Dr. Adrienne Scheck – Tumor Metabolism and the Ketogenic Diet

– That one’s throat. Okay. Disclosures really isthat really that we use a human formulation of the ketogenic diet announced KetoCal, it’s donated to us, uh that’s the only dislosure I have. So, the very first thing we did when we started going into the ketogenic nutrition is we tried this in vitro. We cell string listed AO2V4, there it is, which was a cell line which was derived from the fourth braintumor that a young man had.Very, terribly vigorous tumor. This person was treatedwith everything there was to be treated with back inthe seventies I guess, and it makes any growthfactor they knew to look for. It was grown in twenty-eightmoly moly glucose that’s very high glucose, that’s what the media had. And what we did was we, without reduce glucose, we threw in five moly moly each beta-Hydroxybutyrate macedo acetate, and we likewise tried one of the pharmaceuticals that this patient was treated with, which is … … I don’t have to stand there, now which is bcnu carmistine. That was actually the treat of pick for brain tumors at the time. And what you can see is, the cells that are nottreated grow very nicely, the cells treated with BCNU they lag and then they start to grow.That’s normal, when you consider cadres in vitro with chemotherapy like that. The ketones, without reduce glucose hindered the growth, or slow-going it. But the two togetherwiped different cultures out. And frankly, that wasthe first experiments that we did that represented mechange the part tendency of my laboratory to the ketogenic diet. Because my thought was the ketogenic diet or anything else was pretty much useless unless it was done in combination with standard of care because no clinician would ever use it.So, we had to come up with a mouse modeling at the time. We were doing that, we got the gene that’s in fireflies given it in a mouse glioma cell indication announced GL261. They know glow and now we can follow the mouse tumor. So this is the GL2 61 luck two framework we use albino C57 Black 6 mice syngeneic, immunocompetent we give them KetoCal, it’s a gunpowder, we desegregate it 2:1 and it’s a adhesive. We propel it in the enclosure and they can eat as much as they crave. They roll in it, they get a little greasy but it doesn’t seem to bother them. They don’t uh…and they’re maids … … so it doesn’t seem to bother them. They’re female mice, uh, we don’t inhibit we don’t do any caloric restraint they can eat whatever they demand they don’t tend to lose weight. And they don’t tend to gain weight beyond the swine thatare on standard diet.This is all been published, which is why I’m going through it really fast. Okay, so, the animalsget surgery on daylight zero. The tumor cadres areintracranially embed. On the third day they are searched, randomized to a treatment arm. We measure BHB and glucose elevations and the swine are sacrificedwhen evidences seem. Symptoms appear in thismodel, reproducibly about twenty four hours before they are going to die. So, I’m an animal lover, I do not intend to let swine suffer. When can do invivo imaging, to follow the growth of the tumor. And “thats what we” spotted. What I’m going to show youin the next couple of slips is a combination of twototally separate ventures. So, standard nutrition survival. KetoCal survival. So there is an increase in survival. When you lend temozolomide which is the chemotherapy of choicefor brain tumors now, survival is better, standarddiet of chemotherapy than it was, standard food alone. But, the KetoCal obliges that better. But, this was what blew our heads and started to get cases announcing on me and that might be okay for you clinicians but as a scientist whena patient requests you it’s a little interesting( titters) um, because all I can tell them is well, I can tell you it helps our mice.Y’know, um, so regardless this is is wholegrain radiation and the radiated animals do better than, uh, than the standard diet alone. But when we supplemented KetoCal mostly we medication nine of eleven mice. And the reason that weknow that this happened is we can follow the growth of the tumor this is just one of the swine, and here is one of the swine that was on ketogenic diet. I don’t know how well youcan see it in the back but this is a extremely, very bright tumor cell line.The tumor was there, starts to disappear. Comes up a little but by around era forty, all of the mice you could not see anything. And this is published work its in open access publications so feel free to look at it. Right around day one hundred and one, I decided that rather than continue to ruminated our budged tohave these little noblewomen running around their cage happy let’s permutation them back to standard diet and see what happens? And we did that foranother two hundred epoches and the tumors didn’t come back. And, uh, the pathologist said there was no evidence of any active tumor in these animals whenthey were relinquished. So, well unbelievablyexcited about this. It does not mean wethink we can cure humen but in the very leastthis was a step forward.This really kind of charges us up for ogling more specifically at radioactivity plus chemo, plus uh, ketogenic food. One of the things we is located within our very first experiment was that it increases ross. And we determined this usinga different technique where we could only look at one timepoint and it was late. And I’m thinking this stuffmakes radiation work better but radioactivity wreaks through ross, this fixes no sense. Let’s find another way to look at ross, maybe the ross goes up early and then it comes down? So we did, we use thisL0 12 chemiluminescence and the closest we could come to looking was twenty four hours but at every timepoint the ketogenic food abbreviates ross. So for years I’d stand up, give a talk and say if anyone inthe audience understands this please come talk with me.Cos we don’t. Uh, but we do now. We thoughts. The other thing that we think helps is that it does shorten inflammation based on, uh, looking at cyclooxygenase 2 all the other speakers inthe past that mentioned anything about an irritation said here today did increase swelling. I’ve got some fairly tall shoulders to stand on with the previous speakers. So you can see KetoCal abbreviates the COX2. Radiation, it goes up, as you would expect. But the animals on KetoCal it holds that COX2 fromgoing up after radioactivity. So, we really think that in addition to helping the radiation work better it might actually do a lot of things that would help a patient. Okay, so, things I don’t have time to talk about is the pluripotentnature of ketogenic diet is unbelievable. When I firstly started Ithought it was snake oil. I didn’t believe it to be perfectly honest. It hinders tumor rise. It increases radiation in at least part of the chemotherapies we’ve looked at and others of course areshowing other chemotherapies are, um, also, made to work better.We’ve published this. It shortens peritumoral edema. It reduces irritation. It reduces antigenesis. It reduces hypoxia. It shortens the speech of uh, hif1alpha and phosphorylated nf kappa b. It increases the antitumor immune response. So, we decided to focus on the radioactivity. And one of the things we have to do is say, okay, we have to go back to the invitro representations, and investigate can we recapitulateit so we can start to tease apart what’s happening. And it is about to change that surely we can. These are the mouse cells. We use a low-pitched quantity of radioactivity which is too gray, impedes rise. Low amount of beta hydroxybuterate. 2 millimolar not nearlyenough to inhibit growth but the two togetherabsolutely work better. This is a human cell line that’s not the patient’s initials. It’s a random two letter code and this is a recurrenttumor so we compute an’ R’ to the end. So, this is a human cell line, again these are grown in high glucose. I’m taking glucosetotally out of the picture for the purposes of what we’re saying. And again, this green line is what’s important.Ketones plus radiationbetter than either alone. So, the mechanisms of action of the ketones beyond glucose reduction. We think it has to do withepigenetic modifications. So, everything I’ve depict you up to this point has been published. All of this now has not been published. My grad student justgot his PHD last month, and had his firstly baby, and moved to North Carolina, in one month. So we’re finishing upsome of those studies and looking to publish hopefully a bit later this year. But, the epigenetic the consequences of the ketogenic food that’s been shown by the rest are: Conversions in histone acetylation. Beta hydroxybuterateis an H-Stack inhibitor as you’ve heard beforeby some very elegant talks in this meeting. There are changes in the non-coding RNAS solely adaptations in micro-RNAS, and there’s also probably modifications in DNA methylation which I won’t actually get a chance to tell you about.So for any of you whoare not bio-chemists, I am certainly not, which is why I need delightful, easy caricatures. When DNA is de-acetylated, it’s closed, I think in caricatures, I think of DNA like this. And the radiation can’t stumbled it. When it’s open, it’s acetylated. Now there’s lots of gap, radioactivity and chemo can shatter it. So, acetylation, hystoneacetylates go that nature Deacetylates go that highway and BHB blocks that. So, that’s you miss in a cancer cadre. So, there’s lots of, or a number of H-Stack inhibitors that is really in clinical trials.As most cares, theyhave various side-effects. But what’s interestingis that a lot of terms this, the hysto and deacetylates is, they play differently in tumor cadres and regular cells. And that really followswhat we receive with BHB. So, first we decided to look at, does BHB increase hystoacetylation in our pose? And, certainly it does. So, this is just a western blot for acetylated hystone 3K19-14. And “youre seeing”, thisis without radioactivity. This is with radiation. In the presence of BHByou do get an increase in acetylation as you would expect. And that’s deepen even a little bit more when you lend radioactivity into the mix. This is consistent with our mouse cadre way. This is in a glioblastoma cell line.Again, M.E is not the patient’s intials it’s a random two letter code. This is the primary tumorthat runs together with the cells I showed you before, which is the recurrent tumor from the same patient. So, these cells arefrom individual patients before they were treated. Again, BHB growths, hystoacetylation in these cells as well. So, what about DNA damage? Gamma H2AX is a marker for DNA damage.It affixes to the end of damaged DNA and it basically tells everybody else “okay come here guys weneed you to repair this, ” so the more Gamma H2AX there is the more damaged DNA there is. As the DNA is amended the Gamma H2AX goes down. So “youre seeing”, it’s kind of low-pitched in the absence of radiation and the BHB doesn’treally make a difference.Radiation expenses the DNA BHB, there is more damage. So what’s happening we think is the BHB is actuallyinhibiting DNA damage reparation. Oops, sorry, wrong one. Again, the mouse cadres, the human cells, it’s even more pronouncedin the human cells, where there’s very little Gamma H2AX, very little marred DNA. Before radiation, after radiation, there’s quite a bit.And it’s increased in the presence of BHB. What about glioma stem-cells? Glioma stem-cells are thought to be cells in the glioma, or they should say stem-like cadres, thought to be cadres in the glioma that are more resistant to radioactivity, most resistant to Chemo, probably reside in ahypoxic province of the tumor and one of the thought’sis that these cells kind of repopulate thetumor after treatment. Although, that’s upfor a lot of discussion which we can get into later.But we did have a talentedhigh-school student in my laboratory that actually looked at glioma stem-cells. And these cadres were very kindly provided by Dr. Brent Reynolds from Florida. So on Day Zero she seeded the cells. On Day One we treated themwith betahydroxybuterate. The next day they gotfour gray of radioactivity. And then, seven dayslater the neurospheres where divorced and counted. So “its one of” the cadre words, L0 and, i look around at live cell count, so betahydroxybuterate doesn’t punch it. Radiation restraint. But the two together, inhibit better. So betahydroxybuterate is also enhancing the effect of radiationon glioma stem-cells. Oops. It is hard withtwo different buttons. So here’s the secondglioma stem-cell line, from a different patient. And again, the magnitude is just slightly different but the effect is the same. The betahydroxybuteratedoes enhance the effect of radiation in glioma stem-like cadres, as well as human glioma cells, and mouse glioma cells. Here we are looking at DNA damage, again, applying Gamma H2AX and in the presence of betahydroxybuterate we do consider a little bit of an increase but with radiation, there’s a big increase.And the acetylation goes up too. Another marker, well it’s not a marker, but another protein that’s involved in DNA damage restore. So this is looking at, is there DNA damage? And this is looking at one of the proteins took part in the restore of that expense. So, more of this is more damage. Less of this is less repair of damage. So, when you look at RAD5 1 again in these glioma stem-cells it’s definitely downin the presence of BHB. So, looking at all the cell routes I’ve been talking about so far.This is our mouse glioma cadre route. This is our human glioblastoma cell line. This is the recurrent tumor cell line from this patient. So in other words, these cadres are the tumor that came back after radioactivity and temolozide treatment. And this is that first cell line I told you about which is extremely vigorous, came from the fourth tumorthat this young man had. So in all cases, the RAD5 1 is reduced. And the acetylation is increased. MRE wasn’t quite statistically significant but it approached important. So, virtually, what we think is happening in radiation, because I said we wherereally confused about that whole Ross thing, is that we think that we might not be increasing the DNA damage through Ross, but we’re probably reducingthe DNA damage amend. And that also various kinds of utters sense with how it seems to help some of the alkylating agents.Is that we considered that, the epigenetic accomplish is actually motiving a reduction in the genesthat are involved in DNA damage amend and we actually have dated that some of theother proteins took part in DNA damage fixing are also increased. So now to switch gears a little to microRNAS. MicroRNAS are, small , non-coating RNAS. They are about twenty-two nucleotides long so they’re little itty bitty things. They’re involved in RNAS silencing through a variety of mechanisms and, one microRNAS categories, they’re kind of promiscuous and it can actually reduce the speech of a number of microRNAS. Sorry, I apologize, a number of proteins. But the repression usually isn’t extreme. But they’re still reasonably active, in fact some people are actually looking at these as potential therapeutic agents. So in collaboration withan excellent researcher, Dr.Nell Said at theImperial College in the UK we communicated her tumor tissue from our mouse and she did an RNAS seek analysis of all MicroRANS and she came up with a entire bunch of MicroRNAS that aredifferentially carried. When the MicroRNAS are up, the proteins that they, uh, alter are down. Okay, so up means we areturning down proteins. And there’s just a whole bunch of’ em. Uh, and quite a few of these are involved in DNA damage restore, interestingly enough. But what’s really interesting is that when I thinkof meatabolism I reckon, well most of them kind of should be metabolism or maybe epigenetic nonsense. Turns out if you look at the hall-marks of cancer, pretty much every hall-markof cancer is affected.And this is the only change here. This one I’m guessing will be when they figure out more MicroRNAS that areinvolved in genome instability. So, when you think about it we all think of metabolism, well I suspect not in this room, but, most offices, when often talking theythink of metabolism, okay it’s the food, it’s the exertion it’s the glucose it’s all that trash. Well in fact, it’s a whole lot more. Metabolism is gonna hitevery single hall-mark and it’s going to change gene expression pretty much in every single hall-mark.So, to summarize. Ketone and just epigenetic reforms BHB is an H-stack inhibitor. That’s been shown by others, it increases hystoacetylationin our tumors this we think lawsuits a decrease in genes that encode the DNA repair proteins. So, we get an increase in the radiation induced DNA damage, and we think that’s howit’s potentiating radioactivity. We do get the increase expression of about fifty-five different MicroRNAS and again that’s the work of Dr. Nell Said. That’s from tumor material, we are working on this in cell line’s and she’s working on it in cell texts. We’re showing that changing the MicroRNA does indeed change the expres of some of these genes. Uh, including the DNA repair genes. Uh, got some very exciting information. One of the MicroRNAS we know is involved in changing CMYK, which is really interesting because I most recently moved toPhoenix Children’s Hospital and now I’m kind ofturning most of my work towards pediatric tumors so, of course, we’re reallyinterested in that.And, uh, there’s also some data that I don’t have time to talk about, we’ve got really very little of it that advocates, that BHB and NIKD might actually too alter gene methylation. I’d like to acknowledge the people that do this work again. Nell, in the UK. We had immunologists that were working on the immunology project.Various other people that work with me. Erick Wolf was my grad student, who just got his PHD. Alex is uh, was a high school student , now a college student and now actually working as my technician which is great becausehe’s totally qualified. Uh …( laugh) .. by me, so all of his bad wonts are good habits I know about. Lena did the glioma stem-cell work as a high-school student. She’s now in Stamford with more grant moneythan I can ever hope to get for my research.And then there where other beings that were in the lab that have contributed to all of this. Acknowledgements, as youall know when you write to the NIH for funding. Diet is a four word oath. Uh, luckily there are other footings and people that have helped fund this and we’re hoping that now Dr. Cantley has jumped on board, diet will no longer be a four word statement. But uh, I’ve been very fortunate in getting people that have been willing to fund some of this work. And questions will come later, so thank you.( applause ).

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