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PDE5 inhibitors Block MDSC Metabolism in Gastric Adenocarcinoma

May 13, 2024

Yale Cancer Center Grand Rounds | May 10, 2024

Presented by: Dr. Juanita Merchant

ID
11662

Transcript

  • 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.