Yale Pathology Grand Rounds: September 29, 2022

Transcript

• 00:06OK. We are on time 12:30,
• 00:08so we're going to start and
• 00:10so first welcome everybody.
• 00:12This is the first in person brand grounds
• 00:16meeting and we're very happy about it.
• 00:19So we expect to have people joining.
• 00:21We have 20 people online and maybe
• 00:24some more people to come so.
• 00:26So it's a great pleasure
• 00:28today to have our speaker,
• 00:30his doctor Weiping Zou.
• 00:32He's currently the Charles
• 00:34Denman Creek professor.
• 00:36He's a professor of immunology,
• 00:38pathology,
• 00:38biology and surgery at the University
• 00:40of Michigan and he's also the
• 00:41director of the Michigan Center of
• 00:43Excellence for Cancer Immunology,
• 00:45immunotherapy.
• 00:45And he has a number of additional
• 00:48appointments that I will omit now
• 00:50because I think they just add too much.
• 00:55Maybe he's training.
• 00:56He obtained his MD in China
• 00:59in Tongji University,
• 01:00and then he went on to get
• 01:03obtain a PhD in Paris.
• 01:04That was followed by postdoctoral training in
• 01:07France and then postural training in Baylor,
• 01:10Dallas.
• 01:12And then from his academic career,
• 01:15he initiated his career as an
• 01:18assistant professor in Tulane
• 01:20University in New Orleans,
• 01:21where he rose to the rank of
• 01:23associate professor with tenure.
• 01:24And then he moved to University of Michigan,
• 01:27where he became a full professor
• 01:29and director of the program.
• 01:32What is very interesting is that
• 01:35he's a very productive investigator.
• 01:37He has over 190 publications.
• 01:40I could count about 35.
• 01:42Really high impact publications in major,
• 01:46you know, sales, science,
• 01:47nature,
• 01:47type of journals and he's also very
• 01:50productive in the research and I
• 01:52could count 5R1 grants at this point,
• 01:54which as you can imagine is a huge
• 01:57amount of effort and shows to the
• 02:00reflects the quality of his work.
• 02:02He has done major contributions
• 02:03in the fields of tumor immunology
• 02:05and looking at different aspects.
• 02:07And recently he has focused more
• 02:09on the role of the metabolism
• 02:12and how metabolism can actually
• 02:14compromise adaptive immune responses
• 02:16in the tumor microenvironment.
• 02:18I have to also to say that he's
• 02:21a very translationally oriented,
• 02:22so his work focuses on very basic
• 02:25mechanisms but also projects into
• 02:28different tumor types and he has
• 02:30done really prominent contributions
• 02:32in colorectal cancer.
• 02:33Ovarian cancer and then also breast cancer.
• 02:36So it's a very, very diverse profile.
• 02:39And so without further ado,
• 02:41I believe Doctor Weiping Zou and his title
• 02:43is metabolic impact on tumor
• 02:45immunity and immunotherapy.
• 02:47And thank you very much.
• 02:59Right. First of all I would like to.
• 03:04Thank God for your kind
• 03:08invitation and introduction.
• 03:11Year is a pioneer institution
• 03:14of modern immunology,
• 03:16particularly human immunology,
• 03:19innate immunity and T cells.
• 03:24We paid up allergy,
• 03:25so in many ways this institution
• 03:28contributed enormously to our knowledge
• 03:32and also immunology translation.
• 03:35So this is absolutely a great
• 03:38pleasure for me to be here.
• 03:40This is the first time I'm able to give
• 03:43this talk to this prestigious university.
• 03:46As I mentioned yesterday when we had dinner,
• 03:50actually my old son wants to
• 03:52get into the C university.
• 03:55He failed and I emailed him.
• 03:59I said, I'm going to give a talk
• 04:01to this university and he said,
• 04:04OK, you succeeded.
• 04:06Thank you again for this
• 04:09wonderful opportunity.
• 04:10So.
• 04:12See you guys.
• 04:22So use too many minorities when we
• 04:26view our cancer therapy history.
• 04:30We have come a long way.
• 04:34First we have surgery,
• 04:36radiation, chemotherapy.
• 04:37Chemotherapy and the targeted
• 04:40therapy for managers and rotation if
• 04:42these days we do our immunotherapy.
• 04:45As you can see from this summary.
• 04:49Each milestone is really based on the
• 04:53basic celebs scientific discovery.
• 04:55So people always asked what is next.
• 05:01So in terms of immunotherapy,
• 05:05what we have done?
• 05:09Actually, early on we know based
• 05:13on the genetic identification and
• 05:16mutations people have discovered,
• 05:19it's pretty clear cancer
• 05:22is a genetic disease.
• 05:25But all work and many others work.
• 05:30Consider cancer is an immune disorder.
• 05:34And further we have studied the
• 05:37immunosuppressive mechanisms
• 05:38including the P1P1 that was in the
• 05:42human tumor migraine environment.
• 05:44So we believe the human tumor
• 05:48microenvironment holds the key to
• 05:50understanding human immunity and therapy.
• 05:53So at this stage,
• 05:54when we say these two contributions,
• 05:57conceptually speaking, it's easy.
• 06:00But when you talk about this 20 years ago.
• 06:04It's not the same thing.
• 06:07So we have some video articles
• 06:10in this space as we focus on
• 06:13immunosuppressive mechanisms.
• 06:19For those who are relatively new
• 06:22to terminology, as we know a lot
• 06:25of people getting into this area,
• 06:27you may see some of our work
• 06:29because we have not only reviewed
• 06:31the studies from our own group,
• 06:34but also from many others.
• 06:36As you may appreciate actually at least
• 06:392 high profile review articles we're
• 06:42done with Livingston I remarkable
• 06:46faculty are ADO Institution.
• 06:52Who's good mentioned?
• 06:54We are pretty much legislatively in the basic
• 06:58mechanisms as well as the transformation.
• 07:02Therefore. For all those years,
• 07:05we have been working on one concept.
• 07:08Say to again cancer microenvironment
• 07:10host key to understanding too
• 07:12many immunity and therapy.
• 07:14To address this we have several research
• 07:18directions or research angles you may say.
• 07:22For example you know
• 07:25suppressive mechanisms network.
• 07:27Such as PD1PD L one OK.
• 07:30Then cancer athletics and
• 07:33epigenetic reprogramming.
• 07:34And then a key immunologic pathways
• 07:37such as individual pathway,
• 07:39image C and stats.
• 07:41And finally,
• 07:42in the recent years we spent a lot of
• 07:46time working on metabolic pathways.
• 07:49So I guess I'm going to spend a little
• 07:52more time on the fourth direction.
• 07:55I will give you just one
• 07:58slight each for A&B&C,
• 08:00just to show you where I have come from.
• 08:08So and like to share this site with
• 08:10you for the first research direction
• 08:13we have immunosuppressive networks
• 08:15actually almost more than 20 years ago.
• 08:21Under the support and
• 08:23collaboration with Millington,
• 08:24we have published this
• 08:26paper in Nature magazine.
• 08:28It was named it is time PDL one.
• 08:31It's not me PDL one.
• 08:33It's named P 781.
• 08:35These people for the first time
• 08:38demonstrating PDL one of P-70 joint
• 08:40expression recognition and profit in
• 08:43the human cancer microenvironment
• 08:46and human human chain influence.
• 08:48We clearly demonstrated if you
• 08:51broke this pathway you can recover
• 08:54the dysfunctionality cells.
• 08:58This is far before the success of
• 09:02clinic trials, either with PD1PD1
• 09:05blockade or with anti serial four.
• 09:08Of course in these days if you look
• 09:11at the PD L1 you will not be able
• 09:14to find this table because early
• 09:16on when leaving discovered the best
• 09:19way he named this gene as B7H1.
• 09:22Of course, he has many other
• 09:24peaceful family members, as you know.
• 09:27So the second with your generation
• 09:29cancer at genetics,
• 09:30I know you have quite a few folks
• 09:33who are interested in epigenetic
• 09:35recognition in this institution,
• 09:38but we look at this from the
• 09:41immune recognition perspective.
• 09:42So in the tumor microenvironment similar
• 09:45to the TH1 and TH2 reciprocal recognition?
• 09:50We have observed a reciprocal
• 09:54regulation between PRC two complex
• 09:56and Swiss sniper complex in the tumor.
• 10:00So actually this recognition was
• 10:03properly controlled interference
• 10:05zeronine therefore TH one type second
• 10:08production and T cell trafficking
• 10:10and human energy.
• 10:11We have worked out the detailed
• 10:15biochemical and functional mechanisms.
• 10:17We I'm not going to show you
• 10:19the details as I mentioned.
• 10:21Then third, research direction,
• 10:24I know also several investigators
• 10:27including my host code is very
• 10:30interested in this image C interferon
• 10:33signaling sets signaling pathway.
• 10:36As you know these are the key immunogenic
• 10:39pathway in the immune responses,
• 10:40not only just in the tumor
• 10:42migraine environment.
• 10:43I show you one example.
• 10:44We have,
• 10:45we have discovered actually you know
• 10:48mutations in MHC pathway and stat
• 10:52and interferon pathway are considered
• 10:54a celebrity resistant mechanism.
• 10:58But we know the vast majority of the
• 11:00patients do not have those mutations.
• 11:03Therefore we need to find out
• 11:05the other pathways which may
• 11:08contribute to safety resistance.
• 11:10So one pathway we have discovered.
• 11:14Actually,
• 11:14the integrity of interferon
• 11:17signaling pathway is controlled by.
• 11:22Jean quota of January.
• 11:23So this is a auto between septor.
• 11:27It turns out actually all of the nearing
• 11:30can control the stability and the
• 11:33degradation of the film gamma receptor.
• 11:36So as a consequence,
• 11:37this controls the image C expression antigen
• 11:40presentation and the T cell functionality.
• 11:42OK, I don't have time to
• 11:45show you this was published.
• 11:47For those who are interested in this,
• 11:49you may have a look instead.
• 11:51Most of my time we are focused on the force
• 11:55we switch direction metabolic pathways.
• 11:59And I will.
• 12:02Talk about the basically two stories.
• 12:05One is system XC and CS4,
• 12:09its relationship with tumor
• 12:12cell philippoussis.
• 12:13Another is SLC 43A2.
• 12:17So those ACC family members are
• 12:20nutrients or metabolite transporters.
• 12:23There are several hundreds of them.
• 12:25Most of them are poorly understood
• 12:28in the field of immunology.
• 12:30We start to figure out some of it.
• 12:35So before that I want to introduce
• 12:39the concept of ferroptosis.
• 12:41So it has been defined in vitro
• 12:44through the synthetic compounds.
• 12:47It means the cells will die
• 12:50through iron dependent but lipid
• 12:53peroxidation induced cell death.
• 12:56There are several genes or pathways
• 13:00associated or regulated cell biosis.
• 13:03So,
• 13:04including this exit system
• 13:07GX4 and a CSR four.
• 13:10However there is no CBC marker
• 13:14to define Philippoussis.
• 13:15What we usually do we use a few
• 13:19criteria to define electrolysis,
• 13:21so one it's maybe the Rose production,
• 13:24another is expression of all states
• 13:27needed species on the membrane,
• 13:29and finally we need to see
• 13:32the functional activities.
• 13:34So in this case we asked
• 13:37a very simple question.
• 13:39We know when CDA T cells are activated,
• 13:42engage tumor cells.
• 13:45CHT cells we need preparing,
• 13:48makes pores on the membrane,
• 13:50then grant them.
• 13:51We get into the cells activated
• 13:54caspase induce tumor cell able doses.
• 13:58This is text book message.
• 14:02So we asked a simple question means
• 14:05if CDA T cells who kills tumor cells?
• 14:09This is a way how the tumor cells
• 14:11die is Philippoussis involved.
• 14:13So in this case we set up several
• 14:17experiments to test this possibility.
• 14:20So one is a 88 over retention model.
• 14:24We do immunotherapy before you can see PD,
• 14:27one can control the tumor growth.
• 14:31Under this condition before the tumor cells,
• 14:34really it is an 8 stage before
• 14:36the tumor cells die,
• 14:37you give the tumor cells out,
• 14:39you detect relative needs worse production.
• 14:43You see actually the immunotherapy
• 14:46induces liberals production in
• 14:48the tumor cells in PD1 cell.
• 14:51Then we did a T cell therapy model.
• 14:54Be 16 over expression cells and
• 14:58treated in vivo with only one cells.
• 15:01It's not a surprise tumor is controlled
• 15:04and again we see individuals production.
• 15:06So this suggests maybe T cells or immune
• 15:10therapy can promote liberals production.
• 15:14Maybe in May induce Philippoussis
• 15:17tumor cell paralysis.
• 15:19We did in vitro studies in this case
• 15:22to provide direct evidence we cultured.
• 15:2541 cells with All Blacks fashion tumor
• 15:28cells we look it's rose production
• 15:30again we see rose production induced
• 15:33by by only one cells and this antigen
• 15:36specific away can see more but if
• 15:39you look at the tumor cell death.
• 15:43Of course you use a small amount
• 15:45of T cells and in this case you can
• 15:48have space to manipulate the system.
• 15:50You will see you your small amount
• 15:52of T cells. You see T cell cleaning.
• 15:56And under this condition,
• 15:57if you use a small amount of RS3
• 16:01is a dosis inducer,
• 16:03you'll see some levels of human cell death.
• 16:06If you put them together,
• 16:07you see dramatic human cell death.
• 16:09These tumor cells can be
• 16:13completely abolished.
• 16:14By THEODOSIS inhibitor,
• 16:17So what we have here, it means T
• 16:22cells can promote tumor cell factories.
• 16:26However,
• 16:26T cells themselves are not sufficient.
• 16:29You need to have a trigger somewhere.
• 16:32So we will explore further about
• 16:35this phenomenon.
• 16:36But we studied further in vivo
• 16:39in military condition.
• 16:40So it's a classic model.
• 16:43For example, with P-16,
• 16:44if you treat the tumor bearing
• 16:47mice with antiscia 4 and PD1,
• 16:49you'll see very nice tumor control.
• 16:52But if you treat the mice under
• 16:54this condition with liberal statin,
• 16:56it's a Philippoussis inhibitor.
• 16:59The therapeutic efficacy
• 17:01is basically punished.
• 17:02So this is very unusual.
• 17:04This is completely unexpected.
• 17:07Because we all know
• 17:10CD8T cells kill the tumor
• 17:11cells through able process.
• 17:13How come a ferroptosis
• 17:16inhibitor can Polish the effect?
• 17:19To really ensure this possibility,
• 17:22we used another model means we in vitro
• 17:26generate erskin resistant tumor cells.
• 17:29It's similar to chemotherapy resistant cells.
• 17:33You do the individual generates the
• 17:35cells with this to the reverse inducer.
• 17:38Then you do the immunotherapy.
• 17:41OK you see here,
• 17:43parental cells are responsive
• 17:45and resistant cells are no longer
• 17:48responsive to immunotherapy.
• 17:50Indicating actually Ferroptosis is a
• 17:55potential mechanism induced by immunotherapy.
• 18:00So we look it's Morgan mechanisms
• 18:02then to make a non story short,
• 18:05we know it's interfering and other things,
• 18:07but just show you interfering here.
• 18:09If you make a lookout receptor in
• 18:12the knockout tumor cells and your
• 18:15culture with only one cells you will
• 18:18see actually the liberals production
• 18:20in the human cells is basically gone.
• 18:23Under the tumor cell death is
• 18:26also basically gone,
• 18:27so indicating this tumor cell death
• 18:30is controlled through the interference
• 18:32and interference signaling.
• 18:34So then we hypothesized maybe
• 18:38interfering with your stimulate
• 18:40oxygen lipid species and therefore the
• 18:43cells die as we previously mentioned.
• 18:47So we did some individual studies.
• 18:51We cultured tumor cells with our CS
• 18:54refill processing user with or without
• 18:57anything comma you can appreciate.
• 18:59In fact the aim is without
• 19:03show actually interfering.
• 19:05Gamma can induce tumor cell
• 19:08oxides lipid species,
• 19:10so particularly USB 16P18 or induced.
• 19:13So this is increased when
• 19:15you have a small amount of.
• 19:17ISL 3.
• 19:19So it means actually in the film
• 19:23gamma can properly do the job.
• 19:25To directly show this we used a independent
• 19:29comma sensitive human tumor sauna.
• 19:31It's HD human cell line
• 19:33you treat with interferon.
• 19:34You can inhibit tumor growth
• 19:36in the English model,
• 19:38but under this condition if you use
• 19:40liberal studying you will see the effect.
• 19:42It's completely gone.
• 19:44But Earth asset in vitro if you only have
• 19:47interference or you only have 3 cells.
• 19:49The sale gas we are not having,
• 19:51so suggesting something else,
• 19:53not only just in.
• 19:55So but what did anything do in this case?
• 19:59We look at the molecular targets,
• 20:01potential molecular targets of
• 20:03interferon particularly XC system.
• 20:05As you know XC system can transport
• 20:09system into the cells then become
• 20:13system and GSH and this will protect
• 20:16the cells test from the tosis.
• 20:19So it turns out interferon actually we press,
• 20:24we press the exit. System.
• 20:26So this is just the the among a.
• 20:28This shows you the protein.
• 20:30Not only this,
• 20:32it's functionally important as shown here,
• 20:35because the system update is reduced
• 20:38when you have independent treatment
• 20:40and then the GSH synthesis is reduced,
• 20:44and particularly if you have
• 20:46again small amount of erosion.
• 20:48This is a well known reduces GSH
• 20:50when you put them together though
• 20:53reduction of GH is really dramatic.
• 20:56So we extend our studies to humans,
• 21:01not only just we use the human cells,
• 21:03human human cells and we meet a
• 21:06correlation with immunotherapy.
• 21:08As you can see here when the
• 21:11patient received
• 21:12e-mail service called Panini Benefits,
• 21:14the XC expression is done in the
• 21:17tumor of course, the interferon
• 21:19signaling and CTA is increased.
• 21:22So what do we have?
• 21:24At least we can say apart from apoptosis.
• 21:29The interaction between CHT cells and
• 21:32tumor cells in this context fail.
• 21:36Photos may be involved,
• 21:38and interferon gamma can target
• 21:40excision to be involved in this space.
• 21:43This has not been previously
• 21:45appreciated because we don't know.
• 21:47We only think that this is able to process.
• 21:50So now as I mentioned in the film,
• 21:53comma is not enough,
• 21:54T cells are not sufficient.
• 21:56So what else?
• 21:57What else?
• 21:58Because early on when Phil poses
• 22:01as a concept was was established,
• 22:05it is basically based on the
• 22:08synthetic compounds.
• 22:09So you treated the cells with the chemicals
• 22:12and then you see the philippoussis,
• 22:14you see the pathway.
• 22:16So if their process is a intrinsic mechanism.
• 22:20We should have a intrinsic
• 22:22mechanism to induce the fibrosis
• 22:24in the cells because we don't have
• 22:27synthetic compound in our body.
• 22:28So we look for the natural
• 22:31theodosis inducers in.
• 22:33So in this case we come to a fatty acid diet.
• 22:38So the reason is we know they are
• 22:40quite many publications talking about
• 22:42the relationship between bias and the
• 22:45celebrity response to immunotherapy.
• 22:47They are also quite some
• 22:50publications talking about.
• 22:51Micro got microbiota and tumor cell
• 22:55respond where patient responsive
• 22:57responsiveness to immunotherapy.
• 23:00So therefore we were thinking
• 23:02maybe interfering is one thing,
• 23:04maybe some my tablets some
• 23:08metabolic nutrient will be involved.
• 23:10We turned to fatty acids because
• 23:13we know when the cells die through
• 23:15their process,
• 23:16it's because of oxidized lipid species.
• 23:18That's why we look at the fatty acid.
• 23:21So then I invite you to look at
• 23:23several groups of fatty acids.
• 23:25So in fact you have short chain,
• 23:27medium chain,
• 23:28non chain and a very long chain fatty acids.
• 23:31I want you to pay attention on
• 23:34the non gene fatty acids such
• 23:36as POAOA and arachidonic acid.
• 23:38Here we checked all of it.
• 23:40So in this case when we look at the
• 23:43map of fibrosis people have defined as
• 23:47a fibrosis involved genes and one is called.
• 23:51A CSR 4 here.
• 23:52So in fact that you prefer it's
• 23:55an enzyme preferred substrates,
• 23:57it's electronic acid AA.
• 23:59So finally you will see the final product
• 24:03is Poly unsaturated offset lipid species.
• 24:06So in this case we are
• 24:08thinking it should be involved.
• 24:11So what we did,
• 24:12we cultured the tumor cells with interferon
• 24:15pronounced different fatty acids.
• 24:18Long term,
• 24:18short term media change often.
• 24:20Then we look at the cell desk.
• 24:22It turns out that in the presence
• 24:24of a the tumor cell,
• 24:26death is dramatically increased.
• 24:28And keep in mind that these
• 24:31cell deaths can be completely
• 24:34blocked by THEODOSIS inhibitor.
• 24:36So it means this is really theodosis
• 24:38and this is repeated reproducible
• 24:40in P-16 and seven tumor cells
• 24:43in mouse and humans.
• 24:45So finally we want to see what has
• 24:49happened actually with electronic
• 24:51acid in the presence of in the
• 24:54field. So we cultured human cells
• 24:57with interfering with or without.
• 25:00E5 neighbored atonic acid we want
• 25:03to see where electronic acid goes.
• 25:07So in this case we made a CSL
• 25:10knockout and the width of tumor cells.
• 25:13We treat the cells in this way.
• 25:15Then we look at different oxygenated
• 25:18species because you may appreciate
• 25:20here what is the black box and
• 25:22the red bars are all deficient.
• 25:25It is deficient cells.
• 25:27You will see actually interfering,
• 25:29really promote.
• 25:31The incorporation of T5
• 25:34neighboured electronic acid in two
• 25:37different oxides lipid species.
• 25:39So this including PE18B16 PC 18.
• 25:44You can see from the slight ACL 1400.
• 25:48So in this case,
• 25:49what has in the gamma Dong look at the
• 25:53brooding expression of CCL 4 actually
• 25:56in film comma stimulate its expression,
• 25:59so this is slow transcriptional
• 26:02recognition as the cheap essay shows.
• 26:05Actually there's a high I funding
• 26:08in the ACL 4 promoter area,
• 26:11and the cheap shows actually
• 26:14this high occupancy.
• 26:15So we first did some in vivo
• 26:18studies to show the relevance.
• 26:20So in this case we made a ACL 4 knockout
• 26:24tumors in several tumor cell lines.
• 26:26You see a CS4 is gone and tumors are
• 26:29getting bigger and in vivo and when
• 26:32we did the combination therapy Ravi,
• 26:35Classic way we treated mice with AA and
• 26:38AA actually can partially control the
• 26:41tumor progression in several models,
• 26:44but keep in mind the A.
• 26:46He's a very small amount of concentration.
• 26:48You you cannot give too much and then
• 26:50you kill the mice because it's quite toxic.
• 26:52So we see the combination therapy can give
• 26:55you some benefits in the mouse model.
• 26:58So when you look at the patient
• 27:01with a CCL 4 expression,
• 27:04in fact high ACC for expression is
• 27:08positively associated with patient survival,
• 27:11suggesting maybe it is a four is relevant in.
• 27:16The tumor microenvironment.
• 27:17So we tested it not only like in tonic acid,
• 27:22we tested them other fatty acids
• 27:24as I mentioned,
• 27:26but what I conclude here don't
• 27:28show you all the details.
• 27:30Apart from AA,
• 27:31OA and POA can also participate in
• 27:36inducing the tumor cell process.
• 27:40All these essays are in the absence
• 27:44of synthetic compound.
• 27:45So indicating what we discovered actually.
• 27:49The effect specific fatty acids plus
• 27:52interferon gamma are the intrinsic
• 27:55philippoussis inducing mechanisms
• 27:57we are able to detect.
• 28:00Of course all the fatty acids
• 28:02species and interference in vivo,
• 28:05they are not synthetic combo.
• 28:07So this is another similar to
• 28:09the concept that we all know,
• 28:12such as H170 cells, not one cytokine.
• 28:16It's not enough to polarize,
• 28:17you need several.
• 28:19Effectors what we have discovered, actually.
• 28:22Tumor cell Philippoussis needs
• 28:25several factors,
• 28:26and interference is one of them
• 28:28and the fatty acids are another.
• 28:31So now that's the the the conclusion
• 28:36we have basically when you have the
• 28:40induction between C8 and tumor cells.
• 28:43Because this is one of the founding
• 28:46father of Tosis is another.
• 28:48I hope this becomes textbook.
• 28:50So fear of loss is is mediated
• 28:53and the recognition
• 28:54through the AC system and CSL 4.
• 28:57Maybe other factors will be involved as well
• 29:00and we are still working on the details.
• 29:04As you know, there are several
• 29:08philanthropic pathways people have defined.
• 29:12So what is the technical message here?
• 29:16You must fear of those is is
• 29:19a mode of action of Syria.
• 29:21And tumor Philippoussis is
• 29:24neural therapy mechanism.
• 29:25So if so we should think about
• 29:29the potential translation.
• 29:31We are thinking about this,
• 29:33many groups are working on this.
• 29:35So now we move to the second part of my talk.
• 29:38It's concerned another ACC family member,
• 29:42it's named SRC 4382.
• 29:45So myself is a immunologist and when you
• 29:49talk to biologists and some other people,
• 29:54there is an idea or thought proposed
• 29:58because the tumor cells are highly
• 30:01proliferative and invasive.
• 30:03The tumor cells need a lot of nutrients.
• 30:05So one way to treat the patient
• 30:07that maybe we can start with the
• 30:10cancer cell death.
• 30:11So that's the way how the
• 30:14biology is maybe some.
• 30:16Pharmacologists think this way,
• 30:17I don't know, so let's see if this works.
• 30:21In that case,
• 30:21I invite you to think about the
• 30:23nutrients and metabolites in
• 30:25the cancer microenvironment.
• 30:27We know when the cells are exposed to
• 30:30different metabolites and nutrients
• 30:32in the particular environment,
• 30:34not only just human cells,
• 30:36but also these cells and disease
• 30:39and Macy's and other cells,
• 30:40they must be subject to the
• 30:43regulation by the environment.
• 30:44Therefore, they are functional.
• 30:45Status must be changed.
• 30:47So it's a very simple way to
• 30:50put so in this case.
• 30:52Early on,
• 30:53some groups have already discovered
• 30:55the T cells are dysfunctional
• 30:57in the tumor micro environment.
• 30:59You may say the T cells are exhausted.
• 31:02That's alright so.
• 31:03But we also know some epigenetic
• 31:05pathways are involved in the regulation
• 31:08of tumor cell dysfunction and T cell
• 31:11dysfunctionality in the tumor environment.
• 31:14So we are thinking maybe in this
• 31:16case a crosstalk between metabolic
• 31:19and apologetic mechanism.
• 31:21This has, uh, evidence actually.
• 31:24People have reported some of them.
• 31:26For example,
• 31:27you know,
• 31:27after H succinate and have a
• 31:29particularly succinate have has
• 31:31been studied in macrophages,
• 31:34minor cells and some others.
• 31:36And we are interested in Sam.
• 31:38So why we are interested in them,
• 31:40you will see why we're interested in them.
• 31:43So in fact,
• 31:44in this case we look at the amino acids.
• 31:48So we did a very simple array,
• 31:52so we cultured basically G cells with
• 31:57different amino acids in the media,
• 31:59but we manipulated the concentration
• 32:01but we admit one by one and then we
• 32:04take the functionality of the T cells,
• 32:06basically the shell gas and the T cells.
• 32:09It turns out if you own it,
• 32:14my theory.
• 32:15So T cells cannot stand,
• 32:17they become very much evolution.
• 32:20And the cells do not express much
• 32:24interference.
• 32:25And then we did another way along
• 32:27means we add amino acids back.
• 32:29So it's a plus experiment.
• 32:32So in this case we calculate
• 32:34the cells with too much
• 32:35to induce the cell or
• 32:38dysfunctionality dysfunctional.
• 32:39They become embodied and reduce stereogram.
• 32:42You will see under this condition
• 32:45if we add methionine pack we
• 32:48will see actually the tumor.
• 32:51T cell F is reduced,
• 32:53T cell function is improved.
• 32:56So indicating actually the T cells
• 33:00are very sensitive to the supply
• 33:04of methionine so and then we look
• 33:07at the methionine metabolic cycle.
• 33:09So in fact my theory can become same
• 33:13and you know Sam is a real donor for
• 33:19methylation so history modification.
• 33:21So that's the reason we want to
• 33:23look at some right so in this
• 33:25case when you cut your.
• 33:27Have to keep cells with two
• 33:29measurement and you supplement
• 33:31with my film and you detect all the
• 33:34Internet intercellular my cabinets
• 33:36you will see if you do so first of
• 33:39all you see reduced intracellular
• 33:41methionine when you don't add in
• 33:44the culture and when you add it
• 33:47comes back and also you don't have
• 33:50them in the SH all those things
• 33:52but you supplement Matheny you can
• 33:55partially and important we cover.
• 33:57They are the intercellular ascend
• 34:00and intercellular other metabolites
• 34:02of of methionine, such as SH.
• 34:06So if so this must affect his notification.
• 34:10So when we in this case we look at this,
• 34:13this is calculated with either tumor
• 34:16cells mouse are not human you will see
• 34:19actually the tumors supernatant reduce
• 34:22its 3K790 resonation dramatically.
• 34:24This is not only the case in in most cells,
• 34:27in too many cells the same thing
• 34:29and you supplement methionine
• 34:31which you can recover it.
• 34:32Other histone markers are less affected and.
• 34:36I I couldn't explain to you why.
• 34:38Then we look at the primary cells T
• 34:41cells in the tumor micro in humans
• 34:44and the mouse and you will see
• 34:47isolated cells from the mouse system.
• 34:49You will see we used H3790 resonation
• 34:52and so is in human CD8T cells
• 34:57in the tumor microenvironment.
• 34:59So in that case,
• 35:01this extreme case 7 and T machination
• 35:03must be functionally important.
• 35:06So to test this possibility,
• 35:08we made a total of 1 specific
• 35:10lookout in T cells.
• 35:12The reason is total one is the
• 35:15only endemic 8719 resolution.
• 35:16So when we made it look out in
• 35:19T cells and then the tumors are
• 35:21getting bigger than the T cells are
• 35:24becoming apoptotic and dysfunctional.
• 35:25So that's one way to go.
• 35:28Another way to go is.
• 35:29We supplement methionine in the
• 35:31tumor bearing mice in this condition.
• 35:34If you supplement then you reduce
• 35:36tumor growth,
• 35:37you will cover histone modification
• 35:40in T cells and also you recover
• 35:43the T cell functionality.
• 35:46So we did it not only in mouse model,
• 35:48we did in patient with cancer.
• 35:50So we supplemented methionine to the patient.
• 35:53Then we see the T cells.
• 35:55It turns out if you do so.
• 35:58My third supplementation can we
• 36:02cover each 3K79 demethylation
• 36:04we cover even Step 5 expression.
• 36:08We checked all the other stuff
• 36:09because step five is most obvious.
• 36:11And furthermore,
• 36:12if you look at the second expression such
• 36:15as IO2, so before therapy, after,
• 36:18before, and after, you will see I2
• 36:21is largely recovered in T cells.
• 36:24You know, somehow step five really
• 36:27controls the expression of.
• 36:29I of O2, then we first look
• 36:32at the possible mechanisms.
• 36:34So it turns out actually it's
• 36:38379 emanation target Step 5,
• 36:40particularly step 5B,
• 36:41and the cheaper essay shows this is the case.
• 36:44In fact, if you cut yourself
• 36:46with supernatant and with
• 36:48the maternal supplementation,
• 36:49methionine supplementation can recover
• 36:52the occupancy in the certified model.
• 36:56So this just show you again,
• 37:00not only we cover the T cell,
• 37:03the the the,
• 37:05the the the the cheaper and also show
• 37:09H3K79 nations we covered and instead
• 37:11of having is recovered and I2 is
• 37:14recovered in both humans and mice.
• 37:17And finally we want to understand.
• 37:20If methionine is there,
• 37:22why the T cells cannot get better?
• 37:27So maybe the tumor cells all
• 37:29compete T cells for methionine
• 37:31in the tumor microenvironment.
• 37:34We turned our attention to methionine
• 37:38transporters so we screened all of them.
• 37:41It turns out actually you will see
• 37:45compare tumor cells and T cells in
• 37:48the same environment and actually
• 37:50the tumor cells express quite a lot
• 37:53of ACC for 3A2 is one of methionine.
• 37:57This product,
• 37:57so this is among A and this is protein.
• 38:01So this is T cells and many
• 38:03other transporters are similar,
• 38:05but they are quite some differences.
• 38:07So we are we continue to work on
• 38:10this space to define the different
• 38:12differences we are able to see and
• 38:15then to see the functionality.
• 38:17So this suggests maybe
• 38:21AC43A2 easy transporter highly
• 38:23expressed in the tumor cells is
• 38:25functionally important if so.
• 38:27We make a knocking down SLC 43A2.
• 38:32In the commercials then we start to
• 38:35capture the human cells with cells.
• 38:36OK, so you can see actually the T
• 38:39cells are becoming less able to
• 38:42reach the functions are recovered.
• 38:44So indicating ACC for this 382 is important.
• 38:49To further demonstrate this possibility,
• 38:51we did in DEVO studies,
• 38:53if you shut down PC police
• 38:55382 in the tumor cells.
• 38:57USC actually the tumor is smaller
• 39:00in the immune competence system.
• 39:03The key cells in terms of their
• 39:05number and their function are better.
• 39:07This is not only in one model.
• 39:09In several models we can see the case.
• 39:11So what we have here is a summary we
• 39:15see in the tumor microenvironment,
• 39:18tumor cells express high levels
• 39:20of transporter.
• 39:22For methionine it's ACC 4382
• 39:25outcompete T cells 4.
• 39:27The only surprise when T cells do not
• 39:30get methionine and the T cells have
• 39:33insufficient sense Earth myself honor.
• 39:36Therefore they cannot successfully
• 39:39do the H3K790 maceration and
• 39:42therefore regulate stats fab.
• 39:44And as a consequence this affect the TCL
• 39:47functionality and the T cell survival.
• 39:49So what we suggest here maybe
• 39:51you know we can either we do
• 39:54mathematics supplementation, we do,
• 39:56we cover the T cell immunity.
• 39:58Maybe we can particularly target
• 40:00the tumor as you C for 3/8 to
• 40:03the rescue T cell functionality.
• 40:05So now it comes back to the question
• 40:09we asked. So can we stop themselves?
• 40:13Can we stop to myself, to this?
• 40:15Yes, we can.
• 40:16You must ask really needed method.
• 40:18Earth one example.
• 40:20But the poverty is if you stop yourself
• 40:23to death, you also stop T cells to death.
• 40:26Under the AIDS and who kills the tumor cells?
• 40:30The T cells?
• 40:31Tumor cells.
• 40:32So that's why what we say.
• 40:33If you want to stop human cells
• 40:36to test using this approach,
• 40:38probably you kill 1000 yourself defeat
• 40:41800 and I would put the opposite way,
• 40:44you kill 800 yourself defeat defeat 101,000.
• 40:50So that's why we need to be really
• 40:53smart to consider not only just.
• 40:56To, to the tumors,
• 40:58but we have also considered the T cells.
• 41:01So now we ask the question again
• 41:03as I put it at the beginning.
• 41:05So what is next in terms of telling what
• 41:09is the next generation of cancer therapy?
• 41:12So in my view,
• 41:15immune therapy we means the basis.
• 41:19Why? Two reasons.
• 41:20Because the military has been
• 41:23successful to cure some patients.
• 41:25We already know this,
• 41:27indicating the powerful reason.
• 41:30The whole powerful is immune
• 41:32system could be second.
• 41:34T cells. Can kill human cells.
• 41:37It's not a surprise.
• 41:39And the further T cells remember to kill
• 41:42tumor cells and then nobody else can do that.
• 41:45So that's why I feel the next
• 41:47generation of cancer therapy,
• 41:49immune therapy, is the basis.
• 41:52So I stop here.
• 41:54I appreciate the contribution from several
• 41:57very tentative federal as I did here.
• 42:00Some of them are faculty,
• 42:02some of them moved to different institutions.
• 42:05And of course I didn't particularly
• 42:08talk about the PD1 video one study and
• 42:11some others it was a collaboration
• 42:13with and I have quite a few,
• 42:16some few other collaborators
• 42:20in the United States and.
• 42:22And in other places,
• 42:23thank you for your attention and looking
• 42:25forward to your comments and questions.
• 42:35Question.
• 42:38Thank you over here.
• 42:40So thank you very much.
• 42:42Much appreciated.
• 42:43Presentation understood correctly,
• 42:45you showed that the rock, correct?
• 42:47Doc acid was a mediator of T cell.
• 42:57You are. Has anyone looked
• 42:59inside the cell, two per cell,
• 43:01that's undergoing the fructose this
• 43:04kind of objectively by the elements for
• 43:06others to see what's elevated threat?
• 43:11It is surveyed, but there
• 43:14are thousands of. Liberal.
• 43:17Right. So in response,
• 43:19So what we have done actually we
• 43:23detected electronic acids in the tumor
• 43:27microenvironment in the tumor floats.
• 43:31So the question is very tricky.
• 43:34You have to have sufficient
• 43:36levels of electronic acids,
• 43:37but if you have very high concentration
• 43:40you kill everything. Right.
• 43:41So, so, so The thing is you have
• 43:44to have two things simultaneously,
• 43:48one is interfering, another is doing acid.
• 43:52So that's a play you have to go.
• 43:55Yeah, we did not systemically to
• 43:58detect all the metabolites in the
• 44:01tumor microenvironment by our own.
• 44:03There are some report in that space.
• 44:05There are technical challenges
• 44:07in in that situation.
• 44:09I guess the question is what do you ask?
• 44:11It's a it's very annoying to to do it.
• 44:13For example,
• 44:15we really dynamically monitor the
• 44:18metabolic environment when the sales
• 44:21either become able to or philanthropic
• 44:25whether there's any difference.
• 44:27In this case, we need to have props.
• 44:31It's a real property.
• 44:33We follow these people.
• 44:34Maybe some of you are smart and and
• 44:37have tools we can we can do that.
• 44:39So one day it could be done.
• 44:41You know, just a trace where it
• 44:44goes and how high the levels are,
• 44:48yeah.
• 44:49Thanks.
• 44:54So I wonder if the opposite guys.
• 45:04Struck.
• 45:16Great question actually.
• 45:18So as far as my understanding is,
• 45:22you know when we look at the Philippoussis.
• 45:27It's all the different ways how
• 45:29the cells die or four for example.
• 45:31You know you you have some
• 45:34executive genes which have been
• 45:36well defined in enable those poses,
• 45:38right but for fear of nosis,
• 45:42it's really about the membrane and
• 45:44damage so mediated by oxygenic
• 45:47species that's what we know.
• 45:49So therefore so very direct question when you
• 45:53asked whether the factors what we studied.
• 45:57Or whoever studied have
• 45:59directly effect on the membrane,
• 46:01let's say maybe the nails and the structures,
• 46:04those things, right?
• 46:05We didn't go that far.
• 46:06I even don't have the expertise to do that.
• 46:09So, so I think it's very nice way to go.
• 46:13So one way to to do it is we have
• 46:15done a little bit means we detect
• 46:18a tumor membrane if it oxidizes,
• 46:20inhibit species, that's what we know.
• 46:21But we don't look at the structure
• 46:24to monitor how the sector changes
• 46:27could be unacceptable.
• 46:28Yeah, but we didn't know that.
• 46:30We even don't know how to do that.
• 46:32So maybe.
• 46:34Which way we go that maybe again,
• 46:36if we have some proxy,
• 46:38it could be useful, right.
• 46:40So maybe you, you,
• 46:41you have some ideas in that space.
• 46:42We were chatting it on maybe this,
• 46:44that that's a good way to go
• 46:46because if people are still some
• 46:48people feel or feel to see if
• 46:51you don't have a executive gene.
• 46:53So what are you talking about, right.
• 46:54But The thing is the pathway is regulated
• 46:57and the pathway can be inhibited,
• 46:59can be activated, it can be regulated.
• 47:01So that's very difficult mechanism, right?
• 47:03So therefore it's.
• 47:04The program still this.
• 47:22Yes, yes, yes.
• 47:23That's also a good point.
• 47:25So you know, when you design experiments,
• 47:28you want to see something,
• 47:29you look at something, right?
• 47:30So therefore we didn't look
• 47:33at some other cells.
• 47:35So my. So right now we know
• 47:38different types of cells.
• 47:40We have given the sensitivities
• 47:42to different stimuli,
• 47:44stimuli, fibrosis stimuli.
• 47:46They may have different
• 47:48mechanisms to control.
• 47:50So that's typically something we are
• 47:53working on including for example,
• 47:55how about megabytes?
• 47:56How about T cells, right?
• 47:58So I guess this is not an mechanism
• 48:01exclusively for tumor cells.
• 48:03There is no such thing.
• 48:05So the mechanism could be functional
• 48:08for other types of cells.
• 48:10The question is?
• 48:11When and how and which one?
• 48:14We are working with this
• 48:15but you can go on details.
• 48:20This idea the only competition
• 48:22out of competition
• 48:23of tumor cells.
• 48:26Do you think it's the growth across
• 48:29towards you know we think about like
• 48:31lung which is sensitive to prepare
• 48:33incorrectly that is not or for example
• 48:35the location of the tool you have
• 48:37the two reason delivered reason the
• 48:39London or different access to metabolic
• 48:41substrate have you what are your thoughts
• 48:45about that? Yes it's it's OK.
• 48:46It's a great question.
• 48:47It's hard to address. OK.
• 48:49So what we started to look
• 48:51at since even in the same we
• 48:53have recently paper just.
• 48:55Eventually you even in the same
• 48:57human parent such as liver right?
• 49:00We reach locate the neighbor metastasis HCC.
• 49:03So in the liver microenvironment
• 49:06you have HC and metastasis.
• 49:09Then we look at even the same
• 49:12macrophage subsets they are
• 49:14metabolic patterns are different.
• 49:17So if they are, metabolic patterns
• 49:20are different, therefore their
• 49:22metabolic needs are different.
• 49:24How does it happen? Right.
• 49:27We have nuclear. You know,
• 49:29but we are still working on those things.
• 49:31So we we work more on the the way how the
• 49:36sales die because we believe this is this,
• 49:40these matters are not.
• 49:42So so the thought is.
• 49:46Different cells have
• 49:48different metabolic pattern.
• 49:50The same cells in different metabolic
• 49:53environment may have to adapt this
• 49:56particular environmental survive #1, right?
• 49:58So then whether they can expand.
• 50:00So I guess a part from the genetic
• 50:05mutations which people have started
• 50:07or not in the space of cancer biology
• 50:09and genetics in the recent days,
• 50:11people really moved to the
• 50:13field of metabolism because the
• 50:15metabolism is somehow universal.
• 50:17It must be regulated in
• 50:19one way or versus another.
• 50:21So that's that's why I we we have
• 50:23high interest in this, right.
• 50:25But the answer is very big.
• 50:27I know I didn't really give you an answer,
• 50:28just what we have.
• 50:33Specificity. Signing on.
• 50:38So why do you think?
• 50:43Yes, that's, uh,
• 50:44it's it's a great question actually.
• 50:46It's. So it turns out this is a, it's a,
• 50:50it's a, it's a biochemical question.
• 50:53OK. So if you look at the constant,
• 50:57it's the lowest among all the
• 51:01other isomorphic modifiers.
• 51:02So that's why it's. So it's sensitive.
• 51:05It's. Yeah, yeah, yeah. Yeah.
• 51:08Yeah. So so this actually this.
• 51:11This information is available.
• 51:12It's not from us,
• 51:14it's from when we figured out that it's
• 51:17needed on and we asked the same question,
• 51:19ask to ourselves why we
• 51:21see this is predominant,
• 51:22the others are not so dramatic and
• 51:24then we know it's publications.
• 51:26It turns out that's the case.
• 51:27Yes.
• 51:31In any of your models you're looking at.
• 51:35The cancer cells undergoing sister type.
• 51:41Um, I working on this.
• 51:44We are working on this.
• 51:46We should though I'm
• 51:48not, I'm not working on the persistent.
• 51:50So, so yes, it's a great question.
• 51:52Actually we initially I was really puzzled.
• 51:56Puzzled by what when you see I show you
• 51:59the picture actually when you treat
• 52:01the mice with PDL one and the CDA 4.
• 52:06And under this condition you treated
• 52:09mice with fair process inhibitor?
• 52:12And as the therapeutic efficacy is gone.
• 52:15This puzzled me so because, I mean,
• 52:18we we we know this is able to see
• 52:19the T cells kill tumor cells.
• 52:21It's even though this is caspase,
• 52:23and it's very well established,
• 52:25you cannot throw away all those
• 52:26things what people have known, right?
• 52:28So the only explanation is these across.
• 52:33So who is first, who is second,
• 52:35who is in the middle and who initiate what?
• 52:37Who emphasis what? Those kind of things.
• 52:39So we worked very hard,
• 52:41but we have no group so far.
• 52:43But we know there must be a gross.
• 52:45Yeah, I'm thinking in terms of like the
• 52:47paper from the green script where, you know,
• 52:49they show that the many monitors slight
• 52:56differences.
• 52:59Yeah, that's a possibility. Uh, actually,
• 53:01I just had a discussion recently.
• 53:04I probably will discuss with him
• 53:06again to see which way we can we
• 53:10can get some insight. Yeah, yeah.
• 53:14So I have another question
• 53:15about practical you know,
• 53:16we ask pathology for always
• 53:18frustrated by PO1 as a biomarker or
• 53:21TMB like there's no good biomarkers.
• 53:25It sounds like you have about a number
• 53:27of potential molecules that could work
• 53:29as biomarkers, you know having the
• 53:31right transporters in the right place.
• 53:34Do you see any, any sort of immediate
• 53:37possibility of some of these as
• 53:39biomarkers for immunotherapy? Yeah, so.
• 53:44So I I guess this is is it's quite a
• 53:48it's quite a depressing I would say.
• 53:50So when you located the biomarkers right,
• 53:53so it could be money you know perfectly
• 53:56well when people started to do the PD1
• 53:59PDL 1 clinical trials and nobody knows
• 54:01it the PD one or PDL one expression.
• 54:04So now after that you know it is PDL
• 54:06one expression and if they approve
• 54:08you know you have certain levels of
• 54:11PD1 expression it's indication right.
• 54:13So it's. It's not the way how how we know
• 54:17initially for it is for Philippoussis.
• 54:20I don't know which one we can
• 54:22we can really say.
• 54:23So the best way is well check all the
• 54:27social associated genes particularly protein
• 54:30levels whether this will give us something.
• 54:34You know so for example we looked at
• 54:38a CSR four expression when you see
• 54:41high ACR four expression may this may
• 54:46help can it is for BA real bellmaker.
• 54:51You get to have something to test it
• 54:53in clinic, in patient and your mouse,
• 54:55you know mechanism, fine.
• 54:58But you if you want to see it directly
• 55:00in patient, that's another story.
• 55:02We need to see the patient.
• 55:04That's why we appreciate your work.
• 55:06We need to see the patient.
• 55:07We need to see the tumors in
• 55:09patient and see what's going on.
• 55:11Yes, but there are ways to go.
• 55:57OK.
• 56:08I'm I I'm afraid I really didn't get
• 56:10any question you asked. Maybe just.
• 56:40So you mean when they express the grammar?
• 56:45Season.
• 56:51Right.
• 56:54So that's why. Our fear of dosis,
• 56:57it's obvious when we do immunotherapy.
• 57:01So that's the system we used.
• 57:03So actually in response to
• 57:05your question early on when you
• 57:08just give tomatoes and mice.
• 57:10So both you still have T cells and T
• 57:13cells are more or less functional.
• 57:15But under this condition we treated
• 57:17myself with ferocity and crypto.
• 57:20We hope, we hope we can see.
• 57:23We thought.
• 57:25So you get to have sufficient levels of
• 57:29interference and electronic acid and so
• 57:32and maybe other BOA&OA in the environment.
• 57:35How do we do that if you don't have
• 57:40a sufficient T cell infiltration?
• 57:43Even so, I guess we can manipulate
• 57:47the system, for example,
• 57:48maybe for example you have some,
• 57:50it's not one way to go, maybe for example,
• 57:52if you have a card in cell therapy, right?
• 57:54So not all the patients are responsive
• 57:57and then we have some cells here,
• 57:59maybe we can manipulate this
• 58:01specially in this, in this way,
• 58:03another way we can do also maybe you know we
• 58:06have ways to improve the teacher trafficking,
• 58:09right?
• 58:10So, so, so far where do?
• 58:15A pure airplus mechanism.
• 58:17In the absence of immune system
• 58:19whether this is a valid approach, we don't.
• 58:22Maybe there's a way to go?
• 58:26Maybe radiation or chemo or something?
• 58:29Yes and a no.
• 58:31And also we have another paper I can
• 58:33mention this here and we have a paper
• 58:35to actually that's the first people
• 58:38talking about the effect of radiation
• 58:41is partially dependent on our fibrosis,
• 58:45but this fabulous especially again
• 58:47recognized by the immune system.
• 58:50So you need to have an immune system.
• 58:53Yes,
• 58:53so that that's a cancer discovery people.
• 58:57We.
• 58:57Properties 4-5 years ago,
• 59:00yeah.
• 59:02Questions?
• 59:06Thank you very much
• 59:06again. Yeah. Thank you.
• 59:07Thank you. Thank you.