Targeting Oncometabolite-induced DNA Repair in Cancer

Transcript

• 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.