PDE5 inhibitors Block MDSC Metabolism in Gastric Adenocarcinoma
May 13, 2024Yale Cancer Center Grand Rounds | May 10, 2024
Presented by: Dr. Juanita Merchant
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- 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&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.