Back Home: Mechanisms of Neural Mimicry in Melanoma Brain Metastasis

Transcript

• 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.