The Nuclear Pore and XPO1: Guardian of the Genome

In this webinar, you will hear from Karyopharm’s Founder, President and Chief Scientific Officer and other renowned subject matter experts on the incredible biology around the nuclear pore complex, including the nuclear export of key proteins via XPO1 and the unique mechanism of action of our lead compound, XPOVIO® (selinexor). Learn how the regulation and location of key proteins in-and-out of the cell’s nucleus can have such a profound impact across so many different disease areas.

Full Transcript Below:

Tina Beamon: Hello and welcome to the inaugural karyotalk webinar, the nuclear pore in XPO1: guardian of the genome. I’m Tina Beamon Vice President and Chief Compliance officer with Karyopharm Therapeutics. We are delighted to have you as a guest for today’s event. Today’s disease state program will explore the role of exportin one protein, also called XPO1 or CRM1, in the nuclear pore complex and in nuclear transport. The nuclear pore complex is made up of a multifaceted network of biological actors, including importins that bring proteins from the cytoplasm into the nucleus and exportins that bring proteins in the other direction from the nucleus out into the cytoplasm. Over the next hour we will learn about the nuclear pore complex generally and how XPO1 protein, one of the key exportins, behaves within this framework including its impact on a variety of different diseases like cancer, Covid, traumatic brain injury, and Duchenne muscular dystrophy.

The presentation will begin with an introduction from Karyopharm’s president and chief scientific officer,
Dr. Sharon Shacham. We will then hear from panelists professor Michael Rout, Professor Cesar Borlongan, Dr. Thomas Walsh, Professor Matthew Alexander, and Dr. Clifton Mo. Please turn your attention to the menu at the bottom of the screen. We will release polls throughout this program. Please vote on the answer that best suits you. Today’s polling will help us understand what parts of the program are most important to you. So, with that let’s get started. Please welcome Karyopharm’s president and Chief Scientific Officer, Dr. Sharon Shacham.

Sharon Shacham: Thank you very much Tina and I should mention Tina is our head of compliance so, this way we are making sure that we are all compliant here. And this is a very exciting webinar, it’s our first webinar in the series of the KaryoTalk. And I would like to take this opportunity to again thank all the panelists for joining us in this very first webinar and to the KaryoNext team that worked and enabled us to have this webinar. We are going to have a very special journey today. So one of the most interesting pathways in biology and together with my colleagues we will show how one protein in the cell can play a very important role in many diseases as Tina mentioned, from cancer to brain trauma to DMD and to viral infections the protein that we are talking about. It is called XPO1 and it’s like a train and although we can’t really imagine our life without an efficient train system and I’m not talking about Amtrak here, but a real train system. The XPO1 train is being kidnapped by the disease and it gets out of control and when that happened it threatened to damage the cell. And today we will discuss what are the situation that the train is getting out of control and we can see the little monster here of the disease and one approach that we develop here at Karyopharm. To bring the train back into control and continue the path of a normal cell, I know some of you might still not understand what am I’m talking about looking at this cartoon but that’s the idea so you’ll stay with us until the end of this webinar and at the end there’s going to be a quiz so I’m sure you’ll all know it by the end.

Sharon Shacham: So, if we can move to the next slide. Karyopharm was founded in 2008 by the peculiar doctor Shacham and this was about by now 12 winters ago and we are just heading to our 13th winter here in Newton, Massachusetts. And the idea was really started by a paper that professor Rout is going to speak after me published, which showed the first high resolution structure. It was a model of the nuclear pore complex and with that the thought came to us that if we can play with what’s going in and out of the nucleus we can really impact one of the pillars of the protein, which is the communication between the nucleus and the cytoplasm. The company had managed to develop small molecule completely from scratch, to be honest some of this work was done on my computer and the compounds were stored in our home kitchen in the refrigerator and we found a few hits we developed them and in 2010 we were able to demonstrate for the first time anti-cancer activity in vivo and individual. And after that we were we started clinical trial in 2012. So just about eight years ago and this was in patients with blood cancer and with solid tumors. Since then we have treated more than 3000 patients, including about 150 kids with Selinexor as single agent and in combination. And recently we also treated about 150 patients with severe Covid-19.

Sharon Shacham: So, this was in the last eight years. We got our first approval for Selinexor just about a year ago in July third of 2019 and that was for patients with penta refractory myeloma. So very refractory multiple myeloma and about a year after that we got our second approval this time in patients with a relapsing refractory DLBCL. And as you can see on the slide and we might be able to mention it a little bit after Dr. Mo will have his section talking about cancer, is that this mechanism function might be applicable to many cancers as well as other diseases as we are going to talk today.

Sharon Shacham: So, if we’ll move to the next slide. So, in this slide we are seeing on the left data from brain tumor from glioblastoma which is the most aggressive form of brain tumor. Now I want to remind to you that cancer, in general, is associated with unregulated cell growth. And on the right, we are seeing data from patients with multiple sclerosis or from a model of traumatic brain injury that is done in vats. And these two models are associated with cell death but what is combined to in to these two situations or two different or three different diseases is that in all of them we see an increase in XPO1 or grim one that’s the other name of this protein. That is coming in the situation of the disease compared to normal cells we see it here in glioblastoma and we see it here in patients with multiple sclerosis compared to patient without. And this in post mortem analysis and we see it also in the first few days after traumatic brain injury in all of these cases other associated with unregulated cell growth or with cell death we see an increase in XPO1 which is very interesting on top of that we can also know and we have seen it in other cancers as well is that the more extra one you have or more qm one you have the survival is lower so higher chrome one is associated with increased mortality. Why is this how can one protein be associated in both cases?

Sharon Shacham: Let’s go to the next slide and try to understand first what is XPO1 and what is it doing. In this carton we see on the right the nucleus of the cell and on the left the cytoplasm they transport of protein through the nucleus to the cytoplasm and vice versa is highly regulated as we can expect. That and for protein to go in and out all protein above 40 kilo dalton they have to go through the nuclear pole complex, one of the most complicated structures in the cell. And professor Rout after me will talk about this in detail however this transport across the nuclear pole complex can only be done through carriers or trains and XPO1 is the main train that takes protein from the nucleus into the cytoplasm. We see it in this gray bagel and the cargo is seen as the green part it releases it in the cytoplasm and it goes back in to pick up another cargo and send it out to the cytoplasm and it can do it multiple times. The more XPO1 we have the more nuclear export we see. So how does it look can we go to the next slide? What is happening or maybe I’ll take one step back. There are about 200 cargos that XPO1 mammalian cargoes that XPO1 one can take from the nucleus into the cytoplasm and there are other cargos like viral protein that professor Walsh will talk about.

Sharon Shacham: But let’s focus for a second just on the mammalian and the cellular cargoes. Amongst this 200 protein it includes all the tumor suppressor protein which we also call them the guardian of the genome because that’s exactly what they do. They sit in the nucleus they, monitor the the dna for dna damage which is the hallmark of cancer. And once they identify dna damage they say okay this audit essentially fail they call the cell they induce a cell cycle of rest and induce apoptosis of the cancer cell. When XPO1 is over expressed, I don’ know if there is a chance to get the cartoon again, it takes the tumor suppressor protein and what we can see in cancer cells that the tumor suppressor protein are actually outside of the nucleus and not where they are supposed to be. So the train takes them out of the nucleus and release the guardian of the genome or the tumor suppressor protein into the cytoplasm where they cannot do their work and that’s how the cancer cell can proliferate.

Sharon Shacham: Our thought was simple if we will block this strain we will block the excessive export nuclear export of this tumor suppressor protein will restore their localization into the nucleus and they will go back to do their job to identify dna damage and send cancer cells into cell cycle arrest. And that’s exactly what the SINE compounds are doing, they bind to XPO1 in the cargo binding domain and inhibits nuclear export. So how did we do that? If we go to the next slide what we see is the x-ray structure of XPO1. The exo structure of XPO1 was published by professor {Unknown} from the University of Texas in Dallas in 2009 really when I started the company and this structure really enable us to identify small molecules that will bind to XPO1 and prevent cargo binding so it’s a very complicated protein we can see it here and in the right side in the upper right side we can see the cargo binding domain so it’s there is a helical structure or a cavity that allows helical structure to bind and many of the natural cellular or other natural cargoes of XPO1 have this helical structure. Interestingly in this upper here what we see at the bottom there is a cysteine residue that is conserved all the way from yeast to eukaryotic cells into our cells as well and this cysteine is not part of the cargo binding domain. It doesn’t participate but it’s still very conserved and there are all sorts of assumptions why is it there but what we did is we use the cysteine and identify a small molecule that will bind to the system and we can see how two examples of sine compounds and by that they block the cargo binding domain and prevent the regular cargos of the strain to bind to XPO1 and they left in the in the nucleus and do their function. So how do we talk in the cellular level? So this work was done by our team with one of the early compounds we see here osteosarcoma cells and we see about four hours of treatment with a sign compound that we squeeze to about 10 seconds of a movie. When we’ll see when the movie starts again is that this protein that we look which is one of the cargo of XPO1 is actually everywhere, except the nucleus. So, this is a tumor suppressor protein that’s supposed to be in the nucleus and in cancer cells being exported to the cytoplasm and cannot do cannot function in the nucleus where it’s supposed to be. Once we treat these cells with our XPO1 inhibitors we can see nuclear localization by this intense color like we see here of foxo the protein that we will follow in the nucleus and now this protein can start functioning and induce apoptosis in cancer cells and and cell death now the same protein in other diseases like in multiple sclerosis for example is essential to prevent cell death and again in the situation of the disease it’s excluded from the nucleus so restoring the same protein function in the nucleus in other diseases will prevent the progression of the disease.

Sharon Shacham: So this is a brief introduction so if we go back now to our cartoon about the train I’m going to give you a few more hints and let’s summarize it when we look so we have the XPO1 train expressing all our cells essential for the function of normal cells, however, indeed in a situation of diseases like cancer, multiple sclerosis, TBI and it’s also essential for viral propagation we see more trains and we see the train being essentially kidnapped by the disease and that sleep the tumor suppressor protein just going to be kidnapped you know carried by this train and move outside where they supposed to be so this is our superheroes here getting out from where they supposed to be into the nucleus and pushed with the train outside of the nucleus and the little figure here is the sine compounds that prevent us and stop the excessive trafficking induced by the XPO1 train. So, I hope that helps and now maybe we’ll take one step backwards and we’ll let professor Rout explain about the entire bigger system of the nuclear pore complex.

Tina Beamon: Thank you so much Dr. Shacham. We are pleased to now introduce Professor Michael Rout of the Rockefeller university who will present on the nuclear pore complex.

Professor Rout:  Hey thank you very much I’ll just see if I can get this to work there we go. Hopefully you can all see that slide. Yes yes great thank you so thank you very much for having me here. So I’m going to take a step back, actually quite a few steps back, sort of 30,000 foot view and a very brief summary of the work of many groups over many years from all over the world trying to piece together what the nuclear pore complex looks like and how it functions in transport of materials into and out of the nucleus.

Professor Rout: So we’re starting as you can see with the nucleus district it turns out it’s not only in the cell it’s a place in Montana. As you can see here but the nucleus is where all the genetic material of the cell is and bounded by a double membrane made of an outer membrane and an inner membrane where those membranes meet in circular grommets. This is called the nuclear pore and there are many of these nuclear pores all over the nucleus but they’re not just open they’re filled with a proteinaceous complex called the nuclear pore complex right here and as you’ve heard the nuclear pore complex’s job is to be the gatekeeper of the nucleus. To bounce out anything that is not supposed to be in there. To keep in things that are supposed to be in there and to make sure that the things that need to go in get transported in, the things that need to go out are transported out. Small molecules like sugars and ions actually can diffuse passively into and out of the nuclear pore complex but macromolecules and in particular, proteins and rnas have to be actively transported in what is known to be a highly selective and highly regulated set of processes.

Professor Rout: So the many different kinds of macromolecules are imported and exported through mutual core complexes. This is an electron micrograph section of a typical u-carriage itself. The nuclear poor complexes are these very very small things here but there’s thousands of them like 2000 or more on an average mammalian cell. For example ribosomal proteins must be made in the cytoplasm, imported into the nucleus where in the nucleolus they get packaged into three ribosomal subunits and those three ribosomal subunits have to be exported again out of the nuclear pore complex where they get assembled finally into the full ribosome and so on of course. Messenger rnas are made in the nucleus. They’re then packaged into rnps and exported through the nuclear pore out into the cytoplasm where they then get translated and the proteins that carried them get recycled back to carry more mrnas and so in fact the central dogma of life dna makes rna makes protein is in fact interrupted by the nuclear pore at the at the rna makes protein stage the rna has to get out into the cytoplasm.

Professor Rout: But what we’re particularly interested in today is the import and export of proteins in order for a protein to get into the nucleus it has to have a little signal on it so the cargos must have a little signal called an nls a nuclear localization sequence. This gets recognized by any one of a member of of a family called carrier ference or importins or exportins because they were independently discovered by several groups they still carry these different names. An importin carrier pharyn binds this nls on the cargo protein and then will carry it through the nuclear pore complex, out into the nucleus. In the nucleus it meets this protein called ran gtp that is it’s ran as a member of the rash super family of gtp aces and it can exist either in a gtp-bound form or a gdd-band form it’s maintained in the gtp bound form in the nucleus and the gdp bioform in the cytoplasm and that energy differential is what powers transport you can think of it like a,  like a hydroelectric dam with the high energy all in the nucleus and the low on the other side and the bleed of that rand energy is what powers the cycle. If you are a protein to export you have a little pro signal on your cargo protein called an nes or nuclear export sequence and there are a cognitive family of carrier therian that recognize those nes and export the cargos out through the nuclear pore when they get to the other side the round gtp that’s bound to them hydrolyzes to run gdp and it’s that burst of energy as I say that then powers transport and iran itself has its own little transport factor that takes it back into the nucleus where it gets regenerated into the ground gtp bound form and the whole cycle then continues. But it’s all centered on this huge macromolecular complex called the nuclear core complex.

Professor Rout: I’m going to show you a quick overview of some of the major structures of the nuclear pore. They’re made of a membrane ring which is in the lumen of the nuclear envelope. So it sort of holds the whole nuclear pore into the curved grommet that you can see here of the membrane where the outer and inner nuclear membranes of the two membranes neutral envelope meet. That attaches to this very dense inner ring complex which is flanked on both sides by an outer ring complex and then attached to those are peripheral machineries on both the cytoplasmic and nucleoplasmic side. In this diagram the cytoplasm is to the top the nucleoplasm is to the bottom and they are used to help assemble and disassemble transport. Cargoes with their transport factors studied throughout the tube in the middle the tube that all the stuff goes through. In and out through the nuclear pore are these anchors for these things called fg repeat nucleophorines they call that because they actually have phenylalanine glycine that’s fg repeats dozens and dozens of them in each protein. And they form this dense wiggly dynamic network of highly dynamic brush like dense region going throughout the whole tube of the new kapoor as you can imagine that forms a barrier that most things can’t get through. But transport factors carrier ferrins and other transport factors with their cargos the transport factor combined the phenylalanines on those fdu repeats. They have a series of little pockets that combine the phenylalanines and they do so very very quickly on and off and by being able to do that they can overcome this dense mesh work that normally keeps stuff out and actually carry their materials across the nuclear pore either into or out of the nucleus.

Professor Rout: So I’ll just give you a quick movie here, just to give you some idea of the assembly the whole thing here is about 100 nanometers across. The hole in the middle filled with this selective meshwork that will only allow transport patches to it’s about 40 nanometers across the whole structure is anywhere from 50 mega Daltons up to over 109 mega Daltons. In most human cells this is a yeast one but the vertebrate one’s even bigger. But the principles are about the same. You can see it’s made of these coaxial rings at different levels and that there are in fact eight spoke units making up these coaxial rings. But what we found by looking at the structure of the nuclear is it’s very much like a reminiscent of a suspension bridge in that the membrane ring I just showed you firmly anchors it into its substrate which is the curved membrane of the nuclear pore  inside each spoke and the at the by the way the view i’m giving you here in these little diagrams is as if you were inside the tube looking out towards the three towards the spokes and you’re seeing three of those spokes just looking out at them so it’s from the view of an actual thing. Going through the central tube you can see that the these folks have these rigid columns just like the supports and tresses of a bridge but that between these rigid supports are flexible joints because like a suspension bridge you need flexing to allow the bridge to breathe and accommodate heavier or lighter traffic. Likewise all the major units in the bridge are connected by flexible connector cables and again they are tends to allow tremendous strength but also allow flexibility as bigger or smaller things go through the nuclear port and of course all of this builds this high density transport pathway anchors. These fg repeats points them into the central tube in a very organized fashion to create pathways highways of binding through the nuclear pore that transport factors can carry their cargos so this all builds a very strong and flexible transport system that can maintain this huge transport flux in both directions. So that’s sort of a quick overview of the nuclear pore complex. But as you said just like a suspension bridge there are many different kinds of traffic going through the nuclear pore complex and different carrier pharynx specialize in carrying different kinds of cargos either in or out of the nuclear pore complex. We’re still understanding trying to  understand what all these different cargos are and we haven’t got a complete list of who is carrying what,  but there are certainly a whole series of import carrier fairings that carry stuff into the nucleus and they’re different kinds of proteins that can be carried so some for example specialize in carrying only ribosomal proteins. Some specializing carrying h and rnp proteins and so on and so forth and export likewise there are some that specialize in for example trnas certain kinds of proteins and of course the one we’re interested today is a bit of a universal carrier creme one or expo one which recognizes any cargo that carries these nes uh typical nes sequences. And that’s the quick overview so we’ll leave it there. Thank you very much.

Tina Beamon:  Thank you so much professor Rout that was a fantastic presentation. We’re getting some questions in the chat feature and we’re going to just ask you to hold your questions until the very end and should we have time they will be addressed at the end of the program. So with that I’m going to invite up our next speaker professor Cesar Borlongan of the university of South Florida, who will present on the role of XPO1 in traumatic brain injury.

Professor Borlongan: Thank you Tina. So I am Cesar Borlongan, I am from the university of South Florida, Morsani College of Medicine and I’m a director of center of excellence for brain repair and aging. My interest in xp01, I would focus more on the traumatic brain injury. Just for disclosure of a conflict of interest I received funding from Karyopharm some years ago for this specific project on traumatic brain injury. Next slide please. Many of the data that I will present today have been published some years ago on this journal and again trying to segway from Dr. Shacham and then Dr. Rout’s previous presentations, I would focus more on rather than trying to sequester the cancer signals in the nuclear pore i am very much interested in trying to sequester the inflammatory signals associated with neurological disorders such as traumatic brain injury.

Professor Borlongan: As you know even though traumatic brain injury is a considered as an abrupt brain insult there is a secondary cell death plagued with inflammation so that’s my bread and butter is to see how we can sequester that secondary cell that, associated with traumatic brain injury. So the impetus for this study as alluded by Dr. Shacham earlier is that in cancer cells such as the thp1 or the hela cells you could see that under control conditions many of these cargo proteins are in the cytoplasm or in the membrane areas following on treatment with the sine or the KPT350 you can see you can appreciate that many of these proteins now get sequestered into the nucleus. So just like what Dr. Rout presented earlier is targeting that nuclear core you are able to control and regulate the export of this proteins in particular the inflammatory proteins. So in the initial communication with Karyopharm, I was presented with this data this, so this is an in-house company data that when you treat prior to traumatic brain experimental traumatic brain injury in mice or in rats you could see that the KPT significantly reduced the brain injury at the gross morphology. And so being a translational researcher, I would rather initiate the treatment post injury rather than pre-injury. So that’s my contribution to this project on the subsequent sets of slides.  Rather than initiating the xp01 inhibitor or sine,  we initiated the treatment post injury and again you can recognize here following the traumatic brain injury which is a cortical controlled impact injury. there’s a significant damage into the cortical region of these rats. Following transfusion or in infusion of the low dose or high dose of the xp01 or inhibitor or KPT350 there’s a significant reduction in the cortical damage and this is not just in the impact injury but if you count the number of cells lost in the peri impact injury so these are like the area surrounding the core injury there is also a significant reduction of cell loss.

Professor Borlongan: So this is quite a significant finding. Again I want to reiterate the treatment of the xp01 inhibitor or KPT 350 was initiated post injury starting at the two hours and then subsequently the animals receive over the next four days once a  day of this treatment. So this is some kind of clinical implication. As you know we cannot predict when tbi will occur so for us to be able to initiate the treatment after the injury I think this will have a significant clinical application.

Professor Borlongan: Now not only did we find that the reduction in brain injury is significant but there’s also a corresponding recovery in behavior. So these are just two of the behaviors that we conducted. As you know we targeted one, you know hemisphere with the cci with the tbi injury so there’s a significant hemi neglect as you can see also in patients. But these are in animals. When you subject the animals to a rotarad test or the tail hang test,  there’s a asymmetry in their body movements. And again we can appreciate with the treatment with the KPT 350 there’s a significant improvement in their asymmetrical behaviors indicating there’s a significant behavioral recovery from traumatic brain injury.

Professor Borlongan: So in terms of the histology not only did we see a significant reduction in the gross morphology but also if you look at the blood brain barrier markers in this case evans blue extravasation, there’s a significant reduction in, in the entry of this compound into the brain. Indicating that the blood-brain barrier integrity remains intact in animals that receive the KPT 350 and other markers of the blood-brain barriers such as the beta-catanium. Also showed that in animals that received this compound the xplo1 inhibitor there’s a significant  preservation of that junction tight junction proteins such as the beta-catenin that would indicate also that the blood brain barrier is  preserved in animals that receive the xp01 inhibitor.

Professor Borlongan: As I mentioned in my introduction my interest is trying to look at the inflammatory signals which is accompanying the secondary cell death of tbi and lo and behold what we found the markers of inflammation here demonstrated by tnf alpha is also significantly reduced following the treatment with KPT 350. So this indicates to us that there’s a sequestration of these inflammatory signals within the nuclear pore and we are able to sequester also the cell death associated with inflammatory response in tbi.

Professor Borlongan: So just to summarize what I presented in this animal models of tbi, there’s a significant reduction in the the cell death that we’re seeing the secondary cell death it’s associated with anti-inflammatory signals as well as the neuroprotective signals within the nuclear pore. This type of sequestration of cell death is leading to functional recovery in tbi animals and this is also associated with blood brain barrier integrity. So that type of mechanistic of actions are correlated with reduction in inflammatory signals and blood brain barrier integrity.

Professor Borlongan: So in the current times we are also interested in trying to extend this type of nuclear sequestration in Covid19 and the next speaker Dr. Walsh I don’t want to steal the thunder from Dr. Walsh but at least from our end we saw that in some of our patients with Covid 19 not only are we seeing inflammation in the lung but also in the in the brain, especially in patients suffering from stroke after Covid19. And so the significant increase in the patients with Covid 19 in stroke incidence could be associated also with the inflammatory response. Our hypothesis is if we could hinder that inflammatory response associated with Covid 19 there is a high likelihood that we may be able also to ameliorate that secondary cell death of stroke that’s associated with Covid 19.

Professor Borlongan: So thank you so much I’ll give it back to Tina.

Tina Beamon:  Thank you professor Borlongan. And as you previewed for us our next speaker will be Dr. Tom Walsh of Weill cancer medicine, who is an expert in infectious disease. Dr. Walsh will be presenting on the role of xp01 in Covid19.

Dr. Tom Walsh: Good afternoon and thank you all for the opportunity to talk and to discuss this important disease especially as it relates to the potential therapeutic intervention from Selinexor and other sine inhibitors. I would preface just by placing in context of the Covid 19. Our mission here at Weill Cornell Medicine is that of developing new therapeutic interventions for life-threatening infections and that in our immunocompromised patient population with the advent of Covid 19. Karyopharm had contacted Dr. Ruben Navetsky who has done landmark work in the area of multiple myeloma with Selinexor. Dr. Novitskiy then contacted Dr. Small and me where we also are aborted not just an infectious disease but also in oncology. Hence a very strong translational partnership developed with the three of us then working very closely with Karyopharm and helping to develop protocol initiatives and helping to further understand the implementation of Selinexor or as a potential anti-Covid-19 compound.

Dr. Tom Walsh: And so with that I’d like to provide you them with that context the background for Covid 19. Where we stand literally as of yesterday, the epidemiology globally shows that a severe the severe acute respiratory virus syndrome which is the virus itself SARS kov2 is causing a global pandemic of coronavirus 2019 the disease being Covid 19 resulting in more than 24 million infections and sadly more and tragically more than 800,000 deaths worldwide. From a practical clinical standpoint we see a very distinctive pattern upon initial presentation manifested initially by cough dyspnea, fever and hypoxemia Covid19 may progress to a lethal potentially lethal cytokine storm, \deadly respiratory disease and multi-system failure. That is characterized by a striking pro-inflammatory response early in the disease we recognize that there are two phases one of which is the early manifestations of what seemed to be consistent with a viral infection but soon evolving particularly in more compromised patients. As we know the elderly very elderly patients with diabetes other comorbidities into a striking inflammatory process that cause multi-organ dysfunction so recognizing those two phases as we’ll discuss Selinexor or the concept of export and inhibition may offer a powerful therapeutic target.

Dr. Tom Walsh: So conceptually if we want to then target nuclear export as a novel mechanism to treat viral infections, considerable work had already been done as with with a number of the sine molecules particularly vertinexor, Selinexor and other agents as antiviral therapeutics in the last several years. Most investigational agents if we come back to Covid 19, however, inhibit either the viral cytopathic effect or the pro-inflammatory response and there’s been,  there have been many protocols that have been brought to bear usually against targeting one element or the other element. Selective inhibitors of mammalian nuclear export, ie sine such as cell and x-ray inverter, nexort, inhibit both the viral proliferation and block especially the nf kappa b mediated pro-inflammatory cytokine release.

Dr. Tom Walsh: In understanding the viral cytopathic effect in this early work that was done with Venezuelan equine encephalitis virus one can see looking at dappy stains and capsid stains and then cytoplasmic one stains one can then see the accumulation with the exporting inhibitors KPT 185 335 and 350. Using leptomycin as a control and dmso as controls you can see the accumulation of viral product within the nucleus but conceptually and if you look at the right the right panel in look into the nucleus and the aqueous blue and the little circle of molecules below to the left you have viral protein cargo. And look at the spectrum of the striking spectrum of viruses from hiv red protein influenza the nuclear capsid protein. From jc jc virus acnoprotein, human cytomegalovirus pp 66, hpv as well as kappa c sarcoma their own distinctive proteins so on the one hand there is through the export and inhibition the potential for blocking these partICUlar viral proteins and shutting down the viral cycle at the same time. to the right there are multiple molecules host molecules that are exported but very critically those involved in the nf kappa b driven inflammatory cascade.

Dr. Tom Walsh: So if we want to then focus for a moment just on the viral replication again over the last several years a number of investigators have done outstanding work in understanding the potential role for selective inhibitors of nuclear export. As we know these compounds attack the nuclear retention of leucine-rich nuclear export signaling proteins. They block the exploit of cargoes for a number of proteins including SARS, cov2 and with that disrupt the viral replication process. In the panel to the right this is a model for example for influenza where we see the nuclear capsid protein as well as viral ribonuclear proteins being inhibited by the export and co inhibition. So here in a series of experiments conducted by Dr. Tripp and his team at university of Georgia, we see in the mirroring model for influenza the h1n1 model.  The study looking at the role potentially in vivo not just in vitro but in vivo of altering the natural course of viral murine influenza. With that we see that this follows a pattern similar to what we might anticipate for some of the other viruses. Respiratory viruses such as rsv, adenovirus, but with a focus on influenza one can see that initially if one targets different points of delay day one two three and four progressively using plaque assay one can demonstrate in the lungs more than a long drop of a virion this translates very nicely then into improved survival, decreased pulmonary injury and decreased burden of influenza in vivo.

Dr. Tom Walsh: So in correlating that is there a way that we could then look potentially at at SARS CoV2 as a potential target? Well Dr. Zhao and colleagues have done elegant work in this in this area and several different studies. But in a functional proteomics study examined the potential for exporting one as one of the proteins with the highest functional connection to SARS co-v and in the little red circle you can see the central location for exporting for multiple viral proteins suggesting that blockade leads to inhibition of coronal viral assembly proteins and indeed earlier work in non, non-SARS CoV2 but SARS CoV2 demonstrated that protein b or six also led to significant reduction in antiviral activity notably also if you then look in separate experiments, one can see anti-inflammatory transcription factors such as i kappa b and ppr gamma are also affected.

Dr. Tom Walsh: So if we then ask the question well can we also demonstrate this effect in size copy 2 viral propagation going back to our bureau e6 cells if they’re pre-treated with cell and x-or SARS cov2? One can see either with treatment or with prophylaxis or with treatment significant reduction in the plaque assays with very tight replication and repeated experiments taken out over the course of as long as 96 hours these in vitro data certainly lay a, I think, a powerful foundation then for going forward experimentally and clinically.

Dr. Tom Walsh: So if we ask the question then well, given the potential antiviral effect what is the potential benefit of the anti-inflammatory effect? Again trying to exploit both mechanisms of both anti-viral and anti-an anti-inflammatory effect of Selinexor or potentially even other molecules of inhibition of exportin one. In looking at the pro-inflammatory response in patients suffering from Covid-19 the easiest way to understand this is to simply look at the pattern of inflammatory cytokines across the bottom il-2 il-4 il-10 interferon gamma tnf alpha and il-6, all of which show a striking and pro-inflammatory response. In healthy controls versus those that are Covid 19 with ICU or no ICU so the last two  rows or columns in each graph demonstrate the significant increase in il2 especially an interfering gamma driven process and il-10 and strikingly sulfur tnf alpha and il-6. so given this vast up-regulation in inflammatory cytokine storm especially associated with intensive care unit therein lies the potential for further intervention.

Dr. Tom Walsh: So is there a way that we can then understand that in an infectious process? And the answer is yes. If we look at again going back to the h1n1 model we can see within lungs following um four days post-infection and then treatment with burton xor in this situation in comparison say to oscetamivir,  there’s significant reduction of intrapulmonary interfering gamma tnf alpha and il-6. And similarly if we want to then take a step back and say well what is the effect on overall sepsis some of our poor patients with Covid 19 that we cared for in the intensive care unit had such striking pro-inflammatory response that an association with the multi-organ system failure there was almost a process reminiscent of that of sepsis so if we look back then at the at the inflammatory lps induced sepsis murray model with 7xor at 15 milligram per meter squared, we see significant reductions and the lps compared and compared to the lps  7x or treated animals significant reductions in il-6 tnf alpha as well as recruitment of intraperitoneal macrophages and neutrophils.

Dr. Tom Walsh: With that powerful pre-clinical background and as well as a safety profile with a substantially lower dose lower than that of the antonio plastic dose.  With 20 milligram tablet, treated on days one three and five we then embarked upon a prospective double bind randomized control study of phase two of hospitalized patients, with who were greater than or equal to 18 years of age. With that patients were randomized one-to-one either to Selinexor on the regimen. Outline for the potential not just of one week but if they were doing well for two weeks versus an oral placebo on the same regimen days one three and five and then potentially for up to two weeks. Our primary endpoints being the ttci which you see here which is a time to clinical improvement. That would include absence of fever 10 less than 38 and one of the following either reduced tachypnea reduced, improved oxygen saturation or hospital discharge. As exploratory secondary endpoints we also evaluated safety, all-cause mortality and several other addition markers both for inflammation and oxygenation.

Dr. Tom Walsh: So in summary Covid 19 may progress to a devastating side of cytokine storm lethal respiratory disease and multisystem failure. Most invested investigational agents against Covid 19 either inhibit the viral cytopathic effect or the pro-inflammatory response Selinexor selective inhibitor of mammalian nuclear export. Sine inhibits both the viral proliferation and blocks nf kappa b mediated pro-inflammatory cytokine release and as an oral agent with extensive pre-clinical rationale for anti-viral and anti-inflammatory activity sine compounds clearly warrant further investigation as potential treatments for Covid 19.

Tina Beamon: Thank you so much thank you Dr. Walsh. I’m sure you’re noticing the time. We’re scheduled to end this presentation at 230 however we have a lot of great content still to come. So if your schedule permits and you can stay on with us we invite you to do so, that being said if you have to go along we completely understand. This session is being recorded, so as with any good superhero movie we do not intend to leave you with a cliffhanger. So to the extent that would you like to access that recording that will be available for you as well. Our fourth speaker is professor Matthew Alexander with the university of Alabama at Birmingham. Professor Alexander will present research on xp01 inhibition in Duchenne muscular dystrophy or DMD.

Professor Matthew Alexander:  Thank you Tina and thank you Sharon for giving me the opportunity to present some of my work. Again, my name is Matt Alexander I’m an assistant professor in pediatric neurology and genetics at UAB in children’s Alabama. And simply put, my labs interested in developing novel therapeutics for Duchenne muscular dystrophy and other rare neuromuscular disorders.

Professor Matthew Alexander: For those of you not familiar with Duchenne muscular dystrophy, it’s x-linked disorder it’s caused by non-functional mutations in the dystrophin gene it results in a loss of myofiber membrane integrity and as a common theme you’ll see over and over again it results also in increased fibrosis and inflammation. Due to the lack of muscle myofiber membrane integrity there’s abnormal calcium regulation. Decreased contraction force dystrophin itself acts as a molecular shock absorber and as a result there’s also impaired glucose metabolism and overall impaired muscle function.

Professor Matthew Alexander: Again you know Caesar and Tom did such a great job I won’t beat a dead horse, but you can see a common theme emerging that if you if you can find ways to block inflammation block fibrosis in the case of DMD you’ll block the disease progression. You’ll keep the myofiber membrane intact to keep the animal or you know end goal the patient walking better because the muscle itself is overall more intact. And so one of the things we decided to do first was test out KPT 350 the sine inhibitor, sine compound, excuse me in zebrafish. Model of DMD and i won’t go into all that data but basically we identified an optimal dose and were able to show that the KPT 350 was able to correct locomotive function and overall viability of DMD zebrafish and so based on those initial studies we decided to expand into another model: the d2 mdx or DMD mouse model by giving it KPT 350 via oral food pellet.

Professor Matthew Alexander: And so we came up with a dosing regimen. I’m giving these oral food pellets these are peanut butter balls three times a week for eight weeks at five makes per gig body weight and we put these mice through a battery of tests. It’s known as the treat nmd or treat neuromuscular disease guidelines. So you can notice that he DMD mice overall have decreased myofiber size as well as this pattern of degeneration and regeneration the myofiber nuclei and so one of the hallmarks of degeneration regeneration is centrally located myonuclei. And that’s shown in panel b and so one of the things we did again through all these batteries tests is look at centralized myonuclei and we observed in the mice the DMD mice treated with KPT 350. We saw decreased levels of centralized mynuclei overall, increased myofiber size and overall decreased areas of myofiber degeneration and fibrosis as shown in the graph below.

Professor Matthew Alexander: And so again one of the things we wanted to look at was different biomarkers for inflammation and fibrosis and we partnered with another lab Dr. Armando Velata at UC Irvine. And so we looked at different populations. We know that in DMD there’s a lot of emerging evidence that there’s a pro-regenerative macrophage that competes with anti-regenerative macrophage population. And so you’re seeing on panel a t sneak plots showing the different populations and what we were able to observe was that when you gave the mice with DMD the KPT 350 they overall shifted from an anti-regenerative macrophage population into a pro-regenerative m1 like macrophage population and we saw also decreases in overall inflammatory cytokines as well. And things like, you know, things that tom mentioned il6, tnf alpha, and so these are drivers of inflammation, these are causing the myofiber membrane to break down in DMD and so this is very exciting to us.

Professor Matthew Alexander: Overall, if we can generate broad conclusions we were able to observe that if you give mice or zebrafish KPT350 or cyan inhibitors you can block my fiber membrane degeneration. And so again through different histological markers and biomarkers we are able to show that if you give these dmv mice uh KPT 350 or a side and heavier wheel ball, we’re able to overall improve locomotive and muscle function. You’re able to overall able to improve muscle force and decrease inflammation as well as promote this anti-inflammatory or pro-regenerative macrophage population in our DMD mouse model. So this work was recently published in molecular therapy in HighTower at all and 2020 and so with that I’d like to then pass it back to Tina.

Tina Beamon: Thank you professor Alexander. Finally, Dr. Clifton Mo of Dana Farber Cancer Institute will be speaking on the role of xp01 in cancer.

Dr. Clifton Mo: Thank you Tina and thanks everybody for listening. I’d just like to briefly touch upon the XPO1 story in oncology which has already been essentially a successful bench to bedside story with plenty of potential remaining for further advances.

Dr. Clifton Mo: So near and dear to my heart within the multiple myeloma space is where a lot of these seminal nuclear export work was done pre-clinically. And on the left here we have something that looks very similar to what you saw in CeSARS discussion but this is a study on the mm-1s of multiple myeloma cell line after exposure to KPT 185. And as you can see the p53 and icap b tumor suppressor proteins demonstrated significant nuclear localization very quickly within as early as two hours after exposure to KPT 185. With more downstream cell regulatory proteins such as p27 and fox03a demonstrating nuclear accumulation within hours afterwards. On the right we have another representation of the mechanism of action of xpo1 inhibition in the cancer cell which involves importantly both the restoration of functionality of multiple tumor suppressor proteins to include, but definitely not limited to, p53 as well as importantly the reduction in translation of multiple oncogenic mrnas which would ordinarily be translated in the cytoplasm and are now trapped in the nucleus to include cmic bcl2 surviving in others

Dr. Clifton Mo: So just another schematic of the function of xp01 in terms of shuttling proteins between the nucleus and cytoplasm. again just to reiterate what was presented in in the previous presentations in normal cells, xp01 is known to maintain homeostatic levels of these proteins and potentially oncogenic mrnas. However in the cancer cell across multiple cancer subtypes as I’ll show in just a second. The action ceases to become homeostatic and becomes frankly oncogenic, XPO1 plays a key role in multiple cancer-related processes to include cell cycle progression apoptosis as well as metastasis of primary solid tumors and drug resistance across multiple cancer subtypes. It is important to note that over expression of XPO1 has been demonstrated across multiple types of cancer both within my realm and hematologic malignancy as well as in multiple solid tumors. So, again within the myeloma space, in a mirror in a mirroring model there was a definitely a lot of promise demonstrated by exposure to sine compounds on the left both in terms of  mouse survival and importantly on the right in terms of decreasing bone damage. So at least within the multiple myeloma space, in addition to the cell regulatory and pro-apoptotic effects of sine compounds, there also seems to be a direct reduction of osteolytic bone damage most likely due to inhibition of osteoclastogenesis.

Dr. Clifton Mo: Importantly, within multiple cancer subtypes it is because it’s becoming evident that although the xp01 inhibition is very toxic to the malignant cell it does not appear toxic to normal hematopoietic cells. In the animal models the example demonstrated here is in the flip 3 internal den internal tandem duplication aml leukemia graft mouse model where you can see exposure to KPT 251 results in a logarithmic reduction in mean bioluminescence as well as a significant increase in mouse survival. And on the right you have a histologic representation of preservation of normal hematopoietic cells after exposure to KPT251. So again this appears to be much more toxic to cancer cells as we definitely want it to be as opposed to more toxic to normal important hematopoietic cells.

Dr. Clifton Mo:Now we are uh towards the right hand side of the bench to bedside story, this is from a seminal phase one study of xp01 inhibitor therapy in multiple myeloma and as you can see pre-treatment on top compared to post-treatment on bottom you see significant increase in nuclear localization and expression of the important p53 gene to the right of that you see a definite increase in the number of apoptotic cells as measured by the apoptag stain as well as multiple other important tumor suppressor protein levels that are expressed increasingly after exposure to xp01 inhibition.

Dr. Clifton Mo: Also very promising in terms of xp01 inhibition is its increasingly demonstrated ability to partner well with existing anti-cancer drugs within the realm of myeloma. It appears that the xp01 inhibition is the potency of it is enhances the potency of the proteasome inhibitor which is one of our staple drugs for multiple myeloma that has uh definitely helped to transform the treatment landscape for this disease. This is thought to be mediated primarily by an enhancement of an f kappa b inhibition likely due to the effects of on the I kappa b nuclear localization as demonstrated in the previous slide. So despite my urge to talk for the next hour about multiple myeloma I should mention that it is not just myeloma that xp01 inhibition is relevant, across multiple blood cancers as well as solid tumors. xp01 overexpression has been demonstrated and importantly has been associated with poorer survival. So again compared to normal non-cancerous cells, cancer cells have greater XPO1 expression but even within a cancer itself a higher xp01 expression seems to be a definite negative prognostic indicator and deleterious to the patient compared to low xp01 expression.

Dr. Clifton Mo: Fortunately in multiple pre-clinical models, sine compounds have demonstrated robust and anti-cancer activity across all of these listed blood cancers and solid tumors. So you know in the oncology space where sometimes there appears to be somewhat of a disconnect between hematologic malignancy and solid tumor oncology this appears to be one target that is shared across multiple tumor types, both solid and liquid. That’s hopefully less than 10 minutes run through xp01 inhibition in cancer today thank you so much.

Tina Beamon: That was fantastic thank you so much Dr. Mo and thank you to all of the panelists for your insights on the role of xp01 as the mechanism in a variety of disease states. This was a very very informative afternoon together. So with that I’m going to turn it back over to Dr. Shacham.

Sharon Shacham: So thank you again for all the panelists and for all our guests today for staying for our webinar that actually was will be just around 75-minute webinar. Something for us to learn for our next KaryoTalk. I want to just summarize again with our cartoon looking at the XPO1 train that has many cargos outside of the nucleus to the side to the cytoplasm which results in the loss of key important superheroes that’s supposed to be in the nucleus and keep an eye on the DNA and make sure that the cell is either not grow as a cancer cell or not fall apart in a neurodegenerative disease and to prevent this out of control train we have the sine compounds here that can stop the train and let the guardians of the genome do their job.

Sharon Shacham: So I hope you were able to explain a little bit about the the communication between any transport between the nucleus and the cytoplasm and about the role of the nuclear pore complex and XPO1 in the different diseases. So thank you very much everybody and have a good afternoon.

Tina Beamon: Thank you and one parting note: we are excited to share that there will be future webinars! So please be sure to follow us on social media and to check back on our website to find out about future events like this one. Thank you!