New insights into the myeloproliferative neoplasms

good morning everyone good morning welcome back to medicine Grand Rounds today we have dr. ku Shanthi dr. Fouch and Steve completed his Bachelor of Science and medical degree at the University of California Los Angeles then traveled northward because internships presidency chief residency and fellowship in the division of hematology all here at the University of Washington after spending some quality time with us here at UW including serving as Section head of the division of hematology dr. Koo shamsky finally found his way to Stony Brook in New York where he’s now senior vice president for Health Sciences and Dean of the School of Medicine his awards are numerous and include president of the American Society of Hematology past present president of the American Society for Clinical Investigation elected member of the National Academy of Sciences Institute of Medicine and fellow of the American College of Physicians he served as editor-in-chief of the journal blood editor for Williams hematology and is on the editorial board for the hematologist his publications are equally impressive in numerous with well over 350 titles bibliography that expands 35 pages or more and is quite long and it length his mentorship is probably what brought most of you here today and he is known for a strong work with many faculty who continue here at the University please join me in welcoming here thank you so much it really is a great pleasure to be here today I was a little overconfident I thought I could find my way from the parking lot to tee 625 just like it was yesterday but then I realized it was over 10 years since I was a faculty member here but it is great to be here seeing old familiar friendly faces to see that this room has received a new paintjob since I’ve been here today I’d like to share with you some new insights into a group of disorders the myeloproliferative neoplasms and my sort of subtitle is it’s beginning to make some sense these are diseases that have been haunting medicine for many many many years without really understand full understanding let me disclose that I have no real conflicts fortunately I get to disclose that I still have a research grant for the National Institutes of Health and I sit on a few scientific advisory board like NIDDK council and let me start with a patient and this is a patient that I saw when I was here on the faculty at the University of Washington patient is a fort was a 44 year old female who was in excellent health except she had a few minor brushes with alcohol and she began noticing episodes of intermittent diffuse abdominal pain following the third sets episode she came here to our emergency room her abdominal exam was markable for ascites and modest splenomegaly but without other stigmata of chronic liver disease being a hematologist of course the most important thing was her blood count her Ematic rate was 35 her white count was normal and her platelet count was mildly elevated at 650,000 she had an abdominal ultrasound and then an abdominal CT scan which was consistent with hepatic vein thrombosis and it’s acute sequelae because of the hepatic vein thrombosis because of the budget Kyary syndrome we embarked on a hypercoagulable workup and it revealed a single allele for factor v leiden d she was treated with heparin for her hepatic vein thrombosis and she was then converted to therapeutic levels of coumadin anticoagulation and she was discharged and I followed her in clinic six months later despite continuously therapeutic INR values she was readmitted with a recurrence of her bud tre syndrome and she was treated with heparin and despite ongoing therapeutic levels of anticoagulation the Claude extended during her hospitalization and we’ve decided that this high platelet count was probably a manifestation of essential thrombocythemia and that was now contributing to her coagulopathy and because of that she was then treated in addition with hydroxyurea to treat her essential thrombocythemia she was discharged home only to experience

significant bleeding complications and despite normal platelet counts she had a very very stormy course and she ultimately passed away from recurring bud curie syndrome so by bukhari syndrome is an interesting hypercoagulable state it is associated with a number of disorders malignancy particularly hepatocellular carcinoma a number of infections and benign lesions of the liver mostly with hypercoagulable states in this list of hypercoagulable states is just a partial list of the types of disorders that can present with like tre syndrome paroxysmal nocturnal hemoglobinuria is another condition but mostly now we are beginning to see the myeloproliferative neoplasms particularly polycythemia vera and essential thrombocythemia are associated as well with by GRE syndrome and that’s what i want to talk about today so the myeloproliferative neoplasms are a group of seven or eight disorders that are defined by the World Health Organization the most common of which are polycythemia vera and essential thrombocythemia idiopathic myelofibrosis chronic myelogenous leukemia and some lesser-known conditions chronic neutrophilic leukemia hyper example syndrome and systemic mast cell disorder their chronic bone marrow disorders characterized by unregulated growth of one or more blood cell lineages which may extend to extra Maziar sites such as the spleen arise in a single Multi lineage hematopoietic stem or progenitor cell and symptoms and signs of the disorder are a manifestation of excess blood cells or their dysfunction now this is a more Technicolor version of the myeloproliferative neoplasms and the reason I have them color-coded like this is that up until about six or seven years ago we understood the molecular lesions that are responsible for those that are in color in blue chronic myelogenous leukemia of course is the Philadelphia chromosome the 9:22 translocation that gives rise to this aqua gene p210 BCR Abel hyper is an a fillip syndrome some people would call it acute is in a pillock leukemias associated with the deletion of chromosome 4 which activates the platelet derived growth factor receptor receptor tyrosine kinase systemic mast cell disorder is another mutation of a gene on chromosome 4 the c-kit receptor which activates another receptor tyrosine kinase and then chronic neutrophilic leukemias associated also a translocation of chromosomes 9 and 22 but a slightly different translocation causing a p230 PCR able now each of these molecular lesions are mutations in cases and what I’m going to try to advance for you is the notion that these are diseases of cellular signaling that signals in cells are have gone awry because of these mutations and what is the evidence that polycythemia vera essential thrombocythemia the more common myeloproliferative neoplasms are in fact diseases of signaling well there’s about a dozen different ways you can make a mouse have a myeloproliferative neoplasm and all of them involve disordered cellular signaling if you enhancer with report–and receptor signaling or lose a phosphatase that blunts signaling you unregulated of the thrombopoietin receptor or mutate the thrombopoietin yourself if you express a tell Jack to fusion gene if you lose another phosphatase ship one a number of mutations of mice lead to myeloproliferative neoplasms there are some lessons from humans that we can learn also there’s a rare condition called familial essential thrombocythemia this is not a Public Health Menace but it’s a proof of principle on the left is the peripheral smear of a patient that we saw with familial essential thrombocythemia on the right the bone marrow full of megakaryocytes this is the pedigree of the family that we evaluated when I was here at the University of Washington the index case was a thirty year old woman who presented with two blood clots during her two pregnancies and she was found to have a platelet count of seven hundred and thirty-eight thousand when she went to her physician to try to figure out why she was having all those blood clots he worked her up and said oh

you’ve got a disorder called a essential thrombocythemia it’s a very uncommon disease you probably haven’t heard about it and she said oh I know all about essential thrombocythemia my mom has essential Zombo psyche mia and the mom in fact at age 48 had T IAS and had a stroke at age 50 and was on treatment for essential thrombocythemia and so we evaluated the family we evaluated the two children a six-year-old son and a four-year-old daughter they two had 700 and 800 thousand platelets and we evaluated this family she turned out to have very high thrombopoietin levels the primary regulator of platelet production a mutation was found in a splice donor site that led to aberrant splicing of the thrombopoietin gene leading to over production of thrombopoietin so again a disease of signaling of familial disease of platelet production which is in fact a disease of signaling there are mutations of the thrombopoietin receptor in people this is an example of one that was described almost 10 years ago where you had a family with essential thrombocythemia when sequencing was done a single amino acid change of serine 505 in the thrombopoietin receptor 2 asparagine 505 virtually everybody in the pedigree who had a spare gene 505 had a platelet count of a million and if you had the wild-type serine 505 you had a normal platelet count in this family so again thrombopoietin thrombopoietin receptor mutations that cause essential thrombocythemia but these are unusual conditions what about sort of more common the acquired myeloproliferative neoplasms a tremendous insight occurred about 40 years ago tell by name of Jeff Kirk all noticed that bone marrow cells from patients with polycythemia vera that bone marrow cells even in the absence of erythropoietin could grow these big juicy red blood cell colonies and they were called endogenous erythroid colonies and for about 3035 years that observation caused a lot of people to sort of scratch their head it actually became a diagnostic test if you will not a very elegant diagnostic test but a diagnostic test nevertheless for polycythemia vera as the years progressed a number of people noticed that it wasn’t just in polycythemia vera and it wasn’t just erythropoietin that that these cells were hypersensitive to that patients with essential thrombocythemia or primary myelofibrosis were hypersensitive to a whole host of growth factors erythropoietin stem cell factor gm-csf thrombopoietin ILO 3 there seemed to me something about these myeloproliferative neoplasm where the cells were hypersensitive to growth factors so let me tell you a little bit about how growth factors work as a way to introduce the concept of abnormal growth factor signaling so receptors like the insulin receptor and the c-kit receptor are well-known and they’re well known to trigger their biological effects by activating an endogenous kindness the insulin receptor has a kinase in its cytoplasmic node domain the c-kit receptor has a kinase in its cytoplasmic domain but cytokine receptors like the erythropoietin receptor that thrombopoietin receptor the g-csf receptor the growth hormone receptor they don’t have any kinase in their cytoplasmic domain like these others and so the question is where where’s the kinase in 1989 an aussie by the name of Andrew Wilkes identified some new kinase is based on homology sequencing he used consensus sequence from a whole myriad of tiny seeds that were known at the time and pulled out to novel kinase ha’s which he termed jak kinase –is and he has a dry sense of humor and it was called just another kinase however just another kinase didn’t seem to fit the name of this kinase because there are now well over 5000 publications obviously it’s more than just another kinase so it was given a more dramatic name Janus kinases the Roman god of gates and passages and the reason it’s called Janus kinase is

because we can keep the acronym Jack jaeik a Jeff Janus kinase but I still know it is just another kinase now how to grow factors and jak kinase has work so the first is to give you the anatomy so on the left I’ll show you a dime Eric cytokine receptor with its tethered jak 2 so when a growth factor like erythropoietin or bramble protein shows up the first thing that happens is a conformational change occurs which allows these two jak kinase is to cross +4 each other that then begins a cascade where a tyrosine in the cytoplasmic domain of the receptor is phosphorylated that allows the kinase to start phosphorylating a number of other signaling molecules such as gabs and and a phosphatase called SHP to an enzyme called pi3 kinase phosphoenol solitary kinase and to latent transcription factors thought stat3 and stat5 once these are phosphorylated they translocate to the nucleus and influence nuclear gene transcription such as the anti-apoptotic g v CL x or the probe proliferative gene cyclin d now there’s a whole nother cascade of genes that become activated by this initiating phosphorylation event here is a molecule called a KT this is a kinase that gets activated by pi3 kinase these molecules activate Rask which leads to the activation of map kinases and all of these secondary signaling molecules now impact on the cell nucleus to drive cell survival cell proliferation and cell differentiation now back to myeloproliferative neoplasms over the years between the observation of these endogenous erythroid colonies and now people have began noticing that a number of these signaling molecules were activated spontaneously in the cells of patients with polycythemia vera or ET in fact four of the molecules were consistently found to be activated suggesting again in acquired myeloproliferative neoplasms suggesting that there was something wrong about signaling that there was an abnormal kinase somewhere that was causing all of this aberrant signaling and in 2005 four groups almost simultaneously found what that kinase was and it Jack – that an acquired mutation had occurred in Jack – that had a number of ramifications virtually every patient with polycythemia vera has a mutation in jack – that’s activating v6 one 7f mutation it forms a spectacular diagnostic test now for patients with polycythemia vera half of patients with a central Rhambo site emia or idiopathic myelofibrosis have the same activating mutation in Jack – the mutation is a valine – 6 valine 617 – phenylalanine this is a valine that is highly conserved between different Jack twos but surprisingly everyone expected it to be in the kinase domain but it’s actually in a domain that is a regulatory domain and the regulation of this kinase is now messed up the proof that Newton jak 2 could cause myeloproliferative neoplasms was shown in the same time when the mutant kinase was put into Mouse stem cells and then the stem cells transplanted into a mouse the mice within a month or two developed what looked like polycythemia vera there haematic rates climb to 60 to 70 to 75 and so mutant jack 2 can drive a myeloproliferative neoplasm in a mouse now one of the perplexing questions and we still don’t have the complete answer is how does this one mutation jack v6 1 7f caused three different diseases polycythemia vera essential thrombocythemia and myelofibrosis so there are several possibilities one is that either prior or subsequent mutations in these cells sort of

speciate the myeloproliferative neoplasm are there other mute Asians that dictate whether a patient has polycythemia or essential thrombocythemia maybe we’re genetically hardwired for one type of a myeloproliferative neoplasm or another maybe i’m an ET kind of person and when I get utin jak2 hopefully I won’t but if I were to get mutant jak2 I would develop you teeth but value over there being an erythroid kind of person salya would get polycythemia vera with the very same mutation there is actually some evidence and I’ll walk you through that evidence maybe all stem cells are not created equally and a stem cell that is sort of primed to become an erythroid stem cell gets a mutation you get polycythemia vera or a stem cell that’s primed to become a megakaryocytes gets Jack v6 one 7f and you get et or maybe it’s the dose of Jack two that are present in your cells more Jack two you get P vera less mutant Jack – you get V T so there’s a number of possibilities and probably others as well as these so what’s the evidence that that there are other mutations that are driving the disease and may be predisposed to polycythemia vera or ET so this is a comparison of how many cells that you have that are clonal belonging to a single lineage and how many of the cells have mutant Jack – the vast majority of people with ET or polycythemia vera have roughly the same number of clonal cells as mutant Jack – containing cells but there are some patients with P vera or ET that have more clonal cells than Jack – baring cells suggesting that whatever led to clone allottee pre-existed before those cells got mutant Jack to and whatever those mutations are might be telling the cell become a rich boy or becoming negative City and here is now a list of other mutations that have been identified in patients with polycythemia vera or ET that can pre-exists predate the acquiring of the mutant jak 2 and so there’s some evidence to suggest that these other mutations might be guiding the cells to erythroid or to here’s the evidence to suggest that we are pre-wired somehow to become poly say female or have et when we develop mutant jak 2 it’s a very clever study that was published in the Journal of cancer cell about two years ago when you saw those beautiful erythroid well beautiful me erythroid colonies on that slide and you look at the bone marrow colonies in a patient from Povera not all of those colonies are from mutant cells some of them are normal erythroid colonies and so what this group did was they plucked out individual erythroid colonies and by pcr they could tell whether the u.s. boy colony came from the mutant clone the jak2 mutant jack to clone or normal so now stem cells and they could separate them and they could do a raise on them and they could do you know sido chemistry on the cells and when they did the array they noticed that it was clear distinction in the genes that were being expressed in the colonies from patients with polycythemia vera versus the colonies from patients with ET and what they found was a whole group of genes that were over expressed in colonies from patients with essential thrombocythemia when they looked at what some of those genes are they found a number of interferon response genes that were over expressed in patients with ET but not so much in patients with polycythemia and one of them in particular caught their attention stat one one of those secondary signaling molecules that drives proliferation in patients with in response to erythropoietin and from a potent but is also mutant the stats are mutant in patients with policy theater and ET and as you can see in this slide there is a lot more cells that have

activated stat one in patients with ET than in patients with polycythemia vera but here’s the really interesting observation these are the mutant colonies these are the colonies derived from the jak ii bearing cells here is the result from the normal colonies the colonies that don’t have new jak 2 patients who have ET have a lot more stat 1 in their normal colonies than patients with pv have in their normal colonies suggesting that we’re somehow pre-wired if we are a high expresser of stat 1 for whatever reason we’re going to get ET when we get Jack to mutation but if we’re a low expressor of stat one will get polycythemia vera and the proof of the pudding here was that when they down regulated stat 1 in the bone marrow cells of patients with ET they got a lot more red cells suggesting that if those patients just had left at one at the time they developed their disease they would have had Peter what about the dose of Jack – what about how much mutant jack – you have in yourselves can they tell you whether you’re gonna develop polycythemia vera or ET this is an observation that was made a number of years ago that the mean level of mutant jak 2 in the cells of patients is about to fold higher in patients with polycythemia vera than in patients with ET so first of all how does that work we all have them all these patients have the mutation how come some have more – than others this is a very interesting genetic phenomenon that has been known so so take take if you will a patient who is normal so hopefully this is my bone now I have normal – normal Jack – alleles in my bone marrow a mutation happens a G – T nucleotide change occurs and that causes me to now have a single allele of mutant Jack – and now because those cells have a proliferative advantage because they have a chronically turned on kinase they come to overtake the bone marrow and so now instead of 0% then 20% and 30% then 40% of the marrow is now becoming overrun by these cells that have a single mutant Jack – allele now a second event happens and this is a crossover between the two chromosome 9 and this is called you need parental die so me a term that I have hard time saying but what happens is there’s an unequal crossover and you end up with one daughter cell that has two mutant alleles and one daughter cell that has no huge alleles now you might imagine this cell with two you and Jack two alleles will grow even better than the cells that have only one mutant allele and that in fact happens and now the bone now gets completely overrun by cells that have two mutant alleles so this is a way that you could actually double your dose of mutant Jack – so is that why some patients have pea vera and some patients have u T so a friend radix Koda wanted to approach this question experimentally and he created a really clever trans gene what he calls a flip-flop gene so he put in the mutant Jack – allele you into a transgene but he put it in backwards and he surrounded it by these Creary combination sites now if you if you have a cell that has a career combinate mutant jack to flips around and starts expressing mutant Jack – and so he called these mice flip-flop mics has nothing to do with politics has nothing to do with changing your opinions on anything it’s just that it has a gene that you can flip around and turn on mutant Jack – in the cell you want to turn it on in and so when he did that with two different query combin a strange he did it with a strain called Valkyrie and he did it with MX creat he got about a two-fold difference in the level of mutant Jack – that was expressed in the bone marrow cells of the two different strains of mice so very much resembling what we see in

patients with P vera versus bt and when he did that the valve cream ice had a relatively normal hemoglobin and had a high platelet count essential thrombocythemia the MX Creed that were expressing two times more mutant Jack tube had high hemoglobins and relatively normal platelet counts ie polycythemia vera so Radek believes that it’s the level of mutant Jack – in your cells that can drive whether you have Guevara or Beatty the aspect of myeloproliferative neoplasms I want to finish with is the other part Milo proliferation is more than myeloproliferative neoplasms are more than just my love proliferation and this is a compilation of some of the coagulation abnormalities that are found in patients with Milo polluted neoplasms patients with et have a substantial risk of major venous and arterial thrombosis and they also have a yes sir but real risk of bleeding patients with polycythemia vera 30 40 percent risk of major thrombosis during their lifetime and a smaller risk but a nonzero risk of pathologic bleeding as well so what is the origin of the clotting and the bleeding that are present in our patients with polycythemia vera and ET so we wanted to use that experimental flip-flop Mouse to begin to address this we had the notion that if we could just express polish just Express the mutant jak2 in platelets we would get et and we would get clotting or maybe if we just expressed it in white cells or maybe when we just expressed it in endothelial cells if we could pick and choose in what cells we express me jack – maybe we get a clotting phenotype or maybe we’d get a bleeding phenotype and we’d be able to study it so what we did was we Radek sent us his his mice his flip-flop mice and we began acquiring various mice with different Creed’s titoo is a gene that is expressed in all hematopoietic cells and in endothelial cells so if you cross tie to with flip flop you get a mouse which expresses mutant Jack – in he man up all hematopoietic cells and in endothelial cells pf4 is a gene that is expressed only in megakaryocytes in platelets so if you cross pf4 Kree with flip-flop you get a mouse that is expressing mutant Jack – only in megakaryocytes and platelets why Sam you get only leukocytes now if you get clever and do a few bone marrow transplants if you transplant normal bone marrow into that tie to creep flip flop Mouse you can express mutant Jack – only in endothelial cells and if you do the converse experiment you can express mutant jack to only in Matawan excels so now we have these five different strains of mice and we can begin to evaluate them did we get a myeloproliferative disease did we get clotting did we get bleeding so the first one I’ll tell you about is the tie to flip-flop Mouse and basically we get a rip-roaring myeloproliferative neoplasm in these mice you can see that the platelet counts are high the neutrophil counts are high the red count isn’t so much when we look at the bone marrow we see that the bone marrow colony numbers of myeloid progenitors and erythroid progenitors and multi lineage progenitors are all elevated the megakaryocytes progenitors are all elevated when you look at the bone marrow and in the spleen you have a rip-roaring myeloproliferative neoplasm you can see the spleen is quite large in these mice compared to the controls so the tie to flip-flop mice represent a very aggressive myeloproliferative neoplasm when we looked at how much you jack 2 is being expressed we found not so much actually so this is the amount of the mutant Jack 2 compared to the mouse the mouse is only jak 2 you can see that in neutrophils about 30% of the jak 2 is mutant jak 2 in endothelial cell is about 20% and in megakaryocytes only about 10% of the jak 2 is mutant

jak 2 so it actually under represents what you might see in a patient in the PF for flip flop mice these in the my citrox we thought we hoped we believe is only expressing mutant jak 2 and megakaryocytes in fact that’s the case there’s a hint in neutrophils of nothing in endothelial cells and there’s this 10% level of expression in the megakaryocytes so how are we going to evaluate these mites we have very sophisticated tests in hematology to evaluate clotting and bleeding you basically cut off into the tail and you see how long it takes to stop bleeding that’s the sophisticated test so this is a tail bleeding assay you have to be very careful in this test for a while there you know the animal handlers were genotyping our mice for us and of course the most common way to genotype the mouse is to take a little piece of the tail and you know expect the DNA and figure out what the genotype of the mouse was but now if you do a bleeding time on that Mouse you’re cutting farther up the tail and they don’t stop bleeding so we had to work that detail out but the normal is about two and a half three minutes to get the tail to stop bleeding as I said very sophisticated actually it’s reasonably reproducible there are other ways to evaluate clotting and bleeding this is a pretty standard model you you dissect out a mouse carotid artery and you take a little pledget soaked in ferric chloride and lay it on one side of the carotid artery now you imagine that the endothelial cells don’t much like being exposed to 10% ferric chloride and the endothelial cells now slough off doesn’t take long about 30 40 50 seconds till the endothelial cells sort of roll up and go away and die now you begin to see platelet adhesion to the underlying sub endothelial tissue you see small rhombi forming after four or five six minutes and after about 15 minutes you create an occlusive clot and the carotid artery closed off and you can follow this with with a flow detector and this is what the data looks like you apply the ferric chloride to the carotid artery and then after about 15 minutes the flow plummets because the clot has extended now across the entire blood vessel so those were the two tools that we would use the tail bleeding time and the carotid artery thrombosis model to evaluate clotting we were fully expecting that we would get a mouse that would clot just like that that as soon as you take ferric chloride and put it on the carotid artery tie to creamed out high to flip-flop Mouse you know you would have a pretty severe robotic phenotype so what did we find well these mice actually breed they have a long bleeding time the tail bleeding time was tripled in these mice they stopped but it took about seven minutes for them to stop bleeding carotid artery occlusion here’s the wild-type there that 15-minute ceasing a blood flow tied to flip-flop mice never aquatic they never occluded flow just completely just kept flowing and flowing and flowing and we were quite surprised so I told Ian Ian Hitchcock the faculty member who I work with oh you must have messed up you must have switched the genotypes or something go do it again and of course he did it again we got the exact same result we now I think we’ve done this like a dozen time and they just don’t clot do they have one Willebrand’s disease and I’m actually working with Jose Lopez on this they’re fun Willebrand factor levels are normal but I don’t know whether the type of von willebrand factor they have is is normal but at least the levels aren’t so well what about those platelet factor for mice where we’ve messed up the platelets with mutant Jack – they have absolutely normal clotting no problems at all and so this was a big blow and I was reminded of a saying there is nothing so tragic and science and it’s laying of a beautiful hypothesis by an ugly fact so we had to really rethink what we were looking at but we wanted to know what is the cell responsible because remember the tide to flip-flop mice it could be the platelets probably not because the pf4 mice were fine could it be the white cells could it be the endothelial cells so we had to

invoke our other mice and so when we looked at stem cell only so these are the mice that we take tie to flip-flop bone and transplant into normal mice so now these mice will have normal endothelial cells but will have stem cells do they bleed do they not clot their v absolutely normal it is not the stem cells that are the culprit in the bleeding phenotype that we found when we did the other experiment to isolate the new jak 2 to just the endothelial cells they have a really lousy clock they develop the clot but it breaks and it breaks loose and so the endothelial mutant jak 2 is why these mice have a bleeding phenotype and you might ask well I told you early on that this is a disease of the bone marrow stem cell what does the endothelium have to do with it well there’s a strong belief that there is a cell of origin of both endothelial cells and stem cells some people call it the hemangioblastoma know what it’s called but there is a common lineage and it’s probable that that’s where the mutation occurs Ron Hoffman at Mount Sinai in New York has shown that in patients with polycythemia vera their endothelial cells have mutant jak 2 and so it’s now beginning to come together and making some sense so what is the endothelial cell doing differently when it has mutant jak 2 in it then if it is normal jak 2 min and so this is my cartoon depiction of a blood vessel with endothelial cells and red cells in white cells and platelets there on the endothelium is vitally important in coagulation we all we all know that there are a number of molecules that the endothelium secretes or has on its cell surface that prevent thrombosis may be mutant jak 2 is up regulating some of these molecules and are preventing these clots there are a number of molecules that endothelial cells have or can secrete that are procoagulant von Willebrand factor tissue factor P selected may be these molecules are being down modulated or altered in some way that makes for this bleeding phenotype that probably translates patients we’ve been looking at these clots now in a careful way we can actually actually sacrifice the mouse we can look at the clot we’ve been section the carotid artery is there no clot is there a partial clock what exactly is going on and when you look at the nor the tie to creep lip loudmouths they have a clot that goes wall to wall it is just pure clot but in fact in the in the in the tied to flip flop mice the clot is only partial where the where the ferric chloride was you see platelet accumulation but it just doesn’t extend and we’re using this as an observation to help us sort out why these mice fun the final thing I want to tell you about is how our understanding of mutant jak 2 has affected treatment of patients with polycythemia vera I have done this I didn’t I didn’t look like this when I did it but I performed phlebotomy on patients with polycythemia vera and this is a long-ago treatment hopefully it’s more scientifically administer now than in the time that this would cut was made but as of just three or four or five years ago there were a whole host of drugs we used to treat patients with poly taking mia vera leaf lobotomized some patients we try not to give people radioactivity but that was a common we try not to give patients these alkylating agents that cause cancer but we do in fact give patients interferon we give them hydroxyurea and some patients get in agra like all to try to control the Milo proliferation with the discovery that there is a mutant jak 2 kinase in these cells obviously why don’t we inhibit the jak 2 now it’s a little trickier of course the poster child for targeted drug therapy is Gleevec I’m sick Gleevec and a CML chronic myelogenous leukemia the able kinase is driving CML we know that and if you inhibit able kinase you can really control chronic myelogenous leukemia if you’re not able out of a mouse nothing bad happens if you knock jak2 out of a mouse the mouse makes it through about embryonic day 9 and then that’s it

jak 2 is much more vital to the survival of a mouse than Abel is and therefore you can completely obliterate Abel if you want with with Woodley back and nothing bad happens if you treat patients with jak2 inhibitors to aggressively their blood cells on it so it may not be quite as effective but let’s try it and a whole host of mutant jack of jak2 inhibitors were proposed for clinical trials and I’ll just tell you about the results of one of them which has made it – which has made it to clinical trials first this is a paper that appeared in the New England Journal last year a double-blind placebo-controlled trial ruxolitinib this is a jak2 inhibitor it’s actually also inhibits jak 1 for patients with idiopathic or primary myelofibrosis does it work well the answer is it works reasonably well these are patients who were treated with a drug and this is the change in their spleen so of course these patients all had splenomegaly and when you treat virtually everybody except these two patients I don’t know what happened to them maybe they didn’t take their drug but virtually every patient had some level of splenic reduction in size compared to the placebo where there was just a very small number of patients who had their spleen reduced and in most patients the spleen continued to get bigger what about symptoms these patients usually have waisting symptoms they feel lousy all the time and the patient on the drug half of them had a significant reduction in their total symptomatology so the drug seems to be working their spleens are getting smaller they’re feeling better in fact they’re feeling better really quickly they’re feeling better long before their spleen gets smaller they’re feeling better long before any change in their blood counts occur and it’s probably working through a different mechanism and in fact most people now believe that it’s probably working at least in part because it’s inhibiting jak1 and all the inflammatory cytokines that are circulating in these patients that’s why this pls getting smaller and that’s why they’re feeling better is because it’s an anti-inflammatory drug but nevertheless it is reducing spleen size it’s having an effect on the my local operations and the outcome of a follow-up study published by the same author a couple months later suggests that the survival there is a survival advantage for this drug in patients with mild fibrosis and if you had very severe aggressive myelofibrosis you had even a greater survival difference if you were on the drug then on placebo so we are beginning to make some insights into the origins of the myeloproliferative neoplasms and we’re beginning now to take those insights and turn them into a drug drugs that can actually begin to impact this disorder so what do we know so far we know that v6 1 7f jak 2 occurs in virtually all patients with polycythemia vera and half of the patients with EP and myelofibrosis but not in other more atypical myeloproliferative disorders the mutation is at least contributory if not causative for the myeloproliferative proliferation and this is a great diagnostic test for myeloproliferative neoplasms and it also may be of some prognostic value we know that additional mutations are occurring in patients with myeloma liftin neoplasms and it probably account for some of the clonality we’re seeing and some of the variable phenotype of the disease we now have a drug we have jak2 inhibitors that are beginning to be used in these patients but i would argue that it would be far more effective if we could find a drug that only treats the mutant form of the jak ii and not all jak 2 because we are going to get into trouble with / / inhibition of wild-type jak 2 and all of the kinase inhibitors that are in clinical trials now inhibit mutant jak2 the same as they inhibit wild-type Tector but there’s still several questions which is good for people who want to write grants and understand this disease ah we still don’t quite know why one mutation gives rise to three disorders what is the role of cytokines

in the symptoms and origins of the myelin neoplasms is this unique parental die so me is this homozygous expression of the mutant kinase detrimental there’s some evidence to suggest that those patients have more complications more bleeding and clotting than do patients who will have only one used allele about a third of all patients with polycythemia vera have two mutant alleles and two-thirds at one either one of the other genetic contributions to acquired myeloproliferative neoplasm and are they targeted or can we go after these other mutations to add to our kinase inhibition strategy and is the thrombotic and bleeding diathesis of patients with these disorders is it due to a people like signaling so I’ll acknowledge former students and fellows several of them are here at the University of Washington some in San Diego when I was chairman there and now some in our current laboratory at Stony Brook some longtime collaborators this one is particularly long-term collaborator on lots of things and I’ll end there and entertain any questions hey Ken does a super super talk so what’s your level of optimism that people will develop mutation specific jak inhibitor just because it’s like it’s it’s that problem that the that the mutation is actually a loss-of-function mutation you’re inhibiting the ability of an inhibitory domain value what it’s supposed to do so so what’s it’s kind of like K wrasse in that regard so how do you you know what’s your level of optimism that you’ll be able to somehow take this mutant protein and make it work better than it does I don’t have a lot of optimism mostly because I don’t know a single drug company that’s even trying this for the many of the reasons that you mentioned there we understand the molecular structure of the activation loop of jak 2 and where this mutation sits so that at least we sort of know the protein-protein surfaces that are being disrupted by this mutation but as you know it’s a lot easier to inhibit an ATP binding site of an enzyme by putting an inhibitor in the ATP binding side kick and ATP out then to disrupt a protein protein interaction which is what you have to change to change a mutant jak 2 into a normally functioning active so I don’t think that anybody has a whole lot of optimism that we’re going to be able to do it with massive you know screening and things like that maybe someone will get really lucky but unclear observation yep so it’s a good good question so the observation is that patients with an elevated white count if that’s the better predictor of who’s going to clot in patients with mild proliferative neoplasms then the platelet counter or other things and that’s probably a it’s been repeated over and again so we wanted to address that that’s why we have that lyse m3 flip flop mouse is to put the mutant Jack – just into the white cells and see if we got a clotting phenotype and we did not so that Mouse didn’t believe didn’t clot just sort of sat there and cost us money it had a modestly elevated white count and of course our tide to flip flop mice have a very high white count so I was hopeful but it another beautiful hypothesis lame um we do know though that hydroxyurea and things like that will drop the white count in patients with myeloma neoplasms and we do know that there clotting propensity goes down modestly but whether it’s actually because the white count goes down or not I’m not sure let’s say I’m very nice time so I just have a quick question about what is the receptor or receptors that is driving gack to activation and endothelial cells so a good question we know that in hematopoietic cells that it’s probably the thrombopoietin receptor on to which the mutant jak 2 is binding now we know that Jack T mutant

jak 2 will bind to almost any cytokine receptor Ipoh receptor g-csf receptor growth hormone receptor but in stem cells none of those receptors except the thrombopoietin receptor exist so in hematopoietic cells it’s the t po receptor that the mutant jak 2 is glomming on to in doing its thing endothelial cells also Express trauma protein receptors and so I haven’t proven that that’s where it’s binding but I do know the receptors there the mutant jak 2 is there so it’s not a huge lead think that that might be at least one of the places where mutant jak2 is binding of course once it’s tethered to the membrane and it’s chronically active whether the substrates are the same substrate or not we don’t know we’ve not started to look at signaling and endothelial cells and where it leads right now as you know we’re going from the other end what is the abnormality in the endothelial cells and then trace backwards for the signaling pathway but my favorite candidate of course is from a poisonous Dahlia right so that’s a good question we worried about that which is why we did the the trench the mouse where we just had it in him at a poetic stem stem cells and platelets and white cells and red cells maybe not so much but that Mouse did not have any phenotype it had a perfectly normal bleeding time it had a perfectly normal it had a high blood count it had a you know platelet count of 2 3 4 million that’s true we we do know the really high platelet counts at least some of those patients bleed because they develop acquired von Willebrand’s disease that they suck up all the von Willebrand is a factor and and so they they could lead for that reason or severe dysfunction of the platelets there have been some reports of that as well but but I wanted it to be the platelet believe me I wanted to be the platelets which is why I really believe these results I obviously didn’t buy a sum because I got it completely abnormal result so you mentioned about the dosing effect of jak2 mutation it’s also possible because one of the they’re what view of these trees in new pathways in their to activation permutation related activation one of them staff family I’m actually determining the set of genes that turn on by these factors and this has been reported in in type of cancer you think that also possible for this second for your jack to marry parasite specific doctor mutation do you detect he didn’t attack the bleeding for the pipe but he have you detect or look at the the hyperactive part of limits so regarding the the first question about what are the stat dimers are they in any way different or otherwise I’m not sure we’ve not looked at that we have seen mostly homo dimers of stat3 and stat5 another group had identified and published that stat one is also being activated downstream of of jackets it’s an intriguing hypothesis that in the presence of the mutant jak 2 we’d get a different array of downstream signaling molecules I I don’t know that there’s any evidence of that is probably worth looking looking at is I’m what was the second yeah so we haven’t been able to demonstrate in agronomy tree and in other more conventional tests of coagulation that the platelets from these mice are you know better able to aggregate there they seem to be spot-on with normal platelet at ofus it took said never a another nonspecific jak inhibitor has just been recently approved for rheumatoid arthritis and as far as I know there hasn’t been any signal for either clotting or bleeding predict that when you’re targeting systemic tack

activation out sort of the primary target on the on the cells themselves if you might find the I I would have sort of predicted leading early on in this story because we do know that in platelets that are undergoing forced coagulation with agonists and things like that that jak2 is becoming activated there are a number of targets downstream in the platelet that are being activated it is contributing we know that thrombopoietin changes the threshold for aggregation in platelets through activation of jak 2 but I think that the doses that you guys are using and that we’re using our front because of the fear of toxicity are probably not sufficient to get into those complications that would be my thank you very much again