Back Home: Mechanisms of Neural Mimicry in Melanoma Brain Metastasis
March 15, 2023Information
Yale Cancer Center Grand Rounds | March 14, 2023
Presentation by: Dr. Eva
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- 00:00Friday, and thank you so much Harriet
- 00:03and Marcus for your kind invitation.
- 00:05I promise I'll go back in person and
- 00:08hopefully with less technical issues.
- 00:11So as all of you know,
- 00:13probably even better than me,
- 00:15Melanoma is the fifth most common
- 00:17cancer in men and sixteen women.
- 00:19And can you please keep clicking?
- 00:21I'm going to go fast through this first
- 00:23slides because it's just background.
- 00:24As you know, the incidence of
- 00:26Melanoma continues to increase.
- 00:27It double s every 20 years
- 00:29and luckily this is like.
- 00:31Unfortunately,
- 00:31it's not a updated the mortality
- 00:34associated with Melanoma is starting to
- 00:37decrease and this is you definitely to
- 00:40the development of better therapies.
- 00:43For the past 15 years,
- 00:46Melanoma has become the poster
- 00:48child for many targeted therapies
- 00:50as well as immunotherapy space.
- 00:52Please keep clicking,
- 00:54but all of you know there is still
- 00:57despite the success of this.
- 01:01Uh,
- 01:01we're developing immunotherapy treatments.
- 01:04We still can keep clicking.
- 01:06We are still facing significant resistance
- 01:11to some of these treatments or a.
- 01:18Year um side effects.
- 01:20So approximately 40% of the
- 01:23metastatic Melanoma patients have
- 01:24not benefited from the advances
- 01:26that have been done over the past
- 01:28years in the treatment of Melanoma.
- 01:30So this takes me, can you please go on to my,
- 01:35the, the focus of my lab.
- 01:37Can you go back?
- 01:39Yeah,
- 01:39which evolves around these questions,
- 01:43right, the fact that we still need
- 01:45better markers for patient selection,
- 01:47understand.
- 01:48Where we check the patients that are
- 01:50going to uh go on to recur after the
- 01:52initial resection of their tumor,
- 01:54which ones are going to metastasize.
- 01:57And also in particular,
- 01:59as Harriet mentioned,
- 02:00we've developed an interest in
- 02:02understanding the biology of CNS metastases,
- 02:05particularly of brain metastasis
- 02:07because they show less level responses
- 02:10to immunotherapy and the incidence
- 02:12is increasing as patients are living
- 02:15longer and surviving from other systems.
- 02:18Astasis, so my lab is focused
- 02:21into aspects of metastasis,
- 02:23the initial steps of early dissemination,
- 02:25we now know that primary tumors,
- 02:28especially in Melanoma.
- 02:29Is that uh,
- 02:31disseminating start sharing cells that
- 02:33go into circulation and metastasize
- 02:36really early on in the process.
- 02:38So we want to understand,
- 02:39please click uh,
- 02:40which are the mechanisms that
- 02:43drive the metastatic behavior of
- 02:46certain melanomas and not others.
- 02:49We know that unity alterations
- 02:51don't explain this behavior because
- 02:53none of the well characterized
- 02:55genetic alterations of Melanoma
- 02:56or combinations of those genetic
- 02:58alterations can explain why.
- 03:00And primary tumors recurred,
- 03:02metastasized and some others don't.
- 03:05And also we want to understand
- 03:07what is the contribution of
- 03:09intratumoral heterogeneity.
- 03:10So we know now that despite the
- 03:13fact that primary tumors can have
- 03:15a relatively homogeneous genetic
- 03:17profile inside those tumors,
- 03:19we can recognize groups of cells with
- 03:22different transcriptional programs,
- 03:23what we call transcriptional states.
- 03:25And there is a recent search for what
- 03:28are the programs for their states.
- 03:30That drive these metastatic behavior
- 03:32and people have pointed to the neural
- 03:35Crest like or the EMT program on the
- 03:37other side of the equation.
- 03:39We are also trying to understand
- 03:40what happens at the end of the
- 03:42process once the cancer cells,
- 03:44in this case Melanoma cells have already
- 03:46extravasated into those distal organs.
- 03:48As you know Melanoma metastasis to the
- 03:50lung delivered in the brain and we are
- 03:53interested in understanding what are the
- 03:55site specific adaptations in those sites,
- 03:57where are the type of metabolic changes.
- 04:01The cell types of the cancer
- 04:03cells are interacting.
- 04:04And what is this crosstalk between the
- 04:06cancer cells and their environment?
- 04:07How is shaping the ability of animal cells
- 04:09to adapt and grow in those environments?
- 04:12And I will have to stories
- 04:15presenting today if time permits,
- 04:17one that focuses more in just the
- 04:20metastatic potential in general and how
- 04:22muscles cops some of the programs of the
- 04:24neural Crest cells from which they arise.
- 04:26And also in the second part of the talk,
- 04:30I will talk more specifically.
- 04:31About to bring metastasis and how Melanoma
- 04:35cells mimic some of the processes
- 04:38that happen during neurodegeneration,
- 04:40particularly to suppress new inflammation
- 04:42and be able to grow within the brain.
- 04:45So diving into the first story,
- 04:47these are the four areas in which my
- 04:49lab has been focused in recent years,
- 04:52epigenetic alterations, noncollinear,
- 04:54a post translational modifications
- 04:56particularly like oscillation
- 04:57and site specific adaptations.
- 04:59So moving on,
- 05:01can you please move these slides one more,
- 05:04the first story that has been
- 05:07recently accepted for publication
- 05:09is the work of three people.
- 05:12It evolve over time and we have.
- 05:15Uh,
- 05:15tightly muscles invoke and you like clear
- 05:18sticky genetic program during metastasis.
- 05:20So the genesis for this project was a very
- 05:24simple premise and it's the fact that,
- 05:26well,
- 05:27we all know that neural Crest cells are
- 05:29the cells of origin of melanocytes.
- 05:30And these are among the most totipotent
- 05:33and among the most invasive and migratory
- 05:36cells of our body and is well known
- 05:40now if you keep clicking that Melanoma
- 05:42cells adopt programs characteristic.
- 05:45You address the cells during the progression
- 05:48from primary to metastatic cancer.
- 05:50So we wanted to investigate which
- 05:52epigenetic changes happen during
- 05:54melanocyte differentiation from the
- 05:55neural Crest to the melanocyte that
- 05:58potentially could be reversed in the
- 06:00transition from primary to metastasis.
- 06:02And we did this by looking
- 06:05specifically at DNA methylation.
- 06:07There are of course many other
- 06:09mechanisms of epigenetic regulation
- 06:10being the enumeration one of them.
- 06:12So if you move to the next slide.
- 06:16This is what we did basically was
- 06:17a comparison of four data sets.
- 06:19So in one hand I cannot use my pointer,
- 06:22but you can see that we have 4 columns.
- 06:25We have neural Crest cells that were
- 06:27obtained from a collaborator in France.
- 06:29These are human cells.
- 06:31We also had the human melanocytes
- 06:33from different donors.
- 06:35And as you can see there is
- 06:36kind of a mirroring pattern.
- 06:38We were looking for CPG islands that
- 06:41were either hypomethylated in the
- 06:43conversion from neural Crest cells.
- 06:46To Milano sites that were progressively
- 06:48hypermethylated from primary to
- 06:50metastatic Melanoma or the converse,
- 06:52if you see at the bottom,
- 06:54we have some CPG islands.
- 06:57If you can click again,
- 06:58you will see there are some CPG
- 07:00islands around the gene called NR
- 07:0222 that are hypomethylated in the
- 07:04neural grass cells that become
- 07:07hypermethylated in melanocytes and
- 07:09progressively hypomethylated from
- 07:11primary to metastatic Melanoma.
- 07:12And this really call our attention
- 07:15because it really represented.
- 07:16Example of a potential gene or or
- 07:19candidate program to be modulated
- 07:22during neural Crest differentiation
- 07:24to melanocytes that was reversed
- 07:27during the progression from primary
- 07:29to metastatic Melanoma and what
- 07:31is first of all an R2 of two,
- 07:33so it's a nuclear receptor
- 07:35is also called cooked.
- 07:37TF2 is an orphan nuclear receptor.
- 07:39We don't know still what
- 07:40is the natural ligand,
- 07:41although retinoic acid can bind
- 07:44it at high concentrations.
- 07:46This is the motif that it binds,
- 07:47and as you can see the NRF 2 full
- 07:51isoform has a DNA binding domain.
- 07:54It has a ligand domain.
- 07:55So it's a conventional nuclear receptor
- 07:58that can form a **** or heterodimers.
- 08:01It is essential if you,
- 08:04yeah,
- 08:05it's essential for development and
- 08:07particularly for the formation of blood
- 08:09vessels and it has been shown to be
- 08:12important for neural test differentiation.
- 08:15Now there have been already some studies
- 08:17showing the role of NRF 2IN cancer,
- 08:20particularly pancreatic and
- 08:22prostate cancer metastasis,
- 08:24whereas in breast cancer it
- 08:25had a controversial effects.
- 08:27So you can say well you know this,
- 08:29this may already be known
- 08:30that this factor is important.
- 08:32Cancer, however,
- 08:32if you click one more slide,
- 08:35what really cool our attention is
- 08:37that the CPG islands that we found
- 08:40to be differential differentially
- 08:42methylated in neural Crest cells
- 08:45versus melanocytes and then later
- 08:47on in primary versus Melanoma,
- 08:49we're actually affecting exactly that
- 08:53region in the transcription start
- 08:56site that controls the expression
- 08:58of an alternative isoform isoform 2.
- 09:02Which is not the one that has
- 09:04been commonly characterized.
- 09:05So most studies have focused on
- 09:06the full length isoform, isoform,
- 09:08one that has the DNA binding domain,
- 09:11the like minded domain.
- 09:13But this methylated region or the
- 09:16methylated region is in atss that
- 09:18gives rise to a truncated isoform
- 09:20that lacks the DNA binding domain.
- 09:22So on the right you can see that as
- 09:25we had shown before the CPG islands
- 09:30controlling this this nuclear receptor.
- 09:32Networks to ISO two are hypomethylated and
- 09:36they appear in green in cells and stem cells,
- 09:40but completely hypermethylated
- 09:41in melanocytes,
- 09:42and this is also described here at the
- 09:45bottom where you can see the beta value.
- 09:47The beta value of 1 means completely
- 09:50methylated and a better value close to
- 09:530 represents a hypomethylated gene.
- 09:55This completely corresponds to gene
- 09:57expression because you can see that
- 10:00whereas isoform one is expressed in ESL.
- 10:021000 monocytes isoform 2 is expressed
- 10:04in your cells and neural Crest cells,
- 10:07but not expressed at all in melanocytes.
- 10:10OK.
- 10:10So in when you look at the human samples,
- 10:13you can see and that even though
- 10:15it's not completely
- 10:16black and white, what we see is that
- 10:18there is an increased percentage of
- 10:21hypomethylation of isotope in the metastatic
- 10:23cases compared to the primary cases.
- 10:26You can see there is from 30% approximately
- 10:29to more than 50% of the samples have
- 10:33hypomethylation for NR2ISO2 and if you
- 10:35keep clicking you will see that you can
- 10:38see there is a progressive increase.
- 10:40In the better value from Levi
- 10:43to primary and metastasis,
- 10:45so more hypomethylated and conversely
- 10:47increase expression from primary to
- 10:50metastasis with a very nice correlation
- 10:52between M RNA expression and methylation.
- 10:55Is exactly, if you look at
- 10:57the protein levels,
- 10:58we were able to develop actually an
- 11:00antibody specific for isoform 2,
- 11:02which was challenging because
- 11:03there are only 15 amino acid symbol
- 11:05amino terminal that that specific.
- 11:06But also two you can see that there is an
- 11:08increase in the expression of isoform 2.
- 11:10There are few cells that are positive
- 11:13in the primary cases and this
- 11:15population expands in the metastasis.
- 11:17This is something that we are actually
- 11:20trying to understand now whether
- 11:22there is a selection for those cells
- 11:25that expressed isoform 2 over time.
- 11:27And again this is this is something
- 11:30we're coming time to investigate but
- 11:32if we can go on we we can see that
- 11:35isoform one the one that is the full
- 11:38length isoform is not modulated by
- 11:40methylation is completely hypomethylated
- 11:42in ES cells you know cells melanocytes
- 11:45and both in primary and metastatic
- 11:47Melanoma and there is no correlation
- 11:50between expression and methylation.
- 11:52So isoform one is always there,
- 11:54but is isoform 2 the one that.
- 11:56Is normally not expressed in melanocytes,
- 11:58but is increasingly demethylated
- 12:01and expressed from primary to
- 12:03metastatic Melanoma.
- 12:05Now of course the question is,
- 12:06is this isoform tool doing
- 12:08anything in Melanoma metastasis?
- 12:10And for that we had to move to cell
- 12:13lines and again we found this pattern
- 12:15in which melanocytes and some melanomas
- 12:17aliens had complete hypermethylation
- 12:19and lack of expression as you can see
- 12:21here on the right and you can also see.
- 12:27Hypomethylation in some of the
- 12:29cell lines and this corresponds
- 12:31to expression and the same can be
- 12:33seen at the level of protein.
- 12:36You can see isoform 2 expressed only in
- 12:37those cells that have hypomethylation.
- 12:39So this represents a good model to study
- 12:41loss of function and general function.
- 12:43But before I go into that you can see that.
- 12:49The methylation uh status can also
- 12:52be seen in short term culture.
- 12:54These are cells isolated from
- 12:56patients and again you have some,
- 12:59some of these short-term cultures
- 13:01have hypomethylation and some
- 13:03of them have hypermethylation.
- 13:05So this is not an artifact of invitro
- 13:08culture and it happens in cells derive
- 13:13very shortly from from patients.
- 13:16So if now we move into cells that.
- 13:20Have hypermethylation like mayor cells.
- 13:22If we treat them with editing agent
- 13:25like 5 ASA you can see that there is
- 13:28an induction of isoform 2 and this
- 13:31is seen also in short term cultures
- 13:34in which treatment with FAFSA results in
- 13:37induction of isophorone to expression
- 13:39with no change in isoform one.
- 13:41Now when we silence isoform to
- 13:43using the specific srnas against
- 13:45these isoform that don't have
- 13:47any impact on the other isoform.
- 13:49Initially we did not see any
- 13:52effect onto the proliferation,
- 13:54but we observed a clear decrease
- 13:56in the ability to form colonies
- 13:59in soft Agar or to form a sphere
- 14:02spheres upon single cells.
- 14:03So these are properties that
- 14:05are characteristic of metastatic
- 14:07cells and basically measure the
- 14:09ability of the cells to grow under
- 14:12very stressful conditions.
- 14:13Now if we overexpressed,
- 14:14sorry before we go into their expression.
- 14:17This is the experiment in in this case.
- 14:20We injected on the cancer cells
- 14:22intracardiac in a conventional
- 14:24in a model of metastasis that
- 14:25we use very often in the lab.
- 14:27And you can see that the silencing of
- 14:30isoform 2 between independent HR and
- 14:32I had a very significant decrease in
- 14:35metastatic potential in this model,
- 14:37which is quantified here on the
- 14:39right by bioluminescence as well
- 14:41as fluorescence intensity when
- 14:43we extract the organs.
- 14:44So it seems like isoform 2 silencing
- 14:48suppresses metastasis in these.
- 14:50A model and now we move into
- 14:52overexpression systems in which
- 14:54ectopic expression of isoform 2IN
- 14:56cells that have hypermethylation has
- 14:59no effect again into the culture,
- 15:01but it has a significant ability to
- 15:04increase the number of colonies and
- 15:06soft tagar as well as sphere formation.
- 15:08In vivo we injected these cells
- 15:10in the flank of the mice and then
- 15:13we did survival surgery.
- 15:14You can see that we cover two more
- 15:16area because there is always a lot
- 15:18of signal that comes even after
- 15:19you have resected.
- 15:20The majority of the tumor is the
- 15:23subcutaneous tumor and you can see
- 15:25that isotonic expression enhances
- 15:28metastasis both by bioluminescence
- 15:30as well as by histological analysis.
- 15:32We did this also by intracardiac
- 15:34injection and again we observed
- 15:36the same effect with an increase
- 15:38in metastasis overall.
- 15:39So it seems like ISO two,
- 15:42this particular truncated isoform of
- 15:44the orphan nuclear receptor and R2F2
- 15:47is able to promote metastasis and is.
- 15:50There's to be required in some
- 15:52models to in most of the models
- 15:53that we tried in lab and the paper,
- 15:55we have three or four different models.
- 15:58You can see a decrease in metastasis when
- 16:01you're silence or knockout this change.
- 16:03So now what are the programs
- 16:05that are modulated by isoform 2?
- 16:07We did RNA sequencing to get to this
- 16:10question and one of the pathways
- 16:12that was significantly modulated was
- 16:14EMT DPL to mesenchymal transition.
- 16:17Now we went on to demonstrate that
- 16:19a lot of the typical genes involved
- 16:22in the epithelial transition where
- 16:24silence in different cell lines
- 16:26when we deplete isoform 2.
- 16:29And here you can see a validation
- 16:32for snail which is reduced both
- 16:35transcriptionally and at the protein
- 16:37level when we silence ice form 2.
- 16:41Importantly, I as I mentioned
- 16:43at the beginning of my talk,
- 16:45now we have an understanding of
- 16:48intratumoral heterogeneity Melanoma
- 16:49and we have seen that most melanomas
- 16:51both in mouse and human display
- 16:54different transcriptional states,
- 16:55intermediate neural Crest
- 16:57like proliferation EMT or more
- 17:01melanocytic or differentiated.
- 17:04So we were curious to see whether
- 17:06NRF 2 and particularly the signature
- 17:08of genes modulated by isophorone.
- 17:11Who were particularly enriched in any
- 17:13of these transcriptional States and as
- 17:16you can see in this heat map, the EMT,
- 17:18in particular the transcriptional
- 17:20state that corresponds to an
- 17:23epithelial to mesenchymal transition
- 17:25seems to be the one that is more
- 17:29enriched in these two signature
- 17:31together with the neural signature.
- 17:33So this goes together with our findings
- 17:36that if two could be regulating the EMT.
- 17:41Um estate and in this additional
- 17:43analysis in which we we used scenic
- 17:46to understand the regulations the
- 17:49the basically the epigenetic and
- 17:51transcriptional factors that control each
- 17:54of these states melanocytic intermediate
- 17:56proliferative neural Crest like an EMT.
- 17:59You can see an I think it's more
- 18:01visible in the next slide that the
- 18:04top regulon controlling the event
- 18:06signature and this is a completely
- 18:08different analysis done in our mouse.
- 18:11Levels of Melanoma you can see that
- 18:14inner 2F2 is at the top is a top regulon.
- 18:18Controlling the EMT signature.
- 18:20So it seems like these transcriptional state
- 18:23of epithelium to mesenchymal transition
- 18:25seems to be mostly controlled by NF2 now.
- 18:29How this happens?
- 18:31Like I told you at the beginning that N2I2,
- 18:34another two ISO two is a truncated isoform.
- 18:38It lacks the DNA binding domain.
- 18:40So how is it possible that it has this
- 18:43capacity to control gene expression?
- 18:45Now when we started to work in this isoform,
- 18:48there were only two other papers that were
- 18:52studying alternative isoforms of two,
- 18:54all the papers that have been put out there.
- 18:58And study only the full length isoform.
- 19:01So there were two papers.
- 19:03One was suggesting that I2 acted as a
- 19:07dominant negative and was not removing
- 19:10or displacing isoform one from chromatin.
- 19:13With us another paper suggested the opposite
- 19:15that I saw two contributed to bind ISO,
- 19:18one to the chromatin and also what
- 19:20complicates more this interpretation
- 19:22of the results is the fact that
- 19:25isoform one has been or inner 2F2 has
- 19:28been described both as a receptor
- 19:30as a repressor of transcription as
- 19:32well as an activator.
- 19:33So we.
- 19:36It tested and the possibility that
- 19:38NRF 2 ISO two was interacting with
- 19:41isoform one and somehow modulating
- 19:43the ability of an artist to to bind
- 19:46chromatin and regulate gene expression.
- 19:47But for this to be true also from
- 19:50two had to have the capacity to
- 19:53bind to isoform one in the nucleus,
- 19:55and isoform 2 lacks the nuclear
- 19:59localization signal present in isoform one.
- 20:02It has an alternative nuclear localization
- 20:04signal that is much less potent.
- 20:06Thought we first show in a A
- 20:11fragmentation analysis that isoform
- 20:132 is able to reach the
- 20:16nucleus. You can see there not
- 20:19only the cytoplasm, right?
- 20:20So this suggests the possibility that
- 20:23it can interact with iPhone one.
- 20:26Then we also did IP analysis
- 20:28where we show that isoform one
- 20:30can pull down isoform two with two
- 20:32independent antibodies and the
- 20:34opposite is also true, we can use.
- 20:36Mile a GFP pulled down because
- 20:39they unfortunately the endogenous
- 20:41IP for I2 doesn't work well.
- 20:44So we use the exogenous construct
- 20:46that has a GP fusion and so we
- 20:49can pull down again isoform too
- 20:51with the isoform 1 suggesting
- 20:53that it's two isoforms interact.
- 20:55And when we look now at Chief of
- 20:59isoform or chromatin IP of isoform one,
- 21:03we can see that indeed when we silence
- 21:05isoform 2 there is a group of peaks.
- 21:07Of isoform one that now lose
- 21:10binding of isoform one,
- 21:11and this is more dramatic in this
- 21:14group of targets is a little bit
- 21:16less prevalent in this subset of of
- 21:19pigs that are bound by isoform one.
- 21:22But we can also observe the opposite.
- 21:24We can see that in a group of peaks there
- 21:26is an increased binding of isoform one.
- 21:28When I do is gone.
- 21:30So definitely the interaction between
- 21:32these two isoforms is complex.
- 21:34We know that the chip works because.
- 21:38Um and active two is their most
- 21:41significantly enriched transcription
- 21:42factor in these pics together with
- 21:45some other transcription factors
- 21:47which are we're also interested
- 21:49in in looking at the potential
- 21:51interaction of MR2 with those.
- 21:53So we can see that N22ISO2 modulates
- 21:57the binding capacity of the chromatin,
- 21:59but it not only in a way seems to be
- 22:03in a in a gene specific manner and
- 22:05when we integrate the cheap data.
- 22:08Our transcriptomic sequencing of cells
- 22:11in which we have depleted ISO two,
- 22:14we found that the majority of the genes are.
- 22:17A significant portion of the genes that
- 22:19are downregulated when we sell in size.
- 22:21Or two that are targets for an R2F2
- 22:24are genes that are involved in the EMT,
- 22:27snail, twist, Peter two, etc.
- 22:30So it seems like a lot of the NR2ISO1
- 22:34targets are actually in the direct.
- 22:38Same teachings.
- 22:39The opposite is that when we see
- 22:42look at the genes that are now
- 22:45upregulated by isoform 2,
- 22:46the majority of those genes are
- 22:49involving differentiation and
- 22:50pigmentation such as tyrosinase,
- 22:52DCT, etcetera.
- 22:53So with all of these,
- 22:55and still with many questions still open,
- 22:58we propose this modeling which
- 23:00in primary tumors isoform 2 is
- 23:02hypermethylated and silence.
- 23:04So the dimers of isoform 1 prevail over
- 23:07the heterodimers of isoform 2 and isoform 1,
- 23:10whereas in metastasis we see a displacement
- 23:13of the equilibrium towards the home,
- 23:16the heterodimer.
- 23:17Sorry,
- 23:17because now isoform 2 is present
- 23:19and is this heterodimer that allows
- 23:21the expression of neural Crest and.
- 23:24Empty genes such as twisted slag etcetera.
- 23:27So what we are now in the process
- 23:30of understanding is how these
- 23:32heterodimer is able to activate
- 23:34this EMT genes and we think that
- 23:37in part could be due
- 23:38to the interaction with third parties
- 23:41like additional transcription factors
- 23:43that are specifically attracted
- 23:45to the complex like isoform.
- 23:47So we are doing our time and other
- 23:49techniques to understand what are the
- 23:52complexes that are attracted in bound
- 23:54to the chromatin where we have the
- 23:56homodimers versus the heterodimers.
- 23:58Now moving on into the second
- 24:01part of my talk,
- 24:02I don't think I have to repeat the
- 24:04conclusions because that's basically
- 24:06the summary that I just have provided.
- 24:08So in the interest of time,
- 24:10I will move to the second part of the story
- 24:13in which we have focused on brain metastasis.
- 24:16This was the work mostly done by.
- 24:20I actually incorrectly stated PhD
- 24:21is an MD PhD from our laboratory,
- 24:24Kevin Kleffman that is now a.
- 24:28Accident at mass general.
- 24:31So we of course are interested in
- 24:34brain metastasis because it's an
- 24:36important and met clinical need.
- 24:38And although these tumors can
- 24:41respond to immunotherapy,
- 24:43we know that these responses are mostly
- 24:45seen in patients that are asymptomatic
- 24:47and in the symptomatic patients the
- 24:49responses are much poorer and these
- 24:51patients have overall very poor survival.
- 24:53So this remains very important
- 24:56clinical question and definitely.
- 24:58Fascinating biological, um.
- 25:00Uh,
- 25:01question.
- 25:01How Melanoma cells adapt to the
- 25:04brain microenvironment and why
- 25:05they have such a profound tropism
- 25:07for the brain is still something
- 25:10that we don't entirely understand.
- 25:12So uh in collaboration with the management,
- 25:15the director of the Melanoma program at NYU,
- 25:17we develop Melanoma short-term
- 25:19cultures that in some cases are
- 25:22derived from the same patients.
- 25:23So we can in some of the cases we
- 25:25were able to obtain a short-term
- 25:27cultures derived from a brain
- 25:29metastasis and extracranial metastasis
- 25:31from the same patient.
- 25:33And again this is a very
- 25:35difficult comparison to make,
- 25:37but we think it's very useful
- 25:39because it reduces some of the
- 25:41inter tumoral heterogeneity.
- 25:42As we observe what genetically
- 25:44and transcriptionally and what was
- 25:47really exciting to us is that when
- 25:49we labeled these cells with GFP
- 25:51luciferase and inject them back into
- 25:53mice with intracardiac injections,
- 25:55we observed that that metastatic the
- 25:59the the short-term culture that has
- 26:01been derived from the brain has in
- 26:05general more metastatic potential
- 26:07than the one that was derived
- 26:08from an extracranial metastasis.
- 26:10This is represented here on the right,
- 26:12but more specifically.
- 26:13The one that derives from the brain
- 26:16has more ability to metastasize to the
- 26:18brain and this is measured here as a
- 26:21ratio of brain to body luminescence.
- 26:23So it seems like this is a short term.
- 26:25Cultures retain some of the properties,
- 26:29some of the ability that they
- 26:31had gained in in people,
- 26:33in the patients of colonizing the brain
- 26:36and therefore could be a good model
- 26:38to study brain specific adaptations.
- 26:40So we went on to conduct.
- 26:44For the Omega analysis of these short-term
- 26:47cultures, in total we profile 25,
- 26:49approximately 12 and 13 brain
- 26:52metastasis versus second metastasis.
- 26:54Only a few of them, of course,
- 26:56were pair,
- 26:56the rest were unfair and the
- 26:58idea was to try to identify proteins that
- 27:00were differentially expressed in the brain.
- 27:02Metastasis input could be potential
- 27:05drivers of the adaptation.
- 27:07The first reply is that we found when
- 27:09analyzing the data is that the majority
- 27:11of the proteins found differentially
- 27:13expressed where proteins involved.
- 27:14Being neurodegenerative disorders
- 27:16such as Parkinson's, Alzheimer's,
- 27:18Oxfords and this was rewarding because
- 27:22our collaborator in at in the Anderson,
- 27:25Mike Davis had previously found that
- 27:27a lot of the proteins involved in
- 27:31different study transcript transcriptional
- 27:33profiling of brain metastases wouldn't
- 27:36reach in Oxford or proteins involved
- 27:39in the respiratory chain mitochondria
- 27:41and so this was confirmed in our.
- 27:45Plans.
- 27:45We found that short-term cultures of
- 27:48brain metastasis had elongated mitochondria,
- 27:51and they also had increased oxygen
- 27:54consumption rate in this seahorse analysis.
- 27:57But what we focus on was in the differential
- 28:00expression of proteins involved in
- 28:03Alzheimer's and Parkinson's disease.
- 28:05In particular,
- 28:06we landed for this study on AP.
- 28:09The amyloid processing protein there
- 28:11is a precursor for amyloid beta.
- 28:15It was induced in pre metastasis
- 28:17compared to metastasis,
- 28:18but not only AP itself,
- 28:20but the proteins that leave AP into
- 28:23amyloid beta like beta secretase
- 28:26or present presently.
- 28:28So we decided to modulate the loss
- 28:29of a P to see if it had an effect
- 28:32on brain metastasis.
- 28:33And initially we found that
- 28:37sorry went too fast.
- 28:39We found that supernatants
- 28:41of brain metastasis, Dr.
- 28:44short-term cultures,
- 28:44had higher secretion of family beta
- 28:47compared to the extracranial brain
- 28:49metastasis not only in our own hands,
- 28:51but also in short term cultures obtained
- 28:55from collaborators that we study institute.
- 28:57Silence.
- 28:58AP Again, we found no effect
- 29:00in proliferation in culture,
- 29:02but when we inject these cells
- 29:05intracardiac in immunodeficient mice,
- 29:06we observed this reduction of
- 29:09the brain to body ratio,
- 29:11suggesting that the loss of AP was
- 29:13particularly affecting brain metastasis.
- 29:15This was confirmed by histological analysis.
- 29:18This is entertaining of NUMA,
- 29:20which is a human marker,
- 29:22and therefore it can perfectly
- 29:25mark the cells that are.
- 29:27The deriving from the from the scenography,
- 29:30from the implant and you can see that
- 29:32there was a very significant reduction
- 29:35of brain and one positive cells but no
- 29:39effect on kidney or liver metastasis.
- 29:41We did ex vivo imaging MRI to conduct
- 29:46volumetric analysis that show us that
- 29:49we're not only less perimeter studies
- 29:52but also smaller brain metastasis and
- 29:55of course we show that this effect.
- 29:57Happens, you know other models,
- 29:59this is not a brain Tropic Melanoma cell
- 30:01line type one and we use national approach,
- 30:04in this case a crisper cast 9,
- 30:06to show again a reduction in
- 30:09brain metastasis.
- 30:10But this of course open a lot of questions.
- 30:12The first one is which step offering
- 30:16metastasis is the one in which
- 30:18is particularly required for the
- 30:20adaptation and the the arrival
- 30:22of Melanoma cells to the brain.
- 30:24So as you know brain metas is
- 30:26a complex process.
- 30:27It involves multiple steps,
- 30:31the intravasation from the tumor
- 30:33into the first into the stroma,
- 30:36then the intravasation into the vasculature,
- 30:38survival in circulation,
- 30:39and when the cells arrive to the distal site,
- 30:41in this case the brain,
- 30:42they have to again extravasate.
- 30:44Many of these cells will undergo
- 30:46cell death or become dormant,
- 30:48but those that are able to proliferate
- 30:51and survive in this environment will
- 30:53form micro and macro metastasis.
- 30:55So when was abeta required for this?
- 30:58For this process,
- 31:00so Kevin embark himself in really
- 31:03a difficult task of monitoring the
- 31:06kinetics of cancer cells injected
- 31:08intracardiac in these mice.
- 31:10So he did brain slice immunofluorescence
- 31:13and a lot of confocal microscopy
- 31:15and was tracking this GFP positive
- 31:18cells in the brain over days.
- 31:20So you can see that just one day after.
- 31:24In the cardiac injection,
- 31:25these cells are stuck in the vasculature.
- 31:27They even have the shape
- 31:28of the blood vessels.
- 31:29This is a tomato, tomato,
- 31:31lectin marking the the blood
- 31:33vessels you can see there.
- 31:35At day three they start extravasation,
- 31:38they start getting out of the vasculature.
- 31:41Some of these cells die.
- 31:43A lot of these cells die in the
- 31:45blood vessels or outside when they
- 31:47are able to extravasate as sustained
- 31:49by Clifton Space Stream and you
- 31:51can see that later on they start
- 31:53crawling through the blood vessels.
- 31:55In these process called Vascular Co option,
- 31:58they can later on form micrometastasis
- 32:00that day 14 and finally micrometastasis.
- 32:03So when you compare the kinetics
- 32:06of control cells here in the
- 32:08black line to those that lack app,
- 32:10you can see that the first steps
- 32:13of the kinetics are really similar.
- 32:15There is this big crisis where most of
- 32:17the cells that are able to extravasate
- 32:20die either in the vessels or right
- 32:23after extravasation but then after they 7.
- 32:25When the control cells are able to start
- 32:28expanding and proliferating happily,
- 32:30the ones that lack APP can no longer
- 32:34grow after the first or second division
- 32:36and they eventually disappear.
- 32:38They are they are dead.
- 32:41So we wonder which effects were required
- 32:45for for this role of ebata in the brain.
- 32:49And remember that this is a very
- 32:51complex environment where there are,
- 32:53you know, the resident myeloid cells,
- 32:55the microglia.
- 32:55Macrophages in some cases will
- 32:57matter derived macrophages that
- 32:59attracted to the tumor and it excels
- 33:01interfiling for sites and astrocytes.
- 33:03So we first look at astrocytes
- 33:05as a potential.
- 33:09Still of interest, because of the
- 33:11literature that had shown previously
- 33:14that activated astrocytes can be Co
- 33:16opted by the cancer cells to support
- 33:18the growth in the brain environment.
- 33:20And this is indeed the case.
- 33:22Also in our models where we see that
- 33:24if you look at the left panels,
- 33:27you can see that over time,
- 33:29as the muscles arrive to the brain,
- 33:31these are the control cells,
- 33:32you can see an increased presence
- 33:35of GFP positive astrocytes.
- 33:36So there is.
- 33:38Some equipment of activated astrocytes
- 33:40we cannot distinguish if it's
- 33:42recruitment versus activation of
- 33:45the surrounding astrocytes to the
- 33:47point that they form this network
- 33:49of active astrocytes that is called
- 33:53active Astro cytosis supporting
- 33:57the Melanoma micrometastasis.
- 34:00So what we observe is that cells
- 34:03that lack APP are unable to
- 34:06trigger these reactive.
- 34:07Cytosis around them.
- 34:08When they arrive to the brain,
- 34:11there is a significant reduction
- 34:13of positive cells of GFP.
- 34:16Positive cells around the cells
- 34:18demand muscles that lack APP,
- 34:20suggesting that perhaps a beta is important
- 34:23in triggering these Astro cytosis.
- 34:26Now, in data that I don't
- 34:28have the time to explain,
- 34:30we also show that Amelia better not only has
- 34:33the capacity to activate the astrocytes,
- 34:36but also.
- 34:38Can suppress the phagocytosis
- 34:40coming from the microglia.
- 34:42So we can see that there is a
- 34:44reduction of neural inflammation
- 34:45in the presence of family beta.
- 34:48So we think that Ali beta secreted
- 34:50by cancer cells can have multiple
- 34:52effects in the brain metastasis,
- 34:54macular vironment, particularly on
- 34:56the astrocytes and the microglia,
- 34:59but also it can influence,
- 35:01as it has been reported in Alzheimer's,
- 35:03the interaction with the endothelial cells.
- 35:06So of course these open,
- 35:09these findings open some
- 35:11possibilities and therapeutic
- 35:13opportunities because of all the.
- 35:16Armamentarium of drugs that have
- 35:20been developed against America beta,
- 35:22some of them beta secretase inhibitors and
- 35:26more recently anti American antibodies,
- 35:29some of which have been developed
- 35:31for clinical use and in some
- 35:33cases even approved by the FDA.
- 35:35So these open the possibility of
- 35:38repurposing some of these drugs which
- 35:40are generally safe for brain metastasis
- 35:42and for proof of principle we've
- 35:44been testing some of these compounds.
- 35:46In collaboration with Eli Lilly,
- 35:48so we obtain beta secretase
- 35:51inhibitors in the diet of the mice.
- 35:54So initially we injected a cancer cells
- 35:57Melanoma cells in these mice and gave
- 36:00them a better second base inhibitor
- 36:02in the food or controlled diet.
- 36:05And you can see how this reduces the
- 36:07number of brain metastasis in this model.
- 36:10This is a short term culture but
- 36:12also in the five one Melanoma cells.
- 36:15Now of course this is more a prophylactic.
- 36:17Model because treatment starts
- 36:18at the time of injection,
- 36:20so we raise the bar a little bit
- 36:23by allowing the cells to establish
- 36:25metastasis first and then after 21
- 36:28days we gave doxycycline to the food
- 36:30and the in the water of the mice
- 36:33to activate docs inducible SH RNA.
- 36:36And again we saw that in this context,
- 36:38even when the treatment is initiated,
- 36:39once metastasis have been formed,
- 36:41we can see a reduction in the number
- 36:43of brain metastases and if we do
- 36:45the same thing with the Secretary.
- 36:47Keep in touch. We can also see again
- 36:50after initiating the treatment.
- 36:52Once the micrometer studies have been formed,
- 36:55we can see a reduction in brain
- 36:57metastasis we are currently trying to.
- 37:02Moving to well before I go into that,
- 37:05we are now testing the antibodies against
- 37:10Emily Beta either as a monotherapy
- 37:12or in combination with immunotherapy
- 37:14to see if we can recapitulate
- 37:17the same effects of surf here.
- 37:19So to summarize this part of the talk,
- 37:21we have shown the proteomic studies
- 37:24have revealed a novel connection
- 37:27between brain metastasis and
- 37:29neurodegenerative pathologies.
- 37:30This has now been confirmed by other studies.
- 37:32We collaborated with a group of men,
- 37:34iser, last year.
- 37:35We got a study done in single cell
- 37:39analysis of primate testis that also
- 37:43recapitulated these these finding
- 37:45that Melanoma cells mimic the.
- 37:49A neuronal pathways in another of the
- 37:53alterations that are seen in you know,
- 37:55degenerative disorders once
- 37:56they reach the brain.
- 37:58We see that Amelia Beta is particularly
- 38:01required for pre metastasis and
- 38:03not other sites of metastasis is
- 38:05acquired for steps that happen after
- 38:07extravasation and early survival
- 38:09in the brain parenchyma and among
- 38:12the multiple functions of family.
- 38:14But in this context we have seen that it
- 38:16triggers an anti-inflammatory response
- 38:18in the astrocytes and suppresses.
- 38:20Your inflammation, as I mentioned,
- 38:23we are also studying now whether
- 38:26these effects of family beta can
- 38:28be seen also in other models.
- 38:29So we have done a spatial
- 38:31transcriptomic analysis.
- 38:32In this case,
- 38:33it's not a Melanoma model,
- 38:34is the 41 model which is a breast
- 38:37cancer triple negative model
- 38:39that also colonizes the brain
- 38:41after intracardiac injection.
- 38:43These are on the top is a is a brain,
- 38:47is half of a brain of a sham
- 38:49mouse and this is the.
- 38:50Mouse,
- 38:51this is a mouse that was injected
- 38:53with the 41 cells and what I want
- 38:55to bring to your attention is that
- 38:58in this the next vision analysis we
- 39:01can see that the gfap positive cells
- 39:04around the areas where the tumors are,
- 39:07you have to probably take me you know
- 39:09take my my word these are the areas
- 39:11where the tumors are circle here
- 39:14and we see a special expression or
- 39:16increased expression of GFP around
- 39:18those tumor cells we also see.
- 39:20This one is 100 which has been seen
- 39:22it used in tumor cells in the brain
- 39:25environment in other cancer types.
- 39:27And interestingly we find these
- 39:29signature that we have that has
- 39:32been previously reported as the an
- 39:35Alzheimer's associated microglia
- 39:37signature that is a combination
- 39:39of 10 markers and is again seen
- 39:43particularly activated around the
- 39:46brain metastasis here in here.
- 39:49So it seems like perhaps these Alzheimer
- 39:52like response in the microglia genes
- 39:54around the brain metastasis cells
- 39:56could be a more general finding and
- 39:58not only characteristic of Melanoma.
- 39:59As I mentioned,
- 40:00we are now very excited by studying
- 40:03whether app genetic and pharmacological
- 40:06inhibition extends to other cancer
- 40:09types and whether the combination of
- 40:12beta secretase inhibitors or antibodies
- 40:14can work alone or in combination
- 40:17with checkpoint checkpoint locate.
- 40:19In immunocompetent models,
- 40:20I was hoping to tell you about a
- 40:24another very exciting story in the lab,
- 40:27but I I see the clock and we are reaching
- 40:29the 1:00 PM. So I will stop here and.
- 40:35This is this data I wanted to present
- 40:38but perhaps at the second occasion
- 40:40and just wanna thank all the members
- 40:43of the lab for their contributions.
- 40:46This work that I presented was
- 40:48mostly done by the first part,
- 40:50which are Veronica and Claudia in
- 40:53NRF 2 and Ali and Maya in CDP one.
- 40:57And Kevin Kleffman led the I'm a
- 41:02little better story and I want to thank
- 41:04of course all our funding sources.
- 41:06And thank you all for your attention
- 41:09and your patience with the technical
- 41:11issues at the beginning.
- 41:12I'll stop here and take any questions.
- 41:14Thank you.
- 41:17Alright, thank you so much, Eva.
- 41:19I'm going to ask folks to
- 41:21put questions in the chat.
- 41:22We only got a couple of minutes,
- 41:24but while people type in the questions,
- 41:26maybe I don't know if Marcus
- 41:28has any or I can ask one.
- 41:30Go ahead Marcus, I've got plenty.
- 41:34So either really Congrats also on the,
- 41:37you know the nature communications paper.
- 41:39And I was wondering with the NR2F2 story,
- 41:42you're probably aware of Chris,
- 41:44Marines work in Nature last fall on TCF 4.
- 41:49And I'm kind of wondering if you think
- 41:53your NR 2F2 is upstream of TCF four if
- 41:55you've seen any role there and there's
- 41:57probably going to be a subsequent
- 41:59story about resistance to therapies,
- 42:01so there's.
- 42:02Multiple parts to this question.
- 42:05A, the upstream downstream part,
- 42:06but then also the heterogeneity
- 42:08because you're talking about these
- 42:10things happening sort of uniformly.
- 42:12But I'm imagining that the epigenetic
- 42:14regulation is kind of cell by cell
- 42:16and that there's going to be a
- 42:17population of cells that have more
- 42:19or less of that along the way.
- 42:22All excellent questions and and
- 42:24these are all the questions that
- 42:25we are trying to address now.
- 42:26So,
- 42:26so we didn't see a total overlap
- 42:29between our population of N2F2
- 42:32with Chris Marines population.
- 42:35It seems like his population is smaller
- 42:39one inner two seems to be more broadly.
- 42:43I expressed in both EMT and also partially
- 42:47in the neural Crest like population.
- 42:49So his population seems to be a more,
- 42:51I wouldn't say minority,
- 42:52but it's a smaller population
- 42:54and not not directly overlap.
- 42:58You also ask the other challenge that
- 43:01we have is that a lot of the single
- 43:04cell analysis that have allowed us to.
- 43:06Distinguish this.
- 43:07Transcriptionist states don't have,
- 43:10don't allow us, don't don't have the
- 43:12death to look into isoforms, right?
- 43:14So you really have to have a different
- 43:17library preparation and pipeline to
- 43:19identify the different types of forms.
- 43:22So when people look at R2,
- 43:24they are just examining an R2 ISO one,
- 43:27the full length and the one that
- 43:29is really switching from the
- 43:32the primary to the metastatic,
- 43:34the one that is really triggering the EMT.
- 43:36Like program is only isoform
- 43:392 because of this.
- 43:41Balance between home and headliners.
- 43:45Yeah. So we we're definitely
- 43:46trying to understand what is the
- 43:48error key between these pathways,
- 43:49where is upstream, it seems to be
- 43:51one of the critical regulators.
- 43:54So again when we do this.
- 43:57Um, analysis is sending analysis,
- 43:59but still we don't know which
- 44:00one is upstream of which, right?
- 44:02Yeah, that's a very good question.
- 44:04And then you ask.
- 44:05The dynamics right of this
- 44:07of this population.
- 44:09So we are now in the process
- 44:11of labeling endogenously this
- 44:12isoform to be able to trace them.
- 44:14We want to understand whether
- 44:17these isoforms are, you know,
- 44:20dynamically regulated during the
- 44:22metastatic process and we have
- 44:24different labeling systems now
- 44:25that allow us to sell to monitor
- 44:28single cells during metastasis.
- 44:30So I hope we will have soon an
- 44:31answer for that, but we don't know.
- 44:33I I also dissipate that it would change.
- 44:36From the primary tumor as the
- 44:38cells switch from the more
- 44:40proliferative to the more invasive,
- 44:42and then back when they arrive
- 44:43to them at the static side.
- 44:45But still we don't have
- 44:46full evidence for that.
- 44:51So, but there were a couple of
- 44:53questions in the chat both relating to
- 44:55Alzheimer's disease and brain metastases.
- 44:58One is there an increased incidence
- 45:00in brain metastases amount Alzheimer's
- 45:02disease patients and the other one was
- 45:05whether a postmortem you see Alzheimer's
- 45:08clogs in patients with brain metastases.
- 45:11Good questions.
- 45:12So we haven't seen brain metastasis,
- 45:15we haven't seen plaques in brain metastasis.
- 45:18We look for for them,
- 45:19we don't think that they get to accumulate,
- 45:22so we don't, we don't think that the
- 45:25processing is wrong is to simply induced.
- 45:28So we see more soluble abeta being produced,
- 45:31but we don't see oligomers and we don't
- 45:33see plaques. We did that staining.
- 45:35That conversion is done by pathologists
- 45:37to to look at the plaques and we couldn't
- 45:39see either in our models nor in human.
- 45:41Examples and that's the reason why we
- 45:45think that the antibodies that we wanna
- 45:47try are antibodies that design against
- 45:49the soluble and libetta and not against
- 45:51the plaques which are the ones that
- 45:53have been now or the oligomers which
- 45:55have been now approved by the FDA.
- 45:57Now you ask the other question,
- 45:59the incidence in in Alzheimer's.
- 46:01So we'll look into that and we didn't see
- 46:06a epidemiological studies and association
- 46:09between Alzheimer's and brain metastasis.
- 46:13But normally remember that in the majority
- 46:15of the neurodegenerative disorders,
- 46:16there is an inverse correlation
- 46:19between cancer incidence and
- 46:21neurodegenerative disorders,
- 46:22particularly Alzheimer's and Parkinson's.
- 46:25And even though there is
- 46:26not a positive correlation,
- 46:27there is no negative association.
- 46:29Now there is a reported association
- 46:32between Parkinson's and and
- 46:34Melanoma brain metastases.
- 46:36And I mean I think Harry,
- 46:39this is skeptical or not, I see you.
- 46:43No, there's a slight increase,
- 46:45but anyway, I don't know that it's brain
- 46:47metastases specifically, it's just.
- 46:50Come on. I'm sorry. Sorry.
- 46:51Yeah, yeah, yeah. I misspoke.
- 46:52I I meant that. I know my kid.
- 46:53Right.
- 46:54Yeah.
- 46:55Yeah.
- 46:56No,
- 46:56but there's a really interesting
- 46:58observation there.
- 46:59So thank you and thank you so much
- 47:01for this amazing presentation.
- 47:02I have more questions,
- 47:04but I'm going to e-mail them to you.
- 47:06I don't believe there anymore in the chat.
- 47:08We appreciate your patience with all
- 47:09the technical challenges and thanks
- 47:11for virtually visiting next time.
- 47:12It'll be in person.
- 47:14Thanks for wonderful talk to
- 47:16really fascinating work.
- 47:17Thank you,
- 47:18Harriet.
- 47:18Looking forward to see you
- 47:20soon and thanks everybody.
- 47:22Bye.