– That one’s throat. Okay. Disclosures really is
that just that we use a human formulation of the ketogenic diet called KetoCal, it’s donated to us, uh that’s the only dislosure I have. So, the very first thing we did when we started getting into the ketogenic diet is we tried this in vitro. We cell line named AO2V4, there it is, which was a cell line which was derived from the fourth brain
tumor that a young man had. Very, very aggressive tumor. This guy was treated
with everything there was to be treated with back in
the seventies I believe, and it makes any growth
factor they knew to look for. It was grown in twenty-eight
moly moly glucose that’s very high glucose, that’s what the media had. And what we did was we, without reducing glucose, we threw in five moly moly each beta-Hydroxybutyrate macedo acetate, and we also tried one of the drugs that this patient was treated with, which is… …I don’t have to stand there, here which is bcnu carmistine. That was actually the drug of choice for brain tumors at the time. And what you can see is, the cells that are not
treated grow very nicely, the cells treated with BCNU they lag and then they start to grow. That’s normal, when you treat cells in vitro with chemotherapy like that. The ketones, without reducing glucose inhibited the growth, or slowed it. But the two together
wiped the culture out. And frankly, that was
the first experiments that we did that made me
change the entire direction of my lab 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 model at the time. We were doing that, we got the gene that’s in fireflies put it in a mouse glioma cell line called GL261. They know glow and now we can follow the mouse tumor. So this is the GL261 luck two model we use albino C57 Black 6 mice syngeneic, immunocompetent we give them KetoCal, it’s a powder, we mix it 2:1 and it’s a paste. We throw it in the cage and they can eat as much as they want. They roll in it, they get a little greasy but it doesn’t seem to bother them. They don’t uh…and they’re women… …so it doesn’t seem to bother them. They’re female mice, uh, we don’t inhibit we don’t do any caloric restriction they can eat whatever they want they don’t tend to lose weight. And they don’t tend to gain weight beyond the animals that
are on standard diet. This is all been published, which is why I’m going through it really fast. Okay, so, the animals
get surgery on day zero. The tumor cells are
intracranially implanted. On the third day they are scanned, randomized to a treatment arm. We test BHB and glucose levels and the animals are sacrificed
when symptoms appear. Symptoms appear in this
model, reproducibly about twenty four hours before they are going to die. So, I’m an animal lover, I do not intend to let animals suffer. When can do invivo imaging, to follow the growth of the tumor. And this is what we found. What I’m going to show you
in the next couple of slides is a combination of two
totally separate experiments. So, standard diet survival. KetoCal survival. So there is an increase in survival. When you add temozolomide which is the chemotherapy of choice
for brain tumors now, survival is better, standard
diet of chemotherapy than it was, standard diet alone. But, the KetoCal makes that better. But, this was what blew our minds and started to get patients calling on me and that might be okay for you clinicians but as a scientist when
a patient calls you it’s a little bit interesting (laughs) um, because all I can tell them is well, I can tell you it helps our mice. Y’know, um, so anyway this is is wholegrain radiation and the radiated animals do better than, uh, than the standard diet alone. But when we added KetoCal basically we cured nine of eleven mice. And the reason that we
know that this happened is we can follow the growth of the tumor this is just one of the animals, and here is one of the animals that was on ketogenic diet. I don’t know how well you
can see it in the back but this is a very, very bright tumor cell line. The tumor was there, starts to disappear. Comes up a little bit but by around day forty, all of the mice you could not see anything. And this is published work its in open access journals so feel free to look at it. Right around day one hundred and one, I decided that rather than continue to chew our budged to
have these little ladies running around their cage happy let’s switch them back to standard diet and see what happens? And we did that for
another two hundred days and the tumors didn’t come back. And, uh, the pathologist said there was no evidence of any active tumor in these animals when
they were sacrificed. So, we were unbelievably
excited about this. It does not mean we
think we can cure humans but in the very least
this was a step forward. This really kind of charges us up for looking more specifically at radiation plus chemo, plus uh, ketogenic diet. One of the things we found in our very first experiment was that it reduces ross. And we found this using
a different technique where we could only look at one timepoint and it was late. And I’m thinking this stuff
makes radiation work better but radiation works through ross, this makes 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 used this
L012 chemiluminescence and the closest we could come to looking was twenty four hours but at every timepoint the ketogenic diet reduces ross. So for years I’d stand up, give a talk and say if anyone in
the audience understands this please come talk with me. Cos we don’t. Uh, but we do now. We think. The other thing that we think helps is that it does reduce inflammation based on, uh, looking at cyclooxygenase 2 all the other speakers in
the past that mentioned anything about an inflammation said it did reduce inflammation. I’ve got some pretty tall shoulders to stand on with the previous speakers. So you can see KetoCal reduces the COX2. Radiation, it goes up, as you would expect. But the animals on KetoCal it holds that COX2 from
going up after radiation. 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 pluripotent
nature of ketogenic diet is unbelievable. When I first started I
thought it was snake oil. I didn’t believe it to be perfectly honest. It inhibits tumor growth. It enhances radiation in at least some of the chemotherapies we’ve looked at and others of course are
showing other chemotherapies are, um, also, made to work better. We’ve published this. It reduces peritumoral edema. It reduces inflammation. It reduces antigenesis. It reduces hypoxia. It reduces the expression of uh, hif1alpha and phosphorylated nf kappa b. It increases the anti
tumor immune response. So, we decided to focus on the radiation. And one of the things we have to do is say, okay, we have to go back to the invitro models, and see can we recapitulate
it so we can start to tease apart what’s happening. And it turns out that indeed we can. These are the mouse cells. We used a low amount of radiation which is too gray, inhibits growth. Low amount of beta hydroxybuterate. 2 millimolar not nearly
enough to inhibit growth but the two together
absolutely 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 recurrent
tumor so we add an ‘R’ to the end. So, this is a human cell line, again these are grown in high glucose. I’m taking glucose
totally out of the picture for the purposes of what we’re saying. And again, this green line is what’s important. Ketones plus radiation
better than either alone. So, the mechanisms of action of the ketones beyond glucose reduction. We think it has to do with
epigenetic modifications. So, everything I’ve shown you up to this point has been published. All of this now has not been published. My grad student just
got his PHD last month, and had his first baby, and moved to North Carolina, in one month. So we’re finishing up
some of those studies and looking to publish hopefully a little later this year. But, the epigenetic effects of the ketogenic diet that’s been shown by others are: Changes in histone acetylation. Beta hydroxybuterate
is an H-Stack inhibitor as you’ve heard before
by some very elegant talks in this meeting. There are changes in the non-coding RNAS specifically alterations in micro-RNAS, and there’s also probably alterations in DNA methylation which I won’t actually get a chance to tell you about. So for any of you who
are not bio-chemists, I am certainly not, which is why I need nice, easy cartoons. When DNA is de-acetylated, it’s closed, I think in cartoons, I think of DNA like this. And the radiation can’t hit it. When it’s open, it’s acetylated. Now there’s lots of space, radiation and chemo can damage it. So, acetylation, hystone
acetylates go that way Deacetylates go that way and BHB blocks that. So, that’s you want in a cancer cell. So, there’s lots of, or a number of H-Stack inhibitors that are actually in clinical trials. As most therapies, they
have various side-effects. But what’s interesting
is that a lot of times this, the hysto and deacetylates is, they act differently in tumor cells and normal cells. And that really follows
what we see with BHB. So, first we decided to look at, does BHB increase hysto
acetylation in our model? And, indeed it does. So, this is just a western blot for acetylated hystone 3K19-14. And you can see, this
is without radiation. This is with radiation. In the presence of BHB
you do get an increase in acetylation as you would expect. And that’s enhance even a little bit more when you add radiation into the mix. This is in our mouse cell line. 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 tumor
that goes along with the cells I showed you before, which is the recurrent tumor from the same patient. So, these cells are
from the patient before they were treated. Again, BHB increases, hystoacetylation in these cells as well. So, what about DNA damage? Gamma H2AX is a marker for DNA damage. It sticks to the end of damaged DNA and it basically tells everybody else “okay come here guys we
need you to repair this,” so the more Gamma H2AX there is the more damaged DNA there is. As the DNA is repaired the Gamma H2AX goes down. So you can see, it’s kind of low in the absence of radiation and the BHB doesn’t
really make a difference. Radiation damages the DNA BHB, there is more damage. So what’s happening we think is the BHB is actually
inhibiting DNA damage repair. Oops, sorry, wrong one. Again, the mouse cells, the human cells, it’s even more pronounced
in the human cells, where there’s very little Gamma H2AX, very little damaged 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 cells, thought to be cells in the glioma that are more resistant to radiation, more resistant to Chemo, probably reside in a
hypoxic area of the tumor and one of the thought’s
is that these cells kind of repopulate the
tumor after treatment. Although, that’s up
for a lot of discussion which we can get into later. But we did have a talented
high-school student in my lab that actually looked at glioma stem-cells. And these cells were very kindly provided by Dr. Brent Reynolds from Florida. So on Day Zero she seeded the cells. On Day One we treated them
with betahydroxybuterate. The next day they got
four gray of radiation. And then, seven days
later the neurospheres where dissociated and counted. So this is one of the cell lines, L0 and, just looking at live cell count, so betahydroxybuterate doesn’t hit it. Radiation inhibits. But the two together, inhibit better. So betahydroxybuterate is also enhancing the effect of radiation
on glioma stem-cells. Oops. It is hard with
two different buttons. So here’s the second
glioma stem-cell line, from a different patient. And again, the magnitude is just slightly different but the effect is the same. The betahydroxybuterate
does enhance the effect of radiation in glioma stem-like cells, as well as human glioma cells, and mouse glioma cells. Here we are looking at DNA damage, again, using Gamma H2AX and in the presence of betahydroxybuterate we do see 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 repair. So this is looking at, is there DNA damage? And this is looking at one of the proteins involved in the repair of that damage. So, more of this is more damage. Less of this is less repair of damage. So, when you look at RAD51 again in these glioma stem-cells it’s definitely down
in the presence of BHB. So, looking at all the cell lines I’ve been talking about so far. This is our mouse glioma cell line. This is our human glioblastoma cell line. This is the recurrent tumor cell line from this patient. So in other words, these cells are the tumor that came back after radiation and temolozide treatment. And this is that first cell line I told you about which is extremely aggressive, came from the fourth tumor
that this young man had. So in all cases, the RAD51 is reduced. And the acetylation is increased. MRE wasn’t quite statistically significant but it approached significance. So, essentially, what we think is happening in radiation, because I said we where
really 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 reducing
the DNA damage repair. And that also kind of makes sense with how it seems to help some of the alkylating agents. Is that we think that, the epigenetic effect is actually causing a reduction in the genes
that are involved in DNA damage repair and we actually have dated that some of the
other proteins involved in DNA damage repair are also reduced. So now to switch gears a little bit 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 species, they’re kind of promiscuous and it can actually reduce the expression of a number of microRNAS. Sorry, I apologize, a number of proteins. But the repression usually isn’t extreme. But they’re still pretty active, in fact some people are actually looking at these as potential therapeutic agents. So in collaboration with
an excellent researcher, Dr. Nell Said at the
Imperial College in the UK we sent her tumor tissue from our mice and she did an RNAS seek analysis of all MicroRANS and she came up with a whole bunch of MicroRNAS that are
differentially expressed. When the MicroRNAS are up, the proteins that they, uh, affect are down. Okay, so up means we are
turning down proteins. And there’s just a whole bunch of ’em. Uh, and quite a few of these are involved in DNA damage repair,
interestingly enough. But what’s really interesting is that when I think
of meatabolism I think, well most of them kind of should be metabolism or maybe epigenetic stuff. Turns out if you look at the hall-marks of cancer, pretty much every hall-mark
of cancer is affected. And this is the only difference here. This one I’m guessing will be when they figure out more MicroRNAS that are
involved in genome instability. So, when you think about it we all think of metabolism, well I guess not in this room, but, most rooms, when often talking they
think of metabolism, okay it’s the food, it’s the energy it’s the glucose it’s all that stuff. Well in fact, it’s a whole lot more. Metabolism is gonna hit
every 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 changes BHB is an H-stack inhibitor. That’s been shown by others, it increases hystoacetylation
in our tumors this we think causes 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 how
it’s potentiating radiation. 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 tissue, we are working on this in cell line’s and she’s working on it in cell lines. We’re showing that changing the MicroRNA does indeed change the expression 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 just recently moved to
Phoenix Children’s Hospital and now I’m kind of
turning most of my work towards pediatric tumors so, of course, we’re really
interested in that. And, uh, there’s also some data that I don’t have time to talk about, we’ve got just very little of it that suggests, that BHB and NIKD might actually also affect 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 because
he’s totally trained. Uh… (laughter) ..by me, so all of his bad habits 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 scholarship money
than I can ever hope to get for my research. And then there where other people that were in the lab that have contributed to all of this. Acknowledgements, as you
all know when you write to the NIH for funding. Diet is a four letter word. Uh, luckily there are other foundations 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 letter word. 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)