Losses of Nuclear Integrity Tie DNA Damage to Innate Immune Signaling

October 20, 2023

Yale Cancer Center Grand Rounds | November 17, 2023

Presented by: Dr. Megan King

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ID: 10882
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00:00Morning. For those of you who don't know me,
00:02I'm Rachel Greenup.
00:03I'm Chief of Breast surgery and newly named
00:06Co director of the SMILO Breast Program.
00:09And I have the honor today of introducing Dr.
00:12Megan King. Doctor King is an associate
00:16professor of cell biology and of molecular,
00:18cellular and development biology.
00:20She's also the Co leader of radio Biology
00:23and Genome Integrity Research program at
00:26the Yale Cancer Center and an Associate
00:29Cancer Center Director for Basic Science.
00:31She did undergrad at Brandeis and
00:33then went on to receive her PhD in
00:37Biochemistry and molecular Biophysics
00:39from the University of Pennsylvania
00:41under the mentorship of Doctor Mark
00:43Lemon and went on to get a post doc
00:46training with at Rockefeller University
00:48where she discovered new mechanisms for
00:52the targeting and function of integral
00:55inter nuclear membrane proteins.
00:57Since founding her own group in 2009,
00:59Megan has continued to investigate the
01:01broad array of biological functions
01:03that are integrated at the nuclear
01:06envelope from impacts on DNA repaired
01:08to nuclear and cellular mechanisms.
01:10She was named a Sarah Scholar in 2011
01:13and is the recipient of the NIH New
01:16Innovator Award and is currently an
01:19Allen Distinguished Investigator.
01:21She's been at Yale for 15 years,
01:23and we're excited to hear about her
01:24work today. So thank you, Doctor King.
01:32Thank you so much.
01:33It's a pleasure to be here.
01:34And I think, you know,
01:38hearing that bio,
01:38it always reminds me of how far I've
01:40come to what I'm going to be talking
01:43about today and how much that is
01:45a consequence of the environment
01:47at Yale and the interactions that
01:49really have been driven initially
01:51by joining what was on the Radio
01:53biology and genome and radio biology
01:56and radiotherapy research program,
01:58which was connected to me by Patrick Sung,
02:00who's no longer here.
02:01But he kind of immediately roped
02:03me into that program and then all
02:05of the relationships I made through
02:07that particularly with Joanne,
02:09Sweezy and Pat Larusso and really
02:11it's that transition that is
02:12really spurred everything that
02:14I'm going to talk about today.
02:15And so I'm really appreciative of
02:18that because I think it's really
02:20going to broaden the the scope of
02:22where this fundamental biology,
02:24which hopefully you'll see today about
02:26the nuclear envelope is really related to,
02:29you know,
02:30a chemotherapy approach that's being
02:31broadly used in which we're hoping
02:33could be used and even more context.
02:35And so that's what I'm going
02:37to talk about today.
02:37And then the surprise to us has
02:39been a connection between this
02:40and innate immune signaling,
02:41which is also not our expertise.
02:43And so I really appreciate anyone
02:46here online now later thoughts
02:49on that because there's so many
02:51people at Yale who do have more
02:53expertise in that area than we do.
02:55OK.
02:55So just my disclosure,
02:57some of this work is funded through the
02:59strategic alliance with AstraZeneca.
03:03So as as many of you are familiar
03:06with PARP inhibitors are really the
03:09canonical example of synthetic lethality.
03:12And it's such a powerful concept because
03:15it really highlights how we might use
03:17approaches that are really specific
03:19to tumor cells and otherwise do not
03:22affect all the normal cells of the body.
03:24And and what is you know, fabulous approach,
03:26right that that would be.
03:28And so the idea is that PARP inhibitors
03:31in particular cause single stranded
03:33DNA damage to persist or at least
03:35that's one of the mechanisms that we
03:38think about as being important here.
03:40And that typically cells can tolerate
03:42this kind of damage because they have
03:45a functional homologous or combination
03:47DNA repair mechanism that can act
03:49in SNG 2 and repair these breaks.
03:51And this leads to cell survival.
03:54However, in the consequence of
03:56defects and homologous recombination
03:57and kind of the classic example of
04:00this are pathogenic mutations in the
04:02BRCA one and BRCA 2 genes.
04:03There's a defect in tolerating this damage
04:06and this will lead to to cell death,
04:09right.
04:09And so this is the mechanism where
04:11it's the combination of the HR
04:13defect on the PARP inhibitor that
04:15drives a tumor cell death.
04:17So I want to just set the stage for
04:19what I'm going to talk about today
04:21by by reminding you about how P53
04:23works because I'm going to use this
04:24as an example of of our kind of
04:27framework for thinking about the story
04:28that I'm going to tell the debt.
04:30So in interface in normal cells, right,
04:33we have when there's DNA damage,
04:35there is the activation of P53 and
04:38P53 is is really this decision point,
04:41It's both activating mechanisms to
04:44repair that damage, right.
04:46So that the first response of the cell
04:48is try to tolerate and repair this damage,
04:51stall the cell cycle,
04:52fix the genome and then go into mitosis
04:56and and and have normal cell growth.
04:59But but if this damage is too deleterious,
05:02if it persists,
05:03if it can't be tolerated,
05:04then this is going to lead to
05:06the stimulation of apoptosis.
05:08And so really this is this
05:10combination of repair and then when
05:13we can't repair driving cell death,
05:15however,
05:16you know,
05:16we know that this is a mechanism
05:18that is dysregulated in the vast
05:21majority of tumors including those
05:23that respond to PARP inhibitors.
05:24And so this is not the mechanism, right.
05:27So we know we can get the synthetic
05:29lethality of PARP inhibitors
05:31with HR defects even in the
05:33context of dysregulated P53.
05:34So what is this mechanism
05:36actually and you might think that
05:38we understand this mechanism,
05:39but what I'm going to tell you about
05:41today is that we we don't and I'm
05:43going to focus today disclaimer on
05:45the tumor cell intrinsic mechanisms.
05:48That is not to negate the fact that
05:50there are other roles for the immune
05:52system for the tumor microenvironment.
05:53But what we know is that in in
05:55HR deficient cells in a dish PARP
05:57inhibitors can cause cell deaths.
05:59So we know that there is at least
06:02a sufficiency in in cells and
06:04culture for a tumor cell intrinsic
06:06mechanism of cell death and IT and
06:09and how do we think about what kind
06:12of surveillance mechanisms might be
06:14akin to P53 that that drive this.
06:17So I just want to highlight a few
06:19of the challenges that we face in
06:21the use of HARP inhibitors because
06:23really this is our motivation for
06:24the kind of fundamental studies
06:26that I'm going to talk about.
06:28You know, it's very clear that
06:31PARP inhibitors specifically
06:32kill HR deficient cells,
06:33but we don't understand
06:34the cell death mechanism.
06:35As I already highlighted,
06:37acquired resistance is a major
06:39challenge and it's really well
06:41explored in preclinical models
06:43through things like CRISPR screens.
06:45But actually the insights from
06:48patient samples is really still
06:50rather limited and understanding
06:51the cell death mechanism that
06:54PARP inhibitors precipitate could
06:56really help in in this area.
06:58A major challenge is that we lack a
07:02robust biomarker that can tell us that
07:04PARP inhibitors are likely to be effective.
07:07So this can either be that
07:08cells are reconstituted,
07:09homologous recombination or there
07:10could be other contexts outside of the
07:13genetic kind of germline mutations and
07:15BRCA one and BRCA 2 or even somatic
07:18mutations where it could be there
07:20is an HR defect that's actionable.
07:22But because we don't have a biomarker
07:24for HR status that is at least dynamic,
07:26right,
07:27There are kind of sequencing
07:28based approaches,
07:29but we don't have a classic kind
07:32of pathological straightforward
07:33psychology kind of approach
07:35and that's a real limitation.
07:39And and lastly, there's a lot of
07:41enthusiasm about combining PARP
07:43inhibitors with immune checkpoint
07:44blockades and indeed a number of
07:47trials that are exploring this.
07:48But we don't actually understand the
07:51underlying mechanisms of why those
07:53combinations might be effective.
07:54And to really understand that,
07:56we have to understand how how carpenters
07:57are working and and This is why we're
07:59really interested in the crosstalk.
08:01I'll talk about today with the innate
08:02immune system and how that might be
08:04contribute to the the rationale for
08:07these combinations and might point
08:09to what the right approaches are.
08:11So as I said,
08:12I'm going to focus on this cell death
08:15mechanism in my talk today and to 1st
08:18to introduce how we've kind of how
08:20we've been thinking about this problem.
08:22I want to just introduce you to this
08:25canonical innate immune surveillance
08:27mechanism in which C Gas shown here
08:30is they are really key player.
08:33So C gas is an innate immune sensor
08:35protein that is in the cytoplasm of cells
08:38and it binds to double stranded DNA.
08:41And the idea is that it can surveil
08:434 viruses and bacterial pathogens,
08:46but there's increasing evidence that
08:48C gas is also capable of surveilling
08:51self DNA that's present within cells
08:53within eukaryotic cells themselves.
08:55So for example,
08:56a distregated mitochondria can lead to
08:59leaking of mitochondrial DNA into the
09:01cytoplasm which can activate C gas.
09:03And today I'm going to be talking about
09:05how actually the chromosomes or the
09:07chromatin or DNA from the nucleus can
09:10be exposed and surveilled by C gas.
09:12C gas works by when it binds to DNA.
09:15Just I'm going to say very clearly,
09:17when it binds to naked DNA,
09:19this drives a change and and molecules
09:21of C gas come together and they produce
09:24the second messenger called C gamp.
09:26But actually binding of C gas to DNA
09:29does not always lead to this response.
09:32And so there's regulation of this that
09:34I'll talk about in more detail in a moment.
09:36So just recruiting C gas somewhere
09:37does not mean that it's actually
09:39producing this second messenger,
09:41but the second messenger is thought to
09:42be key to its downstream mechanisms.
09:45The recipient of the C gamp signal is sting.
09:48Sting is a membrane protein that
09:50is key to the canonical signaling
09:53pathway that C gas activates.
09:55And that is by driving the phosphorylation
09:58of a kinase called TBK one once
10:01it's traffic to the Golgi and then
10:03this phosphorylates IRF 3 which is
10:05a transcription factor that when
10:07phosphorylated goes into the nucleus and
10:10drives interferon stimulated gene expression.
10:12So that's the kind of canonical pathway.
10:14There's also a non canonical roles in
10:17activating NF Kappa B signaling and
10:19and any of these may in addition to
10:23inflammatory genes cause apoptosis.
10:25So this could be a mechanism that
10:27can drive cell death,
10:28although we really don't understand
10:30this terribly well.
10:31In addition,
10:32sting is also involved in some
10:34other non canonical mechanisms that
10:36could also precipitate cell death,
10:38which as I mentioned is what I'm going
10:40to be focusing on today and part of this
10:42actually involves the autophagy mechanisms.
10:45There appears to be some autophagy
10:47dependent cell death mechanism
10:49downstream of sting and this is
10:51independent perhaps of this canonical
10:53interferon stimulated gene signaling.
10:56And so while I'm going to focus kind
10:58of on these upstream steps today,
11:00we really don't know what the key downstream
11:03steps are in terms of which signaling
11:05pathways are going to be most most relevant.
11:07And so that's really kind of ongoing work.
11:10And I'll just close this slide
11:12by highlighting that actually AC
11:13gas is a really ancient protein.
11:15It actually goes all the way
11:16back to prokaryotes.
11:17And so it's played a role in
11:20surveilling foreign DNA long
11:21before the innate immune system.
11:23And so that kind of makes sense,
11:24this idea that it's actually
11:26multiple signaling pathways that
11:27lie downstream of C gas activation.
11:31So. So how do we get thinking
11:33about innate immunity?
11:34There's abundant evidence in the
11:37literature that HR defects on this.
11:40In this particular case on the left,
11:42we're looking at bracket.
11:43In both these cases,
11:44we're looking at bracket to knock
11:46down models that HR defects are
11:49sufficient to trigger an innate
11:50immune response and this is a response
11:53that's actually further pushed by
11:55the addition of PARP inhibitors.
11:57So let me just walk you through
11:59the example of this data.
12:00As I mentioned,
12:00these are BRCA 2 knock down cells.
12:02So with doxycycline we have
12:03suppression A BRCA 2 expression and
12:05you can see that there's a gain
12:07in IRF 3 phosphorylation which is
12:09one of that canonical downstream
12:11outcomes of C gas signaling.
12:12And this also leads in this
12:15model to Stat 1 phosphorylation.
12:17And a similar thing is seen in the in
12:19in breast cancer cells in this 231 model.
12:22Again,
12:22this is a artificial system of
12:25the knock down of BRCA 2 with
12:27regards to how PARP inhibitors
12:29then synergize with this.
12:30I've just pulled out this data
12:32from BRCA 1 deficient,
12:34BRCA 1 deficient breast cancer line
12:36that's commonly used in the lab
12:38to study a BRCA 1 deficiency and
12:40this is now in a xenograft model.
12:43So these are actually now xenographs
12:45looking at how PARP inhibitors affect
12:47interferon stimulated gene expression.
12:49And you can see that all of these
12:52genes that are downstream of C
12:53gas activation are up regulated in
12:55the with PARP inhibitor treatment
12:57in the xenograft model.
12:59So there's been these observations
13:01of innate immune stimulation in
13:03the context of HR deficient cells
13:06that's further pushed by PARP
13:08inhibitors in a number of cases.
13:10But what is the cause of this?
13:13Right.
13:13So what the,
13:14what the signal is,
13:15How we go from HR deficiency to innate
13:18immune signaling has been really unclear.
13:20One other thing that I want to
13:22just alert you to is that when
13:24there is an HR defect in cells,
13:26one of the consequences is
13:28that we accumulate cells,
13:29accumulate mitotic errors.
13:30So this is just one paper I've
13:33pulled out from Steve West,
13:34actually from more than a decade or
13:36probably more than 15 years ago now
13:38where it's been recognized for a long time.
13:40If there are challenges in
13:42maintaining integrity of the genome,
13:45then in mitosis you have these
13:47intermediates that lead to
13:48persistent bridges of DNA and DNA
13:51breaks and these kind of breakage,
13:53fusion breakage cycles that can
13:55actually be precipitated by an HR defect,
13:58by a radiation, by taxol treatments.
14:00You can arrive at these kind
14:02of structures in many ways.
14:03But I would say HR deficiency
14:04is not the way that most people
14:06have thought about arriving at
14:08these kind of structures.
14:12I also just want to remind you,
14:13'cause I'm a cell biologist,
14:15that actually the nuclear envelope,
14:18not only the nuclear envelope is
14:20breaks down every cell cycle. OK.
14:22So I just wanted to keep this in your
14:25mind too as I talk about this because
14:27I just told you there's an innate
14:29immune surveillance protein that is
14:31looking for DNA and yet every mitosis,
14:33the chromosomes are exposed to the cytoplasm.
14:36So we we know that that's not sufficient
14:38to drive an innate immune response.
14:40So we know in mitosis there
14:42are mechanisms to down rate,
14:44down regulate this surveillance mechanisms
14:46are a way to shield these chromosomes
14:48from actually activating this pathway.
14:50And so these recombination intermediates
14:53are interesting in part because
14:55they don't just occur in mitosis,
14:57they persist into the following interface.
15:00And that's going to be important here
15:02because we need to get to the next
15:04interphase in order for this innate immune
15:05surveillance mechanism to be reactivated.
15:09And indeed, there is also evidence in
15:12the literature that for PARP inhibitors
15:14to actually induce cell death,
15:15cells have to transit through mitosis.
15:18This is additional evidence that you know,
15:20unlike P53, which as I mentioned
15:22is acting an interphase,
15:23that it is essential for cells to go
15:26through mitosis for PARP inhibitors
15:27to actually cause the cell death.
15:29This is actually some work again
15:31in a xenograph model and the
15:33absence of functional bracket,
15:34two and cells treated with a laparib and
15:36what you can see is kind of these events.
15:38So we have a a cell that is likely in G2,
15:42it goes into mitosis.
15:44You can see this is an this is an anaphase.
15:46So there are anaphase bridges here and
15:48actually most cells have some degree of
15:51entanglement of chromosomes in anaphase
15:53that are going to be resolved dynamically.
15:56However, if that does not happen,
15:58if cells are unable to resolve
16:00these entanglements of chromosomes,
16:02So what happens is that these cells
16:04will biochemically come out of mitosis.
16:06So they're back in interface and you can
16:08see that because the nucleus is intact again.
16:10But what you can see in this cell is
16:11you now have a doublet essentially,
16:13right?
16:13You have a cell that actually failed
16:15in cytokinesis and it failed because
16:17you couldn't actually generate 2
16:19cells because there was bridging
16:20DNA between these two cells.
16:22But the cell has biochemically
16:23come back into interface and so we
16:25can imagine that the innate immune
16:27system is active again.
16:28And the question is,
16:30is this somehow aware of the fact
16:32that this is a defective mitosis?
16:34Is there some mechanism to know that
16:36and that this would ultimately Dr.
16:37the cell death and that's what we
16:39see happening on the right with
16:40this chromosome condensation.
16:44I just want to highlight that this
16:46is not really new information,
16:48so we can go back.
16:49This is from 2001 and there has been
16:52long been the understanding that these,
16:56the changes in nuclear shape,
16:58nuclear atypia which are used all the time
17:01by pathologists to diagnose and stays,
17:03cancers are tied to these kind of
17:07aberrations that I've mentioned.
17:09So I just want to you know that they've
17:11been called many things over time.
17:12What I want to point out is that all
17:14of these kind of mitotic errors that
17:17are typically associated with altered
17:19nuclear shape are all things that we're
17:21observing in interphase cells again,
17:23so not in cells just in mitosis
17:24that have an anaphase bridge
17:26but they're in in interphase.
17:28So these were called what the structures
17:30that I just described that you can
17:32have persistent DNA that then is still
17:34there as cells reform their nucleus
17:36and go into the next cell cycle.
17:38And this, you know,
17:4025 years ago were called inter
17:42nuclear strings,
17:43but you can also have micronuclei.
17:45And I just want to point out one of
17:47the differences between these two
17:48types of structures is that these
17:50inter nuclear strings are because
17:51of an inability to segregate the
17:53chromosomes because the chromosomes
17:55are literally entangled and
17:57cannot be physically segregated.
17:59Micronuclei are different and that they
18:01predominantly arise from lagging chromosomes,
18:04acentrosomal chromosome fragments
18:05and perhaps extra chromosomal DNA,
18:08right.
18:08So they really are a different
18:10structure than these two structures
18:11are actually quite different.
18:12And I'll come back to that.
18:14The consequence of this can
18:15lead to BI nucleation.
18:16That's what I just showed you in
18:18that particular bracket 2 model.
18:20And I won't really talk about it today,
18:21but you can also get nuclear ruptures
18:24that happen in interface due to a
18:26defect in the nuclear integrity.
18:28But that is not an event that's
18:29tied to mitosis.
18:30So I'm not going to talk
18:31more about that today.
18:32OK.
18:33So let me just show you kind of
18:35the amazing cell biology that
18:37is tied and specifically to
18:39these persistent DNA bridges.
18:41So here I'm showing you a movie.
18:44These are cells that are expressing
18:46a nuclear localization signal
18:47tagged to a fluorescent protein.
18:48So it's exclusively in the nucleus.
18:50And we're going to look at this cell
18:52that is just going through mitosis,
18:54if it will.
18:58Maybe I'm not allowed to do that while
18:59I have the pointer on, Is that possible?
19:05Yep, that's possible.
19:07OK, so we're gonna look at the cell
19:10that is trying to transit mitosis.
19:11We're gonna see it come out of mitosis.
19:13These cells are still linked by
19:14one of these DNA bridges and you
19:16can see there are these flashes,
19:17there are these transient ruptures
19:18of the nucleus and all the nuclear
19:20localization signal will spill out
19:22and then there seems to be some repair
19:24of that event and then the the the
19:26protein can start to accumulate again.
19:27So it's kind of these cycles of
19:30ruptures and then repair events.
19:32So this is just looking in this case,
19:34this is actually a model where there's
19:37a dicentric chromosome, however one.
19:39So one of the questions is what's
19:41the consequence of this innate
19:44immune surveillance mechanism when
19:45you have one of these ruptures.
19:48So these kind of transient ruptures
19:49of the nuclear envelopes, right.
19:51So the nucleus, we've come out of mitosis,
19:53it should be intact, but it's it's unstable.
19:56And so here I'm going to show
19:58you similarly cells,
19:59but these cells are actually now
20:02expressing AC gas that's tagged and
20:04that's going to be in this panel here.
20:06And I just want to again point
20:07out this is not just anaphase.
20:09This is far after anaphase.
20:10These cells have this bridge.
20:11They're trying to break their DNA
20:14and and and segregate it, right,
20:16Not break it, but segregate it.
20:17And what I hope you can appreciate
20:19is that late in this movie,
20:20all of a sudden what we see is
20:23that there's recruitment of sea
20:24gas all over this strand of DNA.
20:27OK. So it's not something that
20:29happens in mitosis.
20:30It's far after mitosis.
20:31There is this bridge of DNA the
20:33nuclear was trying to form around it,
20:35but we get these ruptures and see gases
20:38recruited and this is a persistent bridge.
20:40I just want to point out you also get
20:42this kind of thing to Micronuclei.
20:44Here's a micronucleus and we can actually
20:46see that that micronucleus is intact
20:48and then it's going to rupture and
20:50then there's massive C gas recruitment,
20:52OK.
20:52So any of these losses of nuclear integrity,
20:55whether it's one of these persistent
20:57Dania bridges or it's a micronucleus
20:59can recruit the C gas protein.
21:05So I'm going to focus today on these
21:08DNA bridges and I'm going to just give
21:10you the rationale for why that is.
21:12Now, one of them is that actually
21:17many perturbations will cause both
21:19these DNA bridges and micronuclei.
21:22But there's evidence in the literature
21:24that DNA bridges are actually much more
21:26potent activators of Segamp production.
21:28If you remember, I told you,
21:29the recruitment of of C gas is
21:31not sufficient to activate it to
21:33generate high levels of Segamp.
21:35You know why might that be?
21:37There's evidence actually that one of
21:39the mechanisms that keeps cells from
21:41overreacting to its own genome is the
21:43fact that nucleosomal or chromatized DNA
21:45is a poor stimulator of Segamp production.
21:48Whereas naked DNA,
21:50what you would have in a virus or a bacteria,
21:52is a far more potent activator
21:54of C gamp activation.
21:56And so this would suggest that
21:58really the state of the DNA matters.
22:01And what I'm going to argue here is
22:03that actually micronuclei for the
22:04most part are chromatized substrate.
22:06It was a lagging chromosome
22:08that formed its own nucleus.
22:09It's unstable but still it's nucleosomal
22:13whereas this DNA in these persistent
22:15bridges as you saw in those movies,
22:17the DNA is being pulled apart.
22:19And so one of the ideas is that it
22:21there's so much tension on the DNA
22:23that actually the histones that make
22:25nucleosomes are being evicted and then
22:27the DNA that's left is naked and and that
22:30that is a more potent activator C camp.
22:34And additional evidence from that
22:36for that comes from observations
22:38that Apobac activity it is actually
22:40very high over overstretched DNA
22:42that is present in bridges,
22:44which suggests that it can also become
22:46single stranded and acted on by APOBEC.
22:48Such as additional evidence that
22:50the structure in these in these
22:52persistent DNA bridges is different
22:54than what might be in Micronuclei.
22:56OK.
22:56And then last bit of cell biology
22:58before I get into our own data that
23:00I need to introduce you to is the
23:01idea that like in that NLS movie,
23:03there's also a nuclear envelope
23:04repair mechanism that is looking
23:06for these breaks in the nuclear
23:08envelope and trying to fix it.
23:09And this is something that's been of
23:11interest to our group for a long time.
23:13So remember as I said,
23:15in a normal mitosis,
23:16the nuclear envelope has broken down,
23:17the chromosomes are exposed,
23:19but they don't activate the
23:20innate immune system.
23:21Then we reform the nuclear
23:23envelope at mitotic exit.
23:25When the nuclear envelope is reformed,
23:26you have sheets of endoplasmic
23:28reticulum around the chromosomes,
23:30but it's full of holes actually.
23:31And those holes are particularly
23:33where there are still microtubules
23:35from the spindle that are
23:36attached to the chromosomes.
23:37So there is a machinery that has
23:39to come in and fix all these holes
23:41at the end of every mitosis.
23:43And that machinery is made-up of the
23:45components that I've shown here.
23:47There is an abundant DNA binding
23:49protein called bath,
23:49not to be confused with the chromatin
23:52remodeler bath and this brings in
23:55a protein called LEM 2 which is
23:58an integral membrane protein and
23:59that's shown here in the cartoon.
24:01So this LEM Two is recruited to these
24:03holes in the nuclear envelope and LEM Two
24:05is an adapter for the escort machinery,
24:08particularly CHIM 7 which is a
24:10nuclear envelope specific escort.
24:11So the escorts are a membrane remodeling
24:13machinery that basically can take a hole
24:15in a membrane and they can close it.
24:17And so this machinery is recruiting,
24:20is recruiting escorts to the nuclear
24:22envelope they form these spiral polymers,
24:24and you need this to have one nuclear
24:26envelope at the end of mitosis.
24:27So this is the normal thing
24:29that's always happening.
24:30But there's abundant evidence that this
24:33same exact machinery is recruited anytime
24:35there's a defect in nuclear integrity.
24:38And so I'm just showing you
24:39an example of this here.
24:40This is actually where a rupture in
24:42the nuclear envelope has been induced.
24:43And you can see that there's recruitment
24:46of this escort chimp 7 as well as
24:48recruitment of sea gas, right.
24:50So one way of thinking about this
24:52kind of similar to the P53 story,
24:54you know, you can repair,
24:56you can repair DNA or the cell can
24:58die and you can give up on things.
25:01We have this machinery that sees
25:02a hole in the nuclear envelope.
25:04It can try to fix the hole,
25:06but if it can't fix the hole,
25:07there's a surveillance by the
25:08innate immune system.
25:09And so there's actually a
25:10competition potentially that's
25:11going on between these factors.
25:12And I'll show you some evidence
25:14for that in a moment.
25:16Right.
25:16So here is,
25:16and I'm just going to lay out why we've
25:19done the experiments that I'm going
25:20to describe in the rest of the talk.
25:23I've already walked through
25:25Interphase and the idea, P 53.
25:27So I just want to make the argument
25:29up front for the hypothesis of a
25:32similar surveillance mechanism that's
25:34active post mitosis to Surveil,
25:36the integrity of the mitotic process.
25:39So if cells go into mitosis with
25:41under replicated DNA or unresolved
25:43DNA repair intermediates,
25:45these are things which we're going
25:46to see in an HR deficient cell,
25:48particularly one that's been
25:49treated with PARP inhibitors or
25:51chromosomes that are entangled.
25:53This will initially activate mechanisms that
25:55try to help segregate these chromosomes.
25:57This involves proteins like the Bloom
26:00helicase on the pitch healer case,
26:02Paul Theta, You know,
26:04mediated and joining,
26:06as well as other topo isomerases.
26:10But if those repair,
26:11you know those attempts to
26:13segregate chromosomes fail,
26:14then one of the consequences I've shown
26:16you is that you can have defects in
26:18nuclear integrity and now the cell
26:19has to kind of decide what to do.
26:20So there's a nuclear envelope
26:22repair network And so I showed you
26:23this bath LEM two chimp 7 access
26:25that as I've mentioned our group
26:27has worked on for a long time
26:29understanding the mechanisms of
26:31and that this can promote cell survival
26:34and possibly genome integrity.
26:36On the other hand if they're unable
26:39to repair these breaks in the
26:41nucleus then this will expose DNA.
26:43This can activate C gas and perhaps
26:45this is the mechanism of cell death
26:47that is tied to mitosis and is tied
26:50to these observations of innate immune
26:53signaling that occur as a consequence of
26:55PARP inhibitors in HR deficient cells.
26:58And I just want to point out that, right,
27:01we're going to push these further if we any,
27:04any time we disrupt the checkpoint, right.
27:06So if cells are going into mitosis
27:08when they have not repaired their DNA,
27:10these are more likely to happen if
27:12you have an HR defect and if you
27:14treat cells with a PARP inhibitor.
27:17The very last thing I'll talk about is,
27:19is there a way that we might use this
27:22nuclear integrity defects as a biomarker
27:24of HR defects or of of of contacts
27:28where PARP inhibitors might be effective.
27:30So I'll come back to that at the end.
27:32And also might this nuclear envelope
27:34repair network be a new target, right.
27:36These are factors which actually limit
27:39the action potentially of agents
27:41that are driving these defects that
27:43we're using clinically.
27:44OK.
27:44So now I'm just going to show you
27:47some of the data from our group.
27:49This initial data is using actually
27:51an ovarian cancer model,
27:53UWB 1280 nines which are a BRCA 1 deficient,
27:55HR deficient cell line.
27:57And so I'm just showing you an example
28:00of what one of these persistent
28:02DNA bridges look like.
28:04This is.
28:04You can think of this as very much
28:06as the end point of that movie
28:08that I showed you that we also see
28:10specifically in HR in this HR deficient
28:12line that's further precipitated by
28:14the addition of PARP inhibitors.
28:16And so like in that example you can
28:19see that this bridge which is all
28:21along connecting these two nuclei
28:23is highly enriched in C gas.
28:25And so we would speculate from this
28:27that this is the region of the
28:29nucleus where the DNA is exposed
28:31to the cytoplasm and where we're
28:33getting C gas recruitment.
28:35And so this is just showing you here what
28:37happens when we treat with PARP inhibitor.
28:39Sorry, I've lost.
28:40Yes, here we go.
28:41Yeah.
28:42So the on the on the left is just
28:44the UWB one, this UW one cell line.
28:46And then when we add elaporib,
28:48interestingly one of the things that
28:50we see is the elaporib increases
28:52the percent of cells that have
28:54these persistent DNA bridges.
28:56But UWB ONE cells have abundant micronuclei
28:59as many tumor cells do in vitro.
29:03And actually,
29:04this is not precipitated by PARP inhibitors,
29:07at least in this context.
29:09And so this is another reason why
29:10we're very interested in these bridges,
29:12because they seem to be the structure
29:14that's most precipitated by PARP inhibitors,
29:15whereas there's just a high rate
29:17of micronuclei all of the time.
29:19But that does not seem to
29:20respond to the addition of,
29:21in this case, a lab rib.
29:24So we also think that for the vast
29:27majority of these persistent bridges
29:29that we observe in response to
29:31PARP inhibitors that there is that,
29:33that there has been a
29:35loss of nuclear integrity.
29:36And so one thing I just want to
29:38point out here is that you know,
29:40one challenge I think in general is
29:42that you cannot see that that these
29:44nuclei have a persistent DNA bridge.
29:47If you just look at DNA stain because
29:49it's too thin essentially or there's
29:51something about the DNA structure
29:52that disrupts the ability of the DNA
29:55stain to intercalate into the bases.
29:56One or the other,
29:57we don't actually know yet.
29:59So actually in order to know
30:00that there's a bridge there,
30:02you need a marker for a bridge.
30:03And actually it turns out that one
30:05of the best markers for a bridge
30:06is this protein called man one,
30:07which is a specific nuclear envelope protein.
30:10And so you know,
30:11you can see quite a beautifully that it is,
30:13you know,
30:14in the nuclear envelope of all cells,
30:15but it really nicely decorates these bridges.
30:17And so this has been a really important tool.
30:19It seems very simple,
30:19but the ability to see the things
30:21that you want to look for is,
30:22is pretty important.
30:23So we've been using this antibody to
30:25this inner nuclear membrane protein,
30:27MAN one in order to surveil this.
30:29And so we can then look at the
30:33coincidence of other factors on
30:35these bridges and I want to focus
30:38specifically on the other elements
30:40of that DNA repair pathway.
30:42So not only is is C gas recruited
30:44and yet we we we interpret that
30:46as ruptured bridges,
30:47but there's also the recruitment
30:48of LEM two and bath.
30:50These are these factors that are
30:51involved in trying to repair these
30:53breaks in in the nuclear envelope
30:54and so this is evidence that that
30:56same kind of antagonism that I
30:58showed you in a induced rupture of
31:00the nucleus is also going on here.
31:03If we identify bridges using this man
31:051 antibody what what we can see is
31:07that all bridges have limb 2 which we expect.
31:10Those are two different inter nuclear
31:12membrane proteins but more than
31:13half of them have C gas recruitment
31:15and so this suggests again that
31:17the majority of the bridges that we
31:19detect are ruptured and that DNA
31:20is likely exposed to the cytoplasm.
31:25I also want to point out that
31:27one of the ideas in that nuclear
31:29envelope reformation is that there's
31:30local recruitment of LEM two and
31:32these escort proteins to try to to
31:34actually seal the nuclear envelope.
31:36And if we kind of zoom in
31:38particularly on LEM two, LEM 2 here,
31:40you can see that there are regions where
31:42there's a really high accumulation of LEM 2.
31:44And so this is likely the region
31:46of this bridge where there's
31:47been a loss of integrity.
31:48And that kind of explains why sea gas
31:50is also seen in this patchy pattern
31:53because there probably is a local effect.
31:55And I'm just showing you here line profiles,
31:57just showing that there's
31:58specific recruitment of LEM two,
32:00this man one protein,
32:01even though it's in a nuclear
32:02membrane protein,
32:02it's kind of distributed throughout
32:04the bridge and that it's not part of
32:06the same repair network as LEM twos.
32:07This makes sense to us.
32:11I also want to point out that while
32:14though that work is in UWB ones,
32:16UDO in the lab has also been looking
32:19at a model of BRCA 1 deficient
32:21triple negative breast cancer.
32:23And so these again are cells
32:24treated with a laparib.
32:25This is just showing you the Dappy staining.
32:27I just want to this I think is a
32:29beautiful example of pointing out that
32:31even when we can't really perceive
32:33these bridges in the DNA stain,
32:35these ones are a little bit earlier.
32:36So you can still kind of see
32:38faintly that there's DNA staining.
32:39You can appreciate the changes in
32:41nuclear shape that are tied to this,
32:43just like those heart-shaped nuclei
32:45in that first movie that I showed you
32:47with the nuclear localization signal.
32:48So there's there's actually 2
32:50hallmarks we think that we can use
32:53as essentially I know proxies for
32:55the presence of these bridges.
32:57One of them is the kind of
32:58orientation of these two nuclei.
33:00But the other is that there are these
33:02classic changes in nuclear shape that
33:03we see that are coincident with this.
33:05And this may become relevant if
33:06we think about whether we can use
33:08the prevalence of these structures
33:09as a biomarker,
33:10which is one of our kind of
33:13long term interests.
33:14This is just showing you that
33:15in this MDA 436 line,
33:17preliminarily what we see is that
33:18there's a dose dependent increase
33:20in the number of cells with these
33:22bridges in response to a laparib,
33:24whereas we don't see this in a
33:26different triple negative line that's
33:28BRCA 1 proficient and HR proficient.
33:32So I've shown you this that we likely
33:35have these persistent bridges,
33:37they accumulate in the context of a laparib,
33:39they recruit C gas.
33:40But is there actually activation
33:42of the innate immune pathway?
33:43Just to remind you that the canonical
33:45pathway is that C gas produces
33:47C gamp which activates sting
33:49which phosphorylates TDK one and
33:51IRF 3 and leads to interferon
33:53stimulated gene expression.
33:55So if we look at the GWB one cells
33:58in the presence of a lab rib
34:00compared to the vehicle control,
34:02we don't actually see the level
34:04of TBK one phosphorylation,
34:05much of an effect.
34:06But if we look at IRF 3 phosphorylation,
34:08we see that there is a stimulation
34:12of the phosphorylation of IRF 3.
34:14And if we look at the downstream consequence,
34:16which is interferon stimulated gene
34:18expression, just picking two of those genes,
34:20we do see that we can stimulate.
34:22We can see stimulation of interferon
34:24stimulated genes with the addition
34:25of ELABORA in this cell line.
34:27And just to point out this,
34:29how much of A signal we get does depend
34:32on how intact the C gasting pathway is.
34:35And many tumors have an activated
34:37C gas expression likely because
34:39there is selection against the
34:41pathway that I'm talking about.
34:43But these cells do as you can see here,
34:44they do express sea gas and sting.
34:47But this is about as much stimulation
34:49as we can probably get in this
34:51line because this is an experiment
34:53where we've just transfected DNA
34:54to drive an innate immune response.
34:56This is the two people using this
34:58in this field all the time.
34:59And we get a pretty similar degree
35:01of stimulation as we get with
35:03the elaborate treatment.
35:04So that may be kind of the top of what
35:06we can get in this particular cell line.
35:08So we do think although this is only
35:10about four fold increase longitude
35:12full change of two that this is that
35:15this is a pretty strong response
35:17for this cell type.
35:19So does the you know does this the
35:22response actually require C gas that
35:25I'm showing you this stimulation
35:27of this innate immune pathway.
35:29So now we're just doing an experiment
35:31where we're knocking down C gas
35:33and you can see the knock down by
35:35qPCR to the C gas gene Here.
35:36I'll just walk you through this.
35:37This is the same stimulation that
35:39we saw of these two genes with
35:41the addition of a lab rib.
35:42If we now knock down C gas,
35:45what we can see is that this does to
35:48some extent limit the activation.
35:50But to what extent that is we're not,
35:52we're not where I would say
35:54where it's unclear yet whether C
35:55gas is completely responsible.
35:57We're trying to be kind of very agnostic
35:58about what is lying downstream.
36:00And so one of the things we're
36:02doing is generating C gas knockout
36:03Isagenix of these cell lines to
36:05really look at how much sea gas
36:07is important for this
36:08response and also of course
36:10for the cell death mechanism.
36:12One of the ideas that I set up was that
36:14this nuclear envelope repair network
36:16could be antagonizing surveillance by
36:17the innate immune system and we have
36:19some evidence that that's the case.
36:21So just to remind you,
36:22the idea is that Bath and this
36:24LEM two protein come in to recruit
36:26escorts to seal these breaks in the
36:28nuclear envelope and this limits
36:29sea gas access and activation.
36:31So here is an experiment where
36:34we have used siRNA to knock down
36:37the bath protein to test this.
36:39So again here you can see the
36:42interferon stimulated gene expression
36:43in a lab with elaborative treatment.
36:45This is again in the UWB 1289 cells.
36:48Interestingly,
36:48and consistent with another study
36:50in the literature,
36:52if you just knock down Bath,
36:53you also get a stimulation of
36:55an immune signaling,
36:57which suggests that just knocking
36:58down bath and removing it can
37:00always stimulate some sea gas.
37:01And that may be as cells are reforming
37:04their nuclear envelope or some other
37:06aspect of the normal cell Physiology.
37:08However,
37:08if we now add a lab rib,
37:10we can boost this even further,
37:12suggesting that there's a synergy
37:14synergistic effect of knocking
37:16down Bath and adding a lab rib,
37:19which suggests that a lab rib actually
37:21precipitates these kind of breaks
37:23in the nuclear envelope because
37:24of these entangled chromosomes.
37:26And then normally Bath would
37:27be suppressing the signaling
37:29downstream of that event.
37:29But when it's not there,
37:30we get more C gas expression.
37:32So this is consistent with
37:34that kind of antagonism.
37:37So I just want to show you just briefly
37:40I because it's just you know we're cell
37:42biologists so we love to look at things.
37:43This is this really cool
37:45reconstruction of what one of
37:46these bridges looks like up close.
37:48And I bring it up because the protein
37:50man one which is in yellow is actually
37:52localized to the mid body and the protein
37:55LEM 2 which is that nuclear repair
37:57protein you can see along this bridge.
37:59But you can see they're
38:00actually in distinct regions.
38:01As I mentioned LEM two is likely to
38:04be along the regions of the bridge
38:07that that are ruptured and actually
38:09man one is sitting on the mid body.
38:12And so one other area that that
38:14we're interested in looking into is
38:16there is a known checkpoint that
38:19controls whether cells do abscission.
38:21That's does seems to be downstream
38:23of surveilling whether there's
38:25been chromosome entanglements and
38:26this is regulated by Aurora B,
38:28which is interesting because
38:29the Aurora kinases have also
38:31been interesting clinically,
38:32although I think I've not been so far
38:35really terribly successful clinically.
38:38But I think that this is one context
38:40where thinking about how Aurora B
38:43might impact these events and be
38:45involved would be very interesting.
38:47So that and that is a reason why you
38:50get these doublet cells that are
38:52binucleate is because there has been
38:54an obscision failure downstream of
38:56the failure to segregate chromosomes.
38:58And so that's something that
39:00we're interested in in pursuing.
39:02OK.
39:02So I just wanted to come back to this
39:06idea that these nuclear integrity defects
39:08are the OR and these mitotic errors
39:11and then nuclear integrity defects.
39:13Could this,
39:14could this be something that we actually
39:16take advantage of as a biomarker?
39:18This is something that we're
39:19really is very preliminary,
39:21but we're very interested in.
39:23So you know as I've already pointed out,
39:25when you have these persistent DNA bridges,
39:27there is this relationship
39:28between the two nuclei,
39:29the result from that mitosis and there
39:32are these changes in nuclear shape.
39:35These are actually H&amp;E from the
39:3910020 trial headed by Pat Larusso
39:42and as well as Kurt Shopper.
39:45And one of the things we've been
39:47looking at is if we look at these
39:49tumors in patients that are bracket
39:51deficient treated with a laparib,
39:53can we see these structures.
39:55And I think what we've been,
39:57we did not expect to be able to see any of
39:59these structures in H&amp;E just to be honest.
40:01But but we're kind of excited that we
40:04think that we can see these kind of
40:07arrangements that are between cells.
40:08You know,
40:08they were not the first people
40:10to ever comment on this,
40:10but I think we're connecting these
40:13observations to an underlying
40:14mechanism that may highlight why we
40:16should be paying more attention to
40:19the prevalence of these structures.
40:21I think particularly because micronuclei
40:22really cannot be perceived in H&amp;E,
40:25this may be a a mitotic error
40:27that's much more
40:28easy to perceive in the
40:30tissue and so might this.
40:32I think and one really interesting
40:34part of this to me is that you
40:36know these there's already an
40:37increase in these bridges just in
40:39HR defective cells that you can
40:41push further with PARP inhibitors.
40:43But this could be a kind of non
40:45genomic way of assessing is there
40:46a homologous or combination or just
40:48DNA repair defect in this cell
40:50line Because I see these mitotic
40:51errors that actually are such,
40:53so large that they can be
40:55perceived even in HNA.
40:57To really validate that we
40:59have to be able to actually,
41:02you know,
41:03convince ourselves that these
41:04really are the structures that
41:05I've been talking about that we
41:06see in tissue culture cells.
41:08And so to be able to do that,
41:09we are working on validating some of
41:11the antibodies that we've raised to
41:13these specific nuclear envelope proteins.
41:15I mentioned it's really hard to see
41:16these bridges even with DNA stain.
41:17You really have to have the right
41:19molecule that you're looking for and
41:21we think that these integral and a
41:22nuclear membrane proteins are exactly that.
41:24And so we're hoping to validate
41:27that these structures indeed
41:29are representative of these DNA
41:31bridges because we can specifically
41:33identify them with these antibodies.
41:35And then in addition,
41:37I think just to be a bit agnostic also,
41:38but other mitotic errors like
41:41Micronuclei LEM two in addition to
41:43being recruited to the ruptured
41:45regions of DNA breaks is also
41:47recruited strongly to ruptured Micronuclei.
41:49And so if we had this molecular tool,
41:51we might also be able to more
41:54accurately quantitate the prevalence
41:55of micronuclei and chemical samples,
41:57which would be fantastic.
42:01And you know why I think that's so
42:03important and I just picked out one example,
42:05I could have picked out many of
42:07them is that there of course is an
42:09interest in expanding PARP inhibitors
42:11beyond you know breast and ovarian,
42:13Braca 1 and Braca 2 deficient patients,
42:15right. So I just pick and picked
42:17out one of these examples of the
42:18fact that there really are some
42:20amazing clinical responders.
42:21This is in pancreatic cancer here.
42:24There has been selection for BRACA
42:26associated pancreatic cancer,
42:28but I think anecdotally,
42:29we know there are triple negative breast
42:31cancers that respond to PARP inhibitors
42:33even if we don't understand why.
42:35There are right very aggressive
42:37prostate cancers,
42:38A subset of which respond to PARP inhibitors
42:40even though we don't understand why.
42:42And so we're hoping that these kind
42:44of biomarkers could potentially
42:46indicate where PARP inhibitors might
42:48be effective even when the molecular
42:50or genetic signature isn't understood.
42:55OK. So just to just to restate
42:58what I've told you today,
43:00while Laparov enhances the prevalence
43:02of these persistent DNA bridges,
43:03there's already more in
43:05an HR deficient context.
43:06But you can push this further with
43:08PARP inhibitors and this does lead to
43:11activation of innate immune signaling.
43:12Their recruitment of bath and
43:15C gas may be antagonistic,
43:17but both are seem to be recruited
43:19to these bridges.
43:20So that suggests that there
43:22many of them are ruptured.
43:24We're interested in whether
43:26just regulating disrupting this
43:28nuclear envelope repair network
43:30could actually further stimulate
43:31the innate immune signaling
43:33downstream of these mitotic errors.
43:36And we're excited about the idea of
43:38these persistent bridges could be an
43:40accessible hallmark of HR deficiency,
43:41which as I said,
43:42we poorly need in terms of what
43:46our next steps are and what
43:48we're focusing on at the moment,
43:49where we really need to understand
43:51if this is really the canonical
43:53ISG expression is relevant here or
43:55perhaps there's some other downstream
43:57consequence that's running in
43:59parallel with the production of Isgs.
44:01That's important.
44:02Again,
44:02you get cell killing in a tumor
44:04cell intrinsic way in a dish.
44:06So we don't know if that's really
44:08a consequence directly of anything
44:10to do with ISG expression.
44:11And so that's something that we're exploring.
44:14We're also taking both candidate
44:16approaches and unbiased screens to
44:18identify what are the factors required
44:21for the cell death in culture.
44:23You in some ways you would have
44:24thought this would have come out of
44:26CRISPR screens which have been done.
44:27But actually I think there are
44:29a lot of reasons to think that
44:30those screens weren't really set
44:31up to identify this mechanism.
44:32And so that's one of the things that
44:35we're setting up to do at the moment.
44:37Again, we're cell biologists,
44:38so we're using correlative light
44:40and electron microscopy to really
44:42understand what's happening in these
44:43DNA bridges and also to and get
44:46information about the DNA structure.
44:48We can do that by looking at accessibility
44:50to the TN 5 transpose ACE as an example,
44:52which is the basis for ATAC experiments,
44:55but you can use that in a microscopy
44:57based experiment as well.
44:59And then we're working with our
45:01partners at AstraZeneca to really
45:03try to test whether we can use
45:06these bridges as a as a biomarker,
45:08you know at the very initial stages
45:11in a really well controlled system.
45:12So one of the things that they have
45:14is that they have xenograft models of
45:16of BRCA 1 deficient tumors which they
45:18then treated those mice with a laparib.
45:20And so we have really nice kind of
45:22ground truth data of HR deficient,
45:24HR proficient,
45:25you know,
45:26with and without treatment with a
45:28laparib or other PARP inhibitors.
45:29And so looking at the H and AE of
45:32those data sets and doing that in a
45:34blinded way will really help us to
45:36understand whether this is something
45:37that's going to be worth pursuing.
45:40All right. So I just like to thank
45:42the people who did the work and then
45:44I'm happy to take any questions.
45:46We have a really great group working
45:49on genome integrity in the lab.
45:50Yuduo is a is a fellow much of what much
45:53of what I showed you today is work from
45:56AJ Kozak who's a PhD student in the lab.
45:59Carrie recently joined the team
46:00and she's going to be working on
46:02these screens for DNA repair.
46:03So we're we're we're almost getting sorry
46:06not DNA repair screens to identify the
46:08the mechanisms of cell death downstream
46:10of PARP inhibitors in the cell models.
46:13And I'll say just joined the lab and
46:15he's going to try to get our our
46:17tissue part of this up and going.
46:18I'd also like to acknowledge Pat
46:21Larusso who who has really been
46:23essential and in all aspects of
46:25getting us involved in this direction.
46:27It would not have happened without her
46:29and I'm happy to take any questions.
46:31Thanks.
46:38Yeah. Have you seen this
46:42type outside of other HRD
46:47such as what do you thinking
46:52Yeah I yeah I think we have not some
46:57of the some of the data that I showed
46:59you from the literature is strongly
47:00suggestive that also in the contents of
47:02bracket two we need mitosis to get cell
47:05death you get innate immune signaling.
47:07We have not, I should ask
47:08Connor actually but I don't,
47:10I don't even think Connor we haven't
47:12stained bracket 2 deficient cells.
47:14So I don't think we've explicitly
47:15done that just cause we've we've been
47:17focused more on BRCA one in our lab.
47:20But I would be highly surprised if
47:22it wasn't the same in a probably
47:242 or a BRCA 2 deficient line.
47:26And and just to make the point you
47:28know others have also seen similar
47:30downstream effects for example of Taxol
47:33treatment and actually have shown that
47:35you know tumor cells that respond
47:37to Taxol have intact C gas stings
47:40signaling and those that don't do not.
47:42So that I think that if this is not going
47:45to be even limited just to HR deficiency,
47:47it's just one of the ways that honestly
47:50TAXOL HR deficiencies of HARP inhibitors
47:53and and even a radiation probably could
47:56all be stimulating the same pathway.
47:58Yeah, as a fault.
48:01So I mean if if you're having an
48:03inhibition of the main signaling,
48:05would these cancers be potentially more
48:07sensitive to alcoholic viruses or kind
48:09of a you know alternative fall strategy?
48:12I think that's a great question.
48:14And I think that as you can
48:16see what we've done,
48:17we've completely ignored right,
48:19any of that, any of that crosstalk.
48:23And I and I think it's if you look
48:26in the literature it's been kind of
48:28challenging and people who've tried
48:29to use this even even not even to
48:31the depth of what you just asked.
48:33But if you look at you know is
48:35sting actually is sting signaling
48:37actually a tumor suppressive or a
48:40tumor driving mechanism, right,
48:42Because inflammation driven by sting
48:44has also been suggested to be a driver,
48:47right.
48:47Is, is actually a tumor driver C gas I think.
48:50And actually if you look at the number
48:53of tumors that have inactivated
48:55C gas versus sting,
48:56very few inactivate sting,
48:58the vast majority have inactivated C gas
49:01if you just look across you know that map.
49:03And so I do wonder if some of the
49:06signaling we're seeing is C gas dependent,
49:08but maybe not strictly through
49:10sting or sting is more complicated
49:12because it's multiple roles and I
49:14think that might be important to
49:15tease out to think about.
49:17Then how is this going to intersect
49:20with approaches like oncolytic viruses.
49:22So I think that's still one of the
49:25confusions at the moment because
49:26honestly there's very high profile papers
49:29saying you know sting agonists would be
49:31great and sting agonists are terrible.
49:32And so that's probably going
49:33to be context dependent.
49:39Oh
49:42go go ahead I'll,
49:42I'll get the Mario and I'll get this.
49:44In the meantime are are there about
49:48cell lines that are HID deficient
49:51where you could look at a lab rib
49:54in one of these cell lines and
49:56compromisers whether there's a role for
50:02the sting activation in in the anti
50:05tumor activity of bilateral because
50:06that that would be an easy way to
50:09determine if if the immune activation,
50:11activation is is important
50:14or not really agree with you.
50:15So I think that that is an
50:17excellent experiment.
50:17It is an experiment that needs
50:18to be done and it you're right,
50:19it's it's obvious and it's achievable.
50:22It hasn't been what our expertise
50:24has been in, but I agree with you
50:25that that's exactly the right.
50:27So we really need some genetic
50:28models to be able to,
50:29to, to do that.
50:30So I I completely agree Jeff has
50:33asked wholexome sequencing is
50:35not as commonly performed as H&amp;E,
50:38but he's curious which degree of
50:40mutational signature derived from
50:41wholexome sequencing indicates the
50:43effective homologous recombination
50:44or is being used as a biomarker.
50:46So to Jeff's point, yes,
50:47this is the only biomarker there is,
50:49is a kind of scoring genomic scarring.
50:52But the challenge I would say is,
50:54and I think Jeff will appreciate this,
50:56is that the cell may be HR defective now,
50:59but then it may become a resistant
51:01to PARP inhibitors because it's
51:02now HR proficient and it will still
51:04have the scarring left from the
51:06period where it was HR deficient.
51:08So that may help us to understand
51:10you know context.
51:12We don't have any reason to think
51:13someone has a germ on or somatic
51:15mutation that they could benefit for
51:16a PARP inhibitor because we see that.
51:18But I'm not sure we're looking for
51:21that signature when there's no reason
51:23to be from the genomics already.
51:25So I don't think we're doing that.
51:26So we're not identifying those patients.
51:27So that's an access issue.
51:29We absolutely are using that when
51:31there's a reason to think that
51:33there is A and HR defect,
51:35but it can't tell us.
51:36It only tells us the history,
51:37it doesn't tell us presently
51:39what's happening in the tumor.
51:40And so I think that's the limitation.
51:43Thanks for your question.
51:47With the bridges, are those all contained
51:49in cytoplasm or do those potentially
51:50contend kind of extra targets for
51:52antibodies and cars or something like
51:54that that would be unique to I think
51:56it's a great question whether you
51:58ever I I think that there's I don't
52:00think we ever see that the plasma
52:02membrane right actually ruptures
52:06although you know escorts also repair holes,
52:10temporary holes in the plasma membrane.
52:12So I won't say that we've actually tested
52:14that and that would be really interesting
52:16to know whether that's the case.
52:18I mean it's interesting that these same
52:21factors actually man one in particular were
52:23all identified as being auto antibody.
52:26They are all tied to auto antibody
52:28to autoimmune diseases as common
52:30targets of many nuclear proteins are.
52:32But I do think that that's interesting
52:34and there's some evidence that that
52:37the Lam 2 protein also probably
52:39in the absence of functional M2,
52:41you do have kind of a prevalence of
52:44autoimmunity which would be consistent
52:46with not being able to do this normal
52:48cycle of Nuvo Con number reformation
52:50does get surveilled through this
52:52mechanism and and can be deleterious.
52:54So I think it's but yeah,
52:55we we don't really what we see is
52:57that you know likely eventually most
52:59of those cells will give up and I
53:01think it's just a just a highlight.
53:03This is why one has to be careful in
53:06assessing in this area particularly facts,
53:08profiles looking at cells that look
53:10like they're G2M cells because you
53:12get these cells that are G2M cells
53:13but they're actually in G1 and that's
53:15because they failed in cytokinesis.
53:17So now they're 4 N but they're
53:20actually no longer mitotic.
53:21And so that's one of the
53:23things that we see here.
53:24So it'll show up in experiments
53:33all right.