Targeting Oncometabolite-induced DNA Repair in Cancer

October 04, 2021

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Yale Cancer Center Grand Rounds | September 28, 2021

Dr. Juan Vasquez

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00:00Everyone for attending this week's
00:02grant Yo Council Center grand rounds.
00:05It's my privilege and pleasure to introduce
00:08Dr Juan Vasquez for this this week talk.
00:12Dr Vasquez is an assistant
00:14professor of Pediatrics.
00:15He received his medical degree
00:16from Brown University and a Master
00:18of Health Science from Yale,
00:19where he also completed his fellowship
00:22in Pediatric Hematology Oncology.
00:23His clinical focus on the
00:25care of children with cancer,
00:26particularly solid tumors,
00:28as reachers research,
00:29is focused on the development
00:31of immunotherapy.
00:32For pediatric tumors,
00:33particularly malignant brain tumors,
00:35he's interested in characterizing
00:36the immune landscape of pediatric
00:38brain tumors and understanding
00:39the interplay between DNA repair
00:41and anti tumor immune response.
00:43One is been an embedded assistant
00:45professor in our laboratory for a
00:47little over two years now and is
00:49really hit the ground running as
00:51rapidly approaching independence.
00:53On that note, he did recently receive his KO,
00:55a career development grant to
00:57fund this project,
00:58which will be talking about today.
00:59So with that I will let one take.
01:02Take the show away.
01:08Great thank you Ranjit Saunders
01:10Commissioner my screen here.
01:29All right? Can you see my screen OK?
01:34Great. Alright, so thank you again.
01:36It's a real honor to be able
01:38to present for you today.
01:39An uncle metabolite induced
01:41repair DNA repair defects.
01:43Uhm? I've got no disclosures.
01:47So today I'll briefly review some
01:50background on Uncle Metabolite
01:52induced DNA repair defects,
01:54which really was established by the
01:56seminal work of my research mentor.
01:58Doctor Ben drove heard from as well as
02:01Doctor Peter Glaser here at Yale and in
02:04collaboration with Brian Shuck at UCLA.
02:06Also present some of our work on
02:08targeting DNA damage response pathways
02:10and uncle metabolite producing tumors.
02:13And then lastly,
02:13I'll touch a bit on the potential
02:15for exploiting these uncle
02:16metabolite induced DNA.
02:17Repair defects in order to promote an
02:20inflammatory tumor microenvironment
02:21and potentially synergized with
02:23immune checkpoint blockade.
02:29So just as a very brief reminder,
02:32the Krebs cycle is very important in
02:34cellular energy production and alpha
02:36ketoglutarate is a is a key intermediate
02:38in the Krebs cycle and Alpha Ketoglutarate
02:41dependent dioxygenase is regulate a
02:43number of key cellular processes.
02:45Mutations in enzymes of the Krebs cycle
02:47result in an excess accumulation of two
02:50hydroxy glutarate succinate and fumarate,
02:52and I'll go through these in
02:54more detail in the coming slides.
02:56But in general,
02:57these uncle metabolites competitively
02:59inhibit alpha ketoglutarate dependent
03:01dioxygenase is by virtue of their structural
03:04similarity there by dysregulating AKI,
03:07variety of downstream cellular processes and
03:09resulting in prolonged congenic signaling,
03:12and this is really why there are
03:14classified now is uncle metabolites.
03:17So focusing first on IDH mutations
03:20or isocitrate dehydrogenase so IDH
03:23catalyzes the oxidation oxidative
03:25decarboxylation of isocitrate
03:27producing alpha keto glutarate.
03:29Uhm, and these heterozygous IDH
03:31mutations result in a new amorphic
03:34activity of that enzyme whereby
03:36alpha ketoglutarate is then further
03:38converted into two hydroxy glutarate.
03:41And most commonly are missense
03:44arginine to histidine mutations.
03:46Make up about 70% of all these
03:48mutations and you can see IDH
03:50mutations in a variety of tumors.
03:51Most most commonly in low grade gliomas
03:54and secondary GBM's as well as AML and
03:58chondrosarcoma and cholangio carcinoma.
04:02Further on down to the Krebs cycle
04:04succinate dehydrogenase catalyzes the
04:05oxidation of succinate to fumarate
04:08and fumarate hydratase catalyzes
04:10the hydration of fumarate to malate
04:12germline heterozygotes loss of
04:14function mutations in these genes
04:16are associated with a predisposition
04:17to cancer formation thought to act
04:20through a two hit hypothesis whereby
04:22tumors have loss of heterozygosity,
04:24leading to excess accumulation
04:26of femur and succinate.
04:28Germline FH mutations predispose
04:30to hereditary leiomyoma ptosis
04:31and renal cancer syndrome.
04:33And germline SDH mutations predispose to
04:36succinate dehydrogenase related hereditary
04:39paraganglioma and pheochromocytoma
04:40as well as renal cell carcinoma.
04:43And, importantly,
04:44renal cell carcinoma in the setting
04:45of both these syndromes is typically
04:47aggressive with a high propensity to
04:49present with metastases early in disease
04:51and once these patients metastasize,
04:52very limited treatment options exist.
04:56So, as I mentioned before,
04:58this is a field really pioneered
05:00by Doctor Benjamin, Dr.
05:01Glaser and former grad
05:03student portal Cylkowski,
05:04and a series of high impact publications
05:07where uncle Metabolites were found
05:09to inhibit homologous recombination
05:11and confer prohibit or sensitivity.
05:14So I'm just going to very
05:15briefly summarize this work,
05:17but what they found is uncle
05:19metabolites inhibit alpha ketoglutarate
05:21dependent histone lysine demethylase
05:23is KTM 4 AMB leading to a Baron
05:26hypermethylation of histone 3,
05:28lysine 9 or HK H3K9 at loci
05:32surrounding DNA breaks.
05:34So they used a really elegant double
05:36strand break chip seek assay,
05:38in which you can see that control cells
05:41there's a spike of H3K9 trimethylation.
05:44That induced double strand breaks
05:46followed by a coordinated recruitment
05:48of double strand break repair factors.
05:50However,
05:50in cells with an uncle metabolite
05:53succinate fumarate and two HG,
05:55there is H3K9 trimethylation
05:57already present at the site before
05:59induction of double strand breaks,
06:01and this really serves to mask
06:03that local trimethylation signal
06:04that's important for triggering
06:06proper recruitment of homologous
06:08recombination proteins,
06:09essentially leading to defective
06:10HR and a bracken NIST phenotype.
06:15So now just to very briefly and generally
06:17introduce the topic of synthetic lethality.
06:20So as you can see here from this figure,
06:21part is really an important enzyme
06:25involved in the repair of single strand
06:27breaks during basic scission repair.
06:29Pop inhibition and results in impaired
06:32based excision repair and converts
06:34single strand then single strand
06:36breaks are converted to double strand
06:38breaks in the in the process of
06:40cellular replication in cells with
06:41an intact homologous recombination.
06:43This DNA damage is effectively
06:44repaired and you have cell survival.
06:46However, in the setting of an HR deficiency,
06:49there's a buildup or accumulation
06:50of unrepaired DNA damage,
06:52ultimately leading to cell death and this
06:53is this idea of synthetic lethality.
06:57So this same synthetic lethality was
06:59found also in the setting of uncle
07:01metabolite induced DNA repair defects.
07:03So just looking at just a snippet
07:05of that data you can see here in the
07:08isagenix model and he LA cells with IDH,
07:10wildtype and I DH R132H mutant.
07:15There's an increased amount of
07:16baseline unrepaired DNA damage,
07:18and this is as measured through a
07:20common tale essay where damaged DNA.
07:23As its nucleus informs US, Comet tail,
07:25which is his representative unrepaired
07:27DNA damage and you can see that IDH
07:30mutant Tumors Harbor an increased
07:32amount of damage at baseline.
07:34Uh, additionally looking here
07:35at a clonogenic survival assay,
07:37you can see that these cells,
07:40these IDH mutant cells have more
07:42sensitivity to irreparably than
07:43their wild type counterparts,
07:45and the same was seen in in vivo.
07:48A study using HTTR human cancer
07:50colon cancer cell line with an
07:53IDH mutation where these tumors
07:54were sensitive to PARP inhibition,
07:57leading to delayed tumor growth.
08:02Similarly, in a subsequent study,
08:04a similar DNA repair defects and
08:06corporate hipper sensitivity were
08:08shown in FH and SDH deficient models.
08:11So looking here now at a collection of human.
08:15Tissues, let's see.
08:18You can see that again compared
08:21to normal tissues.
08:22Those with SDHB mutations in FH
08:24mutations have an increased amount
08:26of baseline DNA repair damage.
08:28I mean sorry baseline DNA damage
08:29and then here looking at a FH
08:31deficient PDX model you can see in
08:33vivo there's delayed tumor growth
08:35with a different park inhibitor.
08:37Here bnes 673.
08:44Based on these findings,
08:45clinical trials have been started,
08:47including here at Yale,
08:48so this is just a report from
08:50our Phase one group here,
08:51showing that there's a subset of
08:53patients with IDH mutated solid tumors.
08:55In this case, chondrosarcoma is that
08:56derives clinical benefit from elaborate,
08:58cheap, elaborate treatment with some
09:00patients showing either stable disease,
09:02or in this case, highlighted here,
09:05partial remission of their tumor burden.
09:09And obviously these trials are continuing
09:11to recruit patients in our ongoing.
09:16Switching now to to our work looking at
09:19targeting DNA damage response pathways
09:22and uncle metabolite producing tumors,
09:25we turned our attention here so we so we
09:27know that monotherapy is unlikely to be
09:29curative and in the majority of patients.
09:31So we set avenues for exploring other
09:33DNA repair pathways that could be
09:35targeted in a combinatorial fashion.
09:37So we turn to the ATR pathway shown here.
09:40So in the setting of DNA damage
09:43ATR phosphorylates, check one.
09:44Which intense intern sets off a cascade to
09:47coordinate several important cell functions,
09:50including the arrest of cell cycle by
09:53activation of intra S and G2M checkpoints.
09:55This allows DNA repair to occur effectively,
09:58and prevents premature mitotic entry
10:00in the setting of ATR inhibition.
10:02Damaged cells are allowed to
10:03proceed past the S phase checkpoint,
10:05thereby promoting the induction of double
10:08strand breaks, premature mitotic entry,
10:10and ultimately, cell death.
10:13As you can see here.
10:16So this is a work led by an
10:18excellent postdoctoral associate,
10:19that term retool,
10:20and as you can see from the
10:22clonogenic survival graph here,
10:25IDH mutant cells were more sensitive
10:27to a combination of a leopard
10:29and the ATR inhibitor Azd 6738.
10:32Compared to the wild type counterparts.
10:35And similarly, in vivo,
10:38using again HCT xenograft flank model,
10:40you can see that the combination
10:43of of a Labrador department,
10:45her elaborate and ATR inhibition resulted
10:47in significantly delayed tumor growth.
10:52Just to get an idea of what mechanisms
10:54might be underlying decided toxicity.
10:56We then assessed for DNA damage as
10:58measured by gamma H2X flow side in
11:01these wild type and mutant cells after
11:04treatment with elaborate is ATR inhibitor
11:07or combination therapy and what you see
11:09is that after 24 hours of treatment,
11:10IDH mutant cells had significantly
11:12increased proportion of damage to X foci
11:15relative to the wild type counterparts,
11:18suggesting increased level of unrepaired
11:20DNA damage after drug treatment.
11:23As I mentioned before,
11:24ATR also plays an important role in
11:27regulating cell cycle progression
11:28in the setting of DNA damage.
11:29So we assessed for the mitotic cell
11:32population looking at phosphorylated
11:33histone 3, which is a marker of mitosis,
11:36and you can see again that with the
11:39combination treatment you see an increase
11:41amount of cells entering mitosis.
11:42So the the idea here is that in the setting
11:45of increased DNA damage and IDH mutant cells,
11:48when you add ATR inhibition,
11:49these cells progressed
11:50through their cell cycle,
11:51enter enter mitosis.
11:52Prematurely and leading to cell death.
11:57Again, turning out to the clinic,
11:59there's actually trials now
12:00ongoing of this combination,
12:02including here at Yale,
12:03where there's a phase two trial,
12:04looking at elaborate and ATR inhibitor
12:07Azd 6738 in the setting of IDH,
12:10even solid tumors.
12:11So we're looking forward to seeing the
12:13results of this in the coming years.
12:18So turning now to the other Krebs cycle
12:20mutations I mentioned before, succinate
12:22dehydrogenase and fumarate hydratase.
12:25So in this study done in collaboration
12:27with Doctor Shep, who's now at UCLA,
12:29he's a urologic cancer surgeon.
12:31We wanted to identify other potential novel
12:33treatment approaches that exploit this uncle
12:36metabolite induced genomic instability
12:38using renal cell carcinoma models.
12:40So here we turned our attention to.
12:45Missoula made, which is an alkylating
12:47agent that mediates its cytotoxic effects
12:49by attaching methyl groups to DNA and
12:51the repair of the N7 methyl guanine adduct.
12:54In particular is needed by mediated
12:56by the base excision repair pathway
12:58in a process that involves park.
13:00Therefore,
13:00we hypothesize that apartment habisch
13:03and will enhance Tim Assamite
13:05induced city toxicity and FHN SDH
13:07deficient renal cell carcinoma models.
13:14To investigate this, we engineered
13:18isagenix FH1 and SDHB knockout cells,
13:21and for this we use the rank a cell
13:24line rank is a pretty well established
13:27mirroring renal adenocarcinoma model
13:29that's derived from balb C mice.
13:32So first by Western blot we
13:34confirmed FH1 and SDHB knockout.
13:37We then also further functionally
13:39validated this knockout using
13:41LCMS or liquid chromatography.
13:43Mass spectrometry to look for
13:44buildup of these uncle metabolites
13:46succinate in fumarate,
13:47respectively, and found that indeed,
13:49our CRISPR mediated knockout does lead
13:51to build up of these uncle metabolites,
13:54as one would expect.
13:56We next performed a seahorse assay
13:58to measure oxidative phosphorylation
14:00and found that again, as expected,
14:02SDHB and FH1 loss of function and the
14:05subsequent Krebs cycle dysfunction
14:07that comes from that leads to
14:09decreased oxidative phosphorylation.
14:12So this helps sort of validate our model.
14:15Next,
14:15we sought to assess the intrinsic
14:17DNA repair capability of Krebs
14:19cycle deficient cells by looking at
14:22markers of DNA damage at baseline.
14:24So here again we turn to phosphorylated
14:26gamma HTX as well as 53 BP,
14:29one which are markers of unrepaired
14:31DNA damage and the cellular response to
14:33DNA double strand breaks, respectively.
14:35We found that similar to our
14:39previous work looking at.
14:42C and A's deficient human tissues we see
14:46an increased amount of baseline DNA repair,
14:49unrepaired DNA damage in the knockout cells
14:51compared to the wild type counterparts,
14:54and as measured by the full site.
14:56Here,
14:56you can see these are the damage
14:58to expose and read and hear the
14:5953 BP one in green.
15:03Next we tested for the ability of
15:05the chemical might of tomorrow
15:07night to potentiate the in vitro
15:10activity of PARP inhibitor BGB 290.
15:13So in this clonogenic survival
15:15assay here cells were treated with
15:18a dose of B GB 290 ranging from
15:20one micromolar to 10 micromolar.
15:22In this, in the presence or absence
15:25of 15 micro molars at Tim's Olamide,
15:27so appear. These two lines.
15:29Here BG be alone and here is with
15:31combined to Missoula might as well.
15:34And what you can see again is that both
15:36in SDHB knockout cells and FH knockout cells,
15:39there's an increased cytotoxicity
15:41with combination and Tim is Olumide.
15:50Lastly, we tested for the in vivo
15:52efficacy of combination treatment,
15:54and these SDH be deficient
15:57rank of flank models.
15:59Of note, one thing that's interesting
16:00here is that in terms of clinical
16:02experience with the combinations of
16:04PARP inhibitor and Thomas Olumide,
16:05which has been tried and
16:07not setting up other tumors,
16:09one of the the limitations of
16:11these trials has been increased.
16:13Set of toxicity with full dose
16:15combination of both of those.
16:17And so typically for in vivo studies.
16:20That is, all my dose is anywhere
16:22between 25 milligrams per kilogram
16:23to 50 milligrams per kilogram
16:25per dose which translate to human
16:27equivalent dose of about 75 to
16:30150 milligrams per meter squared.
16:32So here we were interested to see if
16:34we could find some anti tumor effect
16:37at lower doses of Tim's Olumide.
16:39Which might limit some of those
16:41toxicities so for this study we
16:43used Tim Alumite at three milligrams
16:44per kilogram per dose,
16:46and and did indeed find that even
16:48at such lower doses of temozolomide
16:50we find delayed tumor progression.
16:53And importantly, there were no.
16:55There was no increased toxicity
16:57with the combination treatment,
16:58at least as measured by animal body weight.
17:04So based on this we can say
17:07that the band FH1 knockout Cells
17:09Harbor and increased levels of
17:11unrepaired DNA damage at baseline,
17:13and that the combination of pop
17:15inhibitor Intimes Olamide enhances set
17:16of toxicity in these cells in vitro,
17:18and that the combination with low
17:20dose temozolomide led to delayed
17:22tumor growth in vivo as well.
17:26And turning now to the
17:28clinical setting again.
17:29We recently had an interesting case within
17:31our own department within our own section.
17:33This is a patient cared for by one
17:35of my colleagues Dr pushing car
17:37and this is a patient with GIST
17:40and PARAGANGLIOMAS in the setting
17:42of a germline SDHB mutation.
17:45This is a patient that progressed
17:46through multiple lines of treatment,
17:47including imatinib than that in as well
17:50as a heat shock protein phase one trial.
17:53And so at this point,
17:55having progressive multiple lines of
17:57treatment doctor pushing car up to trial.
18:00Cycles of elaborate with Tim's Olumide.
18:03And this is off any clinical trial.
18:06As you can see here from the pet images.
18:08These are the pretreatment images showing
18:11multiple liver metastatic nodules as
18:14well as Bony lesions along the spine.
18:16And after six cycles,
18:19this patient had a.
18:21Partial remission in remission of all the
18:24Bony lesions as well as partial remission,
18:26multiple liver nodules as well.
18:30Of course this is just anecdotal.
18:33This is an anecdotal case,
18:34so there are trails about
18:36clinical trials currently ongoing,
18:38including a phase two trial that's
18:40currently in development and soon to open,
18:43led by our collaborator Dr.
18:44Shuck at UCLA,
18:46and in this trial they'll be testing
18:49combinations of 290 and low dose
18:52temozolomide in the setting of patients
18:54with refractory or recurrent renal
18:57cell carcinoma that is at FH deficient.
19:05Lastly, I just wanted to touch
19:06a little bit on my work,
19:08focused more on the immune aspects
19:10of Uncle metabolite and DNA repair
19:12defects and potential for leveraging
19:13these defects in order to promote an
19:16inflammatory tumor microenvironment
19:17and even potentially desensitized
19:19to mean checkpoint blockade,
19:21which I know is a topic near and dear to
19:24the heart of many folks on this call.
19:27So as folks on this audience,
19:29I'm sure already acutely aware of only
19:31a subset of patients really benefit
19:32from immune checkpoint blockade and
19:34some of the markers of response
19:36that have been described relate both
19:38to tumor increase amount of tumor
19:41associated mutations and subsequent
19:43neoantigen load as well as a more
19:46inflammatory tumor microenvironment.
19:50So with this in mind,
19:51a lot of attention has really been paid
19:53lately to the role of DNA damage response,
19:54and specifically DNA repair defects
19:56and mediating the tumor immune
19:58microenvironment in response to
20:00immunotherapy and the general idea.
20:01Again, just very generally speaking,
20:03is that the there's a potential in
20:05the setting of DNA repair defects
20:07when you treat these these tumors
20:09with additional DNA damaging agents
20:11that you have an increased number
20:13of mutations and subsequently
20:14increased number of neoantigens
20:16that can be recognized by T cells.
20:18The other sort of a main train of thought
20:21is that these DNA damaged DNA repair.
20:25Defects can also serve to
20:27activate the innate immune system,
20:29for example through activation
20:30of the C gas sting pathway,
20:32which is a double stranded
20:34DNA sensing pathway.
20:36Of course there are now multiple
20:37pathways that are described in
20:39terms of innate immune activation,
20:40including recognition of
20:41double stranded RNA sensing,
20:43which a lot of folks here at Yale
20:44have been working on as well.
20:45But since we're working talking mainly
20:48about double stranded DNA damage,
20:50our focus has mainly been on
20:52the C guesting pathway.
20:55So, as I mentioned before for for
20:57this study we utilized the SYNGENEIC
20:59ranking model and this is a model
21:02that has been characterized before
21:03as being minimally responsive
21:05to immune checkpoint blockade,
21:07and this is our own experiment here,
21:08confirming that at least the
21:09wild type version of this cell
21:11is pretty unresponsive to PD1,
21:12which allows us to to sort of use this
21:15as a model to see if we can increase
21:18sensitivity to immune checkpoint blockade.
21:20And again, this is a very preliminary,
21:23but we've we've been starting to
21:24really explore the immune effects
21:26of these crab cycle mutations,
21:27so this is again a an early experiment
21:29where we performed bulk sequencing
21:31just in the cells looking at wild
21:34type versus knockout cell models.
21:36And there's definitely a differential
21:37gene expression.
21:38But one thing I just want to
21:40characterize a point out here in
21:41terms of a related to the immune
21:43effects of these mutations.
21:45As you can see that one of the
21:47top hits for both of these in the
21:49knockout cells is an increased
21:50expression or upregulation of
21:53the antigen presenting pathways.
21:55We've followed this up with a
21:57separate study looking actually
21:59now at single cell sequencing
22:00and this is just so far had been
22:02done in our SDHP knockout cells,
22:04and thankfully we confirmed SDHP
22:06knockout as we we already did using other
22:09methods and again we see differential
22:12gene expression patterns between
22:13well tape and SDHP knockout cells.
22:15And again this is with work that's been
22:18done and help with help from Doctor Sule.
22:21Interestingly,
22:22we see here as well that the knockout cells
22:26seem to upregulate beta 2 microglobulin,
22:29which I'm sure folks or where is
22:30an important component will is is
22:32a component of the MHC class one
22:33molecule and is really required
22:35for antigen presentation,
22:36and there's been a lot of great
22:38work from folks here at Yale to
22:39show that made it two microalbumin
22:41losses is one of the markers of
22:43immune checkpoint resistance.
22:46We also then went on to look
22:48at differential gene expression
22:49with PARP inhibition and and so
22:51we looked at treatment after we
22:53looked at single cell sequencing.
22:55After 24 hours of treatment
22:57and and what we found so far.
22:58And this is still work in progress and
23:00we're still looking through this data,
23:01but one of the things we've seen
23:03is an increased expression after
23:0424 hours of the labyrinth,
23:06specifically in the knockout cells
23:08with upregulation of interferon
23:10induced protein protein 44,
23:12which is one of the interferon stimulated
23:14genes that has been associated with an.
23:16Interferon related DNA damage signature.
23:19We also saw upregulation of stat one
23:22with elaborate treatment and those
23:24SDHB knockout cells and stat one.
23:26The Jack stat.
23:27One pathway has been shown to be
23:30important for interferon stimulated
23:32gene expression and has been
23:34shown to play a role in mediating
23:37amino therapy response.
23:38So these are interesting.
23:40Sort of very preliminary data
23:42and and gives us a direction to
23:44look for as we go forward.
23:46I also again performed.
23:49Some flow cytometry,
23:50and this is now just looking at the
23:53tumor cells after implantation and
23:54what we see here is that in the SDHB
23:58knockout cells there's an increased
24:00proportion in terms of the percentage
24:02of live cells that are CD3 positive
24:05and of those CD 3 positive cells.
24:07There's an increased proportion that have
24:09PD one expression within the SDHP knockout,
24:11so again very preliminary.
24:13But this is sort of exciting
24:16data to follow up on for us.
24:20Uhm?
24:20Now I will turn to the other part of
24:23our talk from earlier the IDH mutations
24:27because this is also an area that
24:29I'm interested in is not to mention
24:31that have an interest in in the glioma,
24:33tumor immune microenvironment
24:34and have performed some studies
24:36previously looking at that.
24:37So I was really interested
24:38to develop an idea.
24:39Each mutant syngeneic model to allow us
24:42to to explore this a little bit further.
24:46So traditionally the the main
24:48model that's been used for the main
24:50syngeneic model that's been used
24:52for looking at glioma response to
24:54immune checkpoint blockade has
24:55been the steel 261 model which is
24:58chemically induced line with a
24:59moderate degree of immunogenicity
25:01at baseline and as you can see,
25:03this is our own experiment in our
25:05own hands and it goes in line with
25:07previous research that shows that this
25:09about 50% of mice with field to six.
25:11One tumors will respond to anti PD,
25:13one blockade and as a lot of
25:14folks here on this.
25:15So I will know that really doesn't
25:17recapitulate the human experience where,
25:18unfortunately so far clinical
25:20trials looking at I mean checkpoint
25:22blockade in GBM have been have
25:24not shown really much benefit.
25:26So we were hoping to find a model
25:27that maybe might be a little
25:29more translationally relevant,
25:29understanding the limitations that
25:31we're working with that we have to sort
25:33of rely on these syngenetic models.
25:35So we turned to our collaborator
25:36Dale Carter at UCSF,
25:38and his group developed this SB 28 line,
25:41which is a genetically engineered line that.
25:44They've already characterized,
25:45and they found that more more closely
25:48mimics the poorly immunogenic human gliomas,
25:51and so this is a line that.
25:53Intends to have low T cell infiltration,
25:56high number of tumor associated
25:58macrophages and more immunosuppressive
26:01micro micro environment and these
26:03tumors do not really respond to even
26:05dual blockade with PD one and C TL A4.
26:08They've also characterized this line
26:09in terms of the mutational burden.
26:11Showed that again SB 28 cells have a much
26:14lower mutational burden these GL261 line.
26:16So we hope that perhaps this is
26:18this will be a little more of a
26:20translationally relevant model as we go
26:22forward looking at the immune effects.
26:24So in terms of developing this as
26:26an IDH mutant model, specifically,
26:27we've we've used a stable transfection
26:30with an R132H open reading frame,
26:32and again characterized that there
26:34is an expression of the R 138,
26:36two H mutation as well as accumulation
26:38of two hydroxy glutarate.
26:40We've also characterized the in vivo
26:45intracranial growth kinetics of this
26:47model and shown that these IDH mutant
26:51cells form tumors effectively and
26:54characterize the survival with the
26:55IDH mutation. In these in this model.
26:59Again,
26:59we further characterized in vivo as well,
27:01and not just in vitro that in vivo.
27:03These tumors maintain their expression
27:05of the art 132 H mutation seen here
27:08is through the immunohistochemistry
27:10with this rust brown stain here,
27:12as well as again through LCMS looking
27:14for accumulation of two hydroxy glutarate
27:17and tumor tissue and seeing an increase
27:20accumulation in the R132H tumors.
27:24So I really want to take this as
27:25a in terms of future directions.
27:27This is really the the the main project
27:29that my K8 was funded for and I want
27:32to really investigate the impact of
27:33uncle metabolites on both cancer cell
27:35intrinsic immune signaling as well
27:37as the tumor immune microenvironment.
27:39And I want to explore the immunomodulatory
27:41effects of DNA damage response inhibitors,
27:43such as ATR inhibitors in the setting
27:46of uncle metabolite producing tumors
27:47or really extending the findings we've
27:49already had in our flank models to see
27:51how this works in the tumor microenvironment.
27:55I also want to investigate synergistic
27:56interactions between the mean checkpoint
27:58blockade and DNA damage response inhibitors,
28:00and these uncle metabolite producing
28:01tumors and hope to get started on
28:03these preclinical studies in the
28:04next in the upcoming months.
28:08So with that I'll end up and I want
28:10to just thank Doctor Bindra again,
28:12who's my primary mentor and has
28:14really been instrumental in in me,
28:16sort of advancing and receiving my K-8 as
28:18I build my pathway towards independence,
28:20as well as all the members of the
28:22Bingil lab have been instrumental
28:24in helping me sort of progress,
28:26as well as all those folks specifically
28:28who helped with the projects I outlined.
28:30I also want to thank Dr Shuck and his
28:33lab at UCLA, and my many advisors here.
28:36You know, only a few of which.
28:38They're listed here as well as to
28:39all my funders, so thank you again.
28:43Thanks so much. One,
28:44that was a wonderful talk and I
28:46know we're a little bit over but we
28:48don't have a second speaker so if
28:49there are any burning questions,
28:51feel free to put them in the chat.
29:11Crystal clear. You know,
29:14I'll start with one question I might
29:17have missed this of the DDR inhibitors
29:19that you want to look at to combine,
29:22possibly with PD one in the setting
29:24of IDH mutants are is there a
29:26wish list of the DDR inhibitors
29:28they would want to combine?
29:30Maybe you could put one of them
29:32in particular synergized in the
29:34in tablet producing backgrounds.
29:36I mean I think the ATR inhibitors
29:38are really an interesting area to
29:40explore and one that really hasn't
29:41been looked at too much in terms
29:43of the immune effects of these and.
29:44It sort of makes sense conceptually,
29:46that in the setting of these cells
29:48entering sort of premature mitosis,
29:50you'd have a lot of formation
29:52of these micronuclei that could
29:53activate the CSC gas sting pathway.
29:55So certainly I think again, you know,
29:57based on our initial work with Rita
29:59and her findings in the flank model I,
30:02I really want to pursue this more
30:03and see if we can see signs of immune
30:06activation and synergy with PD1 blockade.
30:10Where to ask one more question.
30:11Then we will close up if no others.
30:13Any plans to write up that
30:15wonderful case study with Farzaneh.
30:18We've talked about it and yes,
30:19I would love to know,
30:20so I definitely want to check more
30:22about that because I think that would
30:23be a nice corollary to the you know,
30:25as you know, the aranka work we're
30:27hoping to write that up soon and
30:29submit that as a manuscript.
30:30So I think it would be a
30:32great corollary to that.
30:32So I definitely hope to write that up.
30:35Wonderful,
30:36well this is great.
30:36We had a great turn out today and I
30:38think you just answered everyone's
30:39questions with your slides.
30:40So thanks everyone for joining
30:42us and have a great rest of
30:44your Tuesday thank you room.
30:47Take care bye bye.