The Minor Spliceosome: Exploring Novel Cancer Vulnerabilities

January 17, 2023

Yale Cancer Center Grand Rounds | January 17, 2023

Presentation by: Dr. Mark Rubin

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00:00Yeah, petrol. It's my pleasure
00:02to introduce Doctor Mark Rubin,
00:04who is a professor and director
00:06of Department of Biomedical
00:07Research and Burn Center for
00:09the Precision Medicine at the
00:11University of Bern in Switzerland. Previously
00:13was moving to Europe. Six years ago,
00:15Doctor Rubin was a group leader at
00:17Weill Cornell Medicine Institution that
00:19remained engaged to the prostate spore
00:22as a project leader.
00:24Doctor Rubin is a recognized
00:25world renowned leader and prostate
00:27cancer genomics and pathology.
00:29And in precision medicine,
00:30doctor Rubin's laboratory led a series
00:32of landmark studies defining distinct
00:34molecular features of prostate cancer,
00:36revealing pathways that are
00:38perturbed and drive different types
00:39of different types of this cancer.
00:41Furthermore, he has translated many of
00:43the genomic discoveries into clinical
00:45tests that are currently patented
00:47and Stanley used in the diagnosis
00:49and treatment of prostate cancer.
00:50He founded the Angler Institute for
00:52Precision Medicine and most recently,
00:54the Burn.
00:54Center for Precision Medicine Doctor Rubin
00:57has published around 300 manuscripts,
00:59including those in major top journals.
01:01Today will tell us about his latest
01:03work in the minor spy summit exploring
01:06novel cancer vulnerabilities,
01:07Doctor Rubin.
01:13Well, it's great to. Doctor OK, sure.
01:19Her. No, no. I thought Katie
01:23would want to say something again.
01:28We're OK. So is OK, OK, great.
01:30So it's great to be here and I
01:32think I last time I was here is
01:35definitely over 10 years ago.
01:36So it's it's great to come back and
01:38visit and hopefully I can figure out how
01:41to move the slides forward on this. OK.
01:45There we go. So just the following,
01:48mostly non relevant disclosures.
01:51So a lot of people asked in 2017
01:56when I went to Switzerland, you know,
01:58what I was going to do and why I was going.
02:00And so you heard that there were
02:02some initiatives that I was,
02:04I've been involved in,
02:05so setting up the Burn Center for
02:07Precision Medicine and running a
02:09new department and reorganizing a
02:11department biomedical research.
02:12But one of the decisions I made
02:14when I moved was that instead
02:16of taking projects that I had,
02:18I would start from scratch and.
02:21Really start new projects and just
02:23say I know I see we have a lot
02:26of trainees in the audience and I
02:28just think it's a it was a very.
02:30I think it's been an exciting time.
02:32It didn't count for the pandemic,
02:34but it's certainly been an exciting
02:36time to think about new projects,
02:37what you think is important and
02:40actually going in places that feel a
02:42bit uncomfortable scientifically just
02:44because you need to learn new things.
02:47And so I think I left all the
02:49projects with this prostate spore
02:50and we started a new project.
02:52So I'll tell you a little bit about
02:54two things that we worked on more
02:56recently just as way of background
02:58so many of you are familiar with.
03:00But you know prostate cancer,
03:02but I'll just as a as a means of
03:04background just to remind you that
03:06the landscape for prostate cancer
03:08therapy has really changed dramatically
03:10if we go back ten years ago.
03:13Or 2010, even more than 10 years ago,
03:16it was relatively androgen deprivation
03:19therapy based with the taxanes.
03:22But it's a very different than
03:24the landscape today.
03:25And I'm not going,
03:27I'm not expert oncologists.
03:28I'm a pathologist by training.
03:30And I'm not going to go into all the details,
03:31but just to point out that
03:33there are many choices.
03:35And if you go to clinical meetings
03:37and listen about, you know,
03:38what's going on just all the time,
03:40they're new opportunities.
03:41The one thing I would point out
03:44is that as patients are being
03:47treated with antiandrogen,
03:48various types of more potent
03:51antiandrogen therapies such as
03:54enzalutamide and abiraterone
03:55resistances obviously an inevitable.
03:57So patients can do well that for a short
04:01time or they can do well for long time.
04:03Eventually they'll fail,
04:05but there are other opportunities.
04:07Now there's PARP inhibitors,
04:08so that's quite exciting.
04:09Obviously there's immunotherapy
04:10for such a small subset.
04:13For patients where it seems to be
04:16effective and then there's PSA treatments
04:18or imaging and and and treatment and
04:22these are again really more therapies.
04:26There's problems with all of them,
04:28but there is a lot more opportunity now
04:31for treating advanced prostate cancer.
04:34Resistance remains a major focus of our
04:36labs work and and other labs now to
04:40try to understand why patients are failing.
04:43And what I think is very important
04:45is to think about in all of
04:47these settings what is the key
04:49clinical question,
04:50at what time should we change
04:52therapies or introduce new therapies.
04:54So that's one of the main things
04:56we're focusing on. So I'm going to
04:58make a few comments about lineage,
04:59plasticity and the resistance framework
05:01and I'm going to talk a little bit about.
05:04Some work that may be relevant to this
05:06to the lung spore in the context of
05:09switch sniff and epigenetic modulation.
05:12And then I'm going to talk about a new story
05:14that's unpublished that has connections
05:16with both Yale and and Connecticut.
05:19And it's our minor splicing.
05:21So I have my main goal today will be
05:24hopefully to tell you about something that
05:26you may not know a lot about and maybe,
05:28maybe we'll all learn something
05:30together about minor splicing
05:32and maybe also some insights.
05:34As well and that we can talk about them.
05:36So just a few comments about
05:39resistance and lineage plasticity.
05:40I've been showing these slides for a
05:42few years and I think it it helps me a
05:45lot of giving a framework for thinking
05:46about what we do clinically in the
05:48setting that most in the in the context
05:51that most of the types of therapies
05:54are very index oncogenic pathway driven.
05:57So if you have estrogen receptor,
05:59if you have her too,
06:00if you have EGFR as a main
06:04pathway that's activated.
06:05Braf, et cetera,
06:07the main,
06:08the index oncogenic output is
06:10being targeted and so then you
06:12have resistance related to that.
06:14But there are alternate effectors and
06:16they're also alternate states that can occur.
06:18So in the context of prostate cancer,
06:21the Andrew receptor is the main
06:24oncogenic target for most of the therapy,
06:27whether it's Andrew and deprivation therapy
06:30or targeted ligand targeted therapy or
06:33therapy based on decreasing synthesis.
06:36With androgens or hormones.
06:38So you'd expect and and it is the
06:40case that most of the mutations are
06:42resistance occur in the ANGIOMA receptor.
06:45So there's amplifications mutations and
06:47so this is this is what you expect but
06:51what we also are seeing emerging more
06:53and more frequently with very potent
06:56anti ancient therapy are alternate
06:58pathways and this is getting into
07:01the theme of of lineage plasticity.
07:04So if you switch.
07:05From an adenocarcinoma to something else,
07:08you're no longer driven,
07:09so you're a RH negative.
07:11And what are the mechanisms then that
07:13lead to this resistance is what we're
07:15trying to understand and a number of
07:18other groups are doing the same in prostate.
07:20Now obviously this is very interesting
07:22in other cancers like bladder,
07:24lung and breast.
07:26So just to be specific,
07:28as a pathologist we look under the microscope
07:31and we see this is an adenocarcinoma,
07:33it's very pink.
07:34The cells have a lot of cytoplasm
07:37and after antiandrogen therapy a
07:39certain subset of these patients.
07:42And what I'm showing here is
07:43just the types of therapies that
07:45that patients may receive,
07:46very strong potent anti entrant therapy,
07:48but after these therapies
07:50where most patients,
07:51almost all patients respond initially,
07:54many of the patients will have.
07:56Resistance and there are many
07:57different flavors of resistance,
07:59the most common,
07:59so I don't want to mislead you.
08:01The most common is probably still something
08:03that looks like an adenocarcinoma.
08:05It still can be responsive to more
08:08potent antiandrogens, but a subset,
08:09maybe 10 to 15% will have
08:11something that looks like this.
08:13So it's a neuroendocrine prostate
08:15cancer that's no longer responding
08:17to AR therapy and something
08:19usually these patients have very,
08:21very aggressive disease.
08:23If you look under the microscope
08:25and perform immunohistochemistry,
08:27you'll see that you have the adenocarcinomas
08:30are very positive for Antrim receptor
08:33expression and are not positive for
08:36neuroendocrine markers such as synaptophysin.
08:38And for neuroendocrine cancers,
08:40you lose AR protein expression.
08:43Now that's important,
08:44just one important point, and this is
08:46probably true in many other cancers.
08:47If you block A R and now you
08:49have a neuroendocrine tumor,
08:51the pathways for AR,
08:52so you might see at the transcript.
08:55Level an attempts for Andrew receptor
08:57to produce protein but but protein
08:59levels are are usually very low.
09:01So you don't see active
09:04Andrew receptor protein.
09:06These patients have a very poor outcome.
09:09So patients who are now diagnosed with
09:12neuroendocrine prostate cancer clinically
09:14or by biopsy usually have anywhere
09:17from 7 to 12 months median survival.
09:19So very poor and there are
09:22very few therapy options.
09:24In showing this slide,
09:25I can show that we have an
09:28example of adenocarcinoma all
09:30the way to small cell cancer.
09:32You can see in the adenocarcinoma it's
09:35positive for PSA which is a surrogate
09:37for antron singling and that it's
09:39negative for neuroendocrine markers here.
09:41And then as the tumor seems to,
09:44as we see different tumors where
09:45you have this neuroendocrine tumor,
09:47we see a positivity for the
09:49neuroendocrine markers.
09:49Now I think the important point here is
09:52an additional comment is that this is.
09:54All from the same patient.
09:55This patient had metastatic prostate
09:57cancer and these are from almost
10:00the same lesion in different areas.
10:02And so it's possible that you not
10:04only have this trans differentiation
10:07or lineage plasticity,
10:09but it may be in the context
10:11of mixed Histology.
10:12So I think it's important to just remember
10:16that heterogeneities obviously were is
10:18an important component of resistance.
10:20A few years ago,
10:22probably around 2013 we started
10:24a stand up to cancer.
10:26PCF funded prostate Cancer Foundation
10:28funded trial which was one of the first
10:32precision oncology trials or number of
10:35of PI's leading this Charles Sawyers,
10:38Real Shanian,
10:38Levi Garraway among others if Phil
10:41Kantoff and in this study one of the
10:45things that as pathologist was great is,
10:47is that we're able to look at now
10:50over 1000 cases of patients that
10:52were prospectively collected who
10:53were failing Andrew and decoration.
10:56Therapy and one sort or the other from
10:58multiple institutions and we could
11:00ask the question as a pathologist.
11:02If we used AR signaling as one
11:04score to look how adenocarcinoma
11:06tumor was and neuroendocrine
11:08signaling because we had
11:10transcriptomic data as to how
11:12neuroendocrine tumor was,
11:14we might expect to see 2A division
11:16into 2 camps and we could expect that
11:18there might be a third gradient of
11:21what tumors that are in transition.
11:23This is the real data.
11:25So it looks doesn't look like.
11:27Seven, I think this was at the time
11:29we had around 500 cases, but many,
11:31many of the cases are sitting here,
11:33so high anger receptor signaling
11:36only some of them.
11:38So I mentioned about 15 percent,
11:3910 to 15% of our cases showed
11:42neuroendocrine features.
11:43What was surprising and this is really
11:45the first prospective study that I was
11:47aware of this or have been involved in,
11:49in prostate cancer in this setting
11:51was that when we looked in the this
11:53quadrant here where we expect to see
11:55these very blue cells and neuroendocrine.
11:58So they showed you we see things
11:59that don't quite look like
12:01neuroendocrine prostate cancer,
12:02they look like something else.
12:04So it has a squamous appearance,
12:06take my word for it,
12:07if you're not a pathologist,
12:08but it doesn't look like a
12:10typical neuroendocrine tumor.
12:11And then in area where we see
12:14very strong expression for the
12:16answer and receptor signaling,
12:18not just Andrew receptor but signaling,
12:20we see something that looks
12:21like a neuroendocrine cancer.
12:22These were not mixed up.
12:24We confirmed everything.
12:25So I think what is very important is,
12:27is that the Histology,
12:29so the phenotype and the genotype
12:32don't necessarily correlate that
12:33well and this causes obviously a lot
12:37of confusion for classification,
12:39which is very important for treatment.
12:42This is a case that's in the middle
12:44and it shows a very unusual Histology.
12:46So for somebody doing prostate
12:48pathology for many years now,
12:50we usually see certain morphologies but
12:52we don't see these odd looking nuclei.
12:55And I think this is really related
12:57to a few factors,
12:59but at least one factor is that patients
13:01are getting very potent anti androgens.
13:04Another factor is that they're living longer,
13:06so they're able to survive longer.
13:08So we're seeing changes that
13:10we previously hadn't seen.
13:11So I think this is part of the
13:13natural evolution of the cancer.
13:15It's just that maybe we haven't seen this.
13:17And then in prostate,
13:18maybe in other cancers as well,
13:20metastatic biopsies are not the norm.
13:22So usually we're not actually seeing
13:24what's going on as you treat patients.
13:27So this was eye opening for me
13:29as a pathologist.
13:30When we looked at the molecular
13:32alterations in some of these cases,
13:34they tended to have P53 and RB
13:36alterations in these cases as
13:39well as in neuroendocrine.
13:41I'll talk a little bit more.
13:42About that.
13:42But this leaves us with a picture
13:45where we have a spectrum of disease.
13:47It's very difficult just looking
13:49under the microscope to classify it.
13:51So it really suggested to us that
13:52we need to come up with other
13:54ways of classifying it.
13:55And I would say right now we don't have that.
13:58I mean, we're moving towards that,
13:59but we don't really have
14:01that established to date.
14:03So as I just mentioned,
14:06the phenotype and the scores or
14:08the signaling scores that you have
14:10from transcriptomics don't match.
14:12Perfectly so. And we also recognize
14:14they're probably intermediate states.
14:16So I think those are important observations.
14:18So we start out with a model where we
14:21wanted to look at very discrete differences,
14:24adenocarcinoma and neuroendocrine tumors.
14:27In fact, when we look more carefully,
14:29what we see is a whole collection
14:32of intermediate morphologies,
14:33genotype and also transcriptomic
14:36and probably epigenetic alterations
14:38about the same time we wanted to do.
14:42A very focused study at looking
14:44at these extremes to ask the
14:46question what are some of the other
14:48factors that might play a role.
14:50So genomics but also epigenetics and
14:54transcriptomics were applied then to.
14:58A set of,
14:58I think at the time 50 or 60 cases
15:01where we had bonafide neuroendocrine
15:03cancers diagnosed by pathology and
15:06adenocarcinomas and we compared them.
15:08And I'm not going to go through
15:09these published studies,
15:10but I just want to highlight,
15:11I think the important finding for us
15:14was that we expected to see some some
15:18really exciting genomic alteration that
15:20would characterize the difference,
15:22but we didn't.
15:23What we really see is a commonality
15:25that RB1 and P53 are very commonly.
15:28Altered in the small cell phenotype,
15:30less commonly altered in the adenocarcinoma,
15:34but we do see it and that in general
15:36the overlap in genomic alterations,
15:38copy number alterations is very similar
15:41except for let's say the Andrew
15:43receptor where that would be something
15:45that's very different and altered in
15:47adenocarcinoma but not in the small cell.
15:50When we looked at the data in combination,
15:53so looking at DNA,
15:54RNA and epigenetics and then asking
15:57the question what which element
15:59best explained the differences
16:02between the two phenotypes.
16:04I think importantly the numbers
16:05may differ depending on the
16:07configuration of the of the population.
16:09But certainly epigenetics or methylation
16:11helped explain the difference quite,
16:14quite well in this population.
16:16And I'm not going to say too
16:18much more about that,
16:18but we think methylation or epigenetic.
16:20Alterations are very important.
16:22So here's a figure that we made
16:26for a review and the concept was
16:28that at some point adenocarcinomas,
16:32there's a,
16:32there's an inflection point where
16:34adenocarcinomas maybe go through
16:36some sort of stem like state and
16:37then undergo lineage plasticity.
16:39Now I think for people in the
16:41lung cancer field,
16:42this was something not unexpected,
16:44but this is something we wanted to
16:47explore in prostate as well because
16:49we think that at some point he's
16:51around 10 to 15% of the cases.
16:53Are no longer responding to AR driven
16:56therapies and become a RH negative
16:58in a sense that then they will then
17:02either trans differentiate to small
17:04cell or AR negative neuroendocrine negative.
17:08So another type of air negative
17:10phenotype and that's I think that
17:13was supported by some of the
17:15morphologies we saw from the trial.
17:17I list a number of studies,
17:18I'll just very briefly make a few
17:20comments about the Polycom gene,
17:22so easy H2. And also the switch
17:24sniff work that we've done,
17:26but I I think others are are are quite
17:29interested also for the lung cancer spore.
17:31It's probably quite interesting
17:33thinking about epigenetic regulation
17:35and where we stand today.
17:37So I think this is a very nice review.
17:39They came out after two papers were
17:42published in science suggesting that
17:44there's a stem like state that occurs
17:47before you go to neuroendocrine
17:49or to a negative state and that
17:52one of the key players.
17:53It's probably easy H2 so a Polycom
17:57gene that's responsible for repression
17:59of of a large number of of genes
18:02associated with AR signaling.
18:05And this is just one key experiment
18:07from one of the papers from
18:08David Goodrich's group where they
18:10demonstrate that if you knock out
18:13in a mouse model P53 and RB and
18:16that's what's shown here the the
18:18tumors are no longer sensitive to
18:22the potent antiandrogen enzalutamide.
18:24And so you could see that the tumors
18:26are now continuing to grow even
18:28in the presence of enzalutamide.
18:30If you use EH2,
18:32you can reactivate or resensitize
18:34these tumors to the epigenetic drugs
18:37that are used now clinically for
18:41as easy as two inhibitors and you
18:44can reactivate air sensitivity.
18:46So this is just a concept that this is
18:49a stem like state that's potentially
18:51reversible and I think that's an interesting.
18:54Concept there.
18:55I'm happy to discuss more about those.
18:57There's some controversy a little
18:59bit about that,
19:00but I'll just leave it at that for now.
19:03We were interested in the other,
19:04the flip side of the Polycom complex,
19:07which is a repressive complex
19:08looking at the switch sniff complex
19:10and the things that really were of
19:13particular interest to us were one,
19:14that there's an embryonic switch
19:16sniff complex that goes to other
19:19types of complexes.
19:20So you can have this a stem like complex,
19:23but it also.
19:24A neuronal complex and the idea
19:26was that in development it's very
19:28important that you're able to have
19:31neural development and switch sniff
19:33complex activates and can facilitate
19:35a neural development and playing
19:38an important role that H3K27 marks
19:42of escalation.
19:44So this is important in development,
19:46but in cancer it's also known
19:49that that the switch sniff,
19:52particularly the ATP,
19:53Asus market two and Smart K for are
19:55often altered and a particular interest
19:57in many of the synthetic lethal screens.
20:00So Mark four and smart K2 come
20:02up as sort of key findings and so
20:05there's been a great interest in
20:08translationally developing drugs that
20:11would knockout smarca 2 specifically.
20:14In smart key for ultra tumors and
20:18unfortunately as many of us are
20:21aware and knowing what's happened,
20:23it's very difficult to actually have
20:25a specific smart gate 2 inhibitor.
20:27So most of whether it's an ATP
20:30ACE inhibitor or a protac,
20:32this has been a difficult,
20:33it's been challenging.
20:34So most of the work is is
20:36really focused on knocking out both
20:38and creating a synthetic lethality.
20:42What we found in a sort of as
20:45as is very common in prostate,
20:48so prostate tends to prostate cancer.
20:50Research tends to look at things
20:52differently because for whatever
20:53reason prostate is a little bit
20:55different than other cancers.
20:56And we were interested in looking at
20:59the overexpression of smart Guy 4,
21:01which is not usually the case in
21:03in many of the tumors that have
21:05lost smart gave for expression.
21:07So these are complicated complexes
21:09and I'll simplify it.
21:11By just saying that there's a
21:13working component which is a
21:15TPA and there are two paralogs,
21:17mark four and smart K2 and fortunately
21:19they also have different names.
21:20So there's Brahma and and Berg one,
21:23but I'll just call it smart K4 and smart K2.
21:26And the idea is that most of the work
21:28is going and targeting these and we
21:30were interested to see what happens
21:32in prostate cancer progression,
21:33whether there were mutations or
21:36alterations in these in the ATP Aces.
21:39What we found is,
21:40is that when we look there's as you
21:42look in prostate cancer progression,
21:44we sent to see an increase in smart
21:46K4 and a decrease in smart K2.
21:49So that's a little bit different than
21:51what's seen in some other cancers,
21:54but the other important.
21:55Features as you see as you look
21:58at prostate cancer progression,
22:00we also see an increase in some
22:03of the neural components of the
22:05switch sniff complex that are there
22:08associated with the the neural
22:09complex which is called back 53B as
22:12one of the as one of the proteins.
22:14So just to visualize this as a pathologist
22:16their core complexes don't change.
22:19So going from localized disease or
22:21benign tissue to advanced disease
22:24that's neuroendocrine positive you.
22:26You don't see any differences but what
22:28you do see is the neural marker shown
22:30here back 53B is only expressed in
22:32their endocrine tumor as you'd expect.
22:34And what we see here is that smart K4,
22:37so one of the two ATP A says a
22:40paralogs is very highly expressed
22:42in the neuroendocrine tumors or
22:44maybe the stem like type tumors that
22:46we see smart K2 is not expressed.
22:48So we were originally thinking that
22:50for us would be very interesting,
22:52interesting to modulate smart K4
22:54in the contest of prostate cancer.
22:56And just as an aside,
22:58we don't see any mutations that are
23:00seen in other cancers in either of
23:03the of the paralogs or in any of the
23:06of the Swiss sniff complex members.
23:08When we look at pathology,
23:10again if you look at cases where
23:13you have adenocarcinoma here and
23:15then you have some neuroendocrine
23:17cancer in the exact same tumor,
23:19you can very nicely see some of
23:20the things I'm telling you about.
23:22So the small cell expression of
23:25synaptophysin the scene here.
23:27The BAT 53B,
23:28which again is a neural component,
23:30neural protein component of the Swiss
23:33sniff complex is expressed there,
23:36but not in the adenocarcinoma.
23:38So that's important.
23:39And then here is I think a
23:41very important finding at least in the
23:44published paper where we took an organoid
23:48that was a neuroendocrine organoid
23:50and from a patient and looked at it
23:52now this we're seeing heterogeneity.
23:54So that's one thing that's
23:55important is even in a patient.
23:57Private organoid this passage
23:59many times we see heterogeneity.
24:01And what's interesting here is smart
24:02guy four is expressed here, socks two.
24:05So transcription factor that's very
24:07much involved in stemness is expressed.
24:10But the neural markers that I mentioned
24:13back 53B are not expressed here,
24:14but they are expressed in
24:17the Smart Gate 2 area.
24:19So it looks like the paralogs play a
24:21different role in modulating them.
24:23May do something very different.
24:25And so we think that there's a dynamic.
24:28Activity going on,
24:29you have some cells that are more
24:31poised to be neuroendocrine and
24:33others that are potentially that
24:35are potentially still very stem
24:38like and potentially reversible.
24:45David Goodrich, who and collaborations,
24:48has shared some of the organizers he's
24:50developed through some of these mouse models.
24:52So I mentioned that he's
24:54developed mouse models where
24:55he's done knockout of P53 and RB.
24:58They've also knocked out P-10.
25:00And so in black we have P-10 knocked
25:03out in blue we have both RB and P-10.
25:06And in red we had we include,
25:08he included P53 knockout in
25:10these various mouse models.
25:11And you can see that in the mouse models.
25:13You also have the same observation that
25:16smart K4 goes up when you knock out a RB1.
25:20And a little bit of the controversy is
25:22whether you need P53 or not because we
25:24don't see much change when you add P53,
25:26but that's a sort of another
25:29discussion and then smart.
25:30The two goes down dramatically
25:32when you knock out either
25:35P53RB alone or with P53.
25:38There's some other interesting findings.
25:41The methyl transferases
25:42increase when you do this,
25:45and I'm not going to talk about it today,
25:47but there's I think an interesting
25:49story related to the epigenetic side of
25:52methylation that also occurs in this setting.
25:55One other, I think important
25:56point is in this mouse model,
25:58we then did proteomics on the patient
26:01derived organoids from this model.
26:03And what's interesting and what
26:04you'd expect is if you have these
26:07models where you knock it out,
26:09that we see overexpression of socks too,
26:11which I showed you in the
26:12in the human samples.
26:14We also have smart K fours overexpressed.
26:16But what's quite interesting is,
26:18is that if you take this model out of the
26:20mouse and now put it and just grow it in,
26:23in, in, in.
26:24In vitro should say in vitro,
26:27not in vivo.
26:28What ends up happening is you
26:29don't see these changes anymore.
26:31So just one other comment that environment
26:33we think is very important and I
26:35think when do a lot of us when we're
26:37doing the our organoid experiments,
26:39we have to be mindful that the
26:41results could be very different.
26:42And there's I think more to come about this.
26:45I think Walter Carter who's was
26:47at memorial now is in Lisanne and
26:50others are working very much in this,
26:53in this area.
26:54So just a final comments about switch sniffs.
26:58So we were sort of surprised
27:01at the end of 21,
27:04so beginning of 22 to see a paper
27:06from rural Shanes group because we
27:08were we've been following this field
27:11for awhile and we've known about the
27:13toxicity for the Protex if you try
27:15to target switch net and they have
27:18very nice paper where they developed
27:20a protech that was not a toxic.
27:23So they showed not a lot of.
27:24Very nice toxicology data from their
27:27in their paper that's not toxic
27:29and at least the mouse models that
27:32they show and that the switch sniff
27:35protect for a combined smart A4
27:39and smart K2 are exquisitely potent
27:42against AR sensitive prostate cancer.
27:46And so we found that quite interesting.
27:48We we have been following up we
27:50had actually been working on
27:52something similar and and have.
27:55Come to a similar conclusion,
27:56but extend it a little bit and just
27:59point out that our collaborators Uchen
28:01and Ekta Karana who was formerly a
28:04trainee and Mark Burstein is here.
28:07I had a very nice paper in
28:09science where they used a taxi,
28:11so an epigenetic approach to
28:13classify prostate cancer and I
28:14won't go into all the details,
28:16but against essentially in addition to
28:19AR sensitive advanced prostate cancer,
28:21they also came up with the wind
28:24signaling pathway, a stem.
28:25Like and neuroendocrine.
28:26So I think it's a good working
28:28classification for these advanced
28:30cancers more than just air negative,
28:32but air negative could be stem like it
28:34could be when singling or neuroendocrine.
28:36I'm sure this will change,
28:38but I think it's a very nice study.
28:39So when we applied this classification
28:42and used a protect that we had
28:46acquired in collaboration with
28:48Genentech that was recently published
28:50for lung cancer and very nice study,
28:53we were also able to demonstrate.
28:56Exquisite sensitivity to air positive,
28:58but also to some of the other subclasses,
29:01so extending it beyond air sensitivity.
29:03So we think that these are very useful
29:07approaches of unfortunately that toxicity
29:09at least in our hands is very high.
29:12And so I think the strategy now is really
29:15to try to come up with more specific.
29:18They're smart K2 inhibitors,
29:20but for prostate,
29:22the question is should we also be
29:23looking for smart K4 inhibitor?
29:25And so that's something that's
29:26of great interest.
29:27So just to summarize this
29:29part of the presentation,
29:30I wanted to just give you sort
29:32of the landscape of what's going
29:33on in prostate cancer.
29:35And with regards to resistance,
29:38there's genomic and epigenetic players,
29:41which I I mention are B and P53
29:44being two of the main players.
29:47Which are probably necessary
29:49but not sufficient.
29:50And then there's a very interesting stories
29:53developing in epigenetic regulation.
29:55I didn't talk about rest for the first part.
29:57I'll talk about rest which is an
29:59inhibitor of neural differentiation.
30:01I'll talk about that in the second part.
30:03And then I think that this is
30:05now emerging where based on these
30:08different alterations there may
30:10be different subclasses.
30:11So I just want to highlight,
30:12we've modified a little bit of review
30:14that we had a few years ago where I think.
30:17Microenvironment plays an important
30:19role and there are different
30:22pathways that are taken.
30:23Are they unidirectional, are they reversible?
30:26I think that's going to be a very
30:28important translational issue.
30:29And I think for those treating patients,
30:31obviously wanting to identify the time
30:33point where people will best respond
30:35to novel therapies will be important.
30:38So that's one.
30:39Thing I wanted to tell you about
30:41this morning and now I want to
30:43tell you about something that's
30:45that's entirely new to our group.
30:48And I when I go for bike rides
30:51around my house, I don't know,
30:53there are a lot of bike riders.
30:55I know David Rim might be
30:56listening and he's a bike rider,
30:57but when you are biking and you see
31:00that there's a slope of 27 degrees,
31:02that's really steep.
31:03I mean so in the in the Tour de France,
31:0720 is starting to become extremely.
31:09Challenging.
31:10I've been in races where I can't go up 20.
31:12I'm walking.
31:13So when I see this I'm always thinking
31:15one day I'm going to go down and
31:16try going up this, but I haven't.
31:18I haven't reached that day yet
31:19but I'm going to try that.
31:20Maybe I'll have to wait for an E bike,
31:22but I'm not there yet.
31:24But so I I think in our lab we'd like to
31:27take our challenges and when Anka Outback,
31:30who's a postdoc in my lab who
31:31had been working
31:32in Switch sniff Project, said what she
31:34really wants to work on is splicing.
31:37And she said I'm particularly interested
31:39in minor splicing. I had a problem,
31:42which was one I knew nothing about minor
31:44splicing and I had no idea whether
31:46this was really a good use of her time.
31:48So I think this is always
31:49important point for Pi to decide,
31:51OK, are we really going to?
31:53Take this arm.
31:54So I learned a lot and I'm going
31:56to tell you about what we learned.
31:57It's a it's a work in progress but
31:59I think it's a potentially it's
32:02exciting new area for us and helping
32:04me with my process of education was
32:07Rahul Canadia who's close by so he's
32:09up just up the road at UConn and his
32:11post dot Kyle Drake who very helpful
32:13and this is also a collaboration
32:15with Mark Gerson's lab here at Yale.
32:17So there's a very strong
32:19Connecticut component.
32:20So what I did know about splicing
32:21was I knew from Gunner Rich's group.
32:23The TCGA that splicing is is
32:26occurring very often in cancer
32:28that it can lead to NEO epitope.
32:30So I was more familiar with aberrant splicing
32:33in the context of potentially immunotherapy.
32:36What I was also aware is that the IT plays
32:39initially important role in the regulations.
32:42So this is a hallmark like figure
32:44showing all the different areas where
32:46splicing can play an important role.
32:48But I didn't know anything about minor
32:50splicing so I had to learn from Anka,
32:52Rahul and others.
32:54And so I knew that when gene
32:58genes create messenger RNA.
32:59But in order to do that you have
33:02to take the exons and somehow the
33:04introns have to be spliced out to
33:06get to go from a preeminent M RNA
33:09to an MRA that can be translated.
33:12And so the question is how does
33:14that actually occur?
33:15And I'm not expert but I have
33:17learned that the vast majority of
33:20introns are excised through the major
33:23spliceosome which is U2 splices.
33:26And in this place is home.
33:28There are small nuclear RNA's that
33:31recognize consensus sequences,
33:32both of the three prime and the five prime,
33:35but also branching points that
33:37allow for this placing to occur.
33:39So that's the vast majority of all proteins.
33:42And it turns out,
33:43and this is what Anka was interested in
33:45because of work she had done, her pH.
33:47D,
33:48That there's a minor spliceosome
33:50which recognizes introns that have
33:52a different consensus sequence.
33:54And it's also.
33:55Referred to as the U-12 splicer zone.
33:58And so this is something that's really
34:01very understudied in in homeostasis.
34:03I'll tell you a little bit about
34:05what it we think it does or what
34:07it's it's believed to do.
34:08But it's very important in cutting out
34:11minor introns that make that are part
34:14of of genes that also have major introns.
34:17So I'll show you what that
34:18what that means in a moment.
34:20So the idea is that in a typical gene that
34:22has a minor intron which we're
34:24going to refer to as minor.
34:25Intron gene or Mig.
34:28These genes have one minor intron,
34:31usually not more than one,
34:33and that it requires a specific
34:35machinery to cut this minor intron out.
34:38Now I just will focus for most of the
34:41presentation on one element of the minor
34:43spliceosome which is called Usix attack,
34:45which is one of the catalytic components
34:48of the minor spliceosome and that
34:51in homeostasis believed that under
34:53stress situations the conditions are.
34:56Such that that you six attack
34:58is is not degraded and it allows
35:01for minor splicing to occur.
35:03So whatever this gene is,
35:04it might be important in a stress situation.
35:07It allows now map kinase signaling
35:09occurs and allows for the excision
35:11of this minor intron M RNA to be
35:14produced and then translation
35:16of whatever that protein of that
35:18whatever that protein will be.
35:20So that's an important component.
35:22This is an evolutionary conserved.
35:26Events of minor splicing
35:28is not only in humans,
35:30but also throughout the evolution.
35:34My inner splicing has been maintained
35:37and there's some interesting exceptions.
35:39The main role of minor splicing.
35:44Is seen in development.
35:45So Rahul who's our collaborator is
35:48really an expert in neural development.
35:51And so in patients who have
35:53germline alterations or other
35:55alterations in minor splicing,
35:58they see developmental problems and there are
36:00many developmental diseases that described,
36:02described or attributed to
36:04errors in minor splicing.
36:07For cancer,
36:08there's only two really known diseases,
36:10so put Sieger and myelodysplastic
36:12syndrome that are.
36:13Associated with minor splicing alterations
36:16and in total in Toto they're around 750,
36:19maybe 800 genes that have a minor intron,
36:23which we can refer to as minor intron genes.
36:26So Anka asked some questions that I think
36:29are are pretty straightforward questions to
36:31ask in the in the beginning of this project.
36:34That is,
36:34do we see any evidence of minor
36:37splicing alterations in cancer,
36:39cancer progression?
36:40If so, is it preferentially during?
36:44Disease progression.
36:45So is this something that you see a more
36:48so in advanced or resistant disease?
36:50Is this an active functionally active event?
36:53And that would be very important
36:55if we're going to attribute this
36:57as a key causative role or playing
37:00a part in the cause of cancer
37:02progression or resistance.
37:03And is there what happens if you
37:05inhibit this and what do you can
37:07you reverse some of these features?
37:08And so I'll show you some of
37:09the work we have.
37:10It's a as a,
37:11as I mentioned this is
37:12a work in progress but.
37:14But we do have a first paper in revision,
37:17which seems like it's been a revision
37:18for I think almost a year now,
37:20but hopefully we're getting
37:22closer to that point.
37:23And so first study was in silico.
37:26So one of our collaborators for this
37:30project looked at computationally
37:33at protein protein interactions,
37:36taking 26 of the most well described
37:38prostate cancer genes and ask the
37:41question what is the direct protein
37:43protein interaction with these minor?
37:46And try and containing genes and
37:47as you can see on the right some
37:50very interesting genes and just
37:52highlight BRACA for example here
37:54are kinase a Mick which are genes
37:57that are very much associated with
37:59prostate but also other cancers have
38:01as a very close relationship direct
38:05interactions with minor intron.
38:08Containing genes.
38:09So that's sort of a first interesting hint.
38:12I'm going to tell you about minor
38:15splicing in disease progression,
38:16but before I just want to emphasize why
38:18I'm going to focus on you six attack.
38:20I mentioned that it's a catalytic component.
38:22So if you have a gene that has
38:25a minor intron,
38:26somehow the machinery comes together
38:27and has to cut out this minor intron.
38:31And the reason why U-6 attack we
38:33think is very important is because
38:35it's a dynamic component of this,
38:37of this process where it's
38:39really like the last step.
38:41So it has to come together with you.
38:43Four attack and this catalytic
38:45subunit now cuts out the intron
38:48and that's why it's probably very
38:50carefully regulated in homeostasis.
38:53So that's the reason why we're
38:54focusing on you six attack.
38:56And the idea is,
38:57as I mentioned that in stress we
38:59think that you six attack is used
39:00to help remove the minor intron for
39:02a subset of genes that are probably
39:05important in responding to stress.
39:06That's our hypothesis.
39:07So what happens if you look
39:10in prostate cancer,
39:11this is probably also true for other.
39:13This is, but we focus on prostate
39:15cancer using some of the common
39:17cell lines and patient Dr.
39:19Organoids and arranging them
39:20going from benign all the way
39:23to neuroendocrine disease.
39:24So trying to cover the spectrum,
39:26we see an increase in U-6 attack expression.
39:30As I mentioned there are other
39:31components of the minor spliceosome.
39:33They also show similar overexpression.
39:36We were able to then show this
39:39insight to using RNA ISH and we're
39:41able to show that you can see.
39:43Higher expression of these of the U-6 attack,
39:47but also other components as you
39:50look at primary prostate cancers,
39:53but also higher in primary prostate
39:55cancers that go on to metastasize
39:58and then in metastases even higher.
40:02What's important is,
40:03is this actually functionally doing anything?
40:06And So what Anka did was she used
40:09two vector systems that are designed
40:11so they have either 1 intron and the
40:14intron is either a minor or major intron.
40:17And with luciferous,
40:18with this luciferous assay she's able to
40:21demonstrate expression if it's working,
40:24so if it's working as a minor
40:28intron excising.
40:30Machinery or is there a major
40:33intron excising machinery working?
40:35And I think importantly when she looked
40:37at the major insurance splicing activity,
40:41it remained fairly similar
40:43throughout all the different types
40:45of of model systems she looked at,
40:48but only highly expressed for minor
40:50entrance splicing in the advanced cancers,
40:53which is intriguing suggesting
40:54that this activity is increased.
40:56And this is a very simple vector
40:58system and we've since developed.
41:00More complex vector systems that
41:02I could tell you about.
41:04At the transcript level,
41:05we see and these are just some of the
41:08the the cell lines in prostate cancer,
41:10we see very high expression of U-6 attack
41:13but also other minor splicing components,
41:16but then also the MIGS.
41:18So the minor intron containing
41:20genes are more highly expressed.
41:22So as we might expect now Mark Burstein's
41:26lab helped us with I think a important
41:29part which is also extending this to
41:31other cancers and the question really is?
41:34Are these Migs that we see that are altered
41:38or alternatively expressed in in cancer,
41:41are they potentially very useful in
41:44distinguishing different cancer types.
41:46So we would hypothesize that the
41:48makes are expressed but probably
41:49different in different tissue types
41:51just like we see in progression.
41:53And so in Mark's group is able
41:55to create these silhouette plot
41:57here looking at 23 different tumor
42:00types from a pan cancer analysis
42:02and they were able to show.
42:04That in this experiment where you go
42:07from 0% MIGS and then so there's a
42:10dilution experiment all the way to 100%.
42:13So this is done by doing many,
42:15many iterations.
42:16You can demonstrate that the best model
42:19is a model where you include the mix.
42:21So it shows that they have a very
42:24strong ability to distinguish
42:26different cancer types.
42:27But specifically for prostate,
42:28when we ask the question,
42:30if we look at benign prostate
42:33tissue from GTX.
42:34Database versus localized prostate
42:37cancer or advanced prostate cancer.
42:40We see that the MIG genes do a very
42:43nice job without in any selection
42:45of a subclass of of the Migs to
42:48distinguish the different groups.
42:50I think it's important because
42:52it suggests that I think that
42:53these genes for whatever reason,
42:55for evolutionary reasons,
42:56are important and stress,
42:57but they also are probably reactivated
43:00or useful for cancer progression.
43:03And as I mentioned in the first part
43:06of the presentation is very important
43:08when we think about resistance,
43:10think about probably two types of resistance.
43:13One is still related to AR
43:15signaling active tumors and we
43:17have to find ways to attack them,
43:19but also AR negative tumors.
43:22And I mentioned that there
43:24are these four categories.
43:25So the question really is.
43:28How can we gain any insight into that?
43:31Well,
43:31one of the things that Anka had
43:33read about and was known is that
43:36map kinase signaling is very
43:38important for you 6 attack stability.
43:40And so she asks a question,
43:43I'm using a A an antibiotic
43:45that stimulates map sign kinase
43:48signaling and ask them and also
43:51myosin and was able to demonstrate
43:54that when you activate MAP kinase
43:57signaling or Jack stat signaling.
43:58You see only increased expression of of
44:02minor splicing in the neuroendocrine tumors,
44:06which is sort of interesting,
44:07not in the AR sensitive tumors
44:10when she knocked it down,
44:12it was the same thing.
44:13So I think that's important.
44:15And then in a separate study looking
44:18at the effect of androgen stimulation,
44:22she was able to demonstrate in
44:24different model systems whether
44:25it's a lincat cell line which
44:27is very angry and sensitive.
44:29Or a line cap cell line that
44:32over expresses AR.
44:33She's able to demonstrate that
44:36minor intron activity is increased
44:38when you increase AR and and
44:41can be modulated through that,
44:43but not major entrance splicing.
44:46So basically I'm trying to think about how a.
44:51ASICS attack could be stabilizer or
44:53modulated AR signaling could play a
44:56role but also maps map kinase signaling.
44:58And so she started developing a working
45:01framework of of how to think about
45:04this thinking that neuroendocrine
45:06tumors might require map kinase
45:08signaling to stabilize you six
45:10attack and allow for a minor intron
45:13containing genes to be expressed
45:14whereas AR sensitive tumors might
45:16do it in different way through air
45:19signaling and we have more data.
45:21That helps support this.
45:22One thing that was nice is there are two
45:25high impact papers that came out talking
45:27about a subclass of prostate cancer
45:29that is really driven by Jack stat.
45:31So this is quite interesting.
45:32And these are stem like tumors which
45:35would fit in nicely with with the
45:38model of minor splicing being driven
45:41by a map sign map kinase signaling.
45:43So in just the last part I now I
45:47want to go into what happens if
45:49we try to target you 6 attacks.
45:51Specifically.
45:52And we hypothesize that this
45:55will have an A direct effect on
45:59the ability to to exercise these
46:02introns that are minor introns.
46:04And in fact that's the case.
46:05So when when Anka used a small interfering
46:11RNA's to knock down U-6 attack,
46:14she demonstrated using a a minor splicing
46:17index which looks for misplacing.
46:21So now now the introns are
46:22no longer being spliced.
46:23Out.
46:24And so the data is analyzed and it
46:26consistently shows in all the different
46:28model systems that if you knock out,
46:31if you knock down your six pack it
46:34functionally has the effect of of
46:36not allowing these minor intron
46:39containing genes to to excise out the
46:42entrance for the experts in splicing.
46:44I won't go into that.
46:46I'm not an expert in splicing,
46:47but cryptic cryptic splice site
46:49alterations seem to be the most common.
46:52But here you can actually.
46:54Determine the specific types of splice sites.
46:56So we have experts here,
46:57but I'm not an expert to talk about that.
47:01Anka performed transcriptomics and
47:03proteomics and in the context of
47:05knocking down you six attack the
47:07main finding I think are that in the
47:10different cell lines we saw different
47:12genes that are altered which I think
47:14goes in the to the view that this
47:16is going to be very context specific
47:18what the regulation of minor splicing.
47:20There were certain themes that emerged
47:22and I think the important theme
47:24that should highlight would be 2.
47:26So one would be cell cycle and also
47:29DNA repair were two themes that.
47:31Came out when we look at the
47:33common genes that are altered in
47:35these different model systems,
47:37and so here's an example of the
47:39David analysis where it shows some
47:41of the common themes and cell cycle.
47:45As well as DNA alterations,
47:48came came out as being altered when
47:51you knocked down you six attack.
47:54In single cell sequencing fact fax
47:56analysis not showing all the data
47:58just to just to highlight that we
48:00see at G1 arrest when you knock
48:03down you six attack supporting the
48:05view that that minor splicing plays
48:07an important role in cell cycle
48:10in a series of experiments where
48:12she looked at both cancer cells.
48:14So this is a Antrim receptor sensitive
48:19castration resistant tumor that is still
48:21probably sensitive to AR signaling.
48:24And you knocked down you six attack you
48:26see a decrease in in growth of tumor cells.
48:30We see no change when you look at
48:32either mouse fibroblasts or human
48:34fibroblasts and in cocultures one
48:35of the reviewers I think is a good
48:38point do we see preferential changes
48:40occurring in a Co culture and we we we
48:44actually see only the tumor cells are
48:47affected by by you six attack knockdown.
48:50This is probably the key
48:53therapeutic translational slide.
48:54Which is that in air sensitive
48:56tumors if you treat with enzalutamide
48:58or knockdown usix attack you see
49:01basically the same result which is
49:03a decrease in confluence of cells.
49:05So this is using Incyte.
49:07If you then go to cell lines that
49:09are AR resistant and there's just
49:11two cell lines but we've done it or
49:14more are you see that the tumors are
49:16no longer sensitive to enzalutamide
49:18or the antiandrogen therapy but
49:20continue to be very sensitive to
49:22knocking down you six attack which.
49:24Is A is a promising first step
49:27for thinking about does this have
49:29any therapeutic translation,
49:31although many,
49:32many steps away from actually
49:34having therapeutic translation.
49:35We extended this to the Memorial Sloan
49:38Kettering and Cornell patient Dr.
49:40Organoids that represent a range
49:42of air positive and air negative
49:45prostate cancers and we see the same
49:48effect that you can by knocking down
49:51you six attack you can decrease.
49:54Cell viability and confluence.
49:56I'll skip the videos,
49:58but we can also see this in benign
50:00prostate cell lines that are that we have,
50:03as well as the cancer cell lines.
50:08If you can
50:09move, OK. So the last,
50:11the very last piece I just want to bring
50:14up is a interesting concept and this
50:16is something we call poison peptides.
50:19Maybe somebody's used this in another
50:20context and maybe it's not the right context,
50:22but right now we're just working
50:25title is poison peptides.
50:26And the idea is,
50:28is that when the minor introns
50:31splicing occurs it it performs a
50:34protein that it potentially plays
50:36an important role in cell cycle.
50:39And help stabilize the cancer cells,
50:41but when it's not excised,
50:43the question is what what's happening
50:46with these message and is it,
50:48is it becoming just,
50:49is it just undergoing degradation
50:51or are there some sort of aberrant
50:54or other types of isoforms that are
50:56formed and just want to show you an
50:59example that we were quite intrigued
51:01with which is related to rest.
51:03So rest is a transcription factor plays
51:06an important role in neural fate regulation.
51:09In development and so most
51:11of us know about rest,
51:12rest when rest expression is
51:15present in in cancer types,
51:17we know that it it prevents
51:19neural differentiation.
51:20And when it's down,
51:21we expect that you may see neural
51:24differentiation and this is in cancer.
51:26We see this occurring quite often.
51:28There's also a isoform that's that's
51:31known but little known and that's called
51:35rest four and rest 4 forms a dimer.
51:39With rest one and prevents it from
51:41binding to DNA and therefore it
51:44allows neural differentiation.
51:46So if risk four is present,
51:48you do have neural differentiation
51:49and this is just showing in
51:51a slightly different way.
51:53So there's a small there.
51:56There's a small mini intron here,
51:59a small Exxon here that needs
52:02to be excised in order for you
52:06to go from rest 4 to rest one.
52:09So what I wanted to show you is
52:11that then Anka asks the question,
52:13well what does she see as far as rest
52:16expression as expected it's very low
52:18in neuroendocrine prostate cancers,
52:19this from our stand up cancer data,
52:22but she sees higher expression
52:24of risk for in these cases.
52:27So that's something we had never looked
52:29at because we never thought about
52:30looking at the different isoforms.
52:32And if you think about the
52:34endogenous expression of risk
52:35for in the different cell lines,
52:36we also see the same thing.
52:38So very low risk 4.
52:40Expression in the neuroendocrine tumors
52:42when she knocks down you six attack,
52:44she increases rest rest expression.
52:48But I think what's really interesting
52:50is if you look at the protein
52:52level and you look at the different
52:54isoform she's able to demonstrate
52:56in neuroendocrine model said if you
52:58knocked down Usix attack and again
53:00this is not a perfect but it's a
53:02it's a beginning of a developing
53:04a hypothesis that she sees a
53:07decrease in rest for so.
53:09At least the hypothesis is developing
53:11that we can think that in the normal
53:14state of these advanced prostate
53:16cancers that are neuroendocrine,
53:19they have high levels of rest
53:21four which prevent arrest
53:22from being functional and allow
53:25for neuroendocrine maintenance
53:26or differentiation and that
53:28knocking down you six attack.
53:30But clearly other ways of modulating
53:32rest could also lead to a situation
53:34where you knock down rest for it
53:36and you increase rest anyway.
53:38So I'll I'll leave you with just nice.
53:40Image of Switzerland,
53:41so Nice Lake that we like to go
53:44hiking around and and just a summary.
53:46So I've told you about minor splicing
53:49how there's a it's really a very
53:51small component of the spliceosome
53:53and that we think U-6 attack is
53:56is quite interesting because it
53:58plays an important catalytic role
54:00and potentially represents an
54:02important opportunity for therapy.
54:05And most important slide is just
54:07to make sure I acknowledge all
54:09our great collaborators.
54:10So in addition to.
54:12Bronco with the Ruben Lab members
54:14as well as members from Rahul
54:16Canali's group at at the at the
54:19University of Connecticut and Mark
54:21Gerstein's group here at Yale.
54:22They've been really,
54:23really helpful in letting us think
54:25about this and and really broad terms.
54:28Thank you very much for your attention today.
54:34Happy to take any questions.
54:39OK. Questions. So yeah.
54:43So the floor is open.
54:47Questions for mark.
54:52I I can get started with a
54:54question that was really nice.
54:56Thank you very much and wonderful
54:58to hear about both of those stories
55:00on the Swiss knife complex and on
55:03the the minor intron splicing.
55:04So one of the questions that I have
55:07and it's something that that that's
55:10something that we think about a lot
55:14as well is the sensitivity of of
55:17tumors to smarka for modulation for
55:20example and one of the things that.
55:22You mentioned was or what it seems
55:26like these protax seemed to be
55:29most effective in tumors that
55:32are AR sensitive to which is kind
55:35of interesting if we think about
55:38smart before perhaps exerting its
55:41functions in the resistant tumors.
55:44And I was wondering if you have
55:46if if if you have any thoughts
55:48on sort of that paradox?
55:50Well, if it is a paradox,
55:52yeah. So I'll just expand a little bit.
55:56So I think the things,
55:57just to reiterate what I said
55:59though also and you're well
56:01aware of is that if you do any.
56:04Synthetic lethal screens, smart K4 and
56:06Mark 2 come out as always winners,
56:09they're always there, right.
56:10And so I think that the Protex in effectively
56:14you know hitting both regardless are
56:17you know great targets I think for.
56:20So the question is why is it so?
56:22Why are the AR sensitive tumors.
56:24So you know why are they
56:27exquisitely sensitive to this.
56:29We think that that's not the case.
56:31So we actually think that it extends beyond,
56:33it's not just.
56:35They are and the the complexity we
56:37have is is that we think that AR that
56:41smart K4 is something you want to
56:44decrease and but we know that if we
56:46do that smart K2 has to be maintained.
56:49So there's a problem with the
56:51the Protex is extremely toxic and
56:53so how to get around that.
56:55The other thing also I'm sure you're
56:57aware and you've maybe seen this is
56:59that when you use the protects you
57:01can actually get nice resistance
57:02very quickly and as you expect.
57:04Where would the resistance be?
57:06It's in VHL or and and and one
57:08of the genes associated with how
57:10the protects are are designed.
57:12So we have nice experiments I didn't
57:14show but where we've you know in cycle
57:17through resistance which is going to
57:19be different than the ATP a type resistance.
57:22So in a rules paper I think it
57:24was exciting to see that you can
57:26potentially target cancers.
57:27I think there's a bit of skepticism
57:30in the issue of toxicity because
57:32I think the field and in in Bob.
57:35The box paper from Genentech,
57:36I think they nicely described that
57:38the toxicity is a major issue and
57:41so since a specificity whether
57:43it's for Smarca 2,
57:45but we think it would be interesting to
57:47have smart K4 specificity for prostate.
57:49So I don't,
57:49I don't know I mean it's but we don't
57:51have either so at this point so.
57:55Thank you. Yes. So yes, please go ahead.
58:20You might you might have to
58:21repeat the question for me
58:22because I didn't hear it.
58:26OK.
58:36Do you think you could?
58:37I think it would be hard.
58:38Would you like to come out?
58:39I think you have to say it in the microphone
58:42so people can hear it who are listening.
58:44And also I I can't hear very well, so.
58:49So this is thank you. So what's your name?
58:54So we have a guest speaker here.
58:56My question was about smarka force,
59:00the sensitivity with the protests
59:02and the prostate cancer cells.
59:04I'm wondering if you think that
59:07the smart effort like the the BRG
59:10catalytic subunit is active within us,
59:13why sniff chromatin remodeler?
59:15Enzyme complex in in the context
59:18where it is being affected or
59:20if you think it has a separate.
59:23Activity and I'm wondering because
59:25you mentioned EH2 as well,
59:27I know can have Polycom independent
59:29functions where it associates
59:30with the ENERGEN receptor or
59:32something similar might be going on.
59:34So that's a great question.
59:35So and and the way that I didn't show
59:37data but the way that I think we've
59:40been going at this has been to look
59:43at also there are ATP ace inhibitors.
59:45So we also have data for
59:47the ATP ACE inhibitors.
59:49The protects are more effective,
59:51but they're more toxic and so
59:55but inhibiting a TPA does.
59:59You know make it have a similar
01:00:00effect but again it's not specific
01:00:02to this market force market too.
01:00:04So it's hard to dissociate that and
01:00:06we have SSI data with CRISPR data for
01:00:08knocking out what happens if you knock
01:00:10out smart key forwards market too.
01:00:11So that's one problem.
01:00:13The other problem that we have
01:00:15which didn't discuss at all is
01:00:17that it's really difficult to
01:00:19chip these these proteins,
01:00:20some people can do it.
01:00:21So Cigar Codex Group is is world
01:00:23expert in that we we haven't been
01:00:25able to achieve that unfortunately.
01:00:30Other questions, yes.
01:00:39The role the
01:00:40role of progesterones as
01:00:42well as androgens. Estrogen,
01:00:44OK. So that's a that's
01:00:46one of my favorite topics.
01:00:47I love that topics the role of
01:00:49estrogen in advanced prostate cancer.
01:00:51So I think it's it's it seems like
01:00:53it's paradoxically it shouldn't
01:00:55be that important but estrogen
01:00:57receptor and antrum receptor they
01:00:59have very similar binding sites.
01:01:01So there's a lot of half binding
01:01:03sites that are are are regulated.
01:01:04So I think it's important role and
01:01:07that it's also known that other
01:01:09nuclear hormones are activated and
01:01:11the context of AR depletion so.
01:01:14Do you think it's important and
01:01:15in prior studies we've seen it,
01:01:17it's part of progression,
01:01:18but we didn't specifically focus
01:01:19on that here. So it's.
01:01:29In plasticity it's role in plasticity,
01:01:32so the role of estrogen and plasticity.
01:01:36Or progestin or progesterone.
01:01:39So I think I think in stem in stemness.
01:01:41So I think Charles Sawyers group
01:01:43has looked at that and I think
01:01:46there's the view that it does play
01:01:48a role in resistance and is seen
01:01:50associated with stem like state,
01:01:52but maybe not neuroendocrine state. So
01:01:55yeah. Thank you, Joe.
01:02:01Thank you for coming out.
01:02:07My question is about the therapies for.
01:02:11As you know. Ready for those people?
01:02:17Show that you're.
01:02:23Or is there any incremental attrition? To.
01:02:29Or any.
01:02:34Well, I'm a pathologist.
01:02:35I better not comment on,
01:02:37on clinical oncology therapy.
01:02:38But the only thing I would
01:02:40say is that there's a lot,
01:02:41there's great interest in you know
01:02:44epigenetic regulation which are toxic.
01:02:46And you know close friend of mine Johann
01:02:49Debono was just with him the other day
01:02:51in Basel and we were talking about this.
01:02:53So I think there are a number of
01:02:54studies that are coming down the line,
01:02:55but it may not be you know,
01:02:57so I presented these different categories,
01:02:59it may be for a R negative.
01:03:02But not neuroendocrine tumors that there are,
01:03:04you know, some new therapeutic targets.
01:03:07Her three is the target.
01:03:09There are other targets that are coming up,
01:03:12some common to lung cancer as well,
01:03:15but no winners yet,
01:03:17no successful winners.
01:03:19And then hopefully I pointed out in
01:03:20the first part of the presentation how
01:03:23difficult it is to actually classify these.
01:03:25So once it's air negative,
01:03:27I think there's still complexity.
01:03:29So we're far behind breast
01:03:31cancer and maybe lung cancer.
01:03:33As far as being able to accurately
01:03:35classify what needs to be treated.
01:03:37So I think that in all the and
01:03:39so in the in the advisory boards,
01:03:42I think the common conclusion after
01:03:43two or three days of discussions
01:03:45are always the same,
01:03:46which is that we just need to treat
01:03:49everybody and see who what works.
01:03:51Which is sort of depressing,
01:03:52but it's that's that's sort
01:03:54of where we are I think.
01:03:56I think we have similar issues
01:03:57in lung cancer and resistant
01:03:59tumors trying to figure it out.
01:04:02What pathologist. So that's just
01:04:04my personal opinion, not don't,
01:04:06don't take that. Beyond that,
01:04:08we have one last question in the back.
01:04:12Roll off. Thank you.
01:04:17So like the cluster.
01:04:23I'm sorry, I can't hear from
01:04:25do you think there is a role
01:04:27for differentiation therapies
01:04:28in prostate cancer kind of
01:04:30analogous to the retinoic acid and
01:04:32there would be. So.
01:04:35So one thing I just maybe not
01:04:37directly relate to your question but.
01:04:39Being able to model differentiation
01:04:42would be phenomenal.
01:04:44So if we had model systems where it
01:04:46could show I know like in AML and
01:04:48other cancer and other hematopoietic
01:04:49cancers you can show differentiation
01:04:52of blocking differentiation and I
01:04:54think we don't have those models.
01:04:55I think the closest thing we have right
01:04:58now is develop of like the stem like
01:05:01state and then you know ideally we'd
01:05:03like to flux back between adenocarcinoma.
01:05:05So we're starting to see some
01:05:07of the organoid models and.
01:05:09Mouse, but we don't really
01:05:11have that ability yet.
01:05:12So I do think what you're saying is
01:05:15interesting and we my view would
01:05:16be that if you could tell which
01:05:18tumors are going to transition,
01:05:20you'd want to treat them as early as
01:05:22possible with the other element in
01:05:24addition to AR targeted therapy before
01:05:26it goes down the road to differentiation.
01:05:30Next. By those standards. Yeah.
01:05:39Have you tried like stemness
01:05:41targeted like therapies?
01:05:42Yeah, we haven't, but that's
01:05:44obviously there's a lot of interest.
01:05:46So you saw the, I showed two papers
01:05:49from main paper from Charles Sawyers
01:05:52Group and looking at Jack Stats.
01:05:54So they're very interested in
01:05:57various therapies related to
01:05:59that to targeting stemness.
01:06:01I mean our approach was related to
01:06:03the epigenetic approach and you know.
01:06:07Thank you, I.