The Minor Spliceosome: Exploring Novel Cancer Vulnerabilities

Transcript

• 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.