The p53 Tumor Suppressor Pathway

Transcript

• 00:00Of hosting today and we are incredibly
• 00:04thrilled and delighted that we
• 00:06were able to induce Doctor Gigi
• 00:09Lissonota to be our speaker today.
• 00:12Doctor Lozano graduated from Pan
• 00:14American University in Texas,
• 00:16did some work at Oakridge Laboratories,
• 00:19got her PhD at Rutgers,
• 00:22and then went to Princeton,
• 00:24where she was a postdoc with Arnie Levine.
• 00:28Immediately following that,
• 00:29she was recruited to MD Anderson,
• 00:32and in her long tenure there she has done.
• 00:38Path breaking work on P53.
• 00:41She's recognized for enormous contributions
• 00:43that include the recognition that P.
• 00:4653 works as a transcriptional activator.
• 00:49Many, many findings regarding the ways
• 00:52that MDM two and MDM three regulate P 53.
• 00:57Extensive use of transgenic models
• 00:59to understand these mechanisms.
• 01:01Better definition of specific P53
• 01:04loss and gain of function mutations
• 01:07and and their effects on P53's
• 01:10biology as well as studies of
• 01:14the P53 transcriptional program.
• 01:16She's a member of the National Academy
• 01:18of Sciences and the National Academy
• 01:20of Medicine and at MD Anderson.
• 01:23She is now chair of the Department
• 01:25of Genetics and Hubert L.
• 01:27Olive Stringer,
• 01:28distinguished chair in oncology.
• 01:30In the division of Basic Science
• 01:32Research at MD Anderson,
• 01:34so I don't want to talk for a long
• 01:36time because I'm really eager
• 01:38to hear what you have to say.
• 01:40So thank you very much for joining us
• 01:43and I will just say to the audience.
• 01:45You can use the chat function to
• 01:48enter your questions or we can.
• 01:49I believe we can unmute people
• 01:51at the end if we need to.
• 01:54So with that do welcome.
• 01:56Thank you, thank you very much,
• 01:59Barbara. For that introduction I
• 02:01hopefully now I'm sharing my screen.
• 02:07Can you see it?
• 02:10Weekend OK super Alright so
• 02:12anyway thank you Barbara.
• 02:14It's it's fun to always visit a place
• 02:17and now during covid we're doing these
• 02:19virtual seminars but hopefully at the
• 02:22end I'll be able to address some of
• 02:25the questions that people might have.
• 02:27What I thought I would do today is
• 02:30described some of the models that
• 02:32I think have to find some of the
• 02:35basic real basic understanding of
• 02:37the P53 tumor suppressor pathway.
• 02:39So I'll get started with my disclosures.
• 02:42I am on the Scientific Advisory
• 02:45Board for PMV Pharma.
• 02:47So the P 53 pathway.
• 02:49This is my myopic view of the pathway
• 02:51and it really highlights.
• 02:54I think some of the critical
• 02:56features of the pathway.
• 02:58First and foremost people decree is present
• 03:01in very low levels in normal cells,
• 03:03but any kind of abnormality
• 03:06that the South senses,
• 03:07hypoxia, DNA damage,
• 03:09inappropriate activation of an
• 03:11oncogene will stabilize that
• 03:12P53 protein and then P53 in turn
• 03:15functions as a transcription factor.
• 03:17To activate hundreds of genes
• 03:19and I'll show you some of those
• 03:22experiments in in a little bit,
• 03:24but some of the genes that P.
• 03:2653 is known to activate is P21 for example,
• 03:30which is a inhibitor of the South cycle.
• 03:33It also induces senescence program
• 03:35P53 can activate a slew of gene sick,
• 03:38initiate a pop ptosis and it also activates.
• 03:41Genes are involved in changing
• 03:43the metabolic functions of a cell.
• 03:47Now when P 53 is activated,
• 03:49if the cell is allowed to survive
• 03:52and proceed,
• 03:53P53 has to activate this protein
• 03:56called MDM 2,
• 03:57which is an E3 ubiquitin ligase
• 04:00that targets P53 for degradation
• 04:02and basically removes the Peachtree
• 04:04levels back down to normal.
• 04:07So if you just.
• 04:09Think about what people think we can do.
• 04:11We can do so much in getting arrested,
• 04:14phallic it, killed itself,
• 04:15and it can also induce its own
• 04:18inhibitor to allow the cell to survive.
• 04:21And even though there's so much
• 04:22work in the pizza tree killed,
• 04:25we still don't understand all of the
• 04:27cues that determine which of these
• 04:29pathways Peabody Creek and activate.
• 04:31But because it has all these functions,
• 04:34it is a critical tumor suppressor,
• 04:36and it is the most doctor
• 04:38mutated gene human cancers.
• 04:39So what I showed here is what the
• 04:42field called the Manhattan Plot,
• 04:45and it was developed by Magali Olivia.
• 04:47So across this axis are 125 genes
• 04:50that are commonly mutated and cancers
• 04:52and then across this axis here are
• 04:5536 different types of cancers,
• 04:57and there's some features that stand out.
• 05:00But this is the one I want to
• 05:03highlight here across the board.
• 05:05These are mutations in the P53
• 05:08tumor suppressor.
• 05:08So almost all cancers mutate.
• 05:1153 but P 53 pathway is inactivated by
• 05:15multiple mechanisms and I show here
• 05:18in some of the different cancers and
• 05:21how they inactivate piece of debris.
• 05:23So high grade,
• 05:25serious ovarian carcinomas,
• 05:26mutations in P53 are the most common.
• 05:29However,
• 05:29in liposarcomas it is upregulation of MDM 2,
• 05:33the P fifty 383 if it could be like a San.
• 05:37About 100% of these liposarcomas inactivate
• 05:40the pathway by overexpressing MD.
• 05:43In the new Asia glioblastoma,
• 05:45big Sweet come interested in glioblastomas.
• 05:48Recently, the P 53 gene is altered,
• 05:51deleted in approximately 1/3 of
• 05:53glioblastomas in the MDM to an Indian.
• 05:57For genes are upregulated in
• 05:59about it order and this is a
• 06:02mutually exclusive relationship.
• 06:04So if P53 is mutant,
• 06:06MDM 2 doesn't have to be upregulated.
• 06:10And the other thing I want to point
• 06:13out in glioblastomas is that we have
• 06:15about half of these tumors that
• 06:17have neither mutations and piece
• 06:19of D3 or upregulation of the MDM.
• 06:22Two and MDM four inhibitors of P53.
• 06:24And so since I really believe that
• 06:26the P 53 pathway has to be undermined
• 06:29in the development of all cancers,
• 06:31I think that there's a big hole here
• 06:34that we have to understand in more detail.
• 06:37So today's start is really going to
• 06:39concentrate on just a few proteins,
• 06:41and I'm not going to have time
• 06:43to show you a whole lot of data,
• 06:46but I thought I would use this slide
• 06:49to highlight some of the important.
• 06:51Functions that I of these proteins
• 06:54that I will discuss with you today.
• 06:56So MDM two is an inhibitor of P53.
• 06:59It's an E3 ubiquitin ligase and
• 07:02target speakers for degradation.
• 07:03MDM Four also inhibits P 53.
• 07:07It doesn't have any E3 ligase function,
• 07:10but it actually facilitates and makes
• 07:12MDM two or better yet riveting ligase,
• 07:15although it also has independent
• 07:17functions of MDM two and can
• 07:20actually bind and inhibit the pizza
• 07:23guy free transactivation domain.
• 07:25This relationship does MDM 2 New Four
• 07:28also conform hetero dimer and that
• 07:30header dimer is critical in embryo
• 07:33development to inhibit P 53 and then,
• 07:35as I indicated,
• 07:37and will discuss in some detail,
• 07:39P53 can activate the Indian two
• 07:42promoter so it can up regulate
• 07:44MDM two in inhibited so levels.
• 07:47Another important concept that
• 07:48I'll mention very briefly,
• 07:50maybe at the end is that MDM
• 07:53two can also inhibit.
• 07:55A mutant P53 protein and that's because
• 07:58these mutant proteins have mutations
• 08:00in the DNA binding domain but retain
• 08:02a transcriptional activation domain.
• 08:05But the important point that I want to
• 08:08make here is even though this mutant
• 08:12can be targeted by MD M2 and MD M4.
• 08:15It is mutant in so can no longer
• 08:18feed back it up.
• 08:20Regulate MDM two so with time in our
• 08:22in vivo models we find that these
• 08:24people period this can become stabilized
• 08:27'cause there's insufficient MDM two
• 08:29to down modulate the protein levels
• 08:31and in a few minutes you'll see how
• 08:35important it down modulation is.
• 08:37So the outline of my talk today is
• 08:39I'm going to talk about some of the
• 08:42models that showed us how exquisitely
• 08:44sensitive P 53 is to inhibition via video 2.
• 08:49To tell you what the molecular
• 08:51responses to people three activation
• 08:54in vivo and last but not least,
• 08:57I'm going to describe some of the new
• 09:00cancer models that were working with
• 09:03that expressed mutant P53 proteins.
• 09:05So let's first talk about the MDM
• 09:08proteins and their innovation P.
• 09:1053. So along time ago and now we.
• 09:15Attempted to make an Indian to know
• 09:17Mouse and it's just not possible
• 09:20and the reason is not possible is
• 09:22because an MDM two null embryo just
• 09:25prior to implantation is APOP totic.
• 09:28This embryo stained with the
• 09:30tunnel essay in every salad.
• 09:32This embryo is is dead.
• 09:35And at the time we knew that
• 09:38MDM two interacted with P.
• 09:3953, but we really didn't know how
• 09:43important interaction was and what
• 09:45we did was test the importance
• 09:47of P53 in this little embryo by
• 09:50crossing 2P53 miles and we completely
• 09:52rescue this phenotype.
• 09:54These Meister born and are
• 09:57perfectly normal except now because
• 09:59they lack P 53 they have it.
• 10:02So with with this experiment indicates
• 10:04is that what MDM two is doing in
• 10:08these embryos is upregulating P53,
• 10:10which is preventing the normal
• 10:12development of these embryos.
• 10:14MDM fours are related MDM,
• 10:16two protein that aren't Johansson discovered.
• 10:19And since MDM two has such a unique
• 10:23relationship with P53 we decided that
• 10:25we would make the MDM for knockout
• 10:28but weren't sure what to expect.
• 10:32But in MD for knockout is also embryo
• 10:35lethal a few days after the Indian
• 10:37to know but again that phenotype is
• 10:40rescued by deletion of P53 and we've
• 10:43also made mice that have no MDM to know
• 10:47Indian for Nokia 53 and these mice.
• 10:51The viable they have Pizza 3 two or
• 10:54phenotypes because they lack P 53.
• 10:56So at least physiologically,
• 10:58in the mouse,
• 10:59the two most important functions of
• 11:02these empty in proteins is to keep
• 11:05P 53 levels low during homeostasis.
• 11:07OK,
• 11:07so we have these MDM two heterozygous
• 11:10in these MDM for headers I customized.
• 11:12They have only one allele of
• 11:14each of these two genes.
• 11:16In these mice are perfectly
• 11:18normal and running around,
• 11:19but as I indicated in my
• 11:22in my introductory slide.
• 11:26P 53 is a DNA damage response protein,
• 11:29and so we wondered if there was
• 11:31any phenotypes in these mice.
• 11:33If we irradiated them,
• 11:35if we damaged their DNA, and sure enough,
• 11:38we saw a beautiful phenotype.
• 11:39So the MDM two in the MDM,
• 11:42two Ambien for headers agasse mice are
• 11:44sensitive to low dose ionizing radiation.
• 11:47So in this experiment what we've
• 11:49done is irradiated mice with six
• 11:51Gray and the black line above.
• 11:53Here is a normal mouse.
• 11:56That for 50 days just ignores.
• 12:00Six grade radiation, but the headers.
• 12:02I guess.
• 12:03Mice MDM four in Red and MDM two and
• 12:06blue are dead by three weeks of age.
• 12:10Importantly,
• 12:10if we now move P53 from this system,
• 12:14we completely rescue these phenotypes.
• 12:15So this is the rescue.
• 12:17The Indium MDM, two heterozygous mouse.
• 12:20And here's the rescue of the
• 12:22Indian for headers ecospace.
• 12:24So even though.
• 12:25The MDM two and MDM for heterozygotes
• 12:29mice have sufficient levels of the Zambian
• 12:32proteins to maintain homeostasis with damage.
• 12:36There's just not enough of these proteins
• 12:40to return degree back to normal levels.
• 12:44The next experiment that I want to
• 12:47tell you about is the importance
• 12:49of this feedback loop.
• 12:51So as I indicated,
• 12:52MDM two is regulated by P53.
• 12:55There are two peaks decree
• 12:57binding sites in the P2 promoter.
• 12:59There are people,
• 13:00three dependent,
• 13:01so people free byansi sequences
• 13:03and activates MDM 2.
• 13:05And So what we decided to do is to
• 13:07ask how important was this feedback
• 13:10loop in regulating P53 levers?
• 13:12OK,
• 13:12so we made point mutations and I show
• 13:15here the different point mutations
• 13:17because we didn't want to disrupt
• 13:19the architecture of the promoter.
• 13:21We just wanted to disrupt MD PhD degree
• 13:24binding to the end game to promote it.
• 13:28So these experiments in the bottom bar
• 13:30chip data that basically show that
• 13:33P53 cannot find this mutant promoter,
• 13:36which we call P2P2,
• 13:37and this is different promoter that
• 13:40shows our assays are working in P53 combined.
• 13:43The pull up remote.
• 13:45OK,
• 13:45so we made these point mutations
• 13:48and we were surprised that are
• 13:50most was perfectly normal.
• 13:52We really thought that this
• 13:54feedback loop is going to be
• 13:56critical for regulation of P53.
• 13:59The mice are fine, but again,
• 14:01as in the previous case,
• 14:03their exquisitely sensitive to radiation,
• 14:05so this is the same experiment that I
• 14:09showed you before we rated it the we
• 14:13irradiated the mice with six grade.
• 14:16Normal mice.
• 14:18MDM two heterozygotes.
• 14:20Don't care about this dose,
• 14:22but you can see that the P2P2 homozygous
• 14:26mice that no longer have this feedback
• 14:29loop are dead for the most part.
• 14:32So what is the phenotype?
• 14:34These animals are actually
• 14:36dying because of the complete
• 14:38ablation of the ball mirror,
• 14:40so here's a heterozygous irradiated mice
• 14:43and you can see that at 12 days you have
• 14:47some disruption of bone marrow function,
• 14:50but it's still viable,
• 14:52whereas in the mice that lack
• 14:55the feedback loop,
• 14:56there's a complete ablation
• 14:58of Humana Pelisses,
• 14:59and this is again a P53 dependent.
• 15:03Process so if we take out
• 15:06just one illegal appeal 53.
• 15:08We completely rescued this phenotype so
• 15:11we can rescue this phenotype with complete
• 15:14deletion of P53 or header zygosity 5053.
• 15:17So with these experiments are beginning
• 15:20to tell us is that there's this.
• 15:24There is this important relationship
• 15:26between MDM two and P53,
• 15:28and then there's an important
• 15:30balance that has to be maintained
• 15:32for survival after DNA damage.
• 15:34The last experiment that I'll show
• 15:37you here is our attempts to try to
• 15:40understand which pathway downstream
• 15:42of P53 is important for this phenotype.
• 15:46So I've already told you that heterozygosity
• 15:49Peachtree rescues the phenotype.
• 15:51We also generated mice with deletion of P.
• 15:5421, which is the cell cycle inhibitor
• 15:57and that had no effect on the phenotype.
• 16:00Those mice are also very
• 16:02sensitive to radiation,
• 16:03and then we also deleted Puma,
• 16:06which is one of the APOP
• 16:08totic targets of P53.
• 16:10And here you can see that
• 16:12there was a complete rescue
• 16:13of this hematopoietic defects.
• 16:16So in this scenario it appears
• 16:18that it is the APOP totic function
• 16:21opekta degree that is killing.
• 16:23These hematopoietic stem cells.
• 16:27So those are just a couple of
• 16:30the numerous experiments being
• 16:32done to evaluate the relationship
• 16:35between MDM24 and P53,
• 16:37and it's just an exquisite relationship.
• 16:40You need sufficient MDM 2 for survival.
• 16:45And.
• 16:47And if you have too much and you
• 16:50too MDM for deletion of 53 deletion
• 16:53of downstream effectors of P53 can
• 16:55rescue those lethal phenotypes.
• 16:58Now come. Because.
• 17:00MDM two is so lethal early.
• 17:05During embryo Genesis,
• 17:06one of the experiments we wanted
• 17:09to do is really ask about an adult
• 17:12mouse and how important is MDM two in
• 17:16different tissues at different times,
• 17:19and we've used,
• 17:20we've generated this conditional
• 17:22allele of MDM two and this is using
• 17:26The LOX P system so these two lacks
• 17:29besides encompass two of the accents
• 17:32that code for the major finding.
• 17:35Region 2P53 so this conditional
• 17:37Leo then allows us to delete MDM
• 17:40two in any tissue that we want to.
• 17:42And we've generated number of
• 17:44tissues and experiments that way.
• 17:46But what I want to show you is
• 17:49what happens when you globally
• 17:51remove MDM two in the whole months.
• 17:55So as the title says,
• 17:56Indian too lost in the adult is
• 17:59results in a lethal phenotype.
• 18:01So what we've done here is we've
• 18:03used a Cree transgene that is
• 18:05tamoxifen inducible so this is
• 18:07this is a mouse that has one of
• 18:10the conditional alleles and it
• 18:12has the other legalism missing,
• 18:14so it's single recombination event is
• 18:16going to create an M2 normals or not sell in.
• 18:20All we do is inject tamoxifen and then
• 18:23we look at what happens to these.
• 18:25Phenotypes,
• 18:26and I think you can see from this
• 18:29graph here on the right that within 5
• 18:31days of treating the mice with tamoxifen,
• 18:34they're all dead.
• 18:36So,
• 18:36so losing MDM two and it's a
• 18:41Peachtree dependent process.
• 18:44Causes this enormous physiological response.
• 18:49These are some of the pathologies we
• 18:53see in these mice the hippocampus has.
• 18:57Less number of cells, the retina is is
• 19:00comprised of multiple beautiful layers
• 19:02and you can see that it all of these cases
• 19:06that it's it's a decreased cell numbers.
• 19:09There's some differences in in the liver,
• 19:12and it's actually if you
• 19:14measure liver function.
• 19:15Liver function is compromised
• 19:17when you have deletions of MDM.
• 19:19Two kidney has all these protein
• 19:22casts and dilated tubules,
• 19:23and then in the spleen we have
• 19:27complete absence of white.
• 19:30And as I indicated,
• 19:31these phenotypes are all P53 dependent.
• 19:34So it just.
• 19:35I mean, I think the important
• 19:37aspect of this slide is that in
• 19:40some of the previous slide is it
• 19:42highlights some of the pathologies
• 19:44that we might see when we use MDM.
• 19:47Two inhibitors in the clinic to it.
• 19:51To inhibit the tumors that have
• 19:54high levels of empathy into it.
• 19:56And the hematopoietic defense
• 19:58is actually observed in humans.
• 20:00It is treated with MDM two inhibitors.
• 20:04Uhm?
• 20:04But now I want to use this model system
• 20:07to understand what piece of degree
• 20:09is doing in these different tissues.
• 20:13In you know one reason for doing
• 20:15this is we would like to be
• 20:18able to reactivate P53 somehow,
• 20:20and it's kind of hard people.
• 20:22A lot of people are trying to
• 20:25to reactivate people to agree,
• 20:27but what we were hoping is that
• 20:29we might be able to identify
• 20:32downstream pathways to P53.
• 20:33There would be better targets
• 20:36for reactivation tours.
• 20:37So let me show you what we did.
• 20:41So again we used our MDM,
• 20:44two conditional mouse and we
• 20:46deleted MDM two in the adult mouse.
• 20:49But we did this acutely and we actually
• 20:52chose a 24 time our time point to
• 20:55ask what P53 targets are regulated
• 20:58in different issues that lead to
• 21:01these pathologies in the adults.
• 21:05OK,
• 21:06so this is now all the different
• 21:08issues that we initially looked
• 21:10at and what I'm showing you here
• 21:13is the percent recombination.
• 21:15So once we treat with tamoxifen,
• 21:17we induce recombination of the locus.
• 21:21And you can see that the pancreas,
• 21:23the heart being tested,
• 21:25had the highest level of recombination.
• 21:27Again,
• 21:27I will point out that we only have one MDM,
• 21:31two allele.
• 21:32The other allele is an allele,
• 21:34so single recombination event
• 21:36will activate P 53.
• 21:38And then on this axis we chose to
• 21:40look at P53 activation by measuring
• 21:42the expression of P 21 which is
• 21:45encodes a cell cycle inhibitor.
• 21:48So you can see in this experiment
• 21:50at the kidney,
• 21:51the pancreas in the intestine
• 21:54where the tissues that expressed
• 21:56the highest levels of P.
• 21:5721.
• 21:58And we were thinking the highest
• 22:02levels of of P53.
• 22:04So we've we've looked at these mice,
• 22:06so in 24 hours we see no.
• 22:09So let me back up just for one second,
• 22:12I hope I can do that,
• 22:14so that issues that we
• 22:15decided to look at where
• 22:17the kidney, the pancreas,
• 22:18the intestine in the heart.
• 22:21And the ovary in the ovary.
• 22:23Just because P 53 is mutant in
• 22:26ovarian carcinoma is at 95%.
• 22:3095% frequency and so we wondered
• 22:32if we might be able to to begin to
• 22:37understand that mutation frequency.
• 22:39So for the heart for the ovary,
• 22:42we saw absolutely no phenotypes after
• 22:4624 hours post deletion of MDM 2.
• 22:49In the intestine we saw a
• 22:52fascinating phenotype which
• 22:54is descript dropout phenotype.
• 22:57In yellow here,
• 22:58I've outlined the ***** of the
• 23:00intestine and one of the phenotypes is
• 23:03the complete absence of of the crypt.
• 23:06Sydney in the lab is quantified
• 23:08the number of ***** in these
• 23:11different animals and you can
• 23:13see the mice that have no MDM.
• 23:152 have about little more than
• 23:18little but half of the number of
• 23:20***** is in normal control mouse.
• 23:24The kidney also had some phenotypes
• 23:26at 24 hours and it had twice
• 23:29the number of protein casts,
• 23:31so you can see here.
• 23:32So this is an early phenotype in the kidney.
• 23:36Again,
• 23:36this is the normal kidney control experiment.
• 23:39And then the pancreas had to be a
• 23:43fascinating phenotype which will
• 23:44delve into a little bit more deeply.
• 23:47But we saw in the pancreas is this?
• 23:52Acinar to ductal metaplasia,
• 23:54so here's a normal pancreas in the top,
• 23:57and here's what the pancreas looks like
• 24:00in the in the animals that have no MDM.
• 24:042 Here we've stained with keratin 19,
• 24:07which is a marker for a ductal cell.
• 24:11And here we've measured the Metaplastic
• 24:14area and we also see an immune
• 24:17infiltration in these in these mice.
• 24:20So within 24 hours we saw this huge
• 24:23plasticity in the pancreas from you know,
• 24:27this acinar to ductal metaplasia.
• 24:30OK,
• 24:30so we've taken these five
• 24:32tissues an we've done.
• 24:34We've looked for expression of P53 targets,
• 24:37so on the left here I show you all of
• 24:40the genes that were regulated in these
• 24:43different issues in our RNA seek data.
• 24:46The dark region is the region that
• 24:49is upregulated in the lighter color,
• 24:51shows the regions that were downregulated
• 24:54in each of these five tissues.
• 24:57On the right here I show the percent
• 25:00of these these dysregulated genes
• 25:02that are actually P53 targets.
• 25:04They have a pizza degree binding
• 25:07site and we used data from the
• 25:10literature to identify these tools.
• 25:12With P53 binding sites.
• 25:14So in the intestine,
• 25:15for example,
• 25:16I think that number is 69% of the
• 25:20channels had pizza pre binding sites.
• 25:22So the the most.
• 25:24Most of the genes dysregulated
• 25:26it in the system by deletion
• 25:29of MDM 2RP53 targets the ****.
• 25:32** the other hand had a
• 25:35huge physiological response,
• 25:36600 for jeans that were dysregulated
• 25:39but only 16% repeated different targets.
• 25:42So what we're capturing here at
• 25:4524 hours is not just you know
• 25:48activation of P53 and P3 targets,
• 25:50But the downstream responds to
• 25:54that P53 activation.
• 25:56So this is now compilation of all
• 25:58those five different tissues to
• 26:00examine the overlap in P53 target genes.
• 26:03And as you can see from this figure
• 26:06on the left there were only 7 jewels
• 26:10that were commonly regulated.
• 26:12By MDM 2 lost that repeat 53 targets.
• 26:16So for example here in the pink is we
• 26:19have 206 genes, 135 of the P53 targets
• 26:23are specific to the pancreas and seven
• 26:26were shared with the other four tissues.
• 26:29So these seven jeans are MDM two cycling,
• 26:32G1 MDM two as we as I mentioned,
• 26:36the very beginning is regulated
• 26:38by P53 and we expect it well.
• 26:41We didn't expect them to be able to,
• 26:45but it's not a surprising result because
• 26:48the promoter is intact in MDM 2.
• 26:52Three of these genes segment you one GST,
• 26:55one piece art, one or cell cycle regulators
• 26:59to these jeans are transcription factors,
• 27:02and this gene EDA 2R herself directions.
• 27:05So these are the six Peachtree
• 27:08targets that have a common.
• 27:10They represent the common signature of of
• 27:13upregulated genes in in these three tissues.
• 27:16We wanted to validate the signature
• 27:19to make sure that they were.
• 27:22Truly, a P53 targets physiologically.
• 27:24And So what we did is we did our our DNA
• 27:28damage ionizing radiation experiment.
• 27:31We treated the whole animal
• 27:33with ionizing radiation,
• 27:34and here's the data for two of the jeans,
• 27:38and we've done it for all
• 27:41of seven cycling G1 E DA2R.
• 27:43Here's the wild type levels
• 27:45of expression of these genes.
• 27:47If we irradiate,
• 27:49you can see that these genes are upregulated.
• 27:52In in both cases,
• 27:55and if we irradiate a P53 null,
• 27:58you see you see no up regulation.
• 28:01So these are P3 target genes that are
• 28:04being upregulated following punishing
• 28:06radiation. So these experiments.
• 28:10Highlight this incredible repertoire.
• 28:13Transcriptional targets that P53
• 28:16physiologically regulates the vivo
• 28:18and I think it also suggests that
• 28:21maybe these specific targets can
• 28:23be used to understand in vivo.
• 28:25If you have.
• 28:27If you can reactivate piece of D3
• 28:30or convert mutant and wild type,
• 28:33these might be great markers to
• 28:37look at for activation 53.
• 28:40OK,
• 28:41I want to now just briefly discuss
• 28:44this this encrypted hypothesis.
• 28:47Acinar ductal hyperplasia that
• 28:49we see within 24 hours in the.
• 28:53In the pancreas.
• 28:55So one of the other experiments that
• 28:59Sidney Moyer in my lab did is we
• 29:02obtained these mice MST one missed one.
• 29:06Cree are OK,
• 29:07so missed one is an SNR
• 29:09specific transcription factor.
• 29:11An upstream of the missed one promoter.
• 29:17There is a criar transgene,
• 29:19which means that you can express create
• 29:21only in the *** and ourselves with the.
• 29:25Pancreas and so Sydney worked out the
• 29:29tamoxifen conditions that gave you
• 29:32a similar percent recombination as
• 29:35our previous experiments with MDM.
• 29:38Two position in the entire pancreas.
• 29:41OK, so similar percentage of recombination
• 29:44and similar activation of P3 targets.
• 29:48So here we use two of our targets,
• 29:52EADE, DA2RG, STT, SC1.
• 29:55To measure people to the activation
• 29:58and you can see in both my sweet.
• 30:01MDM two deletion happens in the
• 30:03home pancreas or mice where it
• 30:05only happens in the acinar cells.
• 30:07You have similar activation of
• 30:09these three of these targets,
• 30:10so we felt we could do.
• 30:14We could actually compare deletion
• 30:16of MDM two in the whole pancreas.
• 30:18The deletion of MDM two jestoni
• 30:21Essen ourselves.
• 30:22And we have absolutely no phenotype.
• 30:25So these pancreas look completely normal.
• 30:29Here we're measuring just we're looking at.
• 30:32I mean,
• 30:33I ageny sections in here in the right.
• 30:37We're measuring the immune
• 30:40component and these these pancreas.
• 30:42These pancreatic perfectly normal. So.
• 30:46The take home message here is that.
• 30:49This esnard ductal hyperplasia that
• 30:52we see is a P53 specific hyperplasia.
• 30:56But it's it's arising from signals
• 30:59outside of the acinar cells.
• 31:02So to me,
• 31:03this is a fascinating experiment
• 31:07because no one's ever noted that.
• 31:11That the environment can can affect
• 31:14the pizza delivery response,
• 31:16and so we'll be delving into understanding
• 31:21this phenotype a little bit better.
• 31:25Pancreas is is one of the tumors
• 31:29with 7075% mutations in P53 an and
• 31:32it always has this very compromised
• 31:35stromal component and so maybe by
• 31:39understanding what P 53 is doing is
• 31:43physiologically important Organism,
• 31:45we might be able to impact our
• 31:49understanding of Peter mutations in
• 31:53pancreatic cancer. OK, so the let out.
• 31:56OK so I'm just going to check my
• 31:59Clock to see how much time I'm doing.
• 32:02Well, OK,
• 32:03so I've shown you a lot of data
• 32:05where we deleted MDM two and an
• 32:08I didn't show you data for MDM 4
• 32:11but you you see these people three
• 32:13dependent physiological phenotypes
• 32:14and that's all fine and good.
• 32:16It showed us how important this
• 32:18relationship is between these proteins but.
• 32:21What happens in human cancers is
• 32:23you've got high expressions of
• 32:25MDM two and this is just yes,
• 32:27mean Valentina Vega in the lab
• 32:29a number of years ago looked at
• 32:32Indian 2 levels in head and neck
• 32:34squamous carcinomas and these are
• 32:36some of her beautiful pictures.
• 32:38So here's MDM,
• 32:39two expressed a very highly in a small
• 32:42region of this squamous cell carcinoma here.
• 32:456 expressed almost across the
• 32:47entire tissue and then here is
• 32:49an interesting example of MDM.
• 32:51To be expressed in the cytoplasm,
• 32:53not the nucleus.
• 32:54So we really don't understand
• 32:56what it's doing in the cytoplasm,
• 32:59but not in all three of these experiments, P.
• 33:0253 is 1 type OK,
• 33:04so I think with this experiment
• 33:07in many others that
• 33:09people have done. Again.
• 33:13Again, show that what MDM two is doing in
• 33:17these tissues is inhibiting P53 activity.
• 33:20Now the I also don't want to leave
• 33:23you with the notion that MDM too.
• 33:27The P53 is the only MDM to target.
• 33:31Physiologically is the most
• 33:33relevant target because of the cell
• 33:36lethal phenotypes that we see,
• 33:38but in several experiments that my lab
• 33:42and Carol previous labs have done is,
• 33:45we've tried to overexpress MDM two in
• 33:48normal cells to understand what it's
• 33:51actually doing in with regards to
• 33:54transformation and tumor evolution.
• 33:56So here's what happens.
• 33:58So this is a normal control and the
• 34:02left these are mouse cells express a
• 34:05normal number of mouse chroma zones,
• 34:08and when we overexpressed MDM two
• 34:11we see this incredibly abnormal.
• 34:14Chromosome instability we can quantify
• 34:17the numbers of fusions here and
• 34:20we have a huge number of fusions.
• 34:22We also have a lot of fragments.
• 34:27So in data from multiple labs,
• 34:29if you overexpress MDM two in a
• 34:32normal cell the cell just dies.
• 34:35It can continue to grow.
• 34:37So and you know some experiments
• 34:39that are ongoing in the lab is OK,
• 34:42trying to understand.
• 34:44OK,
• 34:44so if we can't overexpress MDM
• 34:47two in a normal cell?
• 34:49Why do tumors have very high levels
• 34:51of ambient 2IN in one idea that we're
• 34:54working with is that there are some
• 34:57other rotation in those tumors that
• 34:59allows those tumors to survive with
• 35:02high levels of MDM two and so if we
• 35:05could understand what else MDM two is doing,
• 35:08we might be able to obtain a.
• 35:12A window of vulnerability to try to get the
• 35:16MDM two overexpressing cells to implode.
• 35:20But the screens that we're doing
• 35:23currently are are ongoing.
• 35:25OK then for the last few minutes
• 35:29of my lecture I want to.
• 35:32Move over to our understanding of P53
• 35:36mutations in breast cancer models.
• 35:39Didn't tell you earlier, but pizza degree.
• 35:42Why did tell you the people
• 35:45limitations for the most common,
• 35:47but really it speak into three
• 35:50missense mutations that are is the
• 35:53most common type of genetic lesion,
• 35:55and so my lab and that of Tyler left.
• 35:59Tyler Jacks is lab have made germline
• 36:02mutations in P53IN animal models and
• 36:05we show that these mice are tumor pro.
• 36:08But more importantly in contrast.
• 36:11Green or mice?
• 36:12These mice have a high metastatic capability,
• 36:15so this here is our data from the
• 36:18172 mutation corresponds to the
• 36:20origin 175 mutation,
• 36:21which is one of the hot spot
• 36:24mutations in human cancers.
• 36:26And here you can see a metastasis
• 36:28to the liver,
• 36:29and here stained with the P53 antibody,
• 36:32a metastasis to the brain.
• 36:35And this is in contrast to mice
• 36:38that have deletions of 353,
• 36:40so this really was the first example
• 36:43that suggested that expressing
• 36:44a mutant P53 was much more.
• 36:49Much more aggressive than not having
• 36:51people to create and and in the field.
• 36:53We call this a gain of function.
• 36:55Mutant P 53 is doing something in these
• 36:58cells to make them highly metastatic.
• 37:01So these are germline mice and what we
• 37:04wanted to do is to generate semantic
• 37:07models because the these germline models
• 37:10represent Lee from Many syndrome which is
• 37:14an inheritance of people to mutations.
• 37:16But that's a rare syndrome and we really
• 37:20wanted to understand this metastatic
• 37:23phenotype in a system where the the
• 37:26specific cell type has a Peach limitation
• 37:29and surrounding normal environment.
• 37:31To feel yourself to catch neutral
• 37:34goes to T cells are all wild type
• 37:37for P53 so that mouse did not exist.
• 37:39Tyler Jacks made a beautiful mouse that
• 37:42has been used extensively in the literature
• 37:44that basically is heterozygous for P53.
• 37:47So the entire mouse is missing 1P53 allele
• 37:50and Natalie all can be converted to a
• 37:52mutant P53 in a tissue specific fashion.
• 37:55So we didn't think that that was.
• 37:59Adequate enough to study the tumor,
• 38:01stroma tumor immune interactions because
• 38:03of heterozygosity of the P53 locus.
• 38:05So let me tell you a little bit
• 38:08about how we generate these mice and
• 38:12what our breast tumor phenotype is.
• 38:14OK, so here's how we generated these alleles.
• 38:18So we call these WM allele for
• 38:21wild type to mute P 53.
• 38:23So I'll show you in a minute this
• 38:25is a wild type allele normally and
• 38:28it's wild type because we earned
• 38:31started seeding a sequence upstream
• 38:33of the point mutation and this is
• 38:37this example is the Argentine,
• 38:39once it imitation which
• 38:40we generated previously.
• 38:42So what you have is a wild type P50.
• 38:45Three years of pollination site and
• 38:47then in a cream immediate fashion
• 38:50you can remove the wild type C DNA
• 38:53and basically reconstruct the locus.
• 38:55The arm and Cindy to Locust that we
• 38:58had studied forever in the germ line.
• 39:01With these mice and because it took a
• 39:04very long time to make these animals,
• 39:07we actually decided to make a second
• 39:09hotspot mutation and that's the
• 39:11Argentine 2.5 to double mutation,
• 39:13which corresponds to the 248
• 39:15hotspot mutation.
• 39:16Uhm?
• 39:19So this is just showing me wild type
• 39:21initially and then we committed fashion.
• 39:23You make them into protein and you can
• 39:25make it in akhri dependente manner.
• 39:28So this just shows you how normal
• 39:30those mice are.
• 39:32So here we're comparing wild type 2
• 39:35heterozygous mice with the 172 or the 2.5.
• 39:37We stabilized the mutant protein
• 39:39in response to DNA damage.
• 39:41And when we look at the activation
• 39:44of the three targets be 21 in Puma,
• 39:47they were activated to similar levels.
• 39:51We looked at the ability of these.
• 39:54Of DNA damage to induce labor ptosis?
• 39:57No difference between these
• 39:58two alleles in wild type mice.
• 40:00And again for the ability to rest the
• 40:03cycle in mouse embryo fibroblasts,
• 40:06there's no difference,
• 40:07so these mutant alleles really
• 40:09represent these condition alleles.
• 40:11That column you Tilly's really
• 40:13represent wild type allele.
• 40:15They can become a moot.
• 40:18And this is the last experiment I'll
• 40:20show you about the the actual alleles.
• 40:23What we've done is what we've
• 40:25done here is compared the 172
• 40:27heterozygous mice at 245 headers.
• 40:29I guess mice and wild type mice to
• 40:32each other over more than two years
• 40:34to look at the tumor phenotypes.
• 40:37Mice is the age.
• 40:38Just like people will sporadically
• 40:40get tumors, but what you can see is
• 40:42that there's absolutely no statistical
• 40:44difference between the three alleles.
• 40:46So for all practical purposes.
• 40:48This new allele that we generated
• 40:51expresses a wild type P53 protein.
• 40:54OK, so let me tell you bout two experiments.
• 40:58One is our semantic breast semantic
• 41:02model and what we did is we did
• 41:06use the R172H and the 245 alleles.
• 41:10We injected Adna virus Cree into the duct
• 41:14of the mammary gland and so and then.
• 41:17In addition, we use this TV tomato
• 41:20allele which is also create dependent.
• 41:22So when we inject adeno Korea not
• 41:25show you a picture in a minute.
• 41:28We basically make a mutant P
• 41:3153 and we label the cell red.
• 41:35So here is the.
• 41:38Control experiments to show that we
• 41:41do get recombination when we inject
• 41:44the cream expressing adeno virus here
• 41:46on the left is a low titer injection
• 41:49and you can see the red cells here.
• 41:521 to 5% of the ductal cells become infected.
• 41:57And then here on the right you can see.
• 42:00A high titer virus was used was
• 42:03injected into this gland in about 50 to
• 42:0570% of the ductal cells are checked.
• 42:09I also want to note that these
• 42:12mice are 50% balzi.
• 42:13Normally the P53 field is used to
• 42:16see 57 black, 6 string to study.
• 42:21Peabody create tumor phenotypes,
• 42:23but that strain is resistant to
• 42:26breast cancers for some reason,
• 42:28and the beltsy component brings in
• 42:31more sensitivity to breast cancers,
• 42:34and we really don't know the
• 42:37genetic reasons for this.
• 42:40OK, so here is the data.
• 42:42Let me go through it in detail.
• 42:45So this is the 172 mutation expressed
• 42:48only in a few mammary glands.
• 42:50Memory cells, low titer.
• 42:52We didn't see any tumors.
• 42:55High titer,
• 42:56we actually didn't see any tumors.
• 42:59This one tumor showed up.
• 43:01Post that to your end time
• 43:04point of the experiment.
• 43:07Because we weren't sure we were going
• 43:09to get any tumors by just making
• 43:12people scream Mutant in a few cells.
• 43:15We also.
• 43:17Irradiated with the sub lethal
• 43:19dose of radiation.
• 43:21This is not lethal to the mouse,
• 43:24but it does cause damage and if the
• 43:27damage is in the right or wrong genes
• 43:30that contributes to tumor phenotype.
• 43:33So with a low titer we begin to see tumors.
• 43:37We sell one at a higher titer.
• 43:40We now solve 4 tumors and
• 43:43one tumor metastasized.
• 43:45The 2.5 allele was a much stronger,
• 43:47had a much stronger tumor,
• 43:49phenotype with low titer.
• 43:51We saw four tumors and one of them was
• 43:55meta static with the high tier tighter.
• 43:58We saw nine tumors, so this is about
• 44:0175% and more than half were meta static.
• 44:04So let me kind of summarize all the data
• 44:07that we've done with these animals.
• 44:10So first let's just look at
• 44:13the tumor incidence. The R 172.
• 44:15Each allele all by itself in in.
• 44:17In these experiments, we only use
• 44:20one copy because we didn't want to.
• 44:24We didn't want to do LOH and
• 44:26we didn't want to.
• 44:28We just wanted to figure out what
• 44:30would happen with the minimal
• 44:32number of alterations.
• 44:34So if you compare the ones need two hitters
• 44:37Vegas to the 2.5 low and high titer,
• 44:40there's a huge number of tumors in the 248.
• 44:44Nice if we irradiated the ones only two,
• 44:47we got increased tumor incidence and
• 44:49then this is the experiment where
• 44:51we did mutate the other allele.
• 44:54So existing law supporters I got
• 44:57city and we can see.
• 44:59Increased tumor phenotype with the high dose.
• 45:02And this is a comparison on in the
• 45:04middle panel of the metastatic phenotype,
• 45:06and again the 245 documentation
• 45:08was the most metastatic.
• 45:10And then we looked at lots of header
• 45:14zygosity so the 245 mutation had.
• 45:18Variations in terms of LOH.
• 45:20About 50% of the mice showed LOH
• 45:23and then others retained some or
• 45:26all of the P3 alleles.
• 45:28The irradiated are once again 28.
• 45:31Showed 100%. LOH so to us.
• 45:35While we don't understand why we
• 45:38see 100% outrage with this allele,
• 45:41would it says?
• 45:42Is it that wild type allele is
• 45:44very strong at inhibiting tumors
• 45:47in this winsome need to background?
• 45:52These are the breast tumor subtypes that
• 45:55we saw the irradiated once in need two
• 45:58we saw mostly triple negative breast.
• 46:01There are 172 with the
• 46:03that lacks wild type P53.
• 46:05We sell mostly luminal B and then here
• 46:09with the 2.5 mutation we saw all three.
• 46:14Molecular subtypes to me this is fascinating
• 46:17experiment because we've made one mutation,
• 46:20we made a P53 mutation and yet here in this
• 46:24sample with the 2:45 we see off the tumor,
• 46:28molecular subtypes evolving and so one of
• 46:31the experiments that we're doing now is
• 46:35trying to understand with this 245 mutation,
• 46:38what are the triggers to
• 46:40these different subtypes?
• 46:41Triple negative breast cancer
• 46:43is very hard to treat.
• 46:46But for example,
• 46:47here to enrich tumors you can,
• 46:50you can treat with her two antibodies,
• 46:53so we're trying to understand
• 46:55basically the tumor evolution
• 46:56that initiates with this one, P.
• 46:5953 missense mutation.
• 47:02We've also wanted to so the data
• 47:05I just showed you says that
• 47:08the 248 mutation is much more.
• 47:11Dramatic.
• 47:12Then the 175 mutation.
• 47:14So what I showed here is a comparison
• 47:17of ovarian lung and breast tumors
• 47:20from people and just looking at the
• 47:23kind of mutation that they have and
• 47:26you can see in people that the 248
• 47:29mutation is has the worst outcomes.
• 47:33We couldn't do these data just
• 47:35for breast because the number of
• 47:38samples out there was not enough
• 47:40to give us significance.
• 47:43OK,
• 47:43so the one of the last experiments
• 47:46I'll show you here is just trying to
• 47:49understand tumor evolution because
• 47:52we made a semantic model that
• 47:54develop different kinds of breast
• 47:57cancers that were highly metastatic.
• 47:59And so I'm really interested in
• 48:01understanding the task sees in an in
• 48:05vivo physiologically relevant system.
• 48:07So we did.
• 48:08Here is we took the 2:45 mutant
• 48:11animals and we took.
• 48:13UH-22 memory tumors from these mice
• 48:15we sequenced them in three different
• 48:17regions trying to understand a
• 48:19little bit about the heterogeneity,
• 48:21and then we sequenced three
• 48:24metastases from each of these tumors.
• 48:26OK,
• 48:27and we sequence them to an
• 48:29incredible debt depth.
• 48:31So what we have here then is the
• 48:34comparison of the primary to the
• 48:37metastases and if we just look at mouse #4,
• 48:42there is some overlap in these these
• 48:45this overlap is considered an early
• 48:48gene signature and then you see this.
• 48:52Slate sequences that come up,
• 48:54which is how,
• 48:56which is what,
• 48:57where the metastases is now evolving
• 48:59when it gets into its metastatic site,
• 49:03which in this case was the lung.
• 49:06And so we can compare the early
• 49:09mutations of all three.
• 49:11The task season you see early
• 49:13mutations in both experiments,
• 49:15so these are the mutations that
• 49:17were acquired in the primary and
• 49:19metastatic lesion at the same time.
• 49:22But when you look at late mutations,
• 49:24here's all three mutations for mouse #4
• 49:27there's only one late mutation common.
• 49:30So with these sequencing data,
• 49:33indicate is that these matasa
• 49:35fees left the tumor very early
• 49:38during the metastatic process and
• 49:41then seated and had
• 49:43additional changes. So this was the
• 49:47first suggestion that maybe metastasis.
• 49:50Breast cancer metastasis driven
• 49:52by a new P53 is an early event.
• 49:56So to summarize, this model
• 49:58just briefly, we can make.
• 50:00P 3 point mutation in just a few
• 50:04cells that become a tumor that.
• 50:08Interessee migrate,
• 50:10proliferate and develop these metastases.
• 50:14Where we now have I called it a
• 50:16little factory but we just have
• 50:18these mice now developing tumors.
• 50:20We can isolate the circulating tumor cells,
• 50:22so we're trying to do is understand that
• 50:26the changes that occur for these cells
• 50:29to be able to survive in the blood.
• 50:32To home into an organ and then to develop.
• 50:35OK, if I have a few minutes which
• 50:37I have just a few minutes,
• 50:40I'm going to tell you about the
• 50:42other model that we made because we
• 50:45weren't sure that making a people
• 50:47communication in just a few cells was
• 50:49going to give us a tumor phenotype.
• 50:52So here we use K14 create,
• 50:54which expresses then a mutant P53 in all
• 50:56of the epithelial cells of the mammary gland,
• 51:00and this is a model that develops.
• 51:02Triple negative breast
• 51:04cancer 100% of the time.
• 51:06So again this.
• 51:07This suggests that you know South normal
• 51:10cell tumor cell interactions are altering
• 51:13the kinds of tumors that come up.
• 51:16And in this scenario we also had a
• 51:19cast 9 allele that is Creed dependent,
• 51:23so K14 CRV and allows us to make a
• 51:26mutant people degree in just a few
• 51:29in the epithelium of the mammary
• 51:32gland and to express castanon so we
• 51:35can use CRISPR technologies to to
• 51:39begin to address vulnerabilities.
• 51:41OK,
• 51:41so this vulnerability that we examine
• 51:44this in this model was whether these
• 51:48tumors were addicted to having immune to P53.
• 51:52OK,
• 51:53so here is the use that adnot
• 51:57associated virus that expresses
• 51:59a guide RNA that will delete P.
• 52:0253.
• 52:03So the tumor burden before treatment
• 52:06with a V in the control and the mute.
• 52:11Depleted tumors with similar,
• 52:13but you can see here in in the Purple line
• 52:16that those tumors that had depletion of
• 52:18you piece of degree survived much longer.
• 52:21This is just a picture of the tumor
• 52:24phenotypes that control you can see that.
• 52:27It's very.
• 52:28It's obviously a tumor.
• 52:30These mice die very quickly post
• 52:33identification of the tumors,
• 52:34and then you can see here with the
• 52:38depletion immunity theory this this
• 52:41this gland is looking more normal.
• 52:44OK,
• 52:44this is a whole bunch of data for the
• 52:48individual mice that this is a tumor volume.
• 52:52The controls and green here.
• 52:54Once we identify the tumor,
• 52:56they just keep growing the experimental
• 52:58cohort here in purple are animals
• 53:01that have recombined have basically
• 53:03deleted that mute people free protein
• 53:06in these mice live much longer.
• 53:08On the right is a tumor volume,
• 53:11so you can see that depletion of
• 53:14P53 affects the tumor volume.
• 53:17Greatly and then I'll just point
• 53:20out these two samples in Orange,
• 53:22which appeared not to respond
• 53:24to depletion of Mutant P
• 53:2753, and when we look at
• 53:29these two samples in detail,
• 53:31they did not express a stable mutant P.
• 53:3453, and so we think that
• 53:37these two tumors are actually.
• 53:40Since the P 53 isn't stable,
• 53:42the evolution that's occurring
• 53:44in these tumors is due to the
• 53:47absence of the P53 protein and
• 53:49not to a gain of function.
• 53:52OK, so I'm going to stop there
• 53:54and just I thrown a lot of data
• 53:57at you so I wanted to summarize
• 53:59briefly the first set of experiments
• 54:02really captured the exquisite
• 54:04sentence sensitivity of the MDM,
• 54:06two protein and P53 activity.
• 54:08We were able to.
• 54:12Identify this the molecular response
• 54:13to P53 activation in vivo identified
• 54:15numerous targets that are tissue specific.
• 54:18What are they all doing?
• 54:20I think we have our work cut out
• 54:22for us 'cause there's no way I can
• 54:25delete every one of these targets
• 54:27and see how important they are in
• 54:30vivo and then last but not least,
• 54:33I showed you the generation of of
• 54:36this novel mouse model that really
• 54:38allows us now to make a semantic
• 54:40point potential 53 in.
• 54:42Any cell of origin that we want
• 54:45to an in in the breast model.
• 54:49Specifically,
• 54:49we had a highly metastatic phenotype that
• 54:53we're trying to understand in more detail.
• 54:56OK,
• 54:56so my last slide is just the
• 54:59numerous people in the lab that
• 55:02have contributed to the studies.
• 55:05The Vinodh Pant did the feedback loop
• 55:08studies Roberto Navy MTM Tunicate Johnny DMD.
• 55:12Or not,
• 55:13God Tamera did all the studies
• 55:15with the radiation and with the
• 55:18node to look at the P3 response,
• 55:22Sidney generated the conditional mouse to
• 55:24look at the acute activation of people.
• 55:28Three targets you in terrific postdoc
• 55:30in the lab now has her own independent
• 55:34position generated that conditional
• 55:36mood P53 allele and Donata is the
• 55:40one who's studying the addiction.
• 55:42We're really wondering what the
• 55:44mechanisms is acquire these
• 55:46tumors imploding when they no
• 55:48longer have communities industry.
• 55:50So with that, I'll end in, oh,
• 55:53I'm glad to answer any questions.
• 55:56Thank
• 55:57you so much.
• 55:58That was a wonderful talk.
• 56:00I'm going to ask people to put
• 56:03questions in the in the chat,
• 56:05but I I wanted to ask you, sort of,
• 56:09from from the therapeutics perspective,
• 56:11people have been very interested in
• 56:13compounds like Prima and Cody that
• 56:16assist with re re folding of P53 with
• 56:18disruptive mutation and yet clinically
• 56:21those have been a little bit disappointing.
• 56:25Is is much known about whether or not
• 56:29those refolded P 53's are better,
• 56:32worse the same as as substrates for the MDM?
• 56:36Two MDM four? Yeah, so,
• 56:38so we've done a few studies using some of
• 56:42the drugs that are available, not many.
• 56:47My lab is focused on the genetics
• 56:49because if we take out him to an MP4 we
• 56:53see people three different phenotypes,
• 56:55but you know it's very different.
• 56:58Genetic told us mechanisms,
• 56:59but the drugs are really as you indicated.
• 57:03They're going to tell us
• 57:05whether they work or not.
• 57:07So I agree with you, I don't think
• 57:10that drugs are working very well.
• 57:13And you know,
• 57:15I don't know enough about those
• 57:17experiments to know you know how
• 57:19often you know how often the drug
• 57:21with the level of activation.
• 57:23I think that our.
• 57:26Our.
• 57:28That that that 7 gene signature that
• 57:31we identified would really help in
• 57:34those studies to try to understand
• 57:36what is the pika degree response?
• 57:39I also think that the people
• 57:42agree response required.
• 57:44Will vary in different tissues.
• 57:49We just know from our MDM two studies that
• 57:51some tissues are just much more sensitive
• 57:54to increase P 53 levels versus others.
• 57:57So I think that there's just a whole lot
• 58:00more work to be in to do in the clinic to
• 58:03be able to to understand that response.
• 58:07Yeah, yeah, I I think having a common set of.
• 58:11I mean I think people have just looked
• 58:13at P 21 over and over again and it's
• 58:16probably very inadequate, right?
• 58:18I see a couple questions and
• 58:20we have like a minute left.
• 58:22So first Jeff Townsend wants to know.
• 58:25Whether or not you've considered
• 58:28looking at sequencing of much larger
• 58:30cohorts of tumors and multi sample
• 58:33datasets to understand the temporal
• 58:36order of mutation appearances,
• 58:38that's exactly what
• 58:39we're doing right now I have a postdoc in
• 58:43a graduate student who just generated.
• 58:47A cohort of 100. Nice 'cause we want
• 58:49to understand the sequence of events.
• 58:51We want to understand the
• 58:52different events that lead to the
• 58:54different molecular subtypes.
• 58:55So we are in the midst of those
• 58:58experiments and we're going to do RNA
• 59:00seek and in DNA sequencing to understand
• 59:02both what happens if Arnie level.
• 59:04I think that it's critical we need
• 59:06to understand what's happening
• 59:07at the DNA level because I think
• 59:10that that's what gives rise to the
• 59:12different molecular subtypes.
• 59:13But I think it's the expression
• 59:15that's really going to tell us.
• 59:17What's happening to those cells once
• 59:20they reach home to deliver the line so?
• 59:24So we got all those are in progress.
• 59:28Then, Karen Anderson, who's my Co.
• 59:31Host for having invited you,
• 59:33wants to ask what your thoughts
• 59:35might be for a pro TEC directed
• 59:38against MDM two try to grade that
• 59:41as a therapeutic strategy. I
• 59:43think there's two Protex we
• 59:45should be thinking about.
• 59:46One is MDM 2. Although.
• 59:50Somehow I think for an MDM two
• 59:53inhibitor I think you've got to,
• 59:56you've got to target it
• 59:58to the to the tumor cell.
• 01:00:00Better just because of the
• 01:00:03hematopoetic toxic cities that have
• 01:00:05been seen with the MTM 2 inhibitors.
• 01:00:08But I also think we should
• 01:00:10start thinking about potentially
• 01:00:12doing degrading mutant P.
• 01:00:1353 Taking it out of the picture
• 01:00:16to see to see what happens.
• 01:00:20Particularly with those gain
• 01:00:22of function mutations,
• 01:00:24exactly exactly.
• 01:00:28We are over there more questions but
• 01:00:31we are over the time so I want to be
• 01:00:34respectful of very appreciative of of you,
• 01:00:36having joined us today and if
• 01:00:38anybody wants to ask me a question,
• 01:00:41email that they should feel free to I think
• 01:00:43allowed to answer additional questions.
• 01:00:48Super very much.