When Watson and Crick Get linked: DNA Interstrand Crosslink Repair and Human Disease

Transcript

• 00:00The Cancer Center grand rounds.
• 00:03I'm Barbara Burtness,
• 00:05and I'm really extraordinarily
• 00:07thrilled to be introducing agato
• 00:10smuggler Jessica as our speaker today.
• 00:13She is an associate professor of genome
• 00:16maintenance at the Rockefeller University.
• 00:18She received her PhD from Rockefeller and
• 00:21her MD from Wild Cornell Medical College.
• 00:25Following a residency in
• 00:27clinical pathology at MGH,
• 00:29she joined Harvard Medical School as a
• 00:31postdoctoral fellow in Stephen Elledge's lab.
• 00:34She's the recipient of numerous awards,
• 00:36including the Irma T Herschel Research Award,
• 00:38the Rita Allen Foundation
• 00:40Scholars Program Grant,
• 00:41the Burroughs welcome Fund Career Award,
• 00:43AV Foundation Translational Research Award,
• 00:46and the Herald Weintraub graduate student.
• 00:49Her lab focuses on DNA repair mechanisms,
• 00:52particularly those involved with
• 00:55interstrand crosslink repair pathways,
• 00:57and she's interested in identifying
• 00:59new genes that are involved in this
• 01:01repair process in order to reveal the
• 01:03mechanisms at play in human diseases
• 01:05that result from deficiencies in
• 01:07interest strand cross link repair.
• 01:09This includes Fanconi anemia and
• 01:12kerio megalith interstitial nephritis.
• 01:14To this end,
• 01:15she successfully identified SLX 4,
• 01:18red 51 and U BE2T is interstrand
• 01:21crosslink repair factors,
• 01:23as well as developed a mouse model of
• 01:25Carrie Magalog interstitial nephritis.
• 01:27And recently she's begun studies
• 01:29revolving around how replication
• 01:31stress is addressed by cells
• 01:33to promote genome stability.
• 01:34She also has a deep interest in
• 01:36those cancers which arise in
• 01:38patients who have DNA repair.
• 01:40Defects.
• 01:40And it's because of this that I've
• 01:42had the extraordinary pleasure
• 01:43of beginning to work with her
• 01:45on the stand up to cancer Grant,
• 01:46which looks at Fanconi anemia,
• 01:48associated head neck cancer.
• 01:51So.
• 01:51Like I said,
• 01:53if you wanna come up.
• 01:55This plaque is to thank you for for
• 01:57coming to give grand rounds in person,
• 01:59which many people have not
• 02:01been willing to do recently.
• 02:05And we're really looking
• 02:06forward to your talk.
• 02:07Thank you so much. Yes, thank you.
• 02:10Well, thank you Barbara for first
• 02:12of all for inviting me all of you
• 02:15for coming to hear the lecture and
• 02:17all of you on zoom for joining in.
• 02:20And it's really my pleasure to to
• 02:23be here and it's been fantastic to
• 02:26work with Barbara and and learning
• 02:29from her more about the the clinical
• 02:32aspects of what we do and hopefully
• 02:35bringing some new therapies to
• 02:37these patients eventually, OK, so.
• 02:41I will talk about DNA interstrand crosslinks
• 02:44and I'll just remind you though that oh,
• 02:48these are my disclosures.
• 02:50I'll just remind you that DNA repair.
• 02:55It's really essential for a lot of
• 02:57aspects of of life and depending on what
• 03:01kind of flavor of DNA damage you have,
• 03:04whether it's from the outside,
• 03:07from the UV light creating these dimers,
• 03:10or from within the cell,
• 03:11something that I'm quite interested in,
• 03:14for example, creating mismatches.
• 03:17There is a repair pathway that's dedicated
• 03:21to these and there are a number of genetic.
• 03:24Diseases associated with inability
• 03:27to repair these different lesions
• 03:30and they come again in many different
• 03:34phenotypes resulting in developmental
• 03:37abnormalities and the generate the
• 03:40generative diseases and a lot of
• 03:43them obviously are associated with
• 03:46cancer prone due to mutagenic nature
• 03:49of these lesions if the lesions
• 03:52are not properly repaired.
• 03:54So my favorite lesion is this interest
• 03:57in Cross link and I'll give you a little
• 04:00bit of introduction about these lesions.
• 04:03This is a covalent linkage
• 04:04of two strands of the DNA.
• 04:07It's repaired by the Franco anemia
• 04:10DNA repair pathway and I'll give you
• 04:12more of a of the mechanism of how this
• 04:16is done in in a bit in later slide,
• 04:18but the whole purpose of this pathway
• 04:21which is activated during DNA replication.
• 04:25Is to create 2 pristine.
• 04:27Double stranded DNA that can be then
• 04:31moved to dollars trends during during
• 04:34the cell division and I'll just
• 04:37mention I won't talk much about it
• 04:40but this pathway is also activated
• 04:42at difficult to replicate regions.
• 04:45So repetitive DNA are loops and there
• 04:48is growing evidence that at any time
• 04:52there is stalling of replication forks
• 04:54the pathway can come to to rescue.
• 04:57Those forks so that this this intern
• 05:02crossing is really our model of
• 05:05of how pathways activated.
• 05:07And the outcomes of abnormality in
• 05:10pathway activation or function are
• 05:13these genomic instability that you
• 05:15can heat see here in this metaphase
• 05:17spread where you see number of
• 05:20these abnormal radial chromosomes.
• 05:22So we see that in cells from Franconia
• 05:26anemia patients when they are treated
• 05:30with external internal crosslink causing.
• 05:35Chemicals like mydomain sincere splatsin you
• 05:38see number of abnormalities including gaps,
• 05:41breaks and these radio chromosomes
• 05:43and the radial chromosomes occur
• 05:46because of two double strand breaks
• 05:48that are that find each other and are
• 05:52joined together forming these radios.
• 05:54And obviously these radios come radio
• 05:57chromosomes can cause instability but
• 05:59can also cause death of cells when they
• 06:03are trying to the cell tries to divide.
• 06:07So. The reason why we know that this
• 06:09pathway is particularly important in
• 06:11stem cells are all these phenotypes that
• 06:14we see in patients with Fanconi anemia.
• 06:17There are many developmental
• 06:20phenotypes including these skeletal
• 06:22abnormalities that you see here,
• 06:25but pretty much any,
• 06:26any system can be affected and that's seen in
• 06:31patients who are truly null for this pathway.
• 06:35Majority of patients will present with Pence,
• 06:37Cytopenia.
• 06:38Around age, median age of 10,
• 06:41and even though it's called anemia,
• 06:43all of the.
• 06:46All of the hematopoiesis can be
• 06:49affected and and actually platelets
• 06:52are usually the first to to draw.
• 06:55And then there is very this very
• 06:57interesting phenotype that will spend
• 06:59a lot of time talking about the cancer
• 07:02predisposition MSDS and AML occurs in
• 07:05the setting of pancytopenia and squamous
• 07:08cell carcinoma occurs later in life
• 07:12but still in in at young age of average 31.
• 07:16We have very young patients as
• 07:18I'll I'll show you in in future
• 07:21slides and then depending on the
• 07:23actual path of gene that's mutated.
• 07:25We can also have breast cancer,
• 07:27medulloblastoma and and
• 07:29other embryonal tumors.
• 07:31And there are other phenotypes that are
• 07:34still fairly poorly understood like
• 07:36infertility and endocrine abnormalities.
• 07:38So this is a great model in my
• 07:42mind for studying DNA repair.
• 07:46So today, I'll tell you about the first part,
• 07:49very short part about the mechanism
• 07:51of interstrand crosslink repair.
• 07:53Secondly,
• 07:53I'll talk about the identification
• 07:55of endogenous sources,
• 07:57sources of interstrand crosslink.
• 07:59And today I'll talk about the
• 08:02bone marrow side.
• 08:04But we are very much interested in
• 08:06those in the keratinocytes as well.
• 08:08And this is an ongoing work in my lab.
• 08:11And then I'll talk about the mechanism
• 08:14of tumorigenesis and Fanconi anemia.
• 08:16Especially in the squamous cell carcinoma.
• 08:19So just to give you a flavor of how
• 08:23this pathway actually functions,
• 08:27this is a very regulated process of
• 08:30repair of these of these lesions.
• 08:33So we start with the ACL that you
• 08:36that I've shown you before and this
• 08:39is the structure that we know forms
• 08:42when the replication forks stall
• 08:44at the lesion and some of this work
• 08:46has been done in in human cells,
• 08:48some of it.
• 08:49This work was done in Johannes Walters
• 08:51lab in the Xenopus egg extract system.
• 08:53So biochemically,
• 08:55it's a fairly understood pathway.
• 08:58What's essential in this pathway,
• 08:59and I'll show you the proteins
• 09:01that are involved in a moment,
• 09:02but what has to happen is that
• 09:05the the DNA needs to be broken,
• 09:07but in a very regulated way that then
• 09:10gets repaired through translesion
• 09:12synthesis and then through homologous
• 09:15recombination that requires
• 09:17BRC 2 and other proteins.
• 09:19And this eventually leads to this full
• 09:23DNA repaired repaired DNA with with
• 09:27two strands that are fully repaired.
• 09:31So the proteins that are participating
• 09:34in this pathway really has been
• 09:38identified through studies of
• 09:41Franco anemia patients and.
• 09:43Just go to go back for a moment
• 09:45to the history of this disease.
• 09:48Fanconi anemia has been identified in
• 09:501927 by Guido Fanconi and described
• 09:54as hereditary pancytopenia with
• 09:56short stature and hyperpigmentation.
• 10:00The first gene fancy was identified 92,
• 10:02but even then it was known that more
• 10:05than one gene will be causative
• 10:07for this mutation.
• 10:09And this was Buckwald lab who
• 10:11identified the first gene.
• 10:13And now we know that there are 22 genes
• 10:16that can be mutated in Fanconi anemia
• 10:18and we actually are working on on #23,
• 10:22which fits very nicely in this pathway.
• 10:26And the this data is actually showing
• 10:30the percentage of of patients with
• 10:33different mutations in different genes
• 10:35and this is from our International
• 10:39Franconia registry that I,
• 10:41I run at at the Rockefeller University.
• 10:45And you can see that Frank a mutations franc
• 10:48and Frank G mutations are the most common.
• 10:51More majority of the diseases of this
• 10:54disease is autosomal recessive we have.
• 10:56Language which is excellent and
• 10:58then uh thank our that we've
• 11:01discovered right 51 mutations.
• 11:03These are always the Novo dominant
• 11:05and we have a number of patients now
• 11:08with different mutations and and
• 11:09rat 51 and all of them have the same
• 11:13dominant function and they're all
• 11:15denovo presumably because right 51
• 11:17is necessary during biosis and we
• 11:19would never recover any any patients,
• 11:22any transmission really through
• 11:25through the germline.
• 11:27OK.
• 11:27And this registry that I've mentioned
• 11:29has been really instrumental for
• 11:31our work and it was started in
• 11:341982 by Arlene Auerbach who's still
• 11:37participates in the lab meetings
• 11:39and has a lot to say about about
• 11:42the disease and has been really
• 11:44wonderful in in in helping us through
• 11:47this through some of these studies.
• 11:50So now I'll spend really few just few
• 11:54minutes on the proteins themselves.
• 11:56Just to give you a gift,
• 11:58again a flavor for the for this pathway.
• 12:01So majority of the proteins whose
• 12:04genes are mutated in Fanconi patients
• 12:07form this large core complex.
• 12:10All of the colored proteins are
• 12:12actually those that have mutations
• 12:14in Fanconi in Fanconi patients.
• 12:17And you can see that this core complex
• 12:20is situated that this cross link as
• 12:22if it was identifying the the damage.
• 12:26It's actually unclear.
• 12:27How that's exactly done?
• 12:30The key to this core complex is that
• 12:34it has this E3 ubiquitin ligase fancl
• 12:37and with Frank T, which is the E2,
• 12:41they the whole purpose of this core
• 12:43we we understand now that the whole
• 12:46purpose is to ubiquitinated Frankie
• 12:49and Frankie 2 and that process
• 12:51through beautiful structural studies
• 12:53that have been done in the past.
• 12:55Semoran Pablo Ditches lab
• 12:57shows that the Fanki and Frank.
• 13:00Who forms a clamp around the the crosslink?
• 13:04Actually there are more,
• 13:05probably more clamps around as
• 13:07shown by the Dean slab that sort
• 13:10of decorate this cross link.
• 13:12Eventually,
• 13:13and this is again not quite worked out,
• 13:17the proteins that are important for
• 13:20in making these incisions come in
• 13:23and one of the important proteins
• 13:25here is this SLX 4 which acts as a
• 13:29scaffold for three different nucleases.
• 13:31And it's quite amazing how the three
• 13:35nucleases are associated with one
• 13:37protein and frank P is or a cell.
• 13:40X4 is essential not only for scaffolding.
• 13:43But for the function of the of these
• 13:46other nucleases without this slex
• 13:484 none of the nucleus is actually
• 13:51are functional within the cell.
• 13:53They have full in vitro function
• 13:55but not not cellular function.
• 13:57So it's it's fascinating but in
• 13:59this case XPF is the one that's
• 14:03making the incisions.
• 14:04I'll also mention the protein that
• 14:06that or disease that Barbara mentioned
• 14:09the carrier Magali Constitution
• 14:11nephritis which has mutations.
• 14:13In fan one and Fan 1 gene codes
• 14:17for this another nuclease and this
• 14:20system which can also unhook,
• 14:22but it can unhook this this cross link
• 14:25outside of S phase and we're still
• 14:28very much interested in knowing how
• 14:31how that affects the the the function
• 14:34especially in the kidney and and the liver.
• 14:39Eventually,
• 14:39after these, uh,
• 14:41these incisions are made,
• 14:43you bring in translesion polymerases
• 14:46and they are used to to replicate
• 14:50across this unhooked lesion.
• 14:53And that's actually probably
• 14:56not a mutagenic process.
• 14:59It's mostly a Posada that that can
• 15:03that can repair this this lesion.
• 15:06And eventually, as I mentioned,
• 15:08there's a whole homologous.
• 15:10Combination pathway that is involved
• 15:13in a repair of of the double strand
• 15:16break and here all of your proteins,
• 15:18favorite proteins that are
• 15:20associated with breast and ovarian
• 15:22cancer when they're mutated when
• 15:24the genus mutated in one copy.
• 15:26So BRC 2, power B2B,
• 15:30RC1,
• 15:30rad 51 are are functioning in this in this
• 15:35pathway and eventually we go back to SLX.
• 15:39Or and associated Mercedes one and
• 15:43the selects one where the repair needs
• 15:47to be completed using these these
• 15:51nucleases or a bloom healer case.
• 15:54I will also mention that there is
• 15:56another another pathway that's and
• 15:58the reason why the RCA two and Route
• 16:0051 are necessary at these cross
• 16:02links and that's to protect these
• 16:04cross links from from nucleases and
• 16:07our lab has shown that it's there.
• 16:10There is a protection against DNA to
• 16:13and Warner and other labs before us
• 16:17have shown for especially the Jason
• 16:20lab that there is also protection
• 16:22against MRE 11 at stalled.
• 16:24Forks for example,
• 16:26after hydroxyurea and other other damage.
• 16:29So I this is this is all I I have
• 16:32to say about the mechanism of
• 16:34of function of these proteins.
• 16:36There are still a lot of details that
• 16:39need to be that need to be filled in,
• 16:42but we have an idea of how
• 16:44how this pathway functions.
• 16:46And the only other thing that I say
• 16:48I'll say about this is that it's
• 16:51really a very well regulated process,
• 16:53so you make double strand breaks.
• 16:55But they are immediately being
• 16:57shuttled to the proper repair.
• 16:59OK, so we'll talk about what happens
• 17:01in the in the third part of my talk,
• 17:04we'll, we'll talk about what
• 17:05happens when this repair is abnormal
• 17:07and the brakes are still made,
• 17:08but they are inappropriately repaired.
• 17:13So in the second part,
• 17:15I'll talk about the endogenous
• 17:17sources of interest and cross links.
• 17:19So I showed you that the Franconia pathway
• 17:23is necessary for repair of I CL's,
• 17:26but you may ask, well,
• 17:27where are these IC's coming from?
• 17:30Obviously in the in the
• 17:32clinic we are very much.
• 17:37Aware of cisplatin and mitomycin C and
• 17:40interest rate crosslinks occur after
• 17:43treatment with these with these agents,
• 17:46although there are other
• 17:47lesions that occur as well,
• 17:48right intrastrand crosslinks and others.
• 17:51The exogenous damage also can come from
• 17:55bacterial metabolites, and there are.
• 17:58There are E coli strains that are
• 18:02making ICL inducing. Chemicals.
• 18:04It can come from acetyl,
• 18:06aldehyde, from ethanol.
• 18:08It can come from tobacco smoke,
• 18:11which is full of formaldehyde,
• 18:12acrolein and other mutagens.
• 18:15But formaldehyde and acrolein are
• 18:17very good into strong cross linkers.
• 18:20But we are particularly interested
• 18:23in sources of endogenous DNA damage
• 18:26and really beautiful work that I'll
• 18:28describe a little bit in the two
• 18:31slides that are coming up have shown
• 18:34that endogenous toxic metabolites
• 18:37formaldehyde and acetaldehyde are
• 18:41particularly important in the bone
• 18:44marrow and definitely contribute to bone
• 18:47marrow failure in Franconia patients.
• 18:50And in mouse models,
• 18:52so let me just introduce them.
• 18:56This work was done mostly by
• 18:58KJ Patel's group in the UK and
• 19:02they've defined LDH 2 and ADH 5.
• 19:05These are alcohol and aldehyde
• 19:07dehydrogenases as being important
• 19:09for this first tier of protection.
• 19:11So they remove the toxins from
• 19:14the cell and that prevents I CL
• 19:18DNA damage and of course the.
• 19:21Frankonia pathway is a second
• 19:23tier of protection.
• 19:24Whatever has been incorporated as
• 19:27cross links will be removed as well.
• 19:30So let me just give you a a shore
• 19:33segue and talk about a LDH 2 and ADH 5.
• 19:37So a LDH 2 is necessary for removal
• 19:40of acetyl aldehyde which is which can
• 19:43come from ethanol from the outside,
• 19:46but also from metabolism,
• 19:49intracellular metabolism.
• 19:50And LDH 2 is responsible for
• 19:54detoxifying it to acetate.
• 19:57And a KG Patels group has described
• 20:01LDH 2 knockouts when combined
• 20:04with Fangy 2 knockouts.
• 20:06The mouse for the first time
• 20:08really developed bone marrow fell
• 20:10failure and leukemia.
• 20:12So the mouse model without just fancd
• 20:152 mouse model does not develop.
• 20:19Bank bone marrow failure or leukemia.
• 20:21It has mild hematopoietic dysfunction,
• 20:24but it's nothing that you consider
• 20:27a bone marrow failure.
• 20:29In humans combination of FANK
• 20:32mutations and usually it's frank,
• 20:34a mutation and a LDH 2 star 2
• 20:38which is the dysfunctional.
• 20:40Variant of LDH 2 causes early onset
• 20:43of Fanconi anemia phenotype and there
• 20:47is a really beautiful paper from
• 20:50the Takata group that have that has
• 20:54shown that and that was published
• 20:57in 2013 which really solidified
• 20:59our thinking about this this as a
• 21:03as a modifier of Fanconi anemia.
• 21:05And I also will mention that together
• 21:09with Chris Vakoc's lab we showed that.
• 21:11Somatic Aldh 2 mutation silencing
• 21:16in AML's results in in dependency
• 21:19on the Fanconi anemia.
• 21:22So when when Chris's lab and they
• 21:25are at Cold Spring Harbor did a
• 21:28screen and showed in number of
• 21:31AML's dependency on Fanconi anemia.
• 21:34He called us up and and asked what
• 21:36what should I look at why are they dying?
• 21:38And I said well just look at LDH 2 I
• 21:41bet it's. Yeah, 2 and that's what
• 21:43that's what it turns out to be.
• 21:45We just helped him a little bit but
• 21:47some of some of AML's depend on on
• 21:50Franconia because of the of the
• 21:52silencing of LH2 which I think is quite
• 21:56interesting and has implications for
• 21:59for therapies for these for these AML.
• 22:01So then ADH five came.
• 22:05And that that was also from KJ Patel's
• 22:09work that ADH five is necessary for.
• 22:13I mean this is this has been known
• 22:15that has been known that ADH five
• 22:18is necessary for detoxification of
• 22:20formaldehyde through this through this
• 22:23pathway and again they create a DH,
• 22:26five -, 22 negative mice and they
• 22:28had bone marrow failure but also
• 22:30had gromel glomerular damage.
• 22:32That's actually poorly understood
• 22:34still and there is this.
• 22:36Uh new digenic human disease of
• 22:40LDH 2 ADH 5 double knockouts or
• 22:44the dysfunctional alleles which
• 22:47have bone marrow failure,
• 22:48myelodysplastic syndrome,
• 22:49foot for some reason,
• 22:51foot skeletal abnormalities
• 22:54and also neurodegeneration.
• 22:56So now you have phenotypes that are
• 22:59associated with ACL's and creation of
• 23:02I CL's in multiple different tissues.
• 23:05Including in the brain and we if somebody
• 23:08is interested in knowing why the brain,
• 23:10we can discuss that later.
• 23:12But there that really shows
• 23:14us that there is a lot of.
• 23:18Well,
• 23:19the cells have a lot of different
• 23:21pathways for as this first tier
• 23:24of protection and my lab has been
• 23:27interested in identifying other
• 23:29pathways in different cells as cell
• 23:32types and we started with with cells
• 23:34of jurkat cells which are T cell
• 23:37leukemia cells to figure this out.
• 23:39So our hypothesis was that there
• 23:41would be more of these detoxification
• 23:44path pathways in the cells and
• 23:47by understanding what that is.
• 23:49They might actually be equipped
• 23:51with understanding of what can we
• 23:54increase to have some preventive
• 23:57measures in in Fanconi anemia.
• 23:59So this is work from Munjung Jung
• 24:02who's now a a assistant professor
• 24:05at Hopkins and she was a clinical
• 24:08scholar in my lab and she did
• 24:11a metabolism focus screen.
• 24:13So this is crisper screen or that she.
• 24:18Performed in jurkat cells.
• 24:20And she took Frankie to positive
• 24:23and negative jurkat cells.
• 24:25And the whole purpose was to
• 24:27identify the genes that are dropping
• 24:30out from fangy to negative cells.
• 24:33So these are essential,
• 24:35would be essential for faculty to cells.
• 24:38And you can see that there are
• 24:40lots of genes that that dropped
• 24:42out and some of them were actually
• 24:45consistent with what we know.
• 24:47So these are these cells, SLC 7A.
• 24:5111 and Assoc 3A2.
• 24:54They form a complex that is responsible
• 24:57for cysteine movement across the cell
• 25:01membrane and that feeds into glutathione,
• 25:04which is important for
• 25:06formaldehyde detoxification,
• 25:07so that fit.
• 25:08There was also a we did get a
• 25:11DH5 in the screen,
• 25:13but the gene that we concentrate
• 25:16on concentrated on was a LH9A1.
• 25:20Eight out of 10 guides scored in
• 25:24this in this assay and the rest
• 25:27of the work was really based on a
• 25:29LH9A1. This is a aldehyde dehydrogenases
• 25:33in metabolizes amino aldehydes,
• 25:35at least in vitro and it's highly
• 25:38expressed in liver, muscle and kidney.
• 25:40And the idea here is that it will there
• 25:44will be tax toxic metabolite that is
• 25:47now detoxified by AL DH9A1 and this.
• 25:50If you don't have aldh 91,
• 25:52the toxic metabolite will create
• 25:54DNA damage and you don't have if
• 25:56you don't have Franconia pathway,
• 25:58these cells would die and get transformed.
• 26:00But if you have Franconia pathway,
• 26:02you would have cell survival.
• 26:05So umm munjung has validated
• 26:09this in multiple assays,
• 26:11and there is a bio archives paper that
• 26:14we've we put out there competition.
• 26:17Assays cells that didn't have a
• 26:21two and a LDH 9A1 did much more
• 26:25poorly and growth assays.
• 26:28She also could show that there were
• 26:31increased numbers of apoptotic cells,
• 26:33increased DNA damage through gamma H2AX.
• 26:37And increased chromosome breakage.
• 26:38So this is where we can look at
• 26:42without exogenous DNA damage.
• 26:44Look at numbers of breaks that are
• 26:47occurring in the double knockouts
• 26:50in different different clones and we
• 26:52can see that there is a increase of
• 26:55of chromosome breakage if we don't
• 26:58have a LDH 9A1 Infinity 2 cells.
• 27:02She also used human hematopoietic
• 27:04stem cells depleted Frank A with SH.
• 27:08RNA's did a knockout with of LDH 9A1
• 27:11and those could make fewer colonies.
• 27:14But what happened?
• 27:15We made a mouse expecting some
• 27:18level of of bone marrow failure
• 27:21and we've seen phenotypes so these
• 27:24mice are born smaller than fanki.
• 27:27Negative mice which are already
• 27:28small have increased number
• 27:30of eye abnormalities at birth,
• 27:32which has been seen as a DNA.
• 27:33Damage outcome and they have increased
• 27:37number of variant tumors in aged mice,
• 27:40but really very mild hematopoietic defect.
• 27:43So that tells us that there will be
• 27:46differences also between mice and
• 27:48men in how the these detoxification
• 27:51pathways are working.
• 27:52And This is why we when we are modeling
• 27:55things in the mouse with in this in
• 27:57this pathway I think it's important
• 27:59to to do things and in parallel
• 28:02in human system and in the mouse.
• 28:04And and compare and contrast
• 28:06because there will be differences
• 28:08and whatever we model in the mouse
• 28:11actually might not be as as important
• 28:14in humans and and vice versa.
• 28:18So the last question that
• 28:20we wanted to ask is, well,
• 28:22we've identified the the enzyme,
• 28:25but what's really the,
• 28:27the problem, what's the source,
• 28:30what's the toxic metabolite that these
• 28:32cells are dealing with so much junk
• 28:35did a suppressor screen as we are,
• 28:37we are geneticists at heart.
• 28:39So she took the double negative cells
• 28:43frankly to a LH9A1 double knockouts and.
• 28:47I redid the screen the the same method
• 28:51with using the same metabolism library,
• 28:54but now wanted to see the cells
• 28:56that are actually growing better.
• 28:58So now we are knocking out some other
• 29:02gene that is necessary for production
• 29:05of our of our toxic metabolite.
• 29:10And the Omni gene that we've
• 29:14identified is this ATP 13A3.
• 29:16We've actually identified a LDH 9A1 itself,
• 29:20but that's we think is actually
• 29:22through reversion mutations.
• 29:24So these are now we are creating a mutant
• 29:28and a LDH 91 that reverts the function.
• 29:31So we in a way it shows that
• 29:34our screen worked in both ways,
• 29:37but this gene is quite interesting
• 29:39because this is now.
• 29:41A protein that's necessary codes for a
• 29:44protein that's necessary for polyamine,
• 29:47for example spermine.
• 29:51Movement through the from between
• 29:53different membranes and if we
• 29:56have high level of spermine,
• 29:58we know at least this is
• 30:00what we think would happen.
• 30:02We would get high levels of I mean
• 30:05the proper now and as I mentioned I'll
• 30:08ADH 9A1 is involved in detoxification
• 30:10of amino propanol and propanol
• 30:13can lead to acrolein and cells.
• 30:16We actually haven't yet shown that this
• 30:18these are this is what happens in the system.
• 30:20But everything that we have is consistent
• 30:23with this, with this hypothesis, so.
• 30:28If we have triple mutants, these cells,
• 30:32these cells now can grow much better,
• 30:36presumably because we are sequestering the
• 30:40polyamines outside of the away from the DNA,
• 30:44not creating these these crosslinks.
• 30:49So I think this is a a an example
• 30:53of how we are.
• 30:55Identifying endogenous types of DNA
• 30:58damage that are necessary for crosslink
• 31:01repair for that are necessary to be
• 31:04repaired by franklinia pathway and
• 31:07other pathways of crosslink repair.
• 31:10And really adding to this idea of
• 31:15how the first tier of protection is
• 31:19necessary to to protect the the genome,
• 31:22not creating interstrand crosslinks.
• 31:25And that eventually obviously is necessary
• 31:28for normal cell and organ function.
• 31:31So with that,
• 31:32I'll move to the last topic,
• 31:35which is cancer and Fanconi anemia,
• 31:38children and young adults.
• 31:42And when you think about Franco
• 31:45anemia pathway,
• 31:46there are,
• 31:47I already mentioned that there are
• 31:50different types of of tumors that
• 31:53can form and one of the tumors that
• 31:56we think about are these embryonal
• 31:58tumors and AML that are forming when
• 32:03homology directed repair is absent.
• 32:05So these are patients who
• 32:08have beers biallelic BRC 2.
• 32:11Or probably 2 mutations and they are
• 32:15identified early age you know they
• 32:17they have they developed these tumors
• 32:20within the first five years of of
• 32:23their life and we actually have a
• 32:26mouse model of of medulloblastoma
• 32:28that is quite interesting but
• 32:30not ready for prime time.
• 32:32But the the reason why we are
• 32:35interested again in these tumors
• 32:37is because we want to understand
• 32:39why the granule progenitor.
• 32:42Cells or the cells that lead to
• 32:44neuroblastoma or tulips tumors,
• 32:46why do they really need BRC 2 function?
• 32:50What's so special about these cells
• 32:52that require BRC 2 function or probably
• 32:552 function homologous recombination.
• 32:58So that's something that we are
• 33:00we are continuing to develop.
• 33:03But outside of homologous recombination
• 33:05when the ICL repair is abnormal,
• 33:08so these are patients with mutations in
• 33:11any of the core complex fanki FANGY 2 SLX 4.
• 33:15We don't have yet patients
• 33:17who have tumors with XPF,
• 33:18but that's more of a problem of the PF
• 33:23being necessary also in other tissues.
• 33:26These patients develop AML's and also
• 33:29develop squamous cell carcinomas
• 33:32and today we'll concentrate on
• 33:34squamous cell carcinomas.
• 33:36So this is a knowledgeable audience
• 33:39about squamous cell carcinomas and
• 33:42we'll concentrate on head and neck.
• 33:45And in sporadic cases,
• 33:47you have two types of of head
• 33:50and neck cancers.
• 33:51One of them is HPV associated
• 33:56that we want to spend too
• 33:58much time talking about.
• 33:59The other one is a HPV negative
• 34:03and that those tumors are
• 34:06associated with carcinogens that
• 34:08are present in tobacco and alcohol.
• 34:10And I would also claim that endogenous.
• 34:15Aldehydes will play a role in here as well.
• 34:19And maybe for franconi patients who might
• 34:21not be exposed to alcohol and tobacco
• 34:24as much as the general population are,
• 34:27those indulgence aldehydes will be important.
• 34:32The the truth about head and neck cancer
• 34:35is that the patients are diagnosed late,
• 34:38the survival is still poor and it's not
• 34:41really decreasing and if it's decreasing
• 34:44it's it's just because there's more,
• 34:46there are more HPV positive cancers
• 34:50and again the treatment can be quite
• 34:56horrendous for for the patients and
• 34:59really learning from from Barbara.
• 35:01The the need is really to stratify these
• 35:05patients to also to identify novel
• 35:08therapeutics that might not be DNA damaging.
• 35:11So we, my lab got interested in these
• 35:16tumors because of our registry and tumors
• 35:20and Fanconi anemia patients just observing
• 35:23the patients who are becoming young adults,
• 35:27especially bone after bone marrow transplant.
• 35:29But even without the bone marrow transplant,
• 35:32many of these patients have developed
• 35:35cancer and in 2003 there was a paper
• 35:38from the registry when I wasn't.
• 35:41Yet there but showing that patients with
• 35:45Fanconi anemia had 700 at least 700 fold
• 35:50increase of of tumorigenesis in in the
• 35:53head and neck cancer in head and neck
• 35:56area and for vulvar cancer and for for
• 36:01cervical cancer and anal cancer there
• 36:04were thousandfold increases in these.
• 36:07So these are cancers that are squamous cell
• 36:11carcinomas in Fanconi anemia patients.
• 36:14And they are very there.
• 36:17There are present in the tongue
• 36:21gingiva and buccal mucosa,
• 36:23some pharynx and larynx,
• 36:25but a lot of cancers in in the oral cavity.
• 36:30We do have some esophageal cancers as
• 36:32well and those are quite interesting,
• 36:35but all of them have similar similar
• 36:39genetic or molecular characteristics.
• 36:41So here is our and this is now I'll describe
• 36:46work and I'll go fairly quickly through it.
• 36:49Work through that was recently published.
• 36:53This is our cohort of patients who were
• 36:56whose tumors were sequenced and you
• 36:58can see that the agent diagnosis is.
• 37:00On median of 31 years old,
• 37:03but we have some 13 year old 16 year
• 37:06olds with head and neck cancer that's
• 37:09extremely aggressive and extremely
• 37:11difficult to treat since we cannot use
• 37:14this platin for for these patients
• 37:16and they are radiation sensitive as
• 37:19well as as patients although not that
• 37:22radiation can still be used carefully
• 37:24you can see that these patients don't
• 37:27do well at all much worse than the.
• 37:30Sporadic cancers and patients who are
• 37:34here and survive long usually are
• 37:38patients who had successful surgical
• 37:41resection of their of their primary tumor.
• 37:46Or occasionally radiation therapy
• 37:48that actually was was successful.
• 37:51OK,
• 37:51so we were very much interested
• 37:54in understanding the molecular
• 37:57pathogenesis in these tumors and we
• 38:00showed that majority of these tumors
• 38:03were HPV negative instead majority of
• 38:06them had P53 mutations and the P53
• 38:10mutations were of variety, nonsense,
• 38:13missense, frame shifts and deletions.
• 38:17But P53 was pretty much the only Gina gene
• 38:21that was mutated through point mutations,
• 38:24OK.
• 38:25All the other changes were actually
• 38:28changes that were associated
• 38:30with structural variants.
• 38:32So here you can see that these are
• 38:35number of mutations in across different
• 38:38cancers through TCG data from TCG
• 38:40data and I inserted our Fanconi
• 38:43tumors and they have point mutations,
• 38:45the number of point mutations.
• 38:46It's pretty low and these tumors are lower
• 38:49than in sporadic head and neck cancers.
• 38:52And if we look at the what kind
• 38:55of signatures are present,
• 38:57there is no homology,
• 38:59directed repair or smoking signature and a
• 39:03lot of these are signatures of cell division.
• 39:07So the reason why we think that
• 39:09they have smaller, fewer.
• 39:11Mutations is because they are
• 39:15present in younger patients.
• 39:17And instead what we see is this
• 39:20huge genomic instability where this
• 39:22is a circus plot and all of these,
• 39:25all of these.
• 39:27Lines indicate translocations and
• 39:30structural variants that are present
• 39:33in in these tumors and if we look at
• 39:36number of structural variants across
• 39:39these tumors the there is about 2 to
• 39:43threefold increase when we compare
• 39:45it to HPV negative tumors and HPV
• 39:48positive tumors have very few of
• 39:50these since they already mutated
• 39:52P53 and RB and really don't have to
• 39:56rely on these structural variants.
• 39:59And this is comparable to the
• 40:02structural variants that we see in
• 40:04BRC 2 or BRC 1 tumors which have
• 40:07high levels of structural variation.
• 40:12As far as types of type of of structural
• 40:15variance, we see number of them,
• 40:16we see deletions, translocations,
• 40:19inversions, all of them are increased.
• 40:21And if you take proportion
• 40:23of structural variance,
• 40:25actually the on the there is a slight
• 40:28increase of tandem duplication.
• 40:30So pretty much across all structural
• 40:33variants there is an increase.
• 40:36Which is consistent with breaks being
• 40:39made when the DNA when Fanconi pathway
• 40:43cannot function and these breaks being
• 40:46being repaired inappropriately and
• 40:49it's and and to show you the extent
• 40:52of how inappropriate this repair
• 40:54is we used number of techniques of
• 40:58long read sequencing and you can see
• 41:01that pieces of chromosomes are from
• 41:03different chromosomes chromosome 8.
• 41:06For 1511,
• 41:06they're all sort of strung together
• 41:09in the in in these tumors,
• 41:12creating these completely
• 41:15abnormal abnormal structures.
• 41:18So we also asked how these
• 41:21DNA molecules come together,
• 41:23whether there's something special
• 41:26about them and it's many of you know
• 41:29if you have double strand break you,
• 41:31it can be repaired through
• 41:33three different mechanism,
• 41:34non homologous end joining,
• 41:37microhomology mediated repair
• 41:39and single strand annealing.
• 41:41And all of these,
• 41:44all of these should be functional
• 41:48in Franconia negative cells.
• 41:50And indeed when we look at the junctions
• 41:53in franconi associated squamous
• 41:55cell carcinoma that are shown on
• 41:58the left and sporadic HPV negative,
• 42:01we see the same repair mechanism
• 42:04being active mostly on homologous non
• 42:07homologous end joining and MJ and that's the.
• 42:12The percentages are are identical.
• 42:14So the interpretation of this is
• 42:16that whenever there is a break
• 42:18that's formed in these tumors,
• 42:20it's being repaired just by sticking
• 42:23the the ends together and random
• 42:26fashion that results in these high
• 42:29number of structural variants.
• 42:31We also asked where these breaks
• 42:34occur and specifically we were
• 42:36interested to in in knowing whether
• 42:39they occur at repetitive sites.
• 42:42And there is some increase that's
• 42:46statistically significant in
• 42:48sign elements, so I'll repeat.
• 42:51Had higher number of of breaks,
• 42:55but when we look at the whole genome
• 42:59we can see that the brakes and here
• 43:02I'm showing where all of this breaks
• 43:05out and structural variant ends are.
• 43:10I think when they're being placed in the in
• 43:12the genome across all of the chromosomes,
• 43:15you can see that there is the brakes
• 43:18are occurring throughout the genome.
• 43:20But you do see that parts of some of
• 43:24the genes are being hit multiple times.
• 43:27And that's really the the way we
• 43:30think about it is that there is
• 43:32mutagenesis across and then on top
• 43:35of that there's a selection of
• 43:37particular genes that help the tumors.
• 43:40Growth for example EGFR here is is being
• 43:44amplified and so are other sites you know,
• 43:47but one here.
• 43:48This is the EGFR and and also
• 43:52others the outcome of this.
• 43:56Is that these tumors have very high copy
• 44:00number variance and this is just one tumor,
• 44:03this is Ascot plot.
• 44:04So this is a little specific
• 44:06copy number where total alleles
• 44:08obviously in the cells should be 2,
• 44:11the alternative allele should be one.
• 44:13But you can see that there are
• 44:16number of amplifications and their
• 44:18amplifications and genes that we
• 44:21all know from from tumor pathways.
• 44:24Pick three CA make cycling.
• 44:26One that's that's amplified in many
• 44:29of these tumors and then there are
• 44:32deletions and tumor suppressors
• 44:34like CDK and two way and P53.
• 44:37So this is a pretty representative look
• 44:42at the at the tumor of Fanconi anemia
• 44:46patients and this is a different way
• 44:49of representing it where all of the.
• 44:52The rows are the the genes that are
• 44:56mutated and the columns are single tumor
• 44:59that we've sequenced from Fanconi patients.
• 45:01And you can see that orange or
• 45:04amplifications and deletions in
• 45:06blue and multiple of these pathways
• 45:08are mutated in each of the genes.
• 45:11And if you look at pick three CA and
• 45:14Mick Amplifications 54% of these
• 45:17tumors have Co Co amplification
• 45:20of these two of these two.
• 45:23Oncogenes, so this is a very,
• 45:25this might explain part of why
• 45:27these tumors are aggressive but
• 45:29there are other reasons they they
• 45:31they might be aggressive as well.
• 45:33So to just summarize what
• 45:36happens in franconi tumors?
• 45:39We have.
• 45:41Franconia pathway that protects cells
• 45:43from creating DNA breaks when DNA
• 45:47interstrand crosslinks are present and
• 45:49if you have DNA breaks the structural
• 45:53variants follow and eventually all
• 45:55of them lead to high copy number
• 45:58variation in these in these tumors.
• 46:01And there is also I wanted to mention
• 46:03there is a paper that recently
• 46:05came out from Jean Souliers Lab
• 46:07who looked at AML's and and AML's.
• 46:11They also see structural variant formation,
• 46:15fewer structural variants,
• 46:17and they also see P53 pathway being abnormal.
• 46:22But actually P53 itself is not mutated,
• 46:25it's through the MDM 4 pathway that
• 46:27that there is suppression of P53.
• 46:30So in the bone marrow similar
• 46:35events are happening.
• 46:36Maybe it's at a slightly smaller,
• 46:39smaller scale, which I don't.
• 46:41Alright, understand.
• 46:44And might have to do with the timing of
• 46:47these tumors and time to evolution of
• 46:49of and presentation of these tumors.
• 46:52So knowing what we know
• 46:55about frankonia tumors,
• 46:57we then turned to sporadic cancers
• 47:01and we were wondering whether the.
• 47:05Structural variants that are present
• 47:08in sporadic HPV negative tumors.
• 47:10I showed you that there are still
• 47:12some of them and they're shown
• 47:14here on the right hand side.
• 47:16These are all HPV negative
• 47:18TCG head and neck cancers.
• 47:19You can see that there's
• 47:21plenty of orange and blue.
• 47:22So there are quite a lot
• 47:24of copy number variants,
• 47:25not as many as in in Fanconi tumors,
• 47:27but still.
• 47:30A high number.
• 47:31So we were wondering whether what
• 47:34we find in Fanconi tumors might
• 47:37apply to to head and neck cancer
• 47:41and sporadic and sporadic cases.
• 47:44And what we decided to do is to look at
• 47:49a copy number variants and stratify tumors.
• 47:53So now these are the TCG TCG data
• 47:56HPV negative tumors and stratify
• 48:00them into top quartile.
• 48:02Tumors with a high number
• 48:04of copy number variants,
• 48:06so top quartile,
• 48:07and then compare them to the low quartile,
• 48:10the top 1/4 of tumors with
• 48:14the low copy number variants,
• 48:17and we could correlate the
• 48:20number of pack years with the
• 48:23level of copy number variants.
• 48:25So the higher smoking history,
• 48:28the higher copy number variants.
• 48:31And when we looked at signatures that
• 48:34were present and in these two we could
• 48:37also show that the top quartile.
• 48:39Had about twofold increase in signatures
• 48:45of ID 38SBS4 and others and these
• 48:49are smoking associated in Dells,
• 48:50so that's not surprising,
• 48:52right?
• 48:52That's correlates with the pack history
• 48:56but also non homologous end joining
• 48:59and benzopyrene and acetaldehyde.
• 49:01That suggests something we we already
• 49:04know from epidemiological studies that
• 49:07smoking and drinking go together.
• 49:10So we so these patients probably
• 49:13are also enriched for for
• 49:16increased alcohol exposure.
• 49:18So taking all of this together,
• 49:22we come up with this hypothesis
• 49:25that in sporadic cancers,
• 49:28the tobacco and alcohol exposure creates
• 49:33DNA inter interesting crosslinks.
• 49:36Obviously it creates other mutagenic events,
• 49:39including P53,
• 49:40which is an early event that then
• 49:42allows structural variants to occur.
• 49:45And Franconia pathway even though
• 49:48that it's present in these in
• 49:51these tumors and maybe it might
• 49:54be somatically decreased or maybe
• 49:56there are some genetic factors
• 49:58that that are involved here,
• 50:01but frankonia pathway which
• 50:03is largely efficient.
• 50:05There isn't just enough of it and
• 50:08it's over is overwhelmed and unable to
• 50:11repair all the DNA into crosslinks.
• 50:14It's also important that P53 is is
• 50:16mutated in these so you cannot depend
• 50:19on P53 pathway to to increase the Franconia.
• 50:25Green expression kind of going in
• 50:28gene expression patterns and all
• 50:29of this results in DNA breaks,
• 50:31structural variants and the same
• 50:34pathway leading to copy number alterations.
• 50:38So taken together,
• 50:39you know when we look at the
• 50:42FRANKLINIA pathway function or if we
• 50:45have patients with low pathway function,
• 50:49they have very high levels of head
• 50:51and neck cancers and other squamous
• 50:53cell carcinomas, maybe not 100%,
• 50:55but if they Long live long enough,
• 50:58very high levels and if we have
• 51:02full function we are protected.
• 51:04However, there are a number of
• 51:06genetic modifiers whether it's a.
• 51:08The
• 51:11H2CTA4DC GSTM one and others,
• 51:13as well as these environmental
• 51:16modifiers of alcohol tobacco that
• 51:19is making this pathway functionally
• 51:22inefficient leading to to higher
• 51:24probability of head and neck cancer.
• 51:28So I'll finish and um,
• 51:31I'll just acknowledge my lab.
• 51:34This is Arlene, who's been
• 51:36instrumental in starting the registry.
• 51:39Munjung Jung has worked on a LH9A1.
• 51:44They work on head and neck cancers,
• 51:46was hugely collaborative work,
• 51:48and we were helped by Matthias Sanders.
• 51:53Working with Peter Campbell and Andrew
• 51:56Webster drove this work in my lab.
• 51:58And we had.
• 52:00A lot of collaborators,
• 52:02clinical and patient collaborators
• 52:06and and also other bioinformaticians
• 52:10who've who've helped us.
• 52:13And I'd like to thank my funding agencies,
• 52:18tons of collaborators and
• 52:21especially patients and families.
• 52:23And these are some of the young adults
• 52:25few years ago before the pandemic and
• 52:27I know that at least five of these.
• 52:30Adults are have passed away with with
• 52:33head and neck cancer and during this time.
• 52:36So I'll take questions.
• 52:40Thank you.
• 52:49OK. That was wonderful.
• 52:50For those of you who are online,
• 52:52please use the Q&A function to to bring your
• 52:55questions and I think Jeff. Jeff, yeah.
• 52:59Ohh uh you can have a microphone.
• 53:01I think it's here.
• 53:04And and maybe I'll just start
• 53:06with Nadia, Dimitrova asked.
• 53:08Do you see evidence of extrachromosomal
• 53:10DNA circles and FA tumors to
• 53:13explain the amplifications and
• 53:14we haven't really looked at that,
• 53:17but that would be a good idea.
• 53:19We do see a lot of tandem duplications
• 53:22in the in the especially Mick and
• 53:25other areas that would explain
• 53:27the amplifications as well. Jeff
• 53:29yeah. So that was a great talk,
• 53:30incredibly clear and insightful.
• 53:32I really appreciate it.
• 53:33I really like that last diagram.
• 53:35I know it was meant diagrammatically
• 53:36at least the way you showed it,
• 53:38but it can't.
• 53:39That doesn't stop me from
• 53:41asking do you have a way to
• 53:44quantify Fanconi anemia pathway
• 53:46function on that X axis or not.
• 53:48That's a really,
• 53:49that's a really good question.
• 53:51So we we have some way of doing that
• 53:54within the Franconia population,
• 53:57so something that I haven't talked about.
• 53:59We have. The registry has been
• 54:01amazing and we sequence now
• 54:04pretty much everybody who we had.
• 54:06The DNA for to look for phenotype,
• 54:10genotype correlations and
• 54:11there are a lot of phenotype,
• 54:14genotype correlations that we can tease
• 54:16out and we've started to describe them.
• 54:20And the function we can at the lower
• 54:23end it's hard to tease it out but
• 54:25you can definitely do that with
• 54:28number of foci with ubiquitination
• 54:30of fancd 2 and Frank I it's not great
• 54:33I think we need a better assay for
• 54:36for doing that on the top end I think
• 54:39when we have like what we see what
• 54:42we think fully functional pathway.
• 54:45It's really difficult to say
• 54:47whether it's 95 or or 90%.
• 54:51So we haven't really been able to do that.
• 54:55But as you saw from the second
• 54:57part of my talk, you know,
• 54:59even a little bit of DNA damage
• 55:01can lead to chromosome breakage
• 55:03like the ones that we induce when
• 55:05we don't have a LDH 9A1.
• 55:07So I think there might be ways of of
• 55:10doing better assay development for this.
• 55:16So.
• 55:21So if I can
• 55:23ask a question. So one of the things
• 55:25we're noticing in the virtual tumor
• 55:26board that we do for these young adults
• 55:29with head neck cancer is that they are
• 55:31really not at all responsive to immune
• 55:34checkpoint inhibition which is often
• 55:35been brought forward as as something
• 55:37that might not be so DNA damaging.
• 55:39And I wonder if you could speculate
• 55:42about that both from the perspective of?
• 55:45Sort of this global picture you've been
• 55:47talking about with structural variants,
• 55:48not some so many single nucleotide variants.
• 55:51But also, is there anything that came out
• 55:53in your gene signatures that would speak to?
• 55:56I saw NSDD one which sometimes is
• 55:58associated with immune exclusion.
• 56:00Are you getting a picture from the mutations
• 56:02of what the immune status might be?
• 56:05So from the mutations we we
• 56:08do we do see deletions in.
• 56:13A message. Will say.
• 56:18But frankly the the genome instability
• 56:22probably has a big part in the in the
• 56:27inability to have immune response right.
• 56:29So we are very much interested in
• 56:32that and we are starting to to look
• 56:34at that the tumor microenvironment
• 56:36in these tumors in particular
• 56:38thinking that it's it will be a good
• 56:41model for for understanding that.
• 56:44So we'll, we'll, we'll see.
• 56:45But it definitely looks like it
• 56:47would be too more suppressive, yeah.
• 56:52Tommy.
• 57:05Yes. So that's an interesting question and
• 57:08there is some some data in the literature
• 57:12that implicates mitochondrial DNA damage and
• 57:14Fanconi function within the mitochondria.
• 57:17I'm not sure how the fund Fanconi
• 57:20proteins would get into the mitochondria,
• 57:23but it's something that we have not.
• 57:26Explored. It would be so that,
• 57:29I'm sorry, the question was about the
• 57:31mitochondrial damage and whether there
• 57:33is increased mitochondrial damage.
• 57:35We we haven't really explored that.
• 57:39Um, and there would be.
• 57:42Well, it's it, it would be doable, right.
• 57:45I mean you just we would have to
• 57:47look at mitochondria specifically.
• 57:50The audio. That's right.
• 57:53A lot of these, yes.
• 57:55A lot of these are the
• 57:57detoxification enzymes.
• 57:58Not all of them,
• 57:59but many of them are within the mitochondria.
• 58:04The spermine? Yep.
• 58:11Right. Right.
• 58:18Right. Yeah. So there is a lot,
• 58:20a lot that we could explore in in this
• 58:23disease and mitochondrial function
• 58:24definitely has been brought up as as one
• 58:28of the places where where we should,
• 58:30we should look more.
• 58:34There was one question,
• 58:36is there a bias toward either focal or
• 58:40ARM level copy number alterations or
• 58:43both equally in FA tumors when compared
• 58:46to HPV negative head and neck cancers.
• 58:48So we see, we see both,
• 58:50I don't know if if we really compare them
• 58:54so we can say whether it's equal or not,
• 58:57but we see, we see we see some ARM
• 59:01level copy number alterations as well.
• 59:04But majority I would say,
• 59:06are the focal type.
• 59:14OK, yes. It seems that.
• 59:22Particular these metabolic enzymes.
• 59:27Essentially necessary to collect cells of the
• 59:31dehydrogenases ID. Also very common.
• 59:36Biosynthesis inhibitors.
• 59:42Do you think, well, what are your insights
• 59:44on a particular reason why blood
• 59:46cells would be more susceptible?
• 59:51Right. So one possibility is that
• 59:53they are really not more susceptible,
• 59:56they are just the there are
• 59:58fewer of them and they exhaust
• 60:00faster than any other stem cells,
• 01:00:02right, so that that's let's
• 01:00:04leave that as a possibility.
• 01:00:06The other is we know for formaldehyde.
• 01:00:11That. Very high levels of formal
• 01:00:16formaldehyde can be formed close to the
• 01:00:19DNA during the differentiation process.
• 01:00:22So one hypothesis is that you're
• 01:00:27having differentiation that happens
• 01:00:29during very few replication cycles,
• 01:00:33and that vulnerability of two
• 01:00:36formaldehyde is very high.
• 01:00:39Umm.
• 01:00:41So that's a possibility in coratina sites
• 01:00:44when we do an assay for for example
• 01:00:47whether ADH two or ADH 5 or important
• 01:00:50in with human characteristics sides,
• 01:00:53there doesn't seem to be that dependency
• 01:00:56within the fungi 2 or frank a negative.
• 01:00:59But we when we start playing with
• 01:01:02lipids metabolism they show up.
• 01:01:04And then it makes sense because it's the the,
• 01:01:08the lipid metabolism is very important in the
• 01:01:11in the as a barrier in correcting the sites.
• 01:01:14So some of it,
• 01:01:15I I do believe that some of it will come to
• 01:01:20specific functions or a particular stem cell.
• 01:01:23Um, and I think just more
• 01:01:25needs to be done to understand,
• 01:01:27to understand that one of
• 01:01:29the challenges is that.
• 01:01:31It's very hard to identify how much
• 01:01:34of these aldehydes are present and
• 01:01:37especially within the DNA it's just
• 01:01:40very difficult to to quantify these.
• 01:01:44But it's an I think it's
• 01:01:46a very interesting area.
• 01:01:50OK, Ryan. Yeah, great talk about it.
• 01:01:54This morning, your copy number,
• 01:01:56your copy number, application
• 01:01:57analysis. Have you ever
• 01:01:59have you looked at the sequences
• 01:02:00of those copy numbers?
• 01:02:01Are they mutating in each copy number?
• 01:02:03And what do you think
• 01:02:04driving that copy number,
• 01:02:05especially the amplification?
• 01:02:07Do you think it's like a DNA repair
• 01:02:09pathway, like pull Theta
• 01:02:11or something like that?
• 01:02:12Yes. So let me just,
• 01:02:14I don't know if I left.
• 01:02:16Oh, I can't escape, OK.
• 01:02:19Well, I won't try to find slides for this.
• 01:02:23So we as far as you mean like
• 01:02:27point mutations within the within
• 01:02:30the amplified regions,
• 01:02:32we don't really see any kind of.
• 01:02:36Aggregation of mutations
• 01:02:38like at the junctions.
• 01:02:40Let's say we've looked at that.
• 01:02:42Um, and the way I some of these
• 01:02:46amplifications occur through
• 01:02:48probably or are consistent with
• 01:02:50let's say that with stalling at the,
• 01:02:54at the at the cross link or
• 01:02:57some other lesion and reversal?
• 01:03:00So we do see these inversions that
• 01:03:03that are associated would be expected
• 01:03:06to be associated with replication.
• 01:03:09Some of them are tandem duplications
• 01:03:12and I think a nice system to figure
• 01:03:15this out is actually in a system
• 01:03:18where you have tester sequences
• 01:03:21and stalling through tester that
• 01:03:25that would be one place where you
• 01:03:27could model some of these, I think.
• 01:03:29I guess I was thinking of all these
• 01:03:31reversion mutations that arise
• 01:03:32like in Fanconi anemia perhaps
• 01:03:34you know the copy
• 01:03:35numbers. They're sampling different
• 01:03:36genetic states so they can, you
• 01:03:38know, sort of revert to a.
• 01:03:40I have a lot to say about the,
• 01:03:42the copy, not the the reversions
• 01:03:44in Fanconi because we have a whole
• 01:03:47another paper that's coming soon on
• 01:03:49mosaicism in the blood and a lot of
• 01:03:52these mutations are actually somatic
• 01:03:54recombination events that are UMPD,
• 01:03:58their uniparental disomy events.
• 01:04:01So there are, yes,
• 01:04:02there are point mutations and there
• 01:04:04are other things happening you know,
• 01:04:06splice site.
• 01:04:08New splice site mutations
• 01:04:10and things like that,
• 01:04:11but a lot of them are are full uniparental
• 01:04:15disomy through mitotic recombination.
• 01:04:18Which I think is interesting.
• 01:04:20Thank you.
• 01:04:21Thank you very much.
• 01:04:23Thank you.
• 01:04:27Um, for the fellows I think you're.