Cancer Beads

>>> good afternoon, everyone. glad to see everybody here. welcome to those on the web. very interesting wednesday afternoon lecture from david sabteeny today. involving regulation of growth by the mtor pathway. one of those pathways that

everybody seems to be working on right now. and we have a remarkable opportunity to hear from somebody who really began much of his effort as a graduate student and built much of the foundation upon which at the moment we understand, this

critical path way that regulates cell growth. david got his undergraduate degree at brown, graduating magna cum laudey and then went on to the md-ph.d. program at john's hopkins where he began this effort with his thesis being control of translation by

a novel rapamycin sensitive signaling pathway. sounds like something we might hear about today. his advisor for that was sal schneider. somebody that many of us learned a lot from. he went on to a position at the

whitehead institute as a fellow, a wham which provides particularly creative and independent-minded young scientists an opportunity to be independent at an early stage and a program that we have recently modeled a bit with our own early independence program

that is being supported through the common fund. he was obviously successful in that regard, became a member of the white head in 2002, as well as a professor through the ranks at mit where he is now full professor and member of a coke center for integrative cancer

research as well as being hhmi investigator and a member of the broad institute. his lab has continued to lead in this whole very critical area involving mechanisms of cell growth regulation with the major focus being on the target of rapamycin, the tor pathway and

i'm sure he will be telling us the latest developments in that regard, with all of its consequences for understanding basic science and for applications to many different clinical circumstances. so it's wonderful to have david here with us this afternoon,

please join me in welcoming david sabatini. [ applause ] y. >> thank you very much for organizing this visit. i'm always a little horse. show if someone has trouble hearing me, please let me know.

it is true i have been working on this pathway which will come up pretty soon. for a long time, since i was a graduate student, you could see a good thing, maybe it shows your perseverance and also might say it is remarkably uncreative of me.

for a long time i felt that and tried to stop working on the mtor pathway when i moved from my graduate work to the whitehead. i didn't have been how to do anything else. so at the end of the day, i had to continue on this system.

so i'll tell you a little bit of stories around the tor pathway, particularly a new story which has interesting clinical, one of the major regulators of growth, size control. i think one of the really interesting aspects of this pathway, that distinguishes it

from other pathways that regulate growth, it does it at all different lying call levels, so it's a major cell size. all biological levels. organ size, which i'll share a little bit with you, as well as body size control which we are really interested in but haven't

done almost anything in that area. interesting aspect of the mtor pathway, the first insights come from chemical biology and the study of a molecule, when i used to first talk about this, i had to introduce, although rapamycin is a famous molecule.

this is a bacterial product that comes from easter island off the coast of chile, it has a number of clinical applications which are shown here, particularly lately. a lot of interest in rapamycin which are analogues in the treatment of cancer and although

at the beginning, this was not a very promising avenue, i think as we understand this pathway better, what are the mutations that leads to activation, it is seeming to be a fruitful approach. what took rapamycin from the scientists and the clinicians to

more of the public sphere is the finding listed here at the bottom. this molecule in a number of different organisms, most notably from the public's attention in middle-aged mice prolongs lifespan. this led human beings to start

taking rapamycin low dose off prescription to hopefully prolong their lifespan, ignoring things like this. whether i have given this talk at times, i have had people come up to me say david, police stop listing immunosuppressant therapy because will you

dissuade people from taking rapamycin to improve their lifespan. that is an odd thing. it's an fd akt approved immunosuppressant. this is not a molecule i would take for recreational uses. i'd like to use this old slide

that illustrates whether i think the big picture pointed about this system that kept our interest for so many years. mtor is a large protein kine space responds to anything you give to the cell. so any kind of nutrient, any kind of growth factorux any kind

of stress. that tells you the cell cares a lot about regulating the activity of this system. it pretty much regulates any process that uses or generates large amounts of energy. so for example, translation, biogenesis of ribosomes, large

percent of the cell mass accounts by ribosomes. a number of other processes that are not on this slide, for example lipid synthesis, so this is a major regulator of anabolic and catabolic processes. now because it senses these things, which i think in

aggregate, we like to think of as equivalent to food, so glucose, amino acids, growth factors such as insulin and igf and things that respond to the state and regulates these anabolic and catabolic processes as we move some of our work in the in vivo direction, i'll give

you a little bit of a taste for, we are increasingly thinking of this system as a major modulator of the response or availability or lack of food and whether the organism is an anabolic or catabolic state. depending on where in the life cycle it is, it may lead to

certain kinds of responses nor a growing animal to, animal that is smaller when it reachessa adulthood. now there are two mtor pathways, one we call the mtor complex pathway. the better known pact way -- pathway and another one called

the m tower 2 pathway. it -- mtor 2 can be considered part of the pi3 kinase pathway. so it's a regulator of akt kt and has important rolls in tumors that have hyperactivation i'll toll you about a new connection to this pathway to cancer.

if we think about this pathway in vivo, we manipulated the this is in a mouse liver. a wildtype liver and this is the activity of this pathway. here we inhibited the pathway and here we activated. what is more interesting to look at is what is the response of

this liver in response to fasting or feeding? so the liver loses about 25% of its mass when you fast animal 24 hours b40% if you fast it 48 hours and beyond that the mouse can die by three day fast it is not legal for us to do at mit but if it has been known to be

done, it is not good for the life of animal. when you look at this liver, you can see it no longer shrinks in response to fasting suggesting the response to the fasting, this liver is as small as it is going to get. in contrast, the liver that is

in activative pathway is resistant to these effects although not completely. when we think about the types of signals they need to respond to make the decision to grow or de-grow, which is what is happening in this situation, and the re-feeding situation is the

growing, what we like to think about is the two classes of signals that matter. there are the local nutrient levels. no cell should try to grow in the absence of the building block for mass and to generate energy.

but also in a multicellular organism, all the humoral crew that is tell the cell throughout the body that the organism is in the fed state. so a pathway like the mtor pathway needs to integrate these kinds of signals to make the grow/no grow decision.

we have become particularly interested in what i would say is the signal integration problem for this pathway, which is particularly acute for the mtor pathway because it senses so many different things. so we know a fair amount at the molecular level how this

happens. and a lot of it happens through a tumor suppressor called tsc, mutated in the disease called tumor sclerosis complex. many signals feeding through other genes mutated in cancer talk to tsc and then negatively regulates a small protein called

reb and when it is loaded, it activates mtor 1 kinase activity and drive growth. in our hands, rhew is essential. genetically it's equivalent of deleting this complex in our hands. now the signal that is not illustrated here and we have

become particularly interested in again in the last few years, are amino acids. this is one of the ancestral signals and is quite a bit of evidence it doesn't go through this path way. instead, it appears that amino acids signal a different class

of small proteins, which operate as heterodimers. so these are quite interesting proteins. when we first started studying they were poorly studied. they are part of the ras family. all you can see is the gtp binding site.

they have a gtp akt se domain and a c-terminal domain. we have 4 of them. but the part auras expect of these that is interesting is that they come in two flavors. akt and b flavor that are practically identical. you add amino acids and this

changes and we are understanding how this might happen and now they interact very well with mtor 1. here is data showing that. so minus us plus amino acids, if we make a mutant of rag b in this case, that is constitutively locked to gtp, so

equivalent to a ras activating allele, you have a constitutive interaction, moreover, in cells expressing this protein, the pathway no longer cares about amino acids. what i mean is that if you remove amino acids the pathway doesn't have been.

it's constitutive t lost the capacity to sense the absence of we are worried about those experiments so we knocked in this mutant allele not in the rag b locus which in the mouse is poorly expressed and here are the controls minus or plus amino acids.

this is one of our favorite outputs for mtor 1 activity. you can see nice regulation n contrast, the mess with the activated two copies of the protein, you can see no regulation whatsoever. each though the protein is not well expressed and this is not a

mouse engineering issue. it is a protein stability problem. it seems like the active protein is turned over more quickly. i'll getba can to these might u. mice later on. they are necessary and sufficient to mediate so you

delete them. there is no amino acid signaling. and this is now shown in a number of different organisms. the part of this story where i argue it became more interesting and less like a conventional signaling story, is the answer

to this question. how do they work? because the first assumption we made is that these were activators of mtor one kinase activity like rev and we spent a long time trying to prove this and we had to conclude this was not the case.

the first will you we had as to how this was happening were movies such as this. this is mt tore c1. no amino acids -- mtorc1. there is a dramatic change in localization. you add amino acids and within minutes it becomes punkitate.

you can do minus or plus amino acids all you like. there are for small punk ta and then they become larger. and then it took us a long time to figure out what these are. but they are lysosomes. what is happening is that mt -- mtor 1 comes off the surface

when you remove them. this process is controlled by the rag gtppases. the cellsy showed you, mtorc1 no longer cares about amino and the rags, i haven't shown you this, are also at the lysosomal surface. data led to a model which i'm

illustrating here, in the absence of amino acids, we continuing is a membrane compartment but haven't been able to define what that is. the rags are on the lysosomal surface in this nucleotide state and we have quite good evidence this is the case from our lab

but there will be better evidence coming out suggesting this is the case. when you add amino acids, the nucleotide state now flips and mtorc1 docks here and it can interact with this activator, rheb. you need amino acids to get to

the right place and you need all these other signals to load rheb with gtp. we have done a lot to prove this model in published data. but the key tools we used is a mutant of mtorc1 we can put on the surface of the lysosome in the absence of the rags.

if you do that, you obtain cells that again have constitutive signaling and don't care about amino acids and no longer care about the rags. you can delete them and they don't care at all. but they care about rheb. so we have a lot of confidence

that in a big picture point of view, this model is correct. so some of the questions we are particularly interested in is this pathway deregulating cancer. that's what i'll spend the last half of the talk telling you about.

really the biggest question in this field is what and where is the amino acid sensor. i'll tell you we don't know what the amino acid sensor is. it's not the rags. it's not any of the proteins that we seem to have identified. we all started to become

convinced on the where component. and that is because over at last few years we identified another complex which is also on the lysosomal surface, a complex we call the rag later. this complex severs two functions here.

it teathers the rags to the surface of the lysosome and acts as a gef for the rag a and b versions of the rags. so, this at the end was pointing to something that we couldn't ignore anymore. and that is that so many of these components are on the

lysosomal surface, railings, rag later and rheb and mtor 1. none of these proteins, despite the way they are drawn are transmembrane proteins. so evolution either picked the lysosome to build these structures because they needed somewhere to put them, or there

is something special about lysosomes and amino acid metabolism and we favor that latter interpretation. now i think in retrospect like many things in science, we should have been able to guess this. because lysosomes is the payoff

step for autophagy. mtorc1 is the major regulator. so you want to be right there when you're producing more amino acids to turn off autophagy. in many organisms, rat liver, lysosomes and in the vaccules of organisms such as yeast, amino acids are high concentrations

and some can reach molar amount. we developed a hypotheses then there is something about lysosomes that is key for amino acid metabolism and that is why mtorc1 is in this compartment. now it's easy to illustrate this but there is a simple question that comes out of these kinds of

ideas that turns out to be exceedingly complicated to answer. and that is exactly where are the amino acids sentenced? you can imagine the sensing happens on the outside of the lysosome or that it happens in the inside.

so inside-out type of mechanism. now this is easy to ask, extraordinarily difficult to address particularly in in tact cells. so what we did was develop an in vitro assay where we can take highly purified lysosomes, and highly purified mtor 1 and mix

them and add amino acids and find that these now interact, which to us tells us that at least the basic components of the amino acid sensing machinery are at the lysosomes. so using the system, we have done a number of different experiments and i'm going to

give you a summary. we asked very simple questions. do you need a membrane? none are detergent sensitive. if you poke holes with a detergent, you get no sensing. do you need the amino acids to be in here rather than out here? we have done a number of

different experiments to address the answer is they have to be inside. do you need the v a tp a se? so this is pumping protons in the lysosomal loom in and these protons are used for many things as well as counter ions to bring in other things like amino acids

even when we put the amino acids in in, it is still necessary. it seems to have an additional role we are still trying to understand. so i want to try to prove to you -- sorry, with the proton gradient it turns out not to be important.

once we get amino acids in, we can get rid of the proton gradient but the vatpse still does. i want to prove one of these points, that luminal amino acids matter inside of the cell. so first question is, do amino acids given on the outside of a

cell get inside the lysosome? to do that, we develop a cell line which we can quickly purify lysosomes because they have a tagged lysosomal protein on them. we can feed cells in a short pulse and these are beads that have lysosomes on them and

measure the rate of activity and when you give amino acids, you quickly find in 10 minutes each if you block protein synthesis, you find them inside of lysosomes. if you used detergents or poke holes in the membrane, obviously the amino acids come out.

the more interesting condition is when you overexpress this protein here called pat 1. so what is pat one? it's clearly eliminating the amino acids inside the lysosome. so pat one is turned out to be a very useful tool. it's a protein that is

discovered by others that exports a variety of different amino acids outside of the lysosome presumably when proteins are broken down, the amino acids need to get out. it's completely lysosomal based on the lysosomal marker. and so if our hypotheses is

correct, that lysosome -- you need to get amino acids into the lysosomal lumen, and i just showed you in the previous slide if you overexpress pat 1, you remove amino acids, this should be inhibitor of the mtor pathway. so that's experiment that is

shown here. here is no pat one minus plus nice activation when we overexpress pat one we eliminate this activity. now, whenever you inhibit a signaling pathway, you have to be worried you're not causing toxic effects.

we can express mutants of the rags, the one i showed you before in particular, this gtp locked a or b mutant and before i showed that you rescueses amino acid starvation. so it should then rescue the effects of pat 1 and in deet it not only do you ease sensitivity

tow amino acids but you lose sensitivity tow pat 1 over express. so our interpretation for these results, and i admit one can have other interpretations but the simple vest one needs to get amino as boys this loom toine activate this.

and we are very interested in what the sensor might be. so the model then that we have for the sensing mechanism is something like this. when there is no amino acids, mtor one is an unknown compartment. when you add amino acids, they

get into the lysosome and there are transporters known to do you can imagine other mechanisms, it doesn't go through amino acids but peptides that get degraded or proteins that are integrated in the lysosome. all of these are possible and

probably all going on to differentents and different now the amino acids, once they are here, we know they talk to the vacular atp. we know that the structure changes. there is confirmational changes in the atp ace when you add

the rag later is bound to the vatpase. it's well bound. and when you add amino acids, there is change here which then reflected in a confirmational change in rag. and these are all things that we have looked at.

this seems to activate the geffen so this now becomes loaded with gtp. geffen. and in a part of the model that is speculative, we think the rags come off at that point and they find mtorc1 and bring it back we know this exists as we

can isolate it from cells. and then this gets turn turned on and this drives cell growth. part of the 39 pops off is that some of my colleagues said, david, mtor moves way too quickly to the lysosome to simply bounce around by diffusion to get there.

there must be some active mechanism to bring it there. moreover, we have some data from photo bleaching experiments that the rag seems to be doing some kind of cycling off the surface of the lysosome in an amino acid dependent fashion. now this is of course a very

complicated mechanism and this is where we have gotten. so it's not where we wanted to go necessarily but this is the date telling us. but one of the things that it made us think about is a question that is in the back of our mind for a very long time

and that i feel that if we had an answer to it, we would have a lot of clarity tow what is going on. and that is why are the rags heterodimers? nothing i just told you makes them half to be heterodimers. so if you think about ras, a

prototypical small protein, it could have two states. if you think about these guys, they could have 4 states. so one of the ways we are thinking about this system is that we need to identify which each of these states do. we are confident about these two

at the end. one is an mtor binding state and this is an mtor release state. but we are starting to think that maybe the cycling that i mentioned has something to do with these two other states. so, again, this is a model that

is used to illustrate how we are starting to think about this system. i want to switch gears before i tell you that the cancer story about these mice that i mentioned in which we knocked in -- we have two alleles of the gtp locked form of rag a.

and when we started making these mice, we were having lots of what i would say, retrospect, pipe dreams. we imagined we were going to make a mouse that was resistant to amino acid starvation and do aging and size studies but we never got a live mouse.

so that hurt those plans and he could never find a mouse. he finds pieces of mice but never found a lab mouse. so he started going back in development saying when did these mice die? he never found them dead. they were always there.

they were always at mendelian ratios. so if he differed them by cesarean, no problems. here are the mutant mice, they are in distinguishable from the heterozygous or wildtype. they are a little bit smaller as you can see here, very, very

little but statistically significantly smaller. we have done lots of histology on these mice and cannot find any defect in these animals yet we never had a live mouse. we had to conclude these animals were being born, dying and the mother was likely eating them

before we could find them. and so to do that, to test whether that idea was true, he isolated them by cesarean and resuscitated them as you typically do, and he put them in a cage, humidified cage and asked what happens to these animals?

these are the type of experiments that are routinely done with mice, you put them in the absence of the mother and looking at the longevity of the mouse in a starvation situation. and very, very typically, a mouse will typically live somewhere between 20-24 hours in

this situation. a wildtype mouse. these mice live about half that period of time. very, very consistently. now, this is not some consult defect that e-licits itself postnatally because we can rescue this by giving them

rapamycin. so if you give rapamycin, this line shifts over here. so this is abarent mtor 1 activity. many will recognize that this type of phenotype, this sort of perinatal lethality, difficulty with dealing with starvation, as

well as that small change in size that i mentioned that is significant, that this is characteristic of mice thwart mutant for the autophagy they have these tact two phenotypes. mice born without very much fat or any fat at all, require awe

tough gee to survive the perinatal period to breakdown muscle to generate energy and resources to live for survivor of animal in the wild, being able to live after you're born without feeding might be something that is really selective for quite strongly

because the mother might not be able to feed the pups right away. so, i'll tell thaw these mice have a significant, i would say complete defect in autophagy at this point and we have done all the type of experiments, em. lc3.

all those experiments. i'm not going to show you those. i'm going toy should you the output, amino acids in the blood t breaks down protein and releases amino acids. you can see that times 0 right after birth, cesarean birth, no defects in the amino acids.

doesn't matter on the genotype. by 10 hours, there is a significant defect in the amount of amino acids in the blood of the mutant animals. now by 10 hours, this is starting to get quite close when these animals are going to start to die.

a couple of hours after this. so we decided to go further back and ask, what happens to the activity of the mtor pathway right after birth? and this was quite surprising to us. what you'll see here, this is in the liver.

here are several genotypes. you can see that there a little bit of noise but at thymes noise there is activated pathways. by one hour of fasting, the heterozygous and wildtype animals down regulated their mtor pathway and the mutant has not.

this is in the liver, this is in the heart, pretty much every tissue shows this. so what we have to conclude from this experiment is that within one hoffure life, something has dropped in these animals that the mtor pathway has sentenced and -- sentenced and turned off

the pathway. whatever that thing is, these mice have not sentenced and have not turned off the pathway. so the question became, what is this thing? if you look at amino acid levels at one hour of life, there is not a huge effect.

relatively little effect on we were surprised though when we looked at glucose levels. that is shown here. here are the control animals. you can see that you struggle relatively low amounts of glucose, characteristic for newborn mice.

by one hour, this plummets. this is underestimation of how far it goes because we are using a flew com terwhich has this sensitivityee - glue com ter. if you chemically measure glucose, we had some animals that we can't measure glucose at this point at 24 hours this is

when these mice die. why does this go up? autophagy is producing amino acids and the liver is taking them in and making glucose and releasing it. the glike jen stores in the liver are being broken down to maintain this blood glucose.

if we give them glucose, we can prolong the survival. so we than a defect in glucohomeostasis is leading to their demise. so these are controls. what happens to the mutants? the beginning response is identical.

they plummet. but they never recover. and the reason is they never support the amino acids which is not sufficient to the drive 60s r. sint sis. we can rescuey them by giving them glucose. now, if you have been paying

attention, you'll think there is something wrong with what i said glucose pretty much plummets to zero by one hour of life and yet this mutation in this amino acid sensing pathway is preventing this response to these exceedingly low levels of glucose.

so this senses glucose but we expect it to be off. and we made a mutation. so this struck us as odd. these are resist tonight this facting affect by one hour. we thought that glucose in this pathway was sentenced as an energy store.

but we have a lot of evidence now that this is not only the case, that glucose might be sentenced as a molecule for itself, carbon source and that this is happening through this vatpase rag later rag pathway and this is a slide we hasn't done so much but we actually

know this is the case. so the study revealed a key role for the rags in nutrient homeostasis and revealed the pathway we were being my optic around amino acid sensing and seems to be glucose and amino acid sensor. these animals at this early

stage of life when it is known there is no insulin, so it doesn't come on until they start to suckel, these animals are actings as yeast cells because they are caring for nutrients, glucose and amino acids and they are doing it through what seems to be through a direct nutrient

sensing type of pathway as a single cell organism such as yeast would do it. after weaning and after the introduction of growth fabber signaling, this whole branch might become more important. okay, i'm going to tell you a new story that has been

spearheaded by two really fantastic mit students. they collaborated fullo this as an equal collaboration. and we always have been interested in this question. is the rag pathway deregulating cancer? one doesn't have to be any kind

of sage to pose this question because i have already shown you this slide. every other input to this pathway has a tumor suppressor in it. if there is one on the energy sensing pathway, why shouldn't there be one in the amino acid

sensing pathway? of course we looked for mutations and all the components i told you about, particularly in the rags and there are a couple of very rare mutations in the rag proteins. this started before we discovered this rag later

complex but it moved slowly for a number of different reasons and it started with the identification of this protein. so we have done very extensive proteomics on proteins that interact with the rags. and at the time we identified this protein, there was nothing

known about it. it was identified genetically in a screen, organism that had problems. the sequence told us nothing. the phenotype told us nothing so we didn't know what to do with it. we decided to work on it a

little bit. we generated antibody and started knocking it down and what we found is that quite strongly inhibited the activity of this pathway. so this is in human cells and we did in drosophila cells and knocking down one of the rags it

inhibited this pathway. a characteristic sign we always get when we inhibit the tore pathway in drasophla cells. we had to conclude that this protein was a positive but that's all we knew. and so we did what we really know best how to do and that is

we put this back in cells and said does it bind to anything? i should say, we could never get these to interact inch vitro together. the coip was weak and we could never get interaction. so maybe there are things missing.

there were when we pulled out mios, there were other proteins bound to it. this say complex for reasons we call the gator complex. then we fagged the proteins and asked who they bind to -- tagged -- there are two complexes here.

if you tag any of the red proteins favoring down mostly the red proteins if you dot opposite experiment, tag these proteins, they mostly bring down the red proteins and low amounts of these light blue ones. so the conclusion we drew from this is there are two

subcomplexes this is why we could never get these to so this complex was quite interesting because a similar complex although not exact, has been identified in yeast as having a role in autophagy in certain strains as well in nitrogen sensing.

so this led us to believe that this was an interesting conflict although in yeast it seems to be a complex while here there are two complexes. we had found this one protein as a positive component of this how many all of these others? so we started doing lots of loss

of function experiments here in human cells and knocked down this one again looking like a positive component and here is a knock down of another one and it looks like it's positive. we did all the other ones in human cells. we also did in fly cells and so

at the end of the day, all of these guys act as positive components of this pathway. then we turned to this complex. and here i think things got more interesting for us. we started seeing this a rescue. here it is weak and it's not a great knock down.

so here is wc5 and you can see the knock down is better and we get better rescue and the same thing with the other gene and we get exactly the same thing in fly cells except when we do the fly cells we get hyperactivation in the -state which i'll show you afterwards -- a minus state.

we think this is a fly-specific phenomenon. the conclusion is this is a negative regulator of the pathway and this is positive regulator. so these are interacting sub complex that is have these two properties.

the question becomes what is the relationship between these two? and so the way weed like do do this is where we have mutants of each one but bee don't have that. we had to do the next best thing, double rnai in drosophila cells when you get good

knockdowns and confident that very little amount of the messages are left. this gives meet opportunity to show the you phenotype in drasophla cells. here is when we knocked down a gator one component. this is true of all of them.

there is hyperactivation in the -state. and we think this reflects a feedback loop that is fly specific. you will see a true null human cell afterwards. in contrast, when we knocked down these guys, it's a clear

positive component. now we can combine this rnai reagented with these and ask who wins? and if you do that, you get a clear answer. very clear that the gator 1 knockdowns win. now again, the simplest

interpretation of this is that gator 1 is downstream of gator 2 but it's not the only one. i fully accepted the other interpretations but that's the conclusion that we have gone with is this is the relationship between these two complexes. and using the rag mutants, if

you do those kinds of experiments combining loss of gator with expression of the rags or overexpression of gator which inhibits this pathway, you genfind the rags are downstream of gator 1. so i told you that localization say really important thing in

this pathway so do these gator proteins fit that model? and they do. well and this might be hard to see but here is in the absence of amino arsenides a control knockdown you see this diffuse mtor pattern and add a amino acid -- this is a marker of

knock down these components, you see a diffuse pattern even when amino acids are present in contrast in the control situation. in contrast, if we knock down this positive complex, a component of this, you can start to see these punk that appear

each when there is no amino acids -- furtherra. eye will show you better data in a true null situation. so, what might be the molecular function of this complex? so particularly of the gator 1 complex. this is the first time in this

pathway that we have identified who i would say is a bona fide negative regulator in thises is. now in all small -- in this in all proteins in particular, we know some of the key regulators of the nucleotide state and so for example, gefs, help the protein go from

the gdp state to the gtp state and we know that the rag later is the gef for rag a and b not for c and d. the other side to this is the gap. and these stimulate what can be very low intrinsic gtp activity. moreover, gaps are tra dishy

mutated in cancer pathways. so nf1 on the ras pathway and tsc2 in the mtor reg pathway. so we asked could gator one be the gap for the reg? so we wantedded to ask, do they have high intrinsic gtpase activity? because these are heterodimers,

this complicates the analysis of nucleotide state tremendously. so what we have done is mutate one of them into a binding g protein n-this case rag c. so it doesn't bind the nucleotide we put in that is labeled. here is controlled protein t

also has no activity. you can see when you add gator 1, you get nice activity in this case. this is against rag a and rag b. you have identical as a result no activity against the rag c form so if we make a version of rag a and cree on gtp, we get no

hydrolysis. so we think that wrong of the functions and perhaps not the only function for gator 1 is to act as a gap for the rag a and b. and this is the fact where the name comes from. gator is gap activity towards

rag. not particularly original but gets the point across. okay. so, given how i started this and given the fact that there are a number of different tumor suppressors in all of these pathways, we wanted to ask the

question, is gator one mutating is it appropriate for me to color this in this light blue color such as these other proteins are? and so to do this, we started to interrogate the tcga data in the cancer genome atlas and we did this with mat meyers in his

group and his lab and we had the best data or they had the access to the best data for glioblastoma as well as ovarian that is the data that i'll share with you. and indeed, when we do a rigorous analysis here, we're only looking for mutations that

we are certain will eliminate the activity of the protein. we find that there is a fair number, not huge, about 3% in glioblastoma and 2% in ovarian this is only analyzing these two. the mprl3 is in a location for which they did not have

confidence in the cgl date to know whether this gene was being lost. so i think these numbers will go up a little bit. now interestingly, there has already been clinical trials with rappa mice in in glioblastoma and there were

responders in some of these trials but we don't have responder ideas in that we can use the encyclopedia, incredibly useful resource and there we don't have mutation data because these genes are not studied but we do have very good copy number data.

we interrogated that dataset and looked for cell lines that had double loss. so they were homozygously deleted for these genes. we don't think will be a common mechanism of lots. we think there will be a deletion plus awe mutation.

that's all we could look at and we pulled out of that resource cell lines that were deleted for any of these three genes and confirmed this by genomic pc r and they are gone. so the question becomes, what is the effect on signal transduction when you delete

these genes in a real human cancer cell line? so here are control cell lines. nice signaling. so if the cell line is missing&you can see there is no and there is really two phenotypes we note. one is hyperactivation of the

pathway compared to the control there is really no signaling at all. these look very, very close to those rag agdp mess where we knocked in a gtpase deficient version of rag a. so this is nprl2 and here is 3. the same phenotype.

here is depc5, the same phenotype. these data are very supportive of the loss of function data we did in drasophla cells and human cells suggesting that these are major negative regulators of the amino acid sensing pathway and that when removing them or

naturally have them removed, we don't have sensing of the absence of amino acids. this experiment was done over an hour. we have done really crazy experiments each 24 hours of amino acid deprivation. cells be probably starting to

get sick and even then, they are not sensing the absence of amino this seems to be a key mechanism in the downregulation of this now it's fair to say, have you all these cell lines but they have all the other mutations in not just these. how do you know these mutations

account for this phenotype? well, we don't because we haven't checked all the other mutations so they add back the genes. now it turns out that for many tumor suppressors, it's hard to add back the gene because the cell likes the fact they lost

that gene. this is the case here. we have failed to add back the genes the vast majority of cell lines. we succeeded to add back this. this is barely deductible by an antibody but that a little bit of expression starts to restore

the one cell line we can add it back to is here. is it the defect of this gene causing this phenotype? here is that cell line. we put a controlled protein and no sensing and when we add back this protein, sensing comes back.

so i'm confident saying for this one cell line this gene accounts for this defect and i would bet a lot that this is the case as well. now this is not only at the signaling level but also at the localization level. you can see here in the control,

the parental cell line and in the absence of amino acids, mtor is purchasing indicated. no difference. when we add back this gene, mtor becomes diffuse when you remove amino acids. so is there any potential predictive value in terms of

therapy for tumors that might have these mutations? of course there are a number of different mtor inhibitors there. the rapamycin class. there is lots of work on catalytic inhibitors and those are starting to go into trials.

ofes tho are mtor specific and some are pi3 kinase and mtor as well. we asked, rapamycin, already being used in cancer, do these cell lines that i showed you here, are they particularly sensitive to rapamycin? so, many cell lines don't care

at all about rapamycin. who are two examples of them. so you can see, really the ic50 is in the micromolar range. some cell lines do care a lot. so this is argued to be a biomarker for rapamycin sensitivity. now here in the gator null cells

they are extremely sensitive. so we are hopeful that at least this in vitro experiment may have some correlate in vivo and so we have developed now a deep sequencing approach to sequence these genes particularly starting to collaborate a number of different groups have

patients in respond to rapamycin but we don't know why they respond. so at least some fraction we'll suspect we'll be mutations in these genes. so i told you about this nutrient sensing arm, this glucose and amino acid sensing.

we don't know the sensor yet. our number 1 candidate that the vatpase is the sensor. this is extremely difficult complex to work with so we have not had luck in pure tuifying and i also told you about this new component. we think these are major

regulators of this pathway and i would argue they may be some of the more relevant ones in the cancer situation. the closest to the mtor pathway in regulation and deregulation in cancer. now, there are 8 proteins here and so clearly it can't be that

there is nothing up stream of we have no idea what that might be. moreover, these proteins have nothing in them that tell what they might do. we don't have been how they work at all. so we have a tremendous amount

of work to do. now in this pathway, remember the tsc complex? their three proteins there and we know of 10 different regulators. so i bet these eight proteins there will be a tremendous number of regulatory mechanisms

that come into play with them. so just in conclusion, i told you about this inside-out model of amino acid signaling and about how this point mutation in the rags has a dramatic effect on autophagy and i didn't show you the data. andny natal nutrient

homeostasis. the autophagy pathway is supposed to be regulated by many different pathways but yet one seems to eliminate autophagy induction. and i told you about a complex mutating cancer and we think this is the gap.

it has gap activity towards the rags. it may not be the only gap of course. the work on the rags was initiated by yasemin and is the one who found the rags as part of the mtor pathway. liron discovered the rag later

roberto has done a lot of work on the inside-out model and lynn and alejo discovered the mouse and the complex. i want to thank our funding sources and collaborators as well as all of you for paying attention. thank you very much.

>> thank you for a very interesting pathway. we have time for questions so please use the microphones in the aisles. while peep are -- people are getting there, with notion that there is sensitivity to the amino acid concentration, inside

lysosome, not all are created equal. different shapes. the kidney has different pumps for different classes. do you know whether there is a difference in which amino acids are there and how much response you get?

>> yes, so that's the number 1 question we are trying to sunday which amino acids matter and why. and it turns out that there are many that matter. so the classic son lewis in. so many, many people have shown that it is a key component.

but it's not sufficient. so you can remove many amino acids but you can't add them back and get reactivation. we don't quite understand that. so we can do necessity but we haven't been able to do sufficiency. now we are now doing metabolite

profiling in lysosomes so we can purify them out and ask what is inside them. and it's technically as you might imagine, quite difficult to do. we find lots of amino acids. but in vaccules there is lots of the basics, arginine, lysine,

hist teen. there is less of the acidic amino acids because of the environment. we are interested in that and it could be that the gator pathway senses one amino acid and the rag later arm senses another and there is integration but we

don't know that. >> you showed the changes in amino acids -- [ indiscernible ] i was wondering if it has on toc2 and having effect on the akt pathway. >> so asking whether amino acids affect mtor 2 localization? >> yes.

>> we love to do that experiment. the big problem is that we never ever been able to localize mtor 2. so we don't know where it is. and there is a number of papers reporting -- like bcl2 at one point.

mtor 2 apparently is everywhere. so, i don't think any of that is correct, actually. if you look at null cells, none of the signals disappear. we don't know where it is. we can't do that experiment. but we would very much like to.

we have lots of people have tried that and have not succeeded. >> second question. you also showed that glucose might have two functions in terms of effecting the mtor 1 in terms of metabolism and affecting atpas.

so in the section function in the atpase, does nonmetabolizable carbohydrate or so can function in that pathway? >> that's an interesting idea. so like glucose and stuff like the only problem with that is this system is built to integrate all those signals so

if you give something nonmetabolizable which is be inhibitable, you will inhibit through energy sensing. so with these molecules they would they could have more than one function. it's hard to tease apart those. you might be able to

genetically, but we know of so many ways that this senses energy that that might be quite challenging. but it's an interesting idea. in yeast, there is data suggesting that the vatpase falls apart when you do glucose starvation, it actually

separates. we have tested that in mammalian cells and don't see that happening. so, it doesn't seem that that is the mechanism. >> thank you. >> can you say something about the division of labor between

mtor 1 and 2, you sort of implied that mtor c1 was sentencing glucose locally whereas 2 was sentencing it globally through insulin. is this consistent feature in terms of development, pre-post suckelling and also evolutionarily single cells

versus multicells? >> so lots of interesting ideas so mtor 2 is in yeast it's not sensing insulin in yeast. probably something from nutrients. we have nice data suggesting a sense for membrane stress in yeast.

in mammalian systems, i think mtor 2,a the least in the stuff we have done is quite a bit less interesting than mtor 1. it does look like a growth factor regulated kinase. so a number of different growth factors, insulin being the best study and any growth factor that

seems to turn on pi3 kinase seems to regulate its activity. we have no evidence for sentencing things like glucose in all in any kind of direct way. so that is why we have somewhat focused more on mtor 1. it's weirder.

which is more fun. mtor 2 the big question is how it is regulated. what is up stream of it? it seems mostly fits into growth factors while the other one senses all of this weird stuff, in interesting ways. >> we are going to have a

reception in the library soy those who would like to continue the conversations with the speaker, please come along. there might be some edibles there as well. let us thank once more time our speaker dr. sabatini.