Losses of Nuclear Integrity Tie DNA Damage to Innate Immune Signaling

Transcript

• 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&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&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&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&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.