this program is brought to you by emory university good morning everybody uh welcome to friday fellows conference your speaker this morning as you can see is dr elena dolmatova elena is in our basic science research track she's originally from russia where she went to medical school at moscow state came to united states did a residency at rutgers and then did three years of research in dr grindling's lab and is now in her first year of clinical training as you can see she's going to talk about dr rubinson induced cardiomyopathy dr dylmatopa good morning everybody dr rubison is a chemotherapeutic agent that is been discovered from streptomyces and is most widely known for its use as a treatment of breast cancer triple negative breast cancer but actually is used for a number of different liquid and solid tumors and childhood tumors as well and the mechanism of action is thought to be twofold one of them through the direct dna damage to dna inhalation and topoisomerase two inhibition and the second is by uh reactive oxygen species formation and why those two mechanisms are very helpful in fighting cancer they are also the culprit of all the other complications that we can observe in the chemotherapy with one of the most feared ones being the doxorubicin dyscardiomyopathy that can present even years later after chemotherapy has been already done who is at risk at increased risk for cardiomyopathy and there is a definite dose a relationship with the magic number of 500 milligrams per meter square which uh is debated uh and some of the researchers say it is actually lower than that there is increased risk if you are a woman if a person is older more than 65 if it is a younger child less than four years old if there is also radiation therapy if there are other drugs that are being given at the same time and if person has already pre-existing cardiac condition we may ask what's happening with the genetic predisposition and i also was questioning that unfortunately the studies that are available today suffer from lack of power and even the genome-wide association studies don't have a very good power to detect [Music] polymorphism associated with that that said at this time there is a good evidence of um genomic regions around the genes associated with psychometric structural function topoisomerase 2b expression drug trafficking drug metabolism that are associated with increased risk of uh doxorubicin cardiomyopathy despite we have some evidence towards that none of that met the threshold of being something that we can use in screening of patients prior to chemotherapy as a administration so how do we diagnose uh doxorubicilling discarding life or any sort of chemotherapy in discarding myopathy we are looking at the injection fraction of the heart and it has to satisfy both of the criteria it has to be more than ten percent changing in injection fraction to a value then is less than 53 and uh as i said it has to be both so if somebody had ejection fraction of 65 and after the chemotherapy it became 55 it really doesn't qualify to be a chemotherapy and dyscardiomyopathy it should be confirmed by repeated cardiac imaging within two to three weeks after first detection and toxorubison is a type 1 chemotherapeutic meaning that it's causing irreversible damage by causing damage to the cells in those dependent manner which is in contrast for example with strexus mod which is causing uh reversible damage but in combination they might be even more toxic so the most common way to to assess the function is by 2d echo that is available almost everywhere and you know the most preferred method of ejection fraction calculation is modified by plain simpson technique with the method of disks uh it does have its limitations and if you add uh resting on motion score uh based on 16 segment modeling it increases sensitivity of the method uh if there are two or more segments that you cannot see of the cardio you can benefit from using contrast and unfortunately as we all know there is about 10 percent inter-reader variability between different echoes so if you have this 10 and you want to detect the change of 10 percent that uh becomes not a very sensitive tool for detection of such changes 3d echo is gaining speed and actually is better than to a 2d echo but um because it is um accounting for other things in the left ventricle not assuming its shape but unfortunately it's not readily available everywhere as of this moment we all studied for the boards at some point and we all remember mughas khan as the one that is preferred for the chemotherapy detection when we label the blood and we can assess ov ejection fraction it's actually better than 2d echo in accuracy and reproducibility it's comparable to 3d and actually lacking some of the technical limitations the echo would such as it can be easily used in obese patients patients with poor acoustic windows patients with devices but it has its drawback it's subjects people to radiation which not is not always desirable uh and with motor systems uh there is a debate that it may not allow for the best positioning as the ones that were actually described in the studies first the question is can we benefit from detecting subclinical ov dysfunction and probably yes because then we can adjust and change the chemotherapy before we already have the ov dysfunction there were a couple of things that was suggested to be able to detect a clinical redis function initially diastolic function was suggested but unfortunately it was lacking a positive predictive value then the tissue doppler imaging uh was also thought uh of being useful again you as you know there is some angle dependence in how you position the probe for the mitral analysis tissue imaging and because of that the repeated measures might be affected more by angle than by the change in the tissue doppler values and finally the most novel thing is the global legitimate strain which actually is ango independent and allows for assessment of all the segments of their left ventricular not only mitral analysis at the same time and it is thought that reduction of global issue in australian 15 from percent from baseline is very likely to be abnormal but that number hasn't quite yet made it into guidelines uh and finally uh all of those parameters uh the diastolic function the global student strain or even ejection fraction are load dependent and then when you have a patient on a chemotherapy the volume status of this patient changes quite a bit so strain rate was suggested to be more sensitive because it's relatively low independent compared to the other measurements and finally mri is a go standard for left volume assessment as you might know but it also has a potential for some clinical detection with later gadolinium and t1 mapping when you can look and see if there is early fibrosis or inflammation therefore detect the potential lb dysfunction in the future rather than detecting when the aef already is down biomarkers was suggested to be helpful uh with one of the most obvious ones being a troponin however at this time there is a question about exact timing of troponin measurement where exactly after the chemotherapy would you measure it to have the best result and of course the cut off points or where to increase sensitivity and specificity because not the average opponent elevation will lead to the obedience function and where exactly we draw the line at this time is not quite clear nitrouretic peptides were also suggested but it seems like they reflect the volume status more than the ov dysfunction in the future and as i mentioned earlier volume status of people on chemotherapy can change dramatically so what are the mechanisms of doxorobicin in this cardiomyopathy and if you look at any rna-seq data of any cell type that you dump doxorubicin on what you will be having troubles with is to find a gene that is not changed it doesn't leave a stone unturned and that makes it very difficult to understand what exactly doxorbison does and what is the mechanism of the toxicity with that most of the consensus is around couple of points [Music] a lot of that is revolving around mitochondria and oxidative stress and impaired mitochondrial function and increased mitochondrial iron which is also kind of connected with the disruption of calcium to homeostasis a little bit of a different theory is that uh doxorubicin binds to top poison rice 2b and together they promote dna damage with all of that upregulating expression of death receptors leading to cellular apoptosis oh it's all nice but how can we prevent the doxorbison and dyscardium myopathy and not surprisingly the first things that people thought about were the standard heart failure therapies like bitter blocker ace and arb and those are the trials that try to look at it they're fairly small number trials and the outcomes there might be not always perfect um and they would look at mortality instead of like lp function for example so there is a way to go in terms of having more trials on those there was a lot of pre-clinical trials suggesting that statins may be having anti-inflammatory effects and preventing lv dysfunction but there are only two small clinical trials that are available at this time and if you can see it actually there is a recent method analysis um looking at ace r and beta blockers and patients that were received chemotherapy and it is favoring use of those drugs for prevention of chemotherapy and just cardiomyopathy so we have these drugs but there is one drug that is actually specific to the mechanism of doxorubicin toxicity and what we have we have a lot of iron in the mitochondria and it's being transported in and out there is a whole movement of the iron and what is thought to be happening is doxorubicin may be binding directly to the iron or inhibiting its transport out of mitochondria resulting in accumulation of iron in the micro mitochondria which is toxic and produces a lot of re-oxygen or reactive oxygen species the strasix sand is possible capable of binding this iron and pretty much chelating it out of mitochondria preventing the mitochondrial damage uh but there are some suggestions that it may decrease response to tumor response because of this reduced reactive oxygen species that may be also having chemotherapeutic effect it um has a it increases myosuppression and there is possible risk of secondary malignancy which actually hasn't been proven to be true but because of those uh fears uh right now it's only uh advice to be used in people who would receive a higher doses of doxorubicin or in people who are suspected to receive more doxorubicin in the future uh the one question that bothered me a lot and i didn't see much of a data on that is like if we are working a lot and trying to prevent the cardiomyopathy but whatever drugs we use how would they affect the chemotherapeutic effect of the on the tumor itself because if we're preventing chemotherapeutic effect of the doxorubicin on the tumor we are having a different problem in our hand where we actually not achieving the treatment goals right and all the basic science studies that you can look at they're looking at cardiomyopathy and they're really not focusing on the tumor effect of those drugs at all with the exception of one small study that i was able to find where they looked at metformin carbedo enolopro and control here in black and looked at cytotoxic effects of doxorubicin when these drugs were introduced and luckily for us it seems like there was no effect of these drugs on doxorubicin toxicity meaning that this dr rubicin was still able to have its chemotherapeutic effect on the breast cancer cells the same helped true for the dextrosex and it was also not affecting the chemotherapeutic effect on the cell on the tumor cells and that always in vitro so there isn't really a really in vivo data on that but what it made me think is that if we have things that are cardio protective uh but are not affecting the chemotherapeutic effect there must be some difference in how this [Music] tumor toxicity versus cardiotoxicity are happening and maybe it has something to do with the types of cells they're very different cells right tumor cells are dividing rapidly and they're mostly glycolytic and cardiac cells don't divide pretty much at all and they are mostly dependent on oxidative phosphorylation although they can switch to glycolysis if need be so my thought was that maybe the there is a difference in the toxicity mechanism and with the anti even tumor being mostly affected by the dna uh in the correlation and inhibition of tranquilizer rays too and cardiac muscle being affected by oxidative stress and impaired mitochondrial function now i want to switch gears just a little bit i was working in dr green's lab when it's working on protein named podip2 it's not a very famous protein but it is very multifunctional it was first described as a polymerase ii associated protein and polaris data associated protein and it was involved in dna repair it also is found in other parts of the cells bought the nucleus and it was uh shown to be involved in metabolic switch and reactive oxygen species formation you can see here there is a lot of overlap with what i just said with doxorubicin toxicity in the cardiac toxicity so what i initially thought was that maybe to reduce pole dip if we knock down podip will be able to be protective against oxybacin reduced cardiomyopathy and to test that we had our podip2 knockout mice or wild-type mice and we injected them with doxorubicin and five days later we assess cardiac function serum troponin levels and mortality and here you can see mouse echocardiogram and at baseline there is a nice contraction of the heart and left ventricular involves coming in nicely but after you treat the wild type mouse with doxy ruperts and it develops acute oxybuses cardiomyopathy however in the knockout mouse you don't see this effect it's still contracting nicely and if you are to quantify that you can see that there is significant reduction injection fraction in wild-type mice but we don't see this reduction in knockout mice and we wanted to see if it correlated with troponin levels and similarly wild type of doxorubicin treated mice had significant increase in serum troponin while the knockout mice did not finally when we injected these mice and just observed them over time uh you can see that all of the wild-type mice were dead by the three weeks of [Music] looking at them at the same time all the knockout mice were still alive oh nice but i was still kind of wondering what is the mechanism of protective effect which one of them is important because as podi being multifunctional and doxorubicin having so many different mechanisms which one is the the key and that's when i turned my attention to the recent paper showing that podip2 knockdown is able to switch from the oxidative phosphorylation to glycolysis so if you knock down pole there the cells would become more glycolytic that's when we thought that maybe what's happening is that in cardiac muscle that is normally dependent on oxidative phosphorylation we knock down podip it switches to glycolysis and after treatment of doxorubicin because the mitochondria is not as functional there is not that much oxygen moving around there is less reactive oxygen species formation the same time in breast cancer there's already glycolytic when we knock down podip there would be a little or no effect on the metabolism and after treatment doxorubicin the same effect as we talked about earlier with dna intercalation dna damage would just be still there to test whether that was true i just first wanted to see if knocking down politic would affect the apoptosis and we had our cell culture with the h9c2 cardiac myoblast cells and bt 549 the triple negative breast cancer cells we can knock down pull defusing sir rna and treatment doxorubicin and then assess apoptosis by looking at the cliff caspase as you may remember during apoptosis number of caspares get cleaved and cleave each other and finally uh cleave other proteins so cleave caspase-3 is thought to be a classical apoptosis marker and here you can see the western blotting results uh on cardiac myoblasts and we have a vincolin as a loading control here looking that all of those are loaded equally then with propropolodev we can see that we can knock down paul dip with the cypoli quite successfully and when we looked at cliff caspase you can see that in control cells there is quite a bit of increase in caspase-3 with doxorubicin treatment which we would expect the cells are dying but if we knock down podip we have much less of cleaved caspase indicating that the cells are dying much less when you knock down pole dip and treat with doxorubicin and if you quantify it here you can see that there is significantly less uh of caspase-3 in a cypodium well it's nice that we that we have it in the cardiac myoblast but again my continuous question what happens to the tumor cells and if you can look at the tumor cells i'm sorry i need to fix that because otherwise it's going to be beeping forever and then the tumor cells um again you can see that they're all loaded equally but if you knock down podip uh here and here you can see there is much less podip and you look at the cliff caspase again with the indoctrination quite i see quite a bit of increase in cliff caspase however if you knock down podip and also treat with doxyrubicin you see that in it's not unchanged it's not decreased actually matter of fact it's increased so if you knock down code dip and treat with doxorubicin you actually promote apoptosis in the tumor cells and here's just the quantification so it seems like we are able to kill two birds with one stone uh with the if we knock down pole deep in the heart we're protecting the heart from the oxygen discarding myopathy uh or at least protect the cardiac cells from death from doxy recent toxicity but we are also able to increase apoptosis and increase death in the tumor cells well as iron a is a nice to nice research tool but it's very hard to deliver sirena in the whole organism especially in such a big organisms like a human so we were looking for something more pharmaceutical that we can apply that would work through somewhat similar mechanism and would be able to decrease body and luckily for us there is one drug octo one that is um actually activating copp priorities in the mitochondria and it causes a few effects but one of the ones that we care about is that it actually able to decrease pole dip and by just degradation of that podium so we wanted to see if the addition of this drug operation with this drug that is now being developed this chemotherapeutic drug for glioblastoma uh would actually have similar effects to the ones we observe when we knock down pole dip with sirna and again we try to do it in our cardiac myoblast again it's loaded evenly and we if if we add this onto one to the cells you can see that uh pulled it down opposite to actually does go down if we uh treat the cells with doxorubicin as before there is a increased caspase 3 cleaved caspase 3 indicating initiation of the apoptosis but if we pre-treat cells with octo-1 there is significant less of the cleave caspase meaning that the onco-1 pre-treatment is capable of preventing apoptosis cardiac myoblasts after the oxytocin administration and when we do the same with the breast cancer cells again we can see that onco1 does decrease product2 levels in breast cancer cells but it doesn't decrease the uh apoptotic effect of doxorubicin if anything it maybe increases it slightly um not to the same degree as a psychologist which seems like it does mimic to some degree at least the effects of a cypodium cells and open some other opportunities to try to prevent cardiomyopathy with minimal or even opposite effects on the tumor so in conclusion we have a decreased podip that in cardiac muscle and after you administer doxyrubicin uh would prevent oxybacinous cardiomyopathy while in breast cancer cells it would kill breast cancer cells and as of now we don't have much of a data we have hypothesis and preliminary data here and there but what we suspect is happening is that the prevention of serbian indus cardiomyopathy is uh performed through the inhibition of reactive oxygen species while the promotion of apoptosis of breast cancer is done through the dna intercalation and topoism race too and actually enhance the effect of those effects of doxorubicin because podip2 as i mentioned earlier was involved in dna repair and it's quite possible that if you knock it down the dna repair is happening even less therefore the chemotherapeutic effect of doxorubicin is promoted and finally we do have a drug that to some degree mimics the effects of sipo dip and able of decreasing podip2 and possibly having similar effects on the muscle and breast cancer so of course it's just a very preliminary and what we would like to do is to kind of expand on that thought and expand on those findings and first we would like to figure out what's happening in terms of the mechanism and again we would take our uh cell culture models and we would knock down podip or give unknown one we treat with doxorubicin and we would look we would look further at different other caste spaces that are activated in different apoptosis pathways and see if there is a difference in those uh we would look at mitochondrial function uh reactive oxygen species production and finally look at the the dna damage in those two different cell types ultimately what we would like to do is to do something that would be would be able to assess those two things in in the whole organism because everybody is looking either the tumor treatment or the heart but very little is known about what's happening if you do it in the same organism and to do that we would have two models one of them would uh utilize wild-type type and po-deep knock-out mice and we would inject wild-type oppo dip-knock-out tumor cells we would let the tumor grow and then we would treat the mice with doxorubicin and assess cardiac function serum troponin cardiac fibrosis tumor growth and mortality to see how doxorubicin in under those conditions would act in on both on the heart and the tumor growth and ultimately uh we would like to have just normal biotype mice normal wild type tumor cells and treat the mice with omk201 before administering doxorubicin and look at the same parameters and see if by administration of octo-1 we could prevent doxerbacinidos cardiomyopathy while either not affecting the chemotherapeutic effect or possibly even promoting a chemotherapeutic effect of doxorubicin so in conclusions of this kind of scattered talk uh doxorubicin is a common chemotherapeutic agent that can cause irreversible cardiomyopathy while multiple risk factors have been identified no doxorubicin-specific screening strategy exists at this time early detection is getting easier with improving technologies that we have right now the mechanism of cardiotoxicity likely involved mitochondrial dysfunction oxidative stress the extrason is approved for prevention of doxorbison cardiomyopathy and standard heart failure therapies are also promising and finally the mechanism-specific prevention strategies i think are warranted and more research is needed to try to figure out if we can actually prevent cardiomyopathy while still maintaining the chemotherapeutic effect of doxorubicin on a cancer with that i would like to thank everybody who contributed to this work one way or another with all the very nice collaborative atmosphere in the basic research side and of course the funding that supported this study as well and if you have any questions i would be happy to answer thank you elena very good interesting uh review and and uh both the clinical and the research side um i don't have any questions at the moment any for any questions from the group elena this is dr jerry magoris what is the thought of mechanism of statins in preventing or protecting the heart in chemotherapy induced cardiomyopathy so the the thought was it mostly anti-inflammatory effects okay so and most of that was as i said it's mostly pre-clinical studies in the rat and mice models but yeah their thought was that they inhibit the inflammation in different stages of inflammation like the either the vascular or the immune cell infiltration but an anti-inflammatory part of it okay thank you yeah this is uh andy smith it's great that you're looking at this it's a really big clinical problem that we see in the heart failure world um and and just to comment um you know the patients who develop this they sort of behave in some ways more like a restrictive type cardiomyopathy than necessarily a dilated cardiomyopathy they don't get as much ventricular dilatation as um certainly what we see in non-ischemic idiopathic cardiomyopathy and they may be less likely to tolerate some of the guideline-directed medical therapies in part because of that another comment is that in our heart failure group we're often asked to see patients who develop cardiomyopathy but got the doxorubicin as children um and it's been reported that um children you know who get doxorubicin 20 years later may be diagnosed with um with toxicity and one of the common things that's seen if you look back through their pediatric records is uh that they often have a resting uh tachycardia and a higher heart rate it's it's something to to consider um but uh certainly a a major major problem it's a drug that's used very frequently and and um is is is quite an issue for us in the uh in the advanced heart failure world actually very um i think in part because of what you said with the restrictive um pattern of the cardiomyopathy the strain imaging and strain rate imaging might be bigger value than just standard lv ejection fraction measurement because by the time you develop decrease the ov ejection fraction it might be too late so i think that's probably part of not probably quite definitely part of why people are moving towards more advanced imaging in that in in detection of the cardiomyopathy and of course the uh modeling of delayed cardiomyopathy is very uh very challenging but even acute cardiomyopathy challenging in the animal models but especially delayed dog service and cardiomyopathy is a huge challenge in terms of how to model it in the animal model mouse shall be mouse or rat or anybody so that's one of the things that i'm afraid on the basic science side of things may not necessarily uh evolve as fast as we would like yeah elena as a as a echocardiographer that's usually my involvement with these patients is on the um front side with their their echo pre you know chemotherapeutic or echo they're following along but you're right i mean it's i you know at least uh in the past the guidelines technically had recommended radio nucleotide imaging because it was a more accurate assessment of ejection fraction but you're right at the the echo does provide certain advantages you mentioned lack of radiation you mentioned the the portability you know particularly for an ill hospitalized patient that uh that uh echo um can provide you know as opposed to uh mugga um but also like you said the pre um sort of pre-clinical markers potentially that uh assessment that you could have with echo like you said of strain imaging looking at diastolic function even things of that nature that might manifest abnormalities even before the ejection fraction starts to drop so um and i don't know if the if the guidelines have been updated for this particular chemotherapeutic agent whether they recommend either mugga or echo sort of give the the choice i know here locally i think i feel like it's probably more echo than than muga these days but um is any other thoughts on the the pre assessment of ejection fraction my view on that slightly skewed because i the guidelines i was looking at were mostly from the american society of echocardiography but yes they do recommend the baseline echo all right well very good if um no other comments or questions from the group we'll thank elena for her um uh talk this morning and we'll look forward to seeing everybody next week at conference the preceding program is copyrighted by emory university