PDE5 inhibitors Block MDSC Metabolism in Gastric Adenocarcinoma

May 13, 2024

Information

Yale Cancer Center Grand Rounds | May 10, 2024

ID11662

To CiteDCA Citation Guide

00:00Thank you all for being here this morning.
00:04So today it is my pleasure to
00:08introduce our speaker,
00:10Doctor Juanita Merchant.
00:12Juanita joined the faculty at the
00:14University of Arizona College of
00:17Medicine in Tucson in 2018 as a
00:20Professor of Medicine in the UA
00:24Department of Medicine and Chief
00:26of Division of Gastroenterology
00:28and Hepatology and is currently
00:30a member of the Cancer Biology
00:33Research Program and is currently
00:36also serving as Interim Cancer
00:38Center Director for the Arizona
00:40University of Arizona Cancer Center.
00:43She is coming back home as she earned her
00:47MDPHD hair at Yale School of Medicine,
00:50did her internship and residency
00:53internal medicine at Boston,
00:55MA General Hospital before completing
00:58her Gastroenterology Fellowship
01:01at the University of California,
01:04Los Angeles.
01:06In 2008,
01:07Doctor Merchant was elected to the
01:09National Academy of Medicine and
01:11appointed a member of the National
01:13Institute of Health Council of Councils,
01:16and in 2016 she also joined the
01:18Board of Scientific Counselors for
01:20the National Institute of Diabetes
01:22and Digestive and Kidney Disease,
01:25a unit of the NIH.
01:27Prior to joining UA,
01:28she was on the faculty of
01:30University of Michigan.
01:32She's Board certified in Internal
01:34Medicine and Gastroenterology.
01:36She has written or Co written more than
01:40165 pair reviewed research publication
01:42and is editor or Co editor of two
01:45books and several book chapters.
01:47She is a Co Pi on the NAHAG Forward
01:51program which was developed
01:53to increase the number of
01:56academic gastroenterologists
01:57from underrepresented groups.
01:58Doctor Merchant has remained continuously
02:01funded by the Nah for her work in
02:03Gastric and newer endocrine tumors.
02:06Head Hodge signaling,
02:08gastric cancer,
02:09and transcriptional control
02:11mechanisms in colon cancer.
02:14Please join me in welcoming Dr.
02:17Monita Wharton.
02:22Great. Thank you, Doctor Rogers. Oh,
02:25here we get started.
02:27Well, like to present to you this plaque
02:30in honor of your presentation.
02:32PDE inhibitors, block MDSC
02:35metabolism in gastric endocarcinoma.
02:38Oh, free. Thank you. OK.
02:40So oh fixture, thank
02:46you. OK. Thank you.
02:49Great. Well, I'm,
02:50I'm really excited to be here.
02:52I'd probably come back
02:54about once or twice a year.
02:55I'll be back in November for the
02:57the Dean's Advisory Committee,
02:59but I'm excited to present to the
03:01Cancer Center today because I
03:03really love to get some feedback
03:05from the esteemed oncologists
03:07and faculty here at Yale.
03:10So that so those of us,
03:14so as you know,
03:15I'm a practicing gastroenterologist
03:17and for those of us on the
03:19more the diagnostic side,
03:21gastrogatinal carcinoma is primarily
03:24initiated by an infectious Organism
03:27which I'll review on the next slide
03:30and therefore is largely preventable.
03:33There obviously are some caveats
03:35which we can talk about,
03:37particularly in underrepresented minorities,
03:40but here is the basic summary.
03:44Gastric cancer worldwide has used,
03:46we used to say the second or
03:48third most frequent cancer,
03:49but has now dropped to about the 5th,
03:51probably because more intensive,
03:53particularly in Asia,
03:54in terms of screening and surveillance.
03:57But still,
03:57if that type of diagnosis is still
04:01associated with a high mortality rate,
04:04about 27,000 cases,
04:06new cases will be identified in the
04:09US and it's about 11,000 deaths.
04:13The important point here with respect
04:16to prevention is the infectious
04:18component that can initiate this cancer.
04:20And the person Barry Marshall and
04:23Robin Warren got the Nobel Prize
04:25for discovering the association
04:27of Helicobacter pylori first
04:29with ulcers but then made the
04:31association with gastric cancer.
04:33But also there we need to think about dietary
04:37components such as high salt nitrates.
04:41The other less frequent infection is a viral
04:45infection with Epstein Barr virus or EBV.
04:49So the in the US the prevalence
04:55of gastric cancer has declined.
04:58So from probably from about
05:00the 1920s to about the 1960s,
05:02gastric cancer was probably the
05:05second most frequent cancer,
05:07but then with an improvement in sanitation,
05:10which has not occurred in certain places.
05:12Being in Arizona,
05:13this is a big issue on the
05:16Native American reservations,
05:18but that was what was probably
05:20driving the decline in the US
05:22because Helicobacter is found
05:24in the water table basically.
05:26However,
05:26it is continuing to rise in
05:30minorities and immigrant communities.
05:32The other interesting issue
05:34with respect to gastric cancer,
05:36so that's really the distal cancer
05:37and I don't remember if I I think
05:39I do include a picture of the
05:40stomach for those not familiar
05:42about thinking about the different
05:43regions of the stomach.
05:45But the
05:47cancers that are arising in the cardia are
05:51rising and are thought to be more associated
05:55with the maybe increase in use of Ppis.
05:59I'll come back to that point in a little bit.
06:02But in general the the needle really has
06:06been moved more in Asia where Helicobacter
06:09is pretty much endemic in the population
06:12and certainly on the West Coast where
06:14you see more of the Asian immigrants.
06:16It's still fairly prevalent with
06:19the first degree relatives,
06:21but you can see the highest
06:24incidence tends to be in East Asia.
06:26China, Japan and Korea per capita
06:29is actually Korea's the highest.
06:32So what I'll be covering today is
06:35how we ended up starting to address
06:39this issue or why we started looking
06:42at this question in terms of what is
06:46driving the inflammation to change the
06:49mucosa from chronic inflammation to
06:52the metaplastic changes in the stomach.
06:55And we came at this or I came at this
06:58from the the Hedgehog signaling pathway
07:02which I'll show you why in a few minutes.
07:04And then we started asking questions
07:07in terms of translating from our mouse
07:10models to what can we do in in people
07:14and in moving from Michigan to Arizona,
07:18have been fortunate to start to collaborate
07:20with some of the oncologists there to
07:23begin a phase two clinical trial which
07:25we're very excited about based upon
07:27some of the findings in our mouse models.
07:31So again from a
07:33gastroenterologist perspective,
07:35you know what we typically are seeing
07:38in many instances patients really
07:40don't even come to be seen by the
07:44physician and already have metaplasia.
07:46So I scoped many patients that
07:48just have chronic gastritis,
07:50sometimes metaplasia,
07:51but no helicobacter is nowhere to be found.
07:55What is the connection between metaplasia
07:57in the stomach and the esophagus?
08:00And when I looked into the history
08:02of this term, metaplasia,
08:03what's interesting is that,
08:04so if people remember,
08:06you basically have that goblet cell
08:08that's normally in the small intestine,
08:10but it's showing up in the stomach
08:12or in the esophagus.
08:14So I like to the pathologist like to
08:15say a normal cell in the wrong place,
08:18but that's signifying that the mucosa
08:20is starting to move more toward cancer,
08:24we think maybe more directly in
08:27response to the immune microenvironment
08:30as opposed to the bug being there
08:33pushing the mucosa toward cancer.
08:35But this issue of metaplasia and it
08:38the link of metaplasia to a cancer
08:41was really initially identified in the
08:43esophagus with Barrett's esophagus.
08:46So that metaplastic change in the
08:49esophagus is a precursor lesion and we
08:52actually have surveillance approaches
08:54for patients that have Barrett's esophagus.
08:58So the debate in the GI field is
09:01you know is it going to be worth to
09:05actually start doing surveillance
09:07for gastric cancer based upon
09:09identification of intestinal metaplasia
09:11and the jury still out.
09:12I'm actually going to an NCI think
09:15tank next week we're we're going to be
09:18discussing this whether we can change
09:21the recommendations for gastric metaplasia.
09:24But the reason why that that is,
09:26is because the question
09:27becomes who do we survey,
09:29when do we survey them and how often,
09:32which is where the cost comes in.
09:35So this is a picture of for those
09:38not familiar with the four regions
09:40of the stomach, the cardia,
09:42which is where you see the incidence
09:44of this cardiac cancer is higher.
09:46And this was a nice review article
09:49by the Gastroenterology Group
09:52Samir Gupta leading that from UCSD,
09:55where cancer at the cardia tends to be
09:58higher in lice and less so in minorities.
10:02And it's more strongly associated
10:04with GERD and obesity and not
10:06with socioeconomic status.
10:08Whereas the traditional cancer that
10:12is associated with Helicobacter pylori
10:13infection tends to be in the body of
10:16the stomach where the parietal cells sit.
10:17And the antrum of the stomach,
10:19which is another name for is the
10:21endocrine part of the stomach,
10:22which is where the G cells that
10:24produce gastro.
10:24And the reason I'll be coming
10:25back to that in a second.
10:26And so it's this region here that then
10:29it's connected to the small intestine.
10:32So this is more strongly
10:34associated with socio increase in,
10:36decrease in socio economic status
10:39and underrepresented minorities.
10:42And so definitely in Arizona,
10:44we're seeing high incidence in Hispanics
10:47and the Native American population.
10:50So as I mentioned earlier,
10:54there's a increase in interest
10:56in the tumor microenvironment,
10:58which I think this is well known
11:01to the oncology group here,
11:03which is a very heterogeneous environment
11:07that is comprised of stromal cells,
11:10neuronal endothelial.
11:11But you know most of the work now
11:14is really focused on the immune
11:16cells because this is a the target
11:19for the checkpoint inhibitors.
11:21And so we are coming at this from the
11:23approach that if we can decipher a bit
11:25more about the tumor microenvironment
11:27particularly in gastric cancer where
11:29we already know that the initiation
11:32of the inflammation is from a an
11:35infectious agents most of the time
11:37that we can develop better targets
11:40for treatment and biomarkers.
11:44So this is actually an example of the
11:47what we call the Correa paradigm which
11:51Playa Correa who say epidemiologist
11:54who initially was in Columbia,
11:57South America and made the observation
11:59and published in The Lancet in 75.
12:02This observation that there was
12:04chronic inflammation in the stomach
12:06that progressed on to cancer and
12:08this is looking at obviously if
12:10this is an epidemiologic study
12:12looking at people over time.
12:14But he noticed that there was sort
12:17of this intermediate stage where
12:19some people had loss of the acid
12:22secreting portion of the stomach and a
12:24substitution of the normal epithelium of
12:28the stomach with this mucous phenotype,
12:32what we call metaplasia.
12:34And in the in humans,
12:36the pathologists will read
12:38out intestinal metaplasia,
12:39which is an example here where
12:42you see goblet cells,
12:44even panic cells in the stomach.
12:46This is the intestinal type.
12:48The colonic type is actually more
12:51strongly associated with gastric cancer
12:53and it's more of this foamy type of
12:56mucous metaplasia that
12:57one sees in the stomach.
13:00And so there's about half the world
13:02is infected with Helicobacter and
13:05may develop this chronic gastritis,
13:07but then about 10% will go on to develop
13:11metaplasia and 1 to 3% gastric carcinoma.
13:15So the thought is,
13:17is that this the tipping point in with
13:22respect to the progression toward
13:24the the likelihood of progression
13:26toward cancer is at this step where
13:30there is atrophy and metaplasia.
13:32And we started asking the question
13:35whether hedgehog signaling might be
13:38important in this transition of the
13:40mucosa from chronic inflammation
13:42to the metaplastic change.
13:44And I've added here to emphasize
13:47that the Pilea Correa,
13:49the paradigm was basically formulated
13:52before the discovery of Helicobacter and
13:55then once Helicobacter was identified
13:57that the link was then made to this
14:00chronic and then atrophic gastritis.
14:02But we asked the question about
14:04hedgehog signaling and really is it was
14:07because of a incidental finding by Andy
14:10McMahon's group at Harvard in 2000,
14:13where they published a paper saying
14:15that the Sonic Hedgehog knockout
14:17mouse resulted in gastric metaplasia.
14:19It turned out that probably wasn't accurate,
14:21it probably was more hyperproliferation,
14:24but they didn't have any GI
14:27pathologists reviewing those slides.
14:31So, but based upon that MO El Zatari
14:35at the time who was in my lab,
14:37we actually obtained the mice that
14:41are in which the Laxi reporter is
14:43knocked into the locus of the Glee
14:46one which is the transcription
14:48factor and is the transcriptional
14:51readout for Hedgehog signaling.
14:53So we obtained these mice,
14:55so the knock in of the Laxi molecule,
14:59we're able to maintain these mice in
15:01the heterozygous or homozygous state.
15:02But essentially you have a, you know,
15:05a total body knockout and he infected
15:07those mice with helicobacter.
15:09Now we typically use Feliz in the
15:11mice because you get a much more
15:14aggressive inflammatory response sooner.
15:16We were hoping to save a little bit of
15:18money and and see changes, you know,
15:22and not have to wait six months.
15:24But with pylori itself using
15:26the human pathogen,
15:27it can take a lot longer and the
15:29inflammatory response is not as robust.
15:31So he looked at the mice
15:35at the time of infection,
15:38two months after infection and
15:39then six months after infection.
15:41And so you can actually identify
15:45fluorescently using an antibody.
15:48So in the uninfected mice you see that
15:52the alpha smooth muscle positive cells,
15:54which are the mild fibroblasts in the
15:57stomach are positive for Glee one,
15:59and pretty much those were the only
16:02cells that were expressing Glee one.
16:05So again,
16:06Glee one is in the stroma and typically
16:09what happens is that the epithelial
16:11cells such as the parietal cells will
16:14make the ligand Sonic hedgehog and
16:16it's received by the stromal cells.
16:18So in the uninfected mice,
16:21it's the alpha smooth muscle positive cells.
16:25But during after two months of
16:28infection with Helicobacter,
16:29not surprisingly you have a pro
16:32inflammatory situation where you have
16:34an infiltration of inflammatory cells
16:36and those cells are positive for Glee one.
16:39And when we did flow cytometry,
16:41this is published several years
16:44ago now mostly myeloid cells but
16:47not T or B cells were the cells
16:50that were expressing Glee one.
16:52And so just to summarize that
16:56basically we then we're asking the
16:59question well what are these Glee
17:021 positive immune cells doing.
17:04And what was interesting is that
17:07when we infected again wild type
17:10mice and now what you see here is
17:12an immunofluorescent stain for the
17:15different cell populations in the
17:17corpus of or the body of the stomach
17:20intrinsic factor in the mice mark the
17:23chief cells whereas in human it's
17:26it's normally in the parietal cells,
17:29HKTPAS marks the parietal cells and
17:31this GS2 lectin marks a mucous cell.
17:34So here in the mice that are infected
17:36for six months you see that they're
17:39developing A metaplasia and atrophy.
17:42So these this region should
17:44show parietal cells,
17:45which would be the orange stain,
17:48but you can see here they're kind
17:50of moved off to the side because
17:52they're starting to disappear and
17:54show atrophy and being replaced
17:55by this mucus phenotype.
17:57However,
17:57in the cells that or in the the
18:01mice that were heterozygous for the
18:02Glee, one deletion or because of the Laxi
18:07insertion into the locus or homozygous,
18:10they maintain the normal
18:12architecture of the stomach.
18:13And so we were really surprised by
18:15that because as I mentioned earlier,
18:18it's the stromal cell that's
18:20expressing Glee one.
18:21So this was telling us right there
18:23that there was something going
18:25on in the micro environment,
18:27in the immune environment
18:29that was affecting the mucosa.
18:34So to try to summarize this quickly,
18:37so we started looking at hedgehog
18:40signaling in this transition
18:42from gastritis to metaplasia.
18:44I should also mention one of
18:46the issues with working with
18:48the mouse models is they never
18:50progressed to dysplasia and cancer,
18:52just with an infection from Helicobacter.
18:56So we could only look at this step.
19:00So I've just shown you that
19:02Glee one is important in the
19:06meta formation of metaplasia.
19:07So this like I said is you know
19:102015 sixteen we were doing this
19:12so we did microarrays and we
19:15identified this molecule Schlafen 4.
19:18There certainly were quite a few other genes,
19:21but this one was interesting because
19:23there were some papers of both
19:25Schlafen 2 and Four I should mention.
19:27But the reason why we didn't
19:30pursue the pathogenesis related
19:33to Schlafen 2 is because Two does
19:37not have a ortholog in in humans.
19:40So there is an ortholog for Schlafen 4.
19:44So we wanted to be eventually be
19:46able to translate the work that
19:48we were doing in mice into humans.
19:50So that's why we focus on Schlafen 4.
19:53So this was the further analysis
19:58of this locus which we identified
20:00in the array of the mice.
20:03Comparing wild type mice to
20:05the Glee One knockout mice,
20:06you can see here that there's
20:09a decrease in Schlafman 4,
20:10which suggested that this gene was
20:14regulated by hedgehog signaling.
20:18And so we did chromatin immunoprecipitation
20:23at the time to determine that indeed
20:27Schlafen 4 is a direct target of Glee one.
20:31So you can show that it does sit
20:33on the promoter of Schlafen Four.
20:35However,
20:38I do want to get into in a in a second
20:41what exactly are these Schlafen's.
20:44So the reason why we focused on
20:46them again is because there was a
20:49paper in immunity in 1999 that said
20:52that the Schlafen molecules were
20:55involved in both T cell and and
20:58and myeloid cell differentiation.
21:00So that's why we thought, well,
21:02you know if we're looking at Hedgehog
21:05signaling and it's rolling the
21:06stroma and its effect in mediating
21:09the this metaplast gastritis and
21:11metaplasic transition that that
21:13would be a a good target.
21:15So actually I'm gonna just
21:16give you a quick primer.
21:18The Schlafen locus however is
21:20fairly complicated and this is
21:21what I was kind of getting at.
21:23So we identified Schlafen 4.
21:26There's actually quite a bit
21:28of information from one group
21:30that's looking at Schlafen 2.
21:31We did see this go up in mice,
21:34but you can see that it doesn't
21:36have it's ortholog in humans.
21:38So you'll hear me talk about
21:41as we move to the human data,
21:44the ortholog for Schloffen 4,
21:46that's about 60% similar is Schloffen
21:5012 L So I'm just showing you this now.
21:54Just plant that seed in your brain.
21:57These are what are called
21:59the intermediate schloffens.
22:01And the reason why that's important
22:03is because the longer schloffens
22:05ones in green have another domain
22:08that's a helicase domain that's
22:10thought to bind to nucleic acids.
22:13I will be coming back to this point later,
22:18OK.
22:19So coming back to the mouse model,
22:23we,
22:24as I mentioned,
22:25we're interested in that gastritis
22:27to metaplastic change and we've
22:30identified these immune cells
22:31that are Schlafen positive.
22:33And so to to understand more
22:35of what they did,
22:37we created a very fancy mouse model.
22:40And I know some people are not as
22:42familiar with some of these you know,
22:45kind of mouse tricks that we do.
22:47But essentially we took the mouse promoter,
22:51it was a large back trans gene.
22:53We hook it up to inducible Cree recombinase.
22:57We breed this mouse line to a
23:00reporter mouse line TD tomato.
23:02So this hybrid mouse is expressing
23:08or can be expressed in the presence
23:10when we give it tamoxifen this
23:12reporter so turning the cells red.
23:14But what we also did is to do a
23:18bone marrow transplant and put the
23:21bone marrow from these mice into a
23:24radiated mice so that essentially
23:26only the immune cells are going
23:29to be labeled with TD tomato.
23:31And ask the question,
23:33can we lineage trace this Schlopfen
23:35positive cell from the bone marrow
23:38of these mice that have recovered
23:41and infected with Helicobacter in
23:43waiting four to six months to see
23:46you know how they get to the stomach.
23:48Again this is was published in 2016,
23:51but I just wanted to show you that
23:53it's really been a very powerful
23:55tool for us because you can see
23:58here like stars in the sky and what
24:00I'm showing you here is a wild type
24:02mouse infected with Helicobacter.
24:05But I'm taking we're taking these mice
24:08at four months before we have seen the
24:11cells actually arrive in the stomach.
24:14However, if we breed those mice
24:17onto a background where they're
24:19where the Sonic Hedgehog,
24:21the ligand signal is goosed up.
24:23So it was pretty easy.
24:25It was just a PCMV Sonic Hedgehog Transgene.
24:28We breed those mice, you know,
24:31with the TD tomato signal and you can
24:33see at four months there are these
24:35cells that are TD tomato positive.
24:37Here's a high-powered view.
24:40Since they're fluorescent,
24:41we can pull them out.
24:42You can see they have a granulocytic
24:45nucleus and they are exhibiting
24:47markers of a granulocyte.
24:49Even better.
24:50You can certainly do all sorts of arrays,
24:52which I'll get into a little bit later.
24:54But more importantly,
24:56we could actually isolate these cells
24:58from the infected stomach and show that
25:01they had T cell suppressor activity.
25:04So we did the Co culture and show
25:07that they were really functionally
25:09T myeloid derived suppressor cells.
25:13So I wanted to show you well what's
25:17the connection between Hedgehog and
25:19how this gene is regulated and and
25:22why I think we were I'm happy that we
25:24we decided to kind of stick with this
25:27even though nobody's heard of Stroffen.
25:29So what you see here is where you
25:32can isolate the these cells from.
25:34We basically you know create a a
25:37pus situation by injecting them
25:39with thioglycolate,
25:40take the peritoneal cells and then
25:43we can culture them and incubate them
25:46with recombinant Sonic hedgehog.
25:48About a fivefold induction of
25:50Sonic hedgehog message.
25:51Helicobacter alone threefold but
25:53the two together synergize but
25:56more importantly interferon alpha.
25:59So type 1 interferons, 800 fold induction,
26:03This gene is and that locus is very
26:07strongly induced by type 1 interferons.
26:10However,
26:11if you isolate those cells from
26:14a Glee One null mouse,
26:16you can see that This is why this locus
26:19is still dependent upon hedgehog.
26:22You can get a little bit of induction,
26:24but essentially it's a dead promoter.
26:26So it's like you need 2 keys to unlock
26:30this gene and follow it and we mapped the,
26:35so essentially the hedgehog signal
26:37Glee one is a constitutive signal.
26:40The inducible signal is through
26:43type 1 interferons.
26:44And so then we asked the question,
26:45well you know in the infected Mao
26:49stomach where is we're is type 1
26:53interferons coming from and it turns
26:56out that and we've done some later
26:58work that was published in 2022.
27:01But basically plasma cytoid dendritic
27:04cells are sort of resident dendritic
27:06cells that are the most the cell
27:09population that is probably sensing
27:12the debris field there chronically
27:15and why probably why it takes time
27:18for this to develop.
27:20So really putting putting this
27:21all together and you may want to
27:24look at our gastro paper in 2022,
27:26what we're saying is that Helicobacter
27:28infection is detected not only
27:31by the epithelium,
27:33so the epithelial cells will
27:34also produce type 1 interferon,
27:36but sort of PER on a per cell basis,
27:40it's the plasma cytoid dendritic cell.
27:43There's a certain pathway with activation
27:45of the interferon response factors,
27:48which are the factors,
27:50transcription factors that bind to the
27:53type 1 interferon promoters releasing
27:55type 1 interferons that then will polarize
27:59what we now think is a neutrophil
28:01or granulocytic cell that has been
28:03sitting there and had was recruited
28:05to the stomach at some point in time.
28:07But then this debris field and
28:09threshold must be reached over time.
28:11So these cells are PDL 1 positive
28:14and we were able to show as I
28:17mentioned earlier that they do
28:20have T cell suppressor function.
28:22But analysis of these cells also
28:25reveals that they are producing
28:28other cytokines not surprisingly some
28:31of which that were of particular
28:34interest to us or was IO 1A and Beta.
28:37And we think that and that's why we
28:40think that it's the immune cells that
28:42are really picking up the baton and
28:45really pushing the mucosa more toward
28:48cancer as opposed to the bug itself.
28:52And recently and I didn't put
28:54the reference in here,
28:56we we actually had for other reasons
28:59had generated a triple transgenic
29:02mouse where we can inducibly over
29:05express I-1 beta in the antrum.
29:07So you may ask, well,
29:09why would I bother to do that?
29:10And it's because the Helicobacter infection,
29:12whether it's Feliz or Pylori,
29:15when we infect the mouse,
29:16because the mouse stomach is actually aph
29:18of three or four compared to our stomachs,
29:20which is pH of one,
29:22the the Organism tends to
29:26only infect the corpus,
29:28not the antrum where traditionally
29:30you see it in people.
29:32So we really wanted to understand distal
29:35gastric cancer where we can drive a,
29:37you know,
29:38much more aggressive tumor in
29:40the antrum of the stomach.
29:42And so we took the gastroin,
29:44we made a gastroin Cree ERT two,
29:46crossed it to a TET activator, RTTA.
29:50So these are three different mice
29:53that have to be all bred together.
29:55So a lot of alleles.
29:56And then this mouse is then bred to a
30:00Tet on where we've inserted the IO1 beta,
30:04where it'll generate A secreted form.
30:06And so you give the mice tamoxifen.
30:10So the the TET TET activator will
30:14sit in the cytoplasm until we give
30:16the mice doxycycline in the water.
30:19And so we keep them on doxycycline
30:22and after about six months we about
30:2440% of the mice will develop these
30:27ugly dysplastic looking tumors.
30:29I I caution to call it cancer because
30:31the mouse models never metastasize.
30:34I have yet even the colon,
30:35all the models that people talk about,
30:38they never metastasize.
30:39So you know you can kind of quibble
30:41about what you want to call that.
30:43But I'll I'll just say you can see
30:45there is they're pretty ugly looking
30:47cells and more importantly these
30:49cells are so they do have and have
30:52recruited the Schlafen for positive MDS,
30:54CS into the tumor.
30:55So at least we now have a sort of a
30:58pre clinical model to actually study.
31:02So going back again in time a little bit,
31:05So 2020 we started to do bulk
31:08RNAC which we did with these mice
31:12that were TD Tomato positive.
31:15What I want to point out here that was
31:18quite interesting and coming back to
31:20the Type 1 interferon theme is that a
31:23lot of the genes that we identified.
31:26So this is the heat map happened to
31:28be interferon, strongly interferon
31:31regulated and were these guanalite
31:35binding proteins or GTP aces,
31:40GBP, 2G VIN and they're of the
31:44dynamin class of GTP aces.
31:46This is our the changes in the heat map,
31:53the full log pole change.
31:56But I am comparing it to a paper in
32:002019 where it was really elegant study
32:05of both a mouse and human lung cancer.
32:09So there were seven patients with
32:11lung cancer and they had a mouse
32:14model using Ras and I want to say P53.
32:17There was another gene where they
32:19were able to generate lung cancer
32:22and they did a complete analysis by
32:25single cell sequencing of the of the
32:28tumor microenvironment what they call
32:31in two or neutrophil 2 cells which
32:35we now are thinking those are those
32:38tumor associated neutrophils or Tans.
32:42They the they had the same gene profile
32:46that we identified in our Schloffen
32:49positive MDSCS and I highlight here
32:53that their mouse into was positive for
32:57Schloffen 4 and here this is the human
33:03counterpart for seven patients.
33:04I think one of the problems they I
33:07didn't we didn't see Schloffen 12
33:08L but again when you move to human
33:11you've got a whole variety of stages,
33:14tumor types etcetera.
33:17And so we we they did not observe it in
33:22that but all the other genes were similar.
33:24We've also gone on to show that using
33:29proteomic analysis and using the
33:31Schloffen 4 antibody that we can actually
33:35pull down and show that Schloffen 4,
33:38which I didn't mention is
33:41actually a cytoplasmic.
33:42It's actually an ER membrane
33:45endoplasmic reticular membrane protein.
33:48So I'll come back to that.
33:49So that even adds to the complexity
33:52what are we dealing with.
33:54But interestingly it forms a complex
33:57with at least when we pull down
34:00with many of these genes that we
34:03identified in the bulk RNA seq.
34:07A little bit of a complicated
34:09slide here again it's published
34:11for those that are interested.
34:13So if we take that pull down using
34:18Schlafen for antibody and we wanted
34:22to know whether it had Gtpas activity.
34:25So we take that complex where we
34:29pulled it down and actually show
34:32that it can hydrolyze GTP and so
34:36shown here and it does that here
34:40higher levels in blue of GTP bold
34:44change and the interferon treated
34:47cells where we do the pull down
34:51versus we have also made recently a
34:55Schlafmann for knockout mouse model.
34:57So if we isolate cells from
35:01those versus sildenafil,
35:02now why did I use sildenafil?
35:04I kind of skipped over that and
35:07that's because some of the genes
35:10also were these G cyclic GMP
35:15related phosphodiesterases.
35:17So we already were starting to think,
35:20well, you know, maybe, you know,
35:23there's already an off the shelf.
35:25Oh, did I do that?
35:28There's already an off the shelf
35:30inhibitor of phosphodiesterases,
35:31plus I'm sure the oncologists
35:34are very familiar with,
35:36particularly from the multiple
35:38myeloma field where you can use these
35:43phosphodiesterase 5-6 inhibitors
35:45as a sort of neoadjuvant.
35:47So that was one of the reasons
35:48why we thought, oh,
35:50let's see whether this works.
35:52And indeed it also knocks down the
35:56ability of the this complex to form GTP.
36:00So we put together this model which I'm
36:04showing you here that interferon will induce.
36:08Because remember it's a very
36:10strong inducer of Schlopfen,
36:12so we can mark these cells but along
36:15with Schlopfen there are other
36:17very important type 1 interferon
36:20regulated genes that appear to
36:23be somewhere in this pathway.
36:25And I try you know this is this kind
36:29of a model because essentially what
36:31these myeloid derived suppressor
36:33cells their their ability to inhibit
36:36T cells has to do with their them
36:39being able to gobble up L arginine
36:41out of the the the environment so
36:44that the T cells can't proliferate.
36:47But what are these myeloid derived
36:49suppressor cells are actually
36:51using that L arginine themselves to
36:53what I'm not showing here generate
36:56reactive oxygen species.
36:57Here are some of the
36:59pathways. So arginase making nitric
37:02oxide or No2 make making nitric oxide,
37:07which happens to be a cofactor
37:11for soluble guanillate cyclase.
37:15So guanalase cyclase generates cyclic GMP.
37:22Cyclic GMP, if it hangs around is
37:26a cofactor for protein kinase G,
37:29which can in some cell populations
37:33trigger the cells to undergo cell death.
37:36So if you have high levels of something
37:40that's going to break down cyclic GMP,
37:43you're going to move the
37:46cells away from apoptosis,
37:48regenerate this the sort of
37:51backbone for regenerating GTP.
37:53And so that's why we think and I've
37:56shown you that Schlafen 4 is at least
37:59in a complex with these Guanali binding
38:02proteins which you know need this GTP.
38:05So we think that there's a whole
38:07nother pathway or metabolism that
38:10pulls the substrate away from
38:12maintaining high levels of cyclic GMP
38:15and you can essentially accelerate
38:18that and we'll get back to that,
38:21oops, going too fast if we
38:24inhibit phosphodiesterases.
38:25So you can imagine if we block
38:28phosphodiesterases here,
38:30this is going to build up and you can
38:32trigger the cells to undergo apoptosis.
38:33So that's kind of the hypothesis
38:35that I want to keep in mind.
38:37OK,
38:37so let's move on.
38:39We've moved to the next era where
38:41we're now using single cell sequencing.
38:44And I want to point out again that we're
38:48reinforcing what we initially observed
38:51and I just want you to this is published,
38:54but you can see here in our
38:57go enrichment for this is the,
39:00you know spring plot that won't
39:02bore you with all of that,
39:04but you'll notice that the go enrichment,
39:07Gtpas activity,
39:08GTP binding.
39:09So again a lot of the genes even in the
39:14doing the single cell sequencing seem
39:16to take us to these Gtpas types of proteins.
39:20I want to highlight though this
39:23region here which kind of didn't
39:25blow up quite as big as it should.
39:27But what we were kind of surprised about
39:30is that there's really three groups,
39:35low, medium,
39:36medium,
39:37high and high expressors of Schlafen.
39:40And this is what we're finding
39:42many times as you start to get into
39:45single cell sequencing is that
39:46many of these cells exist in sort
39:49of different activation states.
39:51I we haven't quite gotten to
39:54the pseudo trajectory.
39:55Somebody's working on that 'cause you need,
39:57you need a different program.
39:59But what you can kind of see is
40:01that the Low Expressors Group 3,
40:04which is this blue, actually it has
40:08more of the neutrophil genotype,
40:12so that would be I guess no.
40:15Anyway, I won't point it.
40:17I guess this group here.
40:19And whereas the higher expressing ones,
40:24there's one group number two that
40:29tends to be and so that's this
40:32cluster here higher in nitric oxide
40:352 which is actually a different
40:37group than that express arginate.
40:40So this is just the mouse.
40:42So even that mouse cluster that we are,
40:47we're already thinking that we're
40:49polarizing and becoming myeloid
40:51derived suppressor cells from
40:54a granulocyte or neutrophil.
40:56They actually have different sort of
40:59activation states or different gene
41:02clusters that you can now identify
41:05by single cell sequencing. OK.
41:08So I've given you a lot of information.
41:10So essentially from the mouse model,
41:14what we're saying is that,
41:16and I didn't really give you
41:18the sort of how this all begins,
41:20but essentially when Helicobacter
41:22infects the stomach, it can,
41:25the dying parietal cells or
41:27intraparietal cells actually can
41:30release Sonic Hedgehog into the plasma.
41:32So some of the papers that I didn't
41:35talk about in detail actually you can
41:37pick up Sonic Hedgehog in the plasma
41:39of the mice within two or three days
41:42these cells track to the stomach.
41:44But the first two months or so of
41:46the infection it's we're still in
41:48more of the pro inflammatory stage.
41:50It's not till about when we did a formal
41:53time course about five and a half,
41:55six months of a Helicobacter
41:57infection in mice.
41:58Do you actually see these cells
42:01actually generate enough interferon
42:04alpha in the tissue?
42:06That and I'm the reason
42:07why I'm crossing that out,
42:08is that we actually infuse interferon
42:11antibody in our 2022 paper to show
42:14that we could actually block the
42:17polarization of the Schloffen for MDS,
42:20CS and we did not get the spim.
42:23Is the the term metaplasia that
42:25we use for the mice,
42:27it stands for spasmolytic
42:29polypeptide expressing metaplasia,
42:31but we just call it SPM because in the
42:32mice you actually don't see the goblet cells.
42:35So they had to come up with
42:37another way to market.
42:39And so again what we're proposing is
42:42that if we block the phosphodiesterases
42:45and maybe these along with the GTP
42:48Azes that we can do the same thing.
42:51So what I've shown you is more in vitro data,
42:54but now I'm going to show you what
42:56it looks like with the knockout.
42:59So as I mentioned this is a normal
43:03mouse and like I said we can goose
43:06up the the whole signal and and get
43:10the metaplastic change faster if we
43:12over express with Sonic Hedgehog.
43:15So the green staining you saw before
43:17is the metaplastic change in the mice.
43:20And when we do the conditional deletion
43:24and we're deleting it using Glee one,
43:27Cree ERT two.
43:28So we're deleting it in that those
43:31myeloid cells that we originally
43:34identified the Schlafen cells in.
43:36And you can see that you start to read
43:39the normal architecture of the stomach.
43:41The parietal cells are shown here in white,
43:43are starting to come back.
43:46What about Sildenafil?
43:48Didn't take much.
43:50We did two injections of sildenafil,
43:53same thing.
43:53And here I'm showing you an H&amp;E where
43:56you can really see the parietal cells,
43:59which I'm I'm used to looking at it.
44:01But these big pink cells are
44:04your parietal cells,
44:05starting to return in the presence
44:08of just after two injections of
44:10sildenafil and very recently within
44:12the last couple of months going
44:15back to our aisle 1 overexpressing
44:18mice with those
44:19big ugly tumors.
44:20So here you can see in this low power view.
44:23Here is the villi of the intestine.
44:26Here is the pyloris,
44:28the junction between the stomach or the
44:32antrum and the in the small intestine.
44:35These are Bruner's glands.
44:37Here the tumors develop and
44:39we're able to accelerate it if
44:42you give it the MNU nitrosamine.
44:45So instead of 40% of the mice alone,
44:47we get about 60% of the mice we'll
44:51develop these ugly dysplastic tumors.
44:54But two injections of SILDENAFIL
44:55were able to melt those tumors down.
44:58And
45:01the reason why I put this in here,
45:02this is again kind of hot off the presses.
45:05I want to come back to, OK,
45:07I told you that I mean Schlafen
45:09is AER protein, well guess what,
45:13it's an RNA binding protein.
45:15And so we actually have recently done a
45:20pull down again with the Schlafen antibody.
45:23These are transfer RNAs and what's very
45:27interesting is that it actually binds to
45:31very specifically in an inducible manner,
45:34glycine and tyrosine specific transfer RNAs.
45:38I don't have time to get into it right now,
45:39but we can come back to it at the end.
45:41But I just wanted to start to close the
45:44loop of this is very interesting protein
45:46and why is it so important and why an
45:49ERRNA binding protein is involved.
45:52OK. I'm going to because of time,
45:55I'm going to come back to this diagram
45:57which I know is pretty complicated
45:59because I wanted to show you our
46:01phase two clinical trial.
46:02So this is a collaboration with primarily a a
46:07really talented junior faculty in oncology,
46:11Junaid Arshad,
46:12Rosten Schroff is our Chief of he Monk and
46:17Aaron Scott are the trio of GI oncologists.
46:22And so when I presented this to them,
46:27they Janae suggested,
46:28well you know why not just try,
46:31let's try and see if we
46:32can set up a window trial.
46:34And so essentially I didn't
46:36know what a window trial was,
46:39but he said you know what we can do
46:41because most of these patients are going
46:43to have to go to receive standard of care,
46:45flot therapy and then go for a gastrectomy
46:47if we if there's stage one to three.
46:50So we're only dealing with
46:51stage one to three,
46:52well 1B to to three and so these
46:58are the window trial objectives.
47:01So the primary objective and
47:03I didn't realize this,
47:05we can't just give patients to
47:07Dalafail even though it the safety
47:09profile we know is pretty good.
47:11I'm not supposed to I guess because of CME,
47:13I'm not supposed to say the trade name.
47:15But anyway,
47:16so they're just focused on this
47:19feasibility and safety and but the
47:23secondary objectives shown here,
47:25you know,
47:26to to see whether there's some
47:28pathologic response.
47:29But my interest in what I'll show you
47:32because the study is still ongoing,
47:34I'll just show you that of what
47:36we're looking at in terms of does
47:39tadalafil in a patient with actual
47:41gastric cancer do anything, right.
47:43So remember we've got to follow 12
47:47L these are the exclusion criteria,
47:51study feasibility.
47:54So we've been going for about a year.
47:57We've got six patients enrolled,
47:582 patients have finished the study.
48:00But what I want to show,
48:03so we're our goal is to enroll 10 patients.
48:07When I moved to Arizona,
48:10I set up a repository for our endoscopy lab.
48:13So I or one of the other endoscopists will
48:17do a if if the patient is not referred
48:20in at their not referring him from the
48:23outside that becomes a problem because
48:25we actually want to try to do single
48:27cell sequencing at each of these intervals.
48:30So that really means that we
48:31have to do the endoscopy here.
48:32So just with the biopsies,
48:35jumbo biopsies we can do
48:37single cell sequencing.
48:38And I just wanted to show you that
48:40even in a gastric cancer patient,
48:42we can stain for Schlaf and 12 L So
48:45it's there in the immune cells in
48:48the lamina propria of these tumors.
48:52So I'll just show you a little bit of,
48:56let's see and I'm sorry it ends up
49:00going counterclockwise because of the
49:02way the data gets uploaded into the
49:05cloud for the 10X genomic analysis.
49:07So what we are able to do because
49:11obviously we run into problems with we're
49:14able to capture the 2nd interval endoscopy,
49:18so this one,
49:19but if the patient comes in from the outside,
49:23we basically do single cell sequencing.
49:25We have plenty of normal referrals to
49:28endoscopy that there's nothing there.
49:31They don't have gastritis and so we can do
49:34single cell sequencing on on those patients.
49:37So what I have circled here is the
49:41Myeloid cluster in a normal patient,
49:45one of my patients that I had referred
49:47for endos because I knew that I was
49:50having trouble eradicating Helicobacter.
49:51So they had Helicobacter gastritis
49:55and here intestinal metaplasia.
49:57And then this was one of the
50:00patients that the first patient
50:03that was enrolled in the study.
50:05And so they actually have a lot more
50:09of these Schlafen 12 L positive cells,
50:12which if you look at the just that gene,
50:19you can see here that this is
50:22where the myeloid cells are.
50:24But look at the normal
50:26gastritis intestinal atoplasia,
50:28I'm sort of going in order.
50:30Sorry, I didn't give you the preya paradigm,
50:34but Schlafen 12 L doesn't come on
50:38until you very strong, strongly,
50:40maybe a little bit in the metaplastic stage,
50:44but until these patients are actually,
50:47you actually have gastric cancer
50:50now you're gonna say, well,
50:52what are these other cells?
50:53They're T cells.
50:54So this was the big surprise as we
50:57move and not surprisingly when you
50:59move from mouse models to people,
51:04you know, sometimes all bets are off.
51:07So we now also have to understand
51:11what's going on in these T cells
51:14because you can see again Schlafen
51:1612 LS picked up in the T cells and
51:19gastritis and intestinal metaplasia.
51:21Now I think I have one slide here.
51:24It turns out that
51:27in the actual cancer,
51:31the T cells that are most prominent
51:33that you don't see in the other
51:35groups are the exhausted T cells.
51:37So that's going to be a whole other project
51:41to understand what is this molecule
51:44doing in terms of the metabolism of T cells.
51:49So we have our work cut out for us.
51:51I finally wanted to show you what happens
51:54with Tadalafil and so here's a cancer,
51:59so this was one of the patients where we
52:01the first, this was our first patient.
52:03So we didn't have this was they
52:04were referred in from the outside.
52:06So we only had slides and so this
52:11is sustaining for CD11B myeloid
52:14marker and our Schlofen 12 L Co
52:18localized here in the merge view,
52:20but here the high-powered view in the cancer,
52:23but with with Cialis, oh sorry,
52:25Tadalafil that we are eliminating
52:31the these Schlafen positive MDSCS.
52:37OK. So the take away there seems to be
52:43overlap between the pathways regulating
52:46Schlafen 4 and we also believe 12 L and
52:50cyclic GMP dependent phosphodiesterases
52:53and these inhibitors allow cyclic
52:55GMP to accumulate and induce MDSE
52:58apoptosis and that's the mechanism.
53:01Their elimination we think is we can
53:03at least see it in our mouse model.
53:05The big question will be as we
53:08expand this trial and get past the
53:11safety stage that this potentially
53:13may be a neoadjuvant for gastric.
53:15But again these cells are in a lot
53:18of cancers and we need to you know
53:21think about it in several cancers.
53:23So that I just want to certainly
53:26acknowledge linding who moved with
53:28me from Michigan and has really
53:31carried out all of these studies.
53:33Acknowledge again our HE monk GI
53:37group division and our targets
53:39by a repository and to thank the
53:44patients for their participation.
53:46Thank you.
53:47I'll take any questions
53:53and I should, yes,
53:55I don't know you can.
53:57There's also two questions online.
53:58So thank you for
54:04this education. But my question is the
54:07degree of expression in the myeloid cells.
54:11Is it something innate or is
54:13it acquired? Do we know? Is
54:15it like, is it something that's
54:17hereditary tendency to have higher
54:18expression in certain individuals
54:20and lower in others
54:23maybe
54:26are
54:28people. So your question is whether
54:31people are predisposed because
54:33they have snips or mutations.
54:35I'm just saying is it does it
54:36take like a two hit phenomena
54:38where you have H pylori infection
54:40but there is innate over expression
54:42of certain of these proteins and
54:44then that's when cancer happens? And
54:46I also had a second question.
54:47Do you think that some of the same
54:49pathways are involved in other
54:50types of gastric cancer like
54:52smoking related or others.
54:54So this pathway I think and
55:01is similar. I shouldn't because of
55:05the type 1 interferon regulation,
55:08is it similar to like
55:10the Sting C gas pathway?
55:13I haven't looked to see where
55:15the parallel and the overlap is,
55:17but I would emphasize that you know DAMPS,
55:23but probably even Pamps certainly can
55:26activate these plasma cytodendritic cells.
55:29The reason why I I like focusing on
55:31the Schlafen is because we're able to
55:32take it all the way down to the promoter.
55:35We know why that promoter and
55:37those cells get marked.
55:39So it suggests that you really
55:43need a very strong induction
55:46of Type 1 interferons or maybe
55:49there's mutations in those Irfs,
55:51etcetera constitutive.
55:52I mean it gets pretty complicated where
55:56whether people may be predisposed or not,
55:59we are some of the endpoints that
56:04we're looking at it are so TLR 9
56:07mainly because there is already
56:09information actually related to
56:11gastric cancer and Helicobacter that
56:14patients that have mutations in TLR 9
56:17May have a more aggressive response
56:20to an infection with Helicobacter.
56:22So that's we're starting with more upstream.
56:28Yes, thank you. That was a
56:29great talk. So Tadalphil is,
56:33you know, prescribed for
56:34other things as well.
56:36Do you, have you considered
56:37doing like a retrospective
56:38study and looking at you know,
56:40maybe stomach cancer versus people
56:42who've been prescribed to Dalafil,
56:46a retrospective study? Yeah.
56:48So in other words, it's in wide use.
56:54The problem is and I think we're
56:56going to need AI to do these kinds of
56:58things because it's it's really being
57:00someone has to mine the clinical data.
57:03I'd have to see whether
57:04it's already out there.
57:05Most likely it's not for gastric
57:08cancer maybe for one of the bigger
57:12cancers like lung or colon cancer.
57:15But I still think it's going to take
57:17some energy to pull it out of the
57:19clinical records and really analyze it.
57:21I really offhand I haven't seen any
57:24papers really looking at that but
57:26it's that's an excellent question.
57:28Thank you, Clara. Hello.
57:322 questions.
57:33One, back to the inflammatory cytokine role.
57:36And you showed that overexpressing
57:39is sufficient to contribute.
57:41But if you sort of throw in
57:42inhibitors or utilize cell specific
57:44deletion of Aisle 1 beta TNF,
57:47you had a range of different cytokines.
57:49What's the effect of deletion in
57:51your model on the end outcome?
57:53So deletion of like PNF or we we haven't,
57:58yeah, we haven't gone there.
57:59You can imagine how many mice my
58:01my mouse bill is out of control and
58:04I just it's reading all of those
58:07yeah mice onto those backgrounds
58:08which I I haven't that's why we did
58:11the antibodies a little cheaper.
58:13And then I guess the second question
58:15is a little bit related to the
58:17spectrum of Schlafen expression.
58:18If I know you mentioned in the
58:20mouse model that you can't,
58:21you don't see progression to the to cancer,
58:24it's more than metaplasia.
58:25But in human if you try and sort
58:29of consider the transition between
58:33metaplasia to gastric cancer.
58:36Well, I guess the first,
58:37can you speak to some of the things
58:39that you think are contributing to that
58:42enabling that transition into the in
58:45the 1 to 3% that sort of overlap with
58:48some of the pathways you've highlighted.
58:50In other words,
58:51you got the 10% that have metaplasia and
58:52then one to 3% actual gastric cancer,
58:55right.
58:55And and so in I guess in your
58:58studies that you're doing where
59:00you're looking at are you able
59:02to look at spectrum of Schlafen
59:05expression in that subset that
59:06goes on to gastric cancer relative
59:08to those that stay in metaplasia.
59:11OK. So I'm I'm trying to so have
59:13we looked at so you're taking
59:15gastric cancer or you mean taking
59:18metaplasia patients with metaplasia, I
59:21mean in the pathway are you and and it
59:23can be Schlafen and can be you know
59:25for the full for the full pathway.
59:27In general are you able to see
59:29that those that progress to cancer
59:32are fit on the higher end of of
59:35sort of or on the altered end of
59:36expression of the pathway relative
59:38to those that remain in metaplasia
59:41altered of I'm so I'm sorry
59:45of your can you segregate
59:47like in in in any number of thing can
59:49you segregate the high versus low in the
59:52shop and pathway for those that progress
59:54versus that remain in metaplasia Oh
59:58so but in people or or in the in
01:00:01people in other words we have
01:00:03from bulk RNA or from other
01:00:05types of data sets that may
01:00:07have been done in the stomach.
01:00:10You know it just really hasn't been done.
01:00:12We haven't really segregated the subtypes of
01:00:17Schlafen 12 L cells in the patients at all.
01:00:22I I mean it it's we're just a lot of
01:00:25it is just numbers and we're just happy
01:00:29to be able to to well you know with
01:00:31the repository the logistics is not
01:00:33as tricky because I have to have our
01:00:36inpatient teams let us know there's a
01:00:39patient in house patient has to agree
01:00:41many times they don't come or they
01:00:43come and they we've already gotten
01:00:45the biopsies and our hospital will
01:00:47not release that the even the tissue.
01:00:50So we have a lot of just sort of
01:00:52logistical issues but I'll keep that in
01:00:55mind as we or I can send you the data.
01:00:58Yeah, the data sets out
01:01:00there from the stomach and
01:01:01then look at high versus,
01:01:02you know the high versus lower
01:01:05expressors. Yeah, ends of the
01:01:08I, I, I mean I I will look, I haven't,
01:01:10I just haven't come across it,
01:01:11but it's a good question.
01:01:13You've stumped me.
01:01:16I may have missed this,
01:01:17but is persistent hedgehog sibling in
01:01:19the MDSC cells required to maintain
01:01:22the dysplastic tumors and if so is
01:01:24there a role for smoothened inhibitors?
01:01:26Oh that Doctor Kaplan had
01:01:31raised that issue yesterday.
01:01:33We could think about Vesmotogib to
01:01:37revisit that I it's a good question and
01:01:41I I mean I would try it out in our mouse
01:01:44models probably first I I do know that.
01:01:47So if you use a a Glee one null,
01:01:49we we didn't, we haven't used any
01:01:53hedgehog inhibitors but basically you
01:01:55as I showed you with the in vitro data,
01:01:58you need hedgehog,
01:01:59some kind of hedgehog signalling
01:02:01for that promoter to come on.
01:02:03The assumption is that the
01:02:06cells aren't or polarizing,
01:02:07but we haven't gone back
01:02:09to really explore that.
01:02:14Oh, they were listening.
01:02:17Oh, OK. I thought there was,
01:02:18there was two things on the line,
01:02:20but it was just the CME.
01:02:22OK, no questions. All right.