Welcome, everybody,
wherever you may be.
Our speaker today is
Doctor John Turcci.
John earned a Bachelor of
Science in Biochemistry from
Kemson University in
1985, did his pH.
D in biochemistry at the
University of
Missouri in 1990.
Did his postdoc training at the
University of Rochester with Bom
Bom Bot.
Robert from Bob Bum Barra
studying the million Okazaki
fragment processing.
John began his independent
research career at Wright State
University in 1993,
studying the recognition and
repair of cisplatin DNA damage,
focusing on DNA damage sensors.
In 2005,
he joined the Indiana University
School
of Medicine,
where he is now Professor of
medicine in
the Division of
Hematology and Oncology.
And he's professor of
biochemistry and molecular
biology.
He holds the Tom and Julie Wood.
Family Foundation endowed
professorship in lung Cancer
Research,
and he serves as the executive
director of the Tom and Judy
Wood Center for Lung
Cancer Research at the Indiana
University Comprehensive Cancer
Center.
His research continues to be
focused on DNA damage sensors as
therapeutic targets
for cancer treatment. Now,
before I let John start telling
us about
some of his labs recent work.
I just want to remind all of you
in the audience to put questions
for him.
Any questions you might have in
the chat box?
And we will ask them on your
behalf after the seminar is
finished.
Without further ado,
I'm going to hand things over to
John.
Alright. Thank you
Karen for that.
For that introduction, and to
everyone, tune will Ken and
Karen for the
invitation to present today.
And you've heard the title.
You know who I am from that
intro.
Quick disclosure and disclaimer
1 protects the university for
me.
The other protects me from the
university and thinking about
this presentation,
I was parking back to when I
last presented at the NIH and I
didn't think
it was that long ago,
but in fact it was in.
11 and this is what the group
looked like back then.
You can see me in the back there
a much younger person.
In fact,
this was the President at the
time
looking pretty good as well.
Fast forward to where we are
now. Boy, how things have
changed.
So what I'd like to do is is
take this first slide to
acknowledge the people who
have done the work in case we
don't have time at the end to
get to them.
I'm going to tell you about two
stories today. One.
Focused on the RPA protein.
And much of that work was done
by Matthew Jordan.
Here a postdoc along with Pam
van der Beer Caroza, a new
graduate student.
Jess and Aditi have joined the
lab also on that project as
well.
The second project I'll tell you
about is targeting the coup
protein double strand
break repair largely done by
postdoc Pam Mendoza,
again along with Pam and Joy
Garrett, who does a lot of our
ionizing rad.
Work as well.
We have great collaborators.
We'll do.
A lot of chemistry
along the way.
NAV navandi is our chemist.
He's a Co and many of our
grants and and performs all that
work.
As I mentioned,
Joe Dinlock does all the
radiation with
us and we have a cadre of
physician scientists at the IU
Cancer Center who
really provide nice perspective
for us along the way we.
Also acknowledge Katie Quelzak,
who is the CEO of Nirex
Biosciences,
a startup that.
Way back in 2009,
when we thought we were
targeting the
nucleotide excision repair,
we named the company NIREX.
Reality is we've moved a little
bit past that and we'll talk
about damage centers
in multiple other contexts.
And so one of the questions we
ask at the beginning is can we
target replication,
stress sensors for
cancer therapy?
And just to put this in
perspective, in a typical
mammalian S phase,
we're going to have about 50
million Okazaki fragments
synthesized and
processed.
And that's just a normal
unperturbed replication event
going on.
And if we work at
99.9% efficiency.
Those level of
efficiency, right?
We would still expect 50,
000 potential deleterious events
in the
context of trying to replicate
those chromosomes.
And so this presents a
opportunity where in cancer
cells,
if you have alterations in how
these events are occurring,
you would get even more of these
replication stress induced
events that.
Could potentially be
used for targeting.
The reality when you think about
DNA replication.
This is from cold spring harvest
model and everything is
wonderfully coordinated.
DNA coming into the heel.
A case leading strand being
synthesized
continuously.
Discontinuous lagging strand
incredible orchestration of
events.
Most of us in biology know that
this is often not the case.
And so it got me thinking a lot
about coordination of events and
then these
things called
Phantom traffic jams.
And so if you've seen these,
if you're on I-5 on the West
Coast or
I-65 in the Midwest or I-95 on
the East Coast?
Everything seems to be going
along just fine,
and then all of a sudden one car
taps his brakes.
That causes the car behind and
the taps their brakes.
Ultimately what happens is you
move forward and you say there's
nothing that
actually caused this traffic jam
and you can take this a step
further and say this
is a DNA replication
event and so on.
Your leading strand you
encounter an A basic site or you
encounter a nick in the
DNA or some type of DNA damage.
And that results in this traffic
jam. And if you want to
incorporate the
lagging strand model,
it's just the opposite.
Side of the highway,
where they slow down too,
just because they're watching
the tragedy on the other side.
And so normally you know traffic
stress is not necessarily a bad
thing.
You can get through it.
The real problem is when you
have traffic catastrophe and so
these are the major
events that caused, you know,
loss of life and and dramatic
things that
we'll see in the context of
replication, very similar
phenomenon.
And so when we think about how,
how to control these events.
It really gets down to the DNA
damage and replication stress
signaling pathways,
and it's led by a
series of proteins.
These ones shown in yellow that
are the DNA sensors and these
recognize the
problems in the DNA.
And so for this case a stalled
replication fork.
If you have RPA binding to the
single
stranded DNA that is sensing
that alteration in the DNA
structure and can
signal to.
Downstream kinases
that will then.
Regulate cellular
pathways and processes.
If we look at it in the context
of the coup,
protein similar binds to DNA
double strand ends.
It senses that structure.
So these are structure specific
binding proteins.
It signals to the DNA dependent
protein kinase which then goes
in phosphorylates
all its target proteins to
elicit the regulation of these
pathways and
processes.
Another sensor is in fact the
PARP protein.
It works a little differently in
that it binds to its DNA sense,
a DNA Nic,
for instance,
but instead of signaling to a
downstream
kinase.
It can auto ADP
riboscolate itself.
We'll see a lot of these events
where you're signaling the
sensing paramosolate
itself,
which then leads to its
association
coupled to activation of DNA PK
which auto phosphorylates and
results in its
removal of the DNA to allow the
repair processes to occur.
And so coordinating
all these events.
Is pretty important
and we thought.
The potential exists that we
could target some of these for
therapeutic
interventions.
So the two I'm gonna tell you
about today are in fact the RPA
protein,
its interaction with single
strand DNA will start there and
then move over to
the coup project that we're
we'll discuss as well.
So RPA is the human single
strand DNA binding protein.
This is the pave structure
published decade plus ago,
that shows that DNA single
strand being wrapped around a
series of OB folds and
we focus on this OB fold B.
It's a major component of high
affinity interactions.
With single strand DNA and when
we performed a high throughput
screen a
number of years ago,
we're able to identify a series
of small
molecules that could bind to
that OB fold B and block the
protein DNA interaction.
So we're showing two compounds
here this tdrl 551,
which is the predecessor
to neurx 329.
It differs only by an addition
of this propyl morpholino group
making an amide
bond instead of this
carboxylic acid.
In vitro.
Either one can inhibit the RPA
single strand DNA interaction
and so just in a
test tube reaction purified 30
base single strand DNA purified
hetero
trimeric human RPA with
titrating increasing amounts of
the inhibitor we
can block that RPADNA
interaction this prep was.
Actually purified out of E coli
in an untagged version,
and so we ran through a
traditional purification and
depending on how good or
bad we are in that purific.
We always get contamination of E
coli SSB which is shown as this
bottom shift in
the DNA and what this shows us
is in fact that these compounds
are relatively
specific for the RPA protein and
not necessarily blocking the
single strand E
coli protein
interaction. So we have.
Pretty good specificity,
pretty good potency in a test
tube.
We can actually do the same
experiment in cells and measure
the RPA DNA interaction
through a proximity
ligation assay.
And either leave these cells
untreated or treat them with the
RK inhibitor for two
hours and we can get a
statistically significant
decrease in the total number
of RPA foci as a function of our
RPA inhibitor.
And if we only select the cells
that are PCNA positive, so.
Those cells that are likely in S
phase, you get more foci per
cell,
but we can still decrease that
dramatically as a function of
our
inhibitor.
So the S phase cells or foci for
our PA, as you might imagine.
Associated with replication and
we can decrease that number. You
might imagine.
Then,
as long as we're blocking RPA
interaction
with DNA in these cells,
this is an H460 non small cell
lung
cancer.
As long as those compounds can
get into
the cells, we can induce cell.
Death.
And so in this metabolic
viability assay,
the 551 compound does not get
into the cells very well.
Largely a function of this
carboxylic acid.
But when we put the morpholino
groups on in the 329.
Or the 2004 derivative,
which is essentially replacing
this
iodine with the bromine.
We get very potent cell killing
in a
pretty tight dose
response curve.
And so we can block the RPA DNA
interaction in test tubes in
cells
induced cell death.
Katie Pozak at Nurex did a whole
series of single agent anti
cancer activity
across a number of cell lines.
And the results demonstrate that
the mechanism of action is
largely tumor
agnostic.
And so we can kill lymphoma
cells, bone cells, breast
cancers, lung cancers,
as well as ovarian cancer.
We see a pretty interesting
distribution
along the different tumor types,
with IC 50s ranging from the low
single
digit micromolar up to the 10
and 15 micromolar, suggesting
that there's some.
Factors that are contributing to
differential sensitivity within
any given
tumor type.
The other thing we noticed was
and a lot
of these cells,
we get an incredibly tight dose
response
curve and this is.
Here in the bottom graph with
the A 2780 ovarian cancer cell
line and the GCT 27
testicular cancer cell line
where we can hit these things
with about four
micromolar drug and elicit
hardly any change in viability
and we go up to six
and a.
Half micromolar drug and we
killed all the cells.
And so this incredibly tight
dose response curve is really
indicative of a
threshold event.
And so as we're thinking about
thresholds and you're thinking
about RPA?
Your mind naturally migrates to
Yuri Lucas's work,
and the idea of RPA exhaustion.
And so we have adopted his model
for our own use and essentially
term it chemical
RPA exhaustion.
And so as the model states,
we've got a certain amount of
RPA in the cell that can hold
the amount of single
strand DNA that you generate.
Let's get that back.
There we go.
For unperturbed replication,
you generate a little single
strand DNA.
You have plenty of RPA.
There for that oncogenic
replication stress.
So now you've got.
The amplification.
Maybe an EGFR mutation,
anything that drives replication
stress
generates more
single stranded DNA.
But you still have enough RPA
protection to handle all those
binding events,
and only when you add adna
damaging agent cisplatin, Dr.
Rubicon Campbellsen whatever is
going to generate more single
strand DNA.
Do you get above this threshold
and end up with a replication
catastrophe?
Or cell death phenomenon.
And So what we can do chemically
is treat
with that low level
of our RPA inhibitor.
We lower the threshold a little,
but there's still enough to
cover the
oncogenic replication stress.
So this would be analogous to
that like 4 micromolar treatment
where we haven't
induced much cell killing.
But if we just put in a little
bit more,
take out two more of the RP as
get below that threshold,
this is where we get replication
catastrophe. So Yuri Luc.
Showed this genetically,
we can do it chemically.
And then the reality is the
difference in the therapeutic
window is defined by how
much RPA we can block to keep
the single strand DNA in the
oncologic replication
stress versus the unperturbed.
And so we can actually hit that
therapeutic window in mice.
And so if we take the non small
cell lung cancer cells,
put them subcutaneously into a
mouse, treat them with.
The inhibitor everyday
for a few weeks.
Or a vehicle.
We can get a statistically
significant decrease in tumor
growth and tumor weight
at the end of the experiment.
So we have a safe margin of
treatment for
these mice with an RPA
inhibitor, indicating that we
can hit this
therapeutic window available
through the chemical exhaustion
model we've also.
Taken this at the patient
derived models, the ones I'll
show you here are an
ovarian cancer spheroid model
that we developed.
From patients that present at
the IU Cancer Center.
We get tissue from them largely
from debulking surgery and can
create a series
of.
Tumor models.
Let's see if this, you know,
these movies don't wanna start.
But that's OK.
So what happens here is if these
were going,
you would see that the RPA
treated tumor spheroids induce a
significant amount of
cell death compared to the
untreated. As these are
monitored over time.
So we have both in vivo activity
in non small cell lung cancer
and in patient
derived human ovarian cancer
spheroid models.
And so then the question is,
how is this happening?
And I'm going to put some work
by Matthew Jordan.
We published this last year.
In scientific reports into one
single slide,
and the idea was if we're
blocking our PA interaction with
DNA,
we would necessarily block the
ATR activation and could we
recapitulate this
in in vitro in a
reconstituted essay.
So Matthew purified the proteins
necessary.
RPA top BP1,
which is an interacting protein
that
works with ATR and ATRIP to
signal to all of these
downstream events.
What we looked at is a
downstream event was the
phosphorylation.
Of P53 here,
as well as the phosphorylation
of RPA.
And so Matthew was able to show
very effectively that with the
right
combination of DNA, RPA,
single strand DNA, top BP1,
aatrip,
we could get robust
phosphorylation of P53,
robust phosphorylation of RPA on
serine 33, as well as three
inning, 21 that were.
All sensitive to the
ATR inhibitor VE 821.
And so we have reconstituted the
ATR signaling pathway, largely a
function,
and this is based on azee
sanjar's work at a University of
North Carolina,
he published this decade plus
ago describing this.
We adapted that and then asked
the question how does our RPA
inhibitors work
on these phosphorylation
events in vitro?
We're able to again effectively
show in a reconstituted system.
We could increase the
concentration of either the 551
or the 329 compound and
potently inhibit P53 phos.
Correlations.
So the inability to find that
DNA leads
to the downstream inhibition of
the ATR kinase.
We published similar
results with.
Showing a decrease in
phosphorylation of RPA itself,
both at serine 33 and three in
21, as well as we monitored the
post
translational modification of
RPA via both phosphorylation and
acetylation.
And so they have impacts on the
ability to block its downstream
signaling.
Probably my most favorite
experiment from this was the
analysis where we looked at
inhibition as a function of ATP
concentration,
and this stems from
conversations where when we use
the V EA21 inhibitor,
we have to use it at 10/20/30
times its published I.
50 values and when Matthew went
back and looked at the very
first papers were
Vertex Therapeutics
was developing these.
He came into my office and said,
did you know that when they
developed
these compounds they were
running their kinase?
Case assays for 24 hours at 25
micro molar ATP and I didn't
know that.
And we would argue that's not
tremendously physiological,
nor is a 24 hour kinase assay
spectacular in in the ensomology
field.
But they got very
potent inhibitors.
But the reality is their
inhibitors are very sensitive to
ATP concentration.
So we can get great inhibition
with DEA 21 at 40 micromolar ATP
in the reaction.
But as we increase it.
The effectiveness of that kinase
and it's a competitive ATP
inhibitor.
Goes down as you might imagine,
whereas the ability of our
compound,
the RPA inhibitor,
is largely unaffected by ATP
concentration.
So we really like this model.
We want to inhibit the kinase,
maybe going after the ATP active
site and
and developing these things with
high potency,
but at very low ATP
concentrations, more
physiological might be a
function
worth looking at.
So we have the ability to
inhibit the ATR kinase.
RPA inhibition of
single strand DNA.
The question is what's happening
at the replication forks,
and so Matthew did these single
molecule molecular combing
experiments ultimately
taking the A549 non small cell
lung cancer cells,
treating them with our inhibitor
or without vehicle control and
then doing a
molecular combing
experiment where you?
Do pulse labeling with iodo
deoxyurating for 30 minutes
followed by
Chloroxyuridine.
Isolate the DNA.
Spread them on slides.
Probe for these with specific
antibodies that give you these
tracks and you can
measure.
Track length and what he showed
very effectively was that the
RPA inhibitor
induced replication
fork slowing.
So we got shorter forks as a
function of treatment with the
RPA inhibitor.
It was statistically
significant. Modest. We go from,
you know,
10 microns to 8 microns,
and so maybe a 10 or 20%
difference where
we saw in much more dramatic
effect was in fact the
replication restart assays.
And so in this case you do the
same type of analysis treat with
IOTA the oxygen.
But now we induce a replication.
Installing event with
hydroxyurea and you put in mirin
which is an inhibitor of the
nuclease MRE 11 that degrades
some of these replication forks.
So we're blocking that event and
then 30 minutes after you wash
it out and add
your chloride in plus or minus
our inhibitor.
So the ability to restart is a
function of this chloride
exothermic deoxyurine
ratio.
And what we see is that
inclusion of the RPA inhibitor
in this second step gives
us a dramatic decrease in the
ability to restart.
Those replication forks,
so we have events occurring at
the
replication fork that could
potentially be impacting these.
Downstream events.
So up till now we can inhibit
the DNA
interaction.
We can block ATR signaling.
We get single agent anti cancer
activity but pretty modest in
vivo from that 8549
experiment I showed you.
And so coupled with that and the
60 cell
line screen that Katie did with
those varying IC 50s,
we thought you know is it
possible that we.
Could identify the
genetic alterations.
That would increase sensitivity
to an RPA inhibitor.
And so we conducted a CRISPR
knockout screen.
And it worked wonderfully with
the 329, because we're able to
treat cells with a
low enough dose that they
maintain their viability and
they're right on the
threshold.
So any little increase in the
single
strand DNA generated is going to
give you cell death. So we,
knocked out a whole series of
DNA damage response genes, 230
in total.
17 of them gave
us positive hits.
That showed up in the ability to
increase the sensitivity to an
RPA inhibitor and
so in this case,
the mutations or the alterations
in these
DNA damage response
and DNA repair genes.
Increase the amount of single
stranded DNA such that it
exceeded that threshold
and gave us the cell death and
so the first one is Okazaki
fragment processing.
This is going to take me back
home to work.
I did 30 years ago.
Next is DNA double
strand brake repair.
And there's also DNA
replication. In fact, #4 this is
in fact,
DNA polymerase epsilon.
Really interesting hit and we're
investigating that,
but I'm going to talk about
these first two where we
validated these by making
CRISPR knockouts.
And so the Okazaki fragment
maturation
function is in fact
the Fen 1 gene.
And this goes back now to my
time in Bob Bambara's lab where
we reconstituted
Okazaki fragment processing.
With fan one and I'm not sure
Bob has ever.
Forgiven me for the contributing
to the incredibly poor name
selection.
When we first found this
protein, we called it the calf.
5 to 3 nuclease,
which didn't really stick.
And then it went by RTH rather.
Only when it was called Fen One
did it really catch on what
we're able to show
here is and Bob and I decades
ago implicated it in Okazaki
fragment process.
A lot of balakrishnan's work a
little later really clarified
the mechanism for
help and one is involved in
removing the RNA DNA,
hybrids and extended fragments
from polymerase,
epsilon and Okazaki
fragment processing.
And So what we did was create a
series of N1 homozygous shown in
red and
heterozygous knockouts.
Each one gives you an increased
sensitivity to the RPA
inhibitor. And so in our model,
we're generating more single
strand DNA as a dysregulation of
these Okazaki.
Fragment processing events.
You can do it through
other mechanisms.
DNA2 and PIF,
one that can ultimately maintain
viability,
but when we block the RPA in
activity,
we get an increase in a
significant increase in
sensitivity.
And so that's consistent with
being at the replication for DNA
replication,
stress increasing sensitivity to
an RPA inhibitor, the second
one.
Double strand, break repair.
We publish this with Sharon
Cantor in her paper a number of
years ago.
Where we demonstrated that in an
ovarian cancer model isogenic
that where the
patient presented with abraca
null mutation,
we get very good sensitivity to
the RPA inhibitor.
When we complemented those
cells, actually we didn't do it.
Someone else complemented those
cells and we showed that that
increased resist.
To the RPA inhibitor and so at
this point, it positions you
right at the lagging
strand positions you in double
strand, break repair and really.
The model of
synthetic lethality.
And gap protection driving the
PARP inhibitor sensitivity in
Bracha deficient
cells.
And so the model that Sharon can
originally espouse and
discovered in his
tremendous data supporting to
date is the idea of the single
strand DNA gaps that
are generated.
If you lose Bracha one or Bracha
2,
or if you inhibit PARP and then
you require other ways to.
Protect those types of gaps,
and RPA is an important feature
in that.
So then we ask the question,
how would an RPA inhibitor
combine with
paap inhibition in a Bracha
deficient cell line?
So we took this directly into
mice having the inhibitor in
hand.
So we did the MBA 436 Bracha 1,
deficient triple negative breast
cancer
implanted these into mice and
then treated them daily with
either the RPA
inhibitor alone,
the PAP inhibitor elap rib or
the
combination.
And we are able to essentially
flatline tumor growth,
as Katie Poilzak likes to say.
With the combination in vivo in
mice indicating that therapeutic
window exists
to exacerbate park inhibitor
activity and infect stimulate
the anti cancer activity
of RPA. In this combination,
I'll show this picture on the
right here,
which is essentially a similar
model, the same exact cells but
now in.
A live cell imaging experiment
where we can use a single agent
activity in
combination where we monitor
confluence by taking pictures
every four hours.
Over A7 day period and you get
very very similar results.
Cells grow bot
fine on their own.
You hit them with anhibitor.
They grow fine for about two
days,
at which point they then level
off the RPA inhibitor works a
little faster
initially,
and then actually loses
activity.
We know this is a function of
metabolism, but the combination
essentially flatline
cell growth in
this model as well.
And so then the real question
becomes, how are these
combinations affecting
replication for dynamics?
So Matthew went back to the.
Microscope and combing
experiments. And did this
experiment where essentially
we pretreat with a PARP
inhibitor or not we include the
NEURIX 329 RPA inhibitor
or not or mirin then measure
chlorideoxyuridine and then do a
post
replication S1 nuclease event to
look for single stranded D.
That's generated during these
process, so I'll walk you
through a few of the
really important ones.
We're able to show.
A PARP inhibitor induced
increase in replication for
expeat other people.
Have observed this as well in
different cell lines and so in
this MBA 436 barcode
1 deficient we see that increase
in replication for speed.
So you go from about 5 1/2 up to
about 6 1/2,
and that increase is completely
dependent upon RPA.
When we put our RPA inhibitor
in, we drop this back down to
its baseline.
So the PARP induced increase in
fork speed is dependent upon.
RPA.
Next,
RPA inhibition of park induced
fork
acceleration.
Is blocked by Mirren and so
Mirren is that Emery,
11 inhibitor that blocks the
nuclease activity and so that
shows that the fact
that we bring this back down in
the presence of mirin suggests
that there is
a nuclease event that RPA is
inhibiting and so without R.
You get that degradation
to look at that.
We did the S1 dependent
degradation events of the single
strand gaps.
And what you see in untreated
MBA 436 cells?
It's just the Bracha 1
deficiency gives you the
generation of gaps during these
replication events.
We put an RPA
inhibitor in there.
It goes down a little bit of
just the RPA and again you still
get some degradation
but not as significant.
The PARP inhibitor increased in
replication for speed again
gives you a
massive amount of these single
strand gaps which is even lower
than these,
suggesting that those gap events
are occurring.
The real important result was
that the RPA inhibitors renders
the DNA
insensitive.
To S1 and So what that tells us
is that we've already had
events.
Resulting in the degradation of
this DNA by not having RPA there
to protect it.
So RPA protects these gaps from
degradation and we can
chemically block
that protection.
So then the real question is,
can we take these local events
that are
happening at individual
replication forks and can these
lead to global effects?
And what we did was measure
micronuclei and chromosome
pulverization.
As a measure of genomic
instability and got some really
interesting results
because if you can see and hear
these NBA 436,
you get these little micronuclei
indicated by the red dots in a
fairly
high frequency when you put in
olapy rib, which is a relatively
non.
Specific PARP inhibitor inhibits
part one and Part 2.
We get a significant increase
sarooparib, which is a much more
specific part.
One inhibitor gives us an
increase as well.
The neurix 329 compound doesn't
affect the the fraction of cells
with
micronuclei much at all.
Compared to the control and in
fact, when you do the
combinations,
there's no significant increase
by the addition of the 329
inhibitor.
So you have the same relative
number of the micronuclei that
are forming,
but we know when we add the
combination we get a significant
increase in cell
death.
And so as we measure that
through
chromosome pulverization.
So now the idea of massive
chromosome shattering events
that we can detect in
these metaphase spreads,
if we take a DMSO treated cell,
you see nice chromosomes.
329 RPA inhibitor doesn't change
it much at all.
Elap rib starts to give you a
certain degree of chromosome
shattering.
The percentage goes up from
three or four to 10, and the
degree is pretty.
Obvious,
but when the combo of the PARP
inhibitor
plus the 329 we get massive
chromosome shattering events.
And So what this suggests to us
is in fact there are activities
going on in
these micronuclei.
They themselves are not
necessarily giving us.
The cell death,
but the dysregulation of events
within
micronuclei.
Maybe it's repair replication,
RPA protection of those Dna's as
well as
nuclear can give us this larger
effect. And so we think these
local events can
really have dramatic impacts.
Local events at the replication
fork on
larger global things.
So this leads us to the model
for RPA dependent GAAP
protection.
I was at the DNA gap meeting
Keystone Meeting 2 weeks ago.
Go in in Seoul, South Korea.
And I like to say there that,
you know,
we're pretty much
strand agnostic.
And so whether you get a gap in
the lagging strand through
differential
Okazaki fragment processing or
leading strands,
say be running into a damage
event and then having a primpol
protein come in to
generate gaps,
it really doesn't matter to us
necessarily anything that
generates these gaps is.
Going to rely on a higher
concentration of RPA to protect
it,
and that gives you a
therapeutic window.
So in the classic model,
it's Bracha deficiency gives you
gaps.
You had a PARP inhibitor that
gives you even more gaps.
You're even more reliant on RPA.
We think without RPA protection
then you get degradation either
of the single
strands through the MRI,
11 tip nucleases or potentially
the
single strand DNA.
That's that template and
ultimately giving you DNA double
strand breaks.
Probably a lot of people in the
audience have contributed to
this entire table
that Matthew put together of.
The different mechanisms by
which single strand gap
induction can occur.
And so top on the list is spend
one, but April Beck and Paul
Theta and a whole
series of other things.
You go down the bottom and
there's
alcalaading agents and platinums
and any of these things that
give you DNA damage
events and a reliance on RPA to.
Single strand DNA we think is
worthwhile to try to treat with
an RPA inhibitor.
So that's where we stand on the
RPA project.
I'm going to switch gears in the
last half of the talk and and
jump to our coup
project where essentially we've
taken a very similar role.
We want to understand how the
pathways work and is it possible
to target this
protein DNA interaction?
So we're showing it here in,
in the Walker.
Model.
Published 20 some years ago.
Now of how KU interacts with a
double strand DNA and literally
the DNA threads
through this molecule to bind to
that end of DNA.
The question we're asking here
is can we target these specific
events in lung
cancer therapy? And so again,
just to throw out a few numbers,
there's going to be about 225,
000 people in the US alone that
are going
to be diagnosed with L.
Cancer this year,
160 thousand of those are going
to get
radiation treatment as part of
their therapy.
135,
000 and get platinum based
chemotherapy
and if you put them together
about 60 will get combined
platinum.
IR.
That combined chemoradiation
therapy is about the same number
that are eligible
for IO therapy based on the 30%
of patients that can be treated
with that.
So the reality in this treatment
scheme of patients with lung
cancer is about 200,
000 patients are going
to be treated with.
Therapeutics that
induce DNA damage.
Whether it be IR or platinum,
you're getting adna damaging
therapeutic.
And so it becomes important to
understand how these work and a
lot of this was done
in collaboration with our
clinical colleagues. Shadi Jalal
and Katie Sears,
thoracic oncologist and a
pulmonologist to see and treat
lung cancer.
All the time they actually make
fun of this picture because they
say none of
their patients that have lung
cancer show up. As you know,
25 year old muscular individual
with massive tumors.
Understanding how these double
strand breaks induced by
ionizing radiation are
important,
and clearly we're going to focus
on the
non homologous end
joining pathway.
But I put this up and just
Kersey, the graduate student,
made this slide for us to really
highlight the advances we're
seeing in
understanding homologous
recombination, great advances in
micro homology mediated
or feta mediated repair.
These alternate end joining
pathways in single strand
annealing.
There's been really a lot of
activity and we're very
interested in understanding
how all of these proteins.
Play a role in that.
Prepare as well,
but if we're targeting the coup
protein
that focuses on non homologous
end joining pretty closely and
it got the
idea of if cou is binding to DNA
ends, you can absolutely get one
through a
ionizing radiation induced
double strand break.
But there are a lot of other
ways to get things that look
like ends of DNA that
who could bind to.
And so one of them is if you're
on a
leading strand
replication, you hit.
A.
Some type of damage,
say a platinum lesion.
And then you get fork,
regression and creation of the
reverse
chicken foot model.
This thing here looks like a
double
strand.
End that coupon.
Bind to if you're replicating in
the leading strand here and
going through a
nick,
a single break in the phosphorus
backbone.
This creates A1 ended or single
sided double strand break.
This end essentially creates
something that KU could bind to,
and if you consider lagging
strand gaps in degradation of
those to create double
strand breaks.
Again this thing looks like a
single
strand.
I mean a double strand end.
That the coup combined to and
any of these can be processed by
nucleases to
generate single strand overhangs
that then RPA may bind to as
well.
And so this idea of keeping
everything separate from double
stranded single
strand binding might
not actually be true.
And so while all that's
interesting, we're going to
focus on this ionizing
radiation,
double strand break coup is
going to
slide on to that end and recruit
the DNA PK catalytic subunit,
which then catalyzes an
important auto phosphorylation.
An event that leads to its
dissociation to where the other
proteins like East,
4X or CC-4 xlf can come in to
ultimately rejoin that DNA end.
But this auto phosphorylation is
something we're going to focus a
lot.
And so the question is,
can we make small molecules that
do this?
The answer is yes.
Again, our chemist.
We identified an incredibly
horrible compound, X-80 with low
affinity,
low potency, low solubility.
And so now we made
about 400 derivatives.
Largely modifying this
carboxylic acid here to create a
compound 68 and 1:49
that in a very similar assay we
can show blocks the protein DNA
interaction of
coup with a double strand DNA in
a test tube and that necessarily
blocks the
kinase activity so in.
An in vitro kinase activity we
can show a very potent
inhibition of that
phosphorylation event catalyzed
by the DNA dependent protein
kinase.
So we showed that these
compounds bind to the protein.
And not to the DNA.
We're not looking for DNA
interaction agents at this
point.
And so another derivative 245
which contains a tetrazol here
and replacing
this carboxylic acid.
Shows very nice increase in
stability, the thermal stability
of that protein as
a function of treating with this
this drug.
So the compound binds to the
protein and makes the protein
more stable,
so that when you increase the
temperature you get less
denaturation and you get a
thermal stability shift showing
that the compound.
Absolutely interacts with the
protein. This has been confirmed
using isothermal
calorimetry from
some colleagues.
Charbonnet in Paris,
who has done this on some of our
more
recent inhibitors.
Let's add a copy at University
of Illinois. Chicago showed that
we can,
in a reconstituted assay
measuring non homologous end
joining so purified
proteins in a test tube can
block the joining.
That results in these multimer
formations with increase in
concentration.
Block not joining and in a
reconstituted system in vitro we
can block the auto
phosphorylation of DNA dependent
protein kinase and this is
measured through a
phospho specific antibody made
by Susan Lees Miller's group at
the Serine 2056
and so as the
experiment goes in.
The absence of any DNA you get
no phosphorylation.
We put in our positive control,
which is just adding DNA to the
reaction
and the phosphorylates
wonderfully. We can put any one
of our inhibitors in
and they knock them
out completely.
Nu 7441 is in fact the catalytic
subunit inhibitor of DNA PK and
that blocks it as
well.
And just as a negative control
or neorex
329,
the RPA inhibitor has no effect
on the
phosphorylation events.
And so we can block the DNA
inter.
We can.
We know it's binding to the coup
protein and not DNA.
We can block nej and and
reconstitute a damage dependent
auto phosphorylation.
Perfectly reasonable.
And can we do this in cells?
And.
The answer is yes.
In a host cell reactivation
assay
measuring non homologous end
joining. So just transferring
into cells a
linearized plasma DNA.
Then when Circularized gives you
a
fluorescent signal,
we can get good nej efficiency
and each
of our compounds with concurrent
treatment gives us an in.
Of that rejoining event, again,
probably my most favorite
experiment is
this one,
where we can demonstrate on
target coup
inhibition and sensitization to
a double strand break.
Conducing agents.
So now bleomycin is used as a
RATHI, or mimetic,
to induce DNA double strand
breaks, and in these wild type
mifs you get a
pretty decent dose response
curve. When we put in our coup
inhibitor,
we get increased sensitivity to
that double strand break
inducing agent
consistent with.
Blocking the true
DNA interaction.
Blocking DNA PK auto
phosphorylation, blocking non
homologous end joining and
you get a higher level of
persistent double strand breaks
that kill those
cells.
We do this in a knockout KU 80
null method.
So now KU null cell line,
they're already hypersensitive
because
they don't have non homolexen
joining anymore and we add our
compound to these
kunosols and we don't see any
statistical increase in
sensitivity as a function of
inhibiting coup because there's
no coup in those cells. And so
this.
Really tells us that we're
getting on target coup in
activation in these wild
type cells and that's the
mechanism by which we're
increasing sensitivity.
And so Tammy Mendoza here has
done all this work now so very
nicely that the
2:45 I mentioned that tetrazol
derivative, she could put it
into cells and doing the
live cell imaging,
we get no effect of the 245
compound
alone, bleomycin starts to.
Kill cells on its own,
and the combination is in fact
much
better.
That works out good for us and
she showed very convincingly
that.
This effect is a function of
blocking the DNA dependent
protein kinase by measuring
the autophosphorylation.
And now, in an
immunofluorescence assay.
And so you get little punctate
foci when you treat with
bleomycin you get that DNA
damage and double strand breaks.
Jump the gun we put in our 245
inhibitor and we can completely
block that
quantified over here.
You put in that catalytic
inhibitor, you can block that as
well.
Exactly what you'd expect in
collaboration with Joy Garrett
and Joe
Dinlock did an ionizing
radiation experiment where she
showed the same
event.
And whoops that one.
And so I just kind of blow up
the radiation induced DNA PK
foci that she's
able to detect.
And then when we do the
concurrent
treatment of radiation plus our
inhibitor, we get a decrease
both in the number of
foci and the intensity
of the foci and so.
This was really nice because now
we have on target inactivation,
blocking the pathways that
increase sensitivity to these
double strand break
inducing agents.
The reality was those compounds
are very, very difficult to work
with.
The solubility
still wasn't great.
Cellular uptake was incredibly
poor and in fact required
treatment in serum free
media.
So every experiment involved
plating the
cells, letting them grow,
washing off the serum,
then treating with our drug,
then putting back what we need
to keep
the cells alive.
And so Navi went back to the lab
again and made a whole series of
compounds,
replacing that methylparole with
an oxen doll.
And this relatively modest
modification of this oxendole
really dramatically
increased our capacity in a
number of ways.
One,
it docks very nicely using auto
dog Vena,
so I'll model and not a crystal
structure by any means.
Although we are pursuing that.
Right in the active site that
protects that the DNA.
Has to slide through.
You can see these two little
protrubraces here that
essentially the DNA winds in
and this blocks it.
So we block that protein DNA
interaction, we can block.
Kinase activity as well.
Not shown here,
but the most important result
was
cellular uptake in these non
small cell lung cancers.
We're getting about 1500
picamoles per million cells
across the board,
even the bad ones are at 500.
I will tell you the predecessor
compounds the numbers were in
the 10 and 20 peak of
mole.
So we get a
significant increase.
In cellular uptake.
And Pammy measured in the
clonagenic survival assay.
Now either without radiation or
with radiation,
the addition of our compound can
dramatically increase the
sensitivity.
So with greater cellular uptake,
we jumped right into mouse
experiments.
In vivo,
we put the A 549 lung cancer
cells in
there, put them into mice,
treated them with either the
coup
inhibitor or IR or the
combination wait four hours.
Hit em with ionizing radiation
and then two hours later
sacrificed the mice.
We did direct intratumoral
injection, so we didn't want to
wait for PK.
And then wanted to see if we
could get this result and she
showed very nicely in
an DNA PK auto phosphorylation
assay measured by Western blood
from the
extracted tumors.
After this two hour post
radiation
treatment that the vehicle
treatment, there's none as you
might imagine.
The compound.
The CU inhibitor doesn't do
anything on its own.
Ionizing radiation gives us an
increase in phosphorylation.
As you'd imagine.
And the 2:45. I'm sorry,
the 3392 inhibitor is able to
block that
significantly.
So we get a significant
reduction in coup dependent
DNAPK auto phosphorylation.
She took it a step further and
went to the Ki 67 DNA
proliferation marker in
tumor slices from the extracted
tumors of these mice and the
vehicle controlled the
brown spots or.
The Ki 67.
Or CU inhibitor
doesn't do anything.
Thing on it's own.
The ionizing radiation gives us
a little decrease in the
combination is in fact
better than the mall,
and so this really set it up
that we're
having an in vivo effect of
these Co inhibitors with direct
intratumoral
injection.
And so we went further and did
the PK analysis at this point
and were able to
with a relatively simple
formulation, get really decent
pharmacokinetic
parameters.
We get a decent half life of
about 4 1/2 hours.
We get a maximum concentration
that's about twice the IC90
value,
so we can maintain that for at
least two hours as part of the
treatment and we get
a really good dose response
curve in terms of increasing
doses in the mice.
We get increase in
concentrations and.
So we can hit some really nice.
In vivo delivery of this drug
and target coverage in terms of
blocking the coup
protein.
So we did the experiment,
took the 549 lung cancer cell.
Put them into the mice.
Treated them with now
intraperitoneally. These are
subcutaneous tumors.
So they're treated IP.
And then two hours later hit
with
ionizing radiation.
And we did this regimen once a
week for
four weeks in a row.
So day Zero, day 714 and 21,
and monitor tumor growth and we
see a
little bit of activity from the
CU inhibitor.
We see a little bit of activity
from the ionizing radiation,
probably more than
we would have liked.
We had a titrate that down,
but the combo in fact was.
Then all of them put together,
and so now we have systemic
delivery of
the coup inhibitor to increase
the sensitivity of these tumors
to ionizing
radiation with relatively little
side effects along the way.
Tumor volume tumor weight goes
down and so we're pretty
convinced we have an
interesting model to understand
how to block that specific DNA
PK dependent non
homologous end joining to
ultimately increase radiation
sensitivity.
And So what I've shown the last
45 minutes or so,
we can block protein DNA
interactions as a viable
mechanism to target DDR
signaling.
So protein DNA interactions are
often thought undruggable.
And that's largely from work in
the transcription factor field,
where you're relying on, you
know, single based differences
to give you
specificity.
These proteins are structure
specific
events.
They they bind in a very
differently and we can
functionally target them with
small molecules and blocking
this can give us.
Anti cancer activity.
Single agent in the case of the
RPA inhibitors combination with
RPA
inhibitors in PARP combination
with coup inhibitors in DNA
binding,
so blocking the RPA we think is
through a gap protection
mechanism and blocking
coup in combination with IR is
through double strand break
repair and non hom.
And joining.
So what I'd like to do is
acknowledge everybody who's done
the work.
I did that previously.
Want to acknowledge our funding
sources? Over the years we've
had significant
support from the NIH and we are
very happy for that.
The reality is it's most of your
tax dollars going to the NIH
that decides who
gets these funding.
And so we thank you for that
support.
And if if we want to keep
funding through the NIH,
these are our tax dollars and we
should make sure we go.
That way.
In the absence of that,
we really do like to thank the
Tom and
Julie Wood Family Foundation.
They have been incredibly
supportive of
Indiana University,
our Simon Cancer Center and our
lung
cancer group.
And when other funding becomes
more.
Interesting.
It's always nice to have a
backup.
And our Simon Cancer Center has
also been very supportive over
the years as well as
near ex Biosciences.
And so the FDA here is for
funding and
without that,
none of this would have been
possible.
So I thank them and I thank you
for the time and I'd be happy to
take any
questions.
Thank you Doctor Turcie for the
for the wonderful presentation
for the for the
audience.
Please put your questions in the
chat and we will go through them
and we have first
one from it's comments from
Doctor Robert Sober.
Sobo is.
He's a fantastic work and.
We have a we have a question
from my colleagues.
Travis tracker from NCI.
He said.
This is a great talk.
Uh, sorry if I if he misses
this, is it possible that RPA
inhibition
prevents uh Primo uh recruitment
and re priming that can promote
gap formation?
This will potentially prevent
gaps from being generated rather
than RPA solely
being needed for
their protection.
Yeah. So I mean that's
a great question.
We haven't done that experiment
and and we agreed.
So there's two ways
this can go, right?
So we know Primpol is stimulated
by RPA and if we can decrease
enough of that
then you wouldn't get the
primpol re priming on the other
side.
So it it necessarily wouldn't
give you a gap because you don't
have a piece of DNA
on the other side.
What you have is a long single
strand piece of DNA where the
prim pole would
have synthesized.
And so we think.
That that would also give you
the potential for degradation.
We can't rule out the potential
of of the Theta mediated events
on there as well.
Well,
so we haven't done those
experiments,
but we are actively pursuing how
primpol re priming events are
regulated by the
RPA inhibitor.
It it's it could go either way
and and we don't know yet,
but that's a it's
a great question.
Report. Do you want to come out?
Come off mute.
Yeah.
Thank you for a really
nice talk, John.
Elegant work. Beautiful work.
I was wondering about these
inhibitors,
the RPA inhibitor inhibitors.
You mentioned that there weren't
so many
side effects in the mice,
but in cells and in mice.
What are the side
effects of using these?
I mean,
you're you're trying to do this
in
therapy, so.
It's relevant.
And how safe these are and for
example, do they cost sellers in
essence?
Do they cause double strand
breaks measured by H2X or
something?
Yeah. So so, single agent,
RPA inhibitory activity doesn't
increase
our H2 ax signal.
So we don't see that when we put
in non cancer cell lines.
So human peripheral blood
mononuclear cells,
you know the IC50 for those come
out to be about five to seven
times higher than
we see for the cancer cells.
We've also done experiments in
in non.
Cancerous.
Perpetual cell lines like HCC
293, we see some variable
activity.
So we really think that there's
a fundamental difference even
with the same
doubling time, for instance, of
a, you know, gut epithelial
cell,
it does S phase differently and
generates fewer single stranded
double stranded
breaks in DNA damage.
Hox is lower in them than just a
cancer
cell.
That's dividing at the same
doubling time, so we think that
really does give us the
window we we don't see.
So there's we
monitor weight loss.
We do see some initial with the
RP inhibitor,
but they regain
it pretty quickly.
There's mild neutropenia.
That we see in the mouse and
hematologic differences,
but we've done
combinations with platinum.
We see no increased kidney
toxicity, liver toxicity,
so it looks like it's relatively
safe again.
You know it's mice and we
haven't gone on to larger
rodents and and others.
But at this point,
there's no reason to not pursue
those
things.
Very short term experiment.
Have you done more
long term experiments?
They are.
Yeah,
we've done some toxicokinetics
or
multiple high dosing over.
I think it's.
Three month period.
And we're able to to dose it at
a level that gives us sufficient
target coverage
without significant toxicity,
but they're really the studies
that are
needed at,
you know for the to getting this
thing to
an induced.
It's GLP tox is the whole game.
And so we are.
Yeah.
Pursuing those those
experiments now.
You're making derivatives.
There may be more safe than or
something.
Yeah,
there's always thoughts of more
chemistry.
The the current 329 molecule has
the requisite characteristics
that we like,
but whether that's the final
clinical candidate or not is is
still up for
debate.
We have other derivatives and
the question is,
do you go with a full scale
resynthesis of new pharmacopores
or do for a first
inhuman first in target
compound. See what we get. And
that's the challenge.
The very strong setup you have
there. Very nice.
Thank you.
OK.
Next question we have Ronit
Yaden from Nhlbi. She says great
talk.
Is there any interest and how do
you plan to increase the potency
of various
compound if there is a global
development of them for clinical
use?
It's a great question and we're
pursuing structure based design.
So we're trying to get the
structures of all of these with
their targets.
You know the challenge with a
protein DNA interaction
inhibitor is they're
necessarily hydrophobic and
solubility is bad.
And so we've, we've done Co,
crystal structures,
cryo am structures and trying to
get very accurate predictions of
where these
things are to give us.
Structure based design to
increase potency.
And so that's really one of the
the mechanisms to do it.
I think it the idea of and and
this is what we did initially,
which was largely random
screening and of compounds that
look similar in creation
of molecules with different
pharmacopoeous and ultimately
ended up
with ones that give us pretty
reasonable, you know, low
micromolar IC 50s in.
DNA binding assays,
when we put them in the kinase
assays,
we can get them into the, you
know, 100 nanomolar IC50 ranges
because they're
catalytic activities
versus just a binary.
Protein interaction.
And so we think we're close,
but there's definitely ways to
go to increase those the
potency.
But we think structure
is the way to go.
At this point,
we also are looking at some AI
versions
of trying to do this.
There's a lot of approaches to
do that, but it would benefit
from knowing the the
actual structure of the
inhibitor with the target as
opposed to trying to model
that and then do the
AI versions of it.
Great.
Next we have a question from
Jimmy Lee
from NCI. She says.
Great talk in the MDA MB436B
BRCA deficient tmbc mouse model
with an RP
inhibitor alone tumor start to
grow again after four weeks and
the combination with
PARP inhibitor can cause a
durable response.
But have you tried tested?
RPA inhibitor alone or RPA
inhibitor plus elaborate
combination?
In a pop inhibitor
resistant model.
Yeah, that's again another great
question. And we are actively
doing that.
Matthew's generated a series of
PARP inhibitor resistant lines
to saroopa rib.
The the part one specific
inhibitor and we have very, very
interesting results.
So we can get 1000 fold increase
in resistance to the PAAP
inhibitor in these
MDA 436 that are resistant and
they are equally sensitive to
the RPA inhibitor.
So RPA inhibition
is not affected.
And so we really
do like that model.
Your your question, the RPA,
resumption of the tumor growth
that
resumes after RPA inhibition is
largely a function of
pharmacokinetics and
pharmacodynamics.
We don't see and I showed you
the increase in PK parameters,
the C Max with increasing dose
of the coup inhibitors.
We don't see that with the RPA
inhibitor at its current
formulation.
So it really is a
formulation issue.
Issue and getting
bioavailability to be retained
after multiple dosing events and
so we think once we crack that
nut, we'll be able to maintain
the pressure on
those tumors in the combination
that experiment again is a great
one
combination PARP RPA and then
just let them go and.
See,
when they come back versus PARP
inhibitor
alone and the PARP RPA
combination, they're long term
mouse studies.
And so we're we're trying to
find somebody to throw us enough
money to do
that.
Hey great.
I have a actually a a follow up
question
with this question.
So I noticed that many of the
validation experiment is done in
a cell line like a.
8459.
But have you tried any of these
inhibitors on the cell line or
model with
intrinsic DNA repair deficiency
like this BRCA deficient model,
so that the inhibitor may?
Act as a synthetically
satity fashion.
Yeah.
So we've done that with the RPA
inhibitors.
The RPA was really pursued in
that MDA
436, which is a BRCA
1 deficient model.
So we published and Pami
published this in the paper last
year.
A variety of cell lines with
differential alterations and and
we published an ATM
mutant cell line and the coup
inhibitor was more potent in
that cell line.
The reality is when you get to
isogenic models, that's really
the test.
And so we see less of a
difference in an isogenic ATM
model.
And so we're actually doing the
CRISPR screens to look for those
genetic
alterations that increase
sensitivity to a coup inhibitor.
But we agree with you.
I think those
experiments are great.
We just haven't done them yet
and and they're in process.
We've got three CRISPR screens
going currently and are cranking
through the
bioinformatics to find out what
is increasing that sensitivity.
Great.
I think we don't have additional
questions,
so maybe we'll just wrap up
here.
And thank you for Doctor Turcie
for the insightful and thought
provoking
presentation on DNA damage and
the DNA replication and we
appreciate everybody's
participation and we're looking
forward to continue this this
session and then
have a have a great, great day.
Thank you so much.
Thank you all.
Thank you.