compare to the plasticity and heterogeneity of patient derived xenograft tumors in the same kind of injection models? Can you classify your avatars into the different, PDXs and which ones they're most alike? That's a great question. Our first paper, we really wanted to address that. Really, the only way to do that is by single cell sequencing, and we saw complete alignment with what's been published out of institutions like Mass General, where at the single cell level, you can describe a tumor cell as pro neural, mesenchymal, OPC-like or astrocytic-like. We were able to do that. It really was based on the mutations. A proneural genetically defined model was different than our mesenchymal genetically defined model in terms of the distribution of these what we call cellular states now for GBM. We get exactly what we see there. We see the same transitions between states that you see also in patient samples. Was that your entire question? I can't remember. Thank you. Let me once again thank Doctors Khalessi and Furnari for joining us today and also to thank them for devoting their lives to something that is so important, and for working to bring new light to what we said was one of the greatest mysteries of human biology and human medicine and I think are really making the difference right now in people's lives. Thank you so much and thank you-all for joining us.