Dr. Adrienne Scheck – Tumor Metabolism and the Ketogenic Diet

Dr. Adrienne Scheck – Tumor Metabolism and the Ketogenic Diet

July 27, 2019 7 By William Morgan


– 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)