2a50-6655-4944-9ef0-0bce368e7fc4.txt

Alright. So we're going through the receptor tyrosine kinase signaling pathway again, kind of start to finish. But the difference this time that we've been going through is instead of just looking how the pathway works in normal cells and how everything is properly regulated, we're taking a look at individual steps in the process, where you can have either the conversion of a proto oncogene to an oncogene, what are the types of genetic alterations that will cause those. And then equally important, looking at how those are then exploited as therapeutic targets to stop cancers that are being driven by these particular oncogenes. And so, couple of terms that you often see when you go through these sorts of analyses is, they'll talk about driver mutations and passenger mutations.

And the difference is is that, within cancer cells, as you're gonna see later on when we get to perturbations in the p 53 tumor suppressor, they're very prone to mutations, because they lack the ability to repair DNA damage, and also then they can't undergo apoptosis. And so you can acquire cancer cells are going to acquire a slew of mutations. In fact, the average tumor cell compared to a normal cell has anywhere from 90 to over a 100 different mutations. But only a small set of those are actually responsible for what we call driving the cancer cell. They're involved directly in tumor genesis.

So one of the challenges you face in oncology is distinguishing between mutations that are driving the cancer state and ones that are just coming along for the ride. They're just advantageous mutations, and they're referred to as passengers. Used when we talk about these driver mutations, is that the cancer cell at that particular time in its development or progression, is what's called addicted to that particular oncogene. And what it means is, is that particular mutant oncogene that's allowing the cell to survive all sorts of different selective pressures the cancer cells are gonna be exposed to. And what you're gonna find is these selective pressures change over the course of tumor progression.

And as you're also going to see is, as soon as you start treating a cell, like we just talked about on Friday, with the drug Herceptin, for example. Okay? So you're immediately then providing cells that are intrinsically mutatable. They have high rates of mutation. As soon as you apply selective, selective pressure on those cells, like a chemotherapeutic drug, you're immediately selecting for adaptive mutations that will confer resistance.

And in many instances, the resistance can be confirmed, but can be confirmed by altering the original target, and you'll see examples of that. But the other way that you're going to find that they can arise is by generating new oncogenes by acquiring mutations that function downstream of the targets you originally went after. We'll go into that this morning, how that can occur. So the key thing is is that the cancer cells are basically very, very, very highly adaptable to be able to evade or attain resistance to a particular chemotherapeutic drugs. Okay?

So we're starting at the top of the pathway with how you could target receptors. And so the first one we went through was Perceptin, which is a monoclonal antibody treatment, and it binds to the extracellular domain of the over expressed HER2. There's many, many different antibody therapies available to stop cancer, and they do so predominantly by binding to extracellular domains in different RTKs. Challenge there is that they cannot enter the cell, but the advantage is they don't need to enter the cell to be cytotoxic. They tend to be relatively specific because they bind to a unique oncogene that's being expressed in those cells.

Okay? The downside is that these antibody treatments, including Herceptin, they're not chillers. They slow down tumor progression, but in and of themselves, they don't really have much of a cytotoxic effect. So it's very, very rare that you find Herceptin. And if any of you have known anyone that might have been treated for breast cancer or ovarian cancer with Herceptin, it doesn't replace the need to also use it as an adjunct therapy with a very, very cytotoxic drug, for example, like Taxol.

And in that case, Taxol is the killer because it can actually kill the cells in addition to stopping proliferation, which your septum does. But as you guys know, the problem with these cytotoxic drugs like Taxol, they're not cell specific. And that is when you're treated with Taxol, the drug is going to get into any cell, and it's going to disrupt mitosis in any cell that's proliferating. Stem cells, hematopoietic stem cells, the follicle cells that grow that hair come from. So the point is, you get all these side effects because the drug itself is not specific to the cancer cell.

So an approach that's being more widely adopted and widely widely utilized is using the specificity of an antibody, not merely to bind to a tumor specific receptor and stop its function, but to also use the antibody as a delivery device. And the term that's used for this are called smart bombs. Okay? One of the things that the cancer literature is full of is it tends to take a very militaristic view of targeting it as a targeting it as a disease. We've got smart bombs.

We've got warheads. And the idea is to kind of think in terms of you're trying to increase the accuracy, the specificity of otherwise cytotoxic agents. And so the approach to being more widely used now, and there's many, many examples of these types of drugs coming out, is to generate what are called ADCs or antibody drug conjugates. Okay? So in essence, in the case of Herceptin, and this is already being done with a derivative of it, and then as you start with your monoclonal antibody.

Now the first thing is is that the antibody does not have to recognize an oncogenic RTK. This approach would work for any tumor specific integral membrane exterior of the cell surface. So in this case, they are using HER 2, but the approach would be adaptable to any tumor specific antigen that's an integral membrane protein. The trick is trying to find that because it's like a needle in a haystack, because 99.99% of the proteins are expressed in a tumor cell, particularly on the plasma membrane, are the same that are expressed in normal cells. So that's the cow that's trying to find one of these proteins that's uniquely expressed on the cancer cell.

Okay? With Herceptin, you've got that. You've got overexpressed HER2. So in principle, what we do to turn this into a delivery device is you take a cytotoxic drug and mechanically link it to the antigen of the antibody itself. Okay?

Now the cytotoxic agent, more often than not, the ones that are being used now, like the one up here, which is this compound called DM 1, they're going to disrupt microtubules. And it just turns out that, like you guys saw with Taxol, they're very good at that, and they're extremely high, they're extremely cytotoxic. So you can take a microtubule disruptor gene drug. And in this case, d m one is a drug that prevents microtubule polymerization. So you can think about that and go, wow.

That's gonna be cytotoxic because it means that as microtubules depolymerize as they will normally, you can't make them again. So a cell, for example, in an interface would not be able to make a mitotic signal. K? So they're very, very cytotoxic. To deliver it to the tumor cell then, you can conjugate using a chemical linker that attaches the small molecule.

So in essence, you're using chemistry to make a linker that will attach to the cytotoxic drug. Now the nice thing here is you can actually load multiple molecules of a given cytotoxic drug to a single antibody. You're not limited to just one molecule of the drug. You can conjugate up to 4, apparently. That seems to be the max you can do.

The bond on the linker that attaches the drug to the antibody, what you're going to see is is gonna be acid levi. And the reason is is that when the antibody binds to the cell surface protein, it promotes its its endocytosis. It's the same mechanism that you guys saw back with the LDL receptor. So LDL binds to the LDL receptor. It gets endocytosed.

It's what PCSK9 does when it binds to the LDL receptor. And antibodies do the same thing. Now the difference here is is that the antibody receptor complex does not dissociate in the late endosome, just like pcsk9 doesn't dissociate from the LDL receptor. It goes to the lysosome. Okay.

So what it means is, yes, that antibody just gave its life by doing that. Okay? And, yes, you are clearing receptors off the plasma membrane and removing them. That also happens. But the key thing is is that in the antibody or in the lysosome, that bond, that linker that attached the cytotoxic drug is laid out, and it's cleaved.

And your cytotoxic agent then diffuses out of the lysosome, goes into the cytosol, and now it can disrupt the micro Okay? So now what you have is this new add on type of therapy, and ABCs are all arranged now. So it's the new way of taking, again, the specificity of your antibody and being able to deliver it to a specific deliver your drug to a specific cell. So that is where the technology is moving forward with antibodies. Okay?

The other approach, and we'll get to the couple of examples of these down the road, is you can envision, instead of killing an RTK by targeting the extracellular domain, well, it is a Tyrosine Kinase. And you're going to see that the alternative approach to going after an oncogenic receptor is to target the kinase domain. And in essence, what you're doing there is you're disrupting the signaling by an essence enabling it to no longer be able to activate autophosphorylate and therefore, transfuse the signal that way. Okay? We'll get into some examples of those down the road.

In either case, though, the minute you start treating a cancer cell with a receptor blocking antibody, Perceptin, cetuximab, a whole slew of them, as soon as you start targeting that cell with an antibody or a drug, a kinase kinase inhibitor, where you're taking out the receptor, the selective process begins. And in essence, the tumor cells are now being selected to acquire resistance, which as you're going to see, is going to come by the conversion of another component further down the signaling pathway into becoming an oncogene. As soon as that happens, the treatment you were using up here to take out the receptor, it doesn't matter anymore because the cell is being driven by an oncogene that comes after that. Okay? So it's almost like whack a mole.

You take down one particular oncogene, another one pops up. And what it's showing you is you cannot continue cancer therapies just by chasing after the oncogenes. And you're gonna see we're gonna have to be a little bit smarter than that. And that is what you start thinking about is what about the altered cellular processes that arise from the presence of the oncogene? K.

We need to be a little bit smarter than just thinking we can just go after oncogene after oncogene after oncogene, because there's only so many of them. Okay? So case in point, here's an example now of how you can acquire resistance. And now we're shifting from breast cancer to colon cancer. And what happens here is that many colon cancers are actually being driven by mutations in RTKs, members of the EGF receptor family.

Some of them are caused by HER 2, some of them are caused by oncogenic HER 1. The point is, these cancers can be treated quite effectively by using a monoclonal antibody. So another one that's up here is cetuximab, which is basically nothing more than another monoclonal antibody that does the same thing Perceptin does. Okay? So you start the antibody treatment, and initially, it works.

And that is the tumors go into remission. Oftentimes, you're having to also use an adjunct therapy, or you may have to be using surgery as well. The point is, it works initially. Over time, though, this is what happens. It's very, very common in this particular type of cancer that, initially, the tumor will stop growing.

Patient's in remission, the tumor starts shrinking, and the typical EGF therapy that you guys can see up here typically lasts about 16 weeks. Okay? So but what you find is is that after the while the therapy is being done, okay, so an individual goes in, and they're going through recurring CAT scans or MRI scans to basically look at the tumor shrinkage. And what you often find is that after about 16 weeks or so of treatment, you go back and you do an MRI, and now you see the tumors come back. Okay?

Well, the tumor has to be an appreciable size or appreciable mass in order for it to be detected by MRI. So the problem is you need something earlier. And the reason it came back is that the therapy no longer is effective. The cells have become resistant to it. Okay?

Well, the problem is if you wait 16 weeks, okay, from when you started the therapy before you can go in and determine, for example, that the tumor has metastasized and grown, now it's gonna be much, much more difficult to treat. So what you need is an earlier, earlier means of being able to diagnose half potential mutations arose that can confer resistance. And that is done by using a technique called circulating tumor DNA or ctDNA. And that is cancer cells are extremely leaky in releasing DNA into the bloodstream. Now you'll see later on, some tumors are more refractory to that than others.

The other thing that cancer cells are often doing is that they're dying for a variety of reasons that we'll get into. But as part of that, they're releasing tumor DNA into the bloodstream. Now that tumor DNA itself is not going to cause cancer, but it's a great diagnostic tool. And the reason is, is that if you went in up here much, much earlier on, and then before you could do an MRI to look at a tumor's progression, you just take a blood sample, and you can do PCR on it. So you can do PCR on a very, very tiny amount of blood and basically screen it for a whole battery.

You can just make primers and screen it for a battery of different oncogenes. And then you could sequence those, and then you use that to determine, have I acquired the acquisition of mutations that can tell me it's time to switch drugs? And that's what's been done predominantly here in colon cancer, but it can also be done in other types of cancer. And here's what you see, is if we go back early on, a gene that, more than any other, gets mutated more quickly and is more prevalent in the vast majority of cancers is the G protein RAS, and in particular, the KRAS oncogene. It turns out we have 3 different types of RAS genes in the human genome.

KRAS, HRAS, and NRAS. They function the same way. They're basically the same protein. The difference among the 3 genes, k RAS, is expressed in the greatest number of cells. H RAS is expressed in a smaller subset.

N RAS is only expressed in neural stem cells. That's why it's called n. Carries out the same all 3 carry out the same function, but the one that is most altered in the greatest number of tumors is what's called KRAS. Okay? And what you find is, you go back, and if you were to do CT scanning and looking for the generation and the increased levels, which would mean more cells expressing a mutant KRAS gene, you can see you can start detecting them as early as after the 1st month of treatment.

Meaning, there's resistant cells already starting to pop up that early. And you can see that the levels continue to come up, so that by the time you're at a point where you can do a biopsy, many, many resistant tumor cells have arisen. Okay? So what that means is if you wait to here to chain wait here to change your therapy to something else that would work downstream of the receptor, you've heard out of luck. Not necessarily out of luck, but the tumor's already spread, and it's gonna become a much more refractory to treat.

So what you wanna do is push this back, or you start using a different therapy much, much earlier on. Okay? So now you can also see, though, this is the undertaker. You switch to a different therapy, and the cycle repeats itself. And that is what you're seeing now.

You start a new drug, and you're gonna start seeing the acquisition of additional mutations that can also confer resistance to it. K? So this is, again, why this is going to happen every single time you go after 1 individual target at a time. Okay? But this is the others this is the reason why RAS is the most commonly mutated oncogene that's associated with the greatest number of cancers.

Obviously, if RAS has been mutated to become an oncogene, it doesn't matter if you're targeting the receptor because the receptor is no longer needed. Because as you guys know, if RAS is a mutant g protein that is always turned on, that's due to the fact that its ability to hydrolyze GTP has been greatly compromised. And as you're gonna see, it doesn't take much at all to RAS to make that happen. Okay? So this is the RAS cycle.

This is just like every other g protein we've talked about through the entire course. Okay? So if we take a look down here, if we've got the inactive RAS okay. This is normal RAS down. Remember, RAS has an intrinsic GTPase activity.

Okay? So one of the things that you can see up here that's worth noting is that a RAS GTP complex so a RAS GDP gets turned on by interacting with SARS, which is what the receptor activates, and we go to active RAS, GTP, which is now going to activate the kinase RAF. Okay? That RAS GTP complex has a lifetime of about a minute. Okay?

That is significantly longer than g alpha s is going to have GTP bound before it hydrolyzes it. Okay? So the point is the RAS family of g proteins are actually very slow at hydrolyzing GTP to GDP, which means they're going to remain active longer than any of the other G proteins that we've talked about. Which means because of that, it means that an active g protein is basically going to activate RAS for a very long period of time. Okay?

Now, RAS has a tumor suppressor assigned to it. The tumor suppressor up here is its gap, which is NF 1. And we mentioned it the other day. NF 1 is a tumor suppressor that is a gap for RAS. So that greatly speeds up its ability to hydrolyze GTP.

So RAS is a tumor suppressor that was first discovered by mutations that inactivated it in what are called the sheath cells that surround neurons. And the absence of the NF one tumor suppressor gives rise to a family of tumors that are called neurofibromatosis. In the extreme state, neurofibromatosis is also known as elephant hand disease. Okay? And those of you that may have seen the movie many, many years ago, by 19th century, patient named John Merrick.

Basically, it causes these horrible blowers and tumors all over the body that are relatively benign, but they're relatively large, and they are very, very distillery. So that was the origin of the NF 1 tumor suppressor gene. Okay? That's a recessive mutation. So what it means is the cells are more prone to proliferate, but they're not totally completely out of control.

That can happen though over here. And that is if you take RAS, which is already a pokey g protein in terms of GTP hydrolysis, you can acquire mutations that reduces GTPase activity even further. And these are dominant active mutations because now what happens is you've got a RAS gene, a mutant RAS gene, that's going to remain in the active GTP bound state for a much, much, much longer period of time. Which means it's gonna be associated with RAF, and it means that signal is gonna continue to be propagated down all the way to get keep the cells proliferating. Okay?

So and these are gonna be referred to as RAS d, dominant RAS mutations. Okay? They're very, very common. And so the question is, well, what is it about RAS that would make it such an optimistic target to acquire these gain of function mutations? Well, it has to do with its structure as a g protein.

Okay? So the problem is, is that what made it become an oncogene is a failure to hydrolyze GTP. Okay? So the first thing to see is that RAS, like most of the small g proteins that we've talked about the entire course, RAS, RAF, RAC, Rho, Cdc 42, the RAS, they're compact little globular proteins. Okay?

And here, if we take a look, this is the structure of RAS, and this is the GTP that's bound. Okay? That is the gamma phosphate. The gamma phosphate is what RAS has to hydrolyze to convert it to GDP. There's 2 amino acids that are juxtaposed near the gamma phosphate.

And the problem is, one of them, glycine 12, the other one is glutamine 61 down here, which basically has an amino group as an amide. It can basically conjugate with phosphate on the on the ATP. Here's the problem. You can make a RAS oncogene very, very easily, particularly if you you mutate glycine 12 to any other amino acid. Okay?

Any missense mutation in glycine 12 inactivates or significantly reduces the GTPase activity of rats, way below what it already is. And that sort of makes sense because RAS is or glycine is the smallest amino acid you've got. Its r group is a proton. So any amino acid substitution here is going to be larger than glycine, and that prevents RAS from hydrolyzing GTP. Glutamine 61 is another one that is a little less common.

If you go through and you look at all the different amino acid substitutions that are most typically found with the glycine 12, alteration, you see that glycine 12 is often mutated to valine. It's also converted to cysteine, and it can be converted to aspartic acid. So those are the 3 main mutations that show up. But the point is is that if you've got an Achilles heel, if you have a protein that could lose its activity, if you have one amino acid and you mutated to anything else, it's gonna compromise its function. And in this case, the way you're compromising it is you're compromising this GTPase activity.

Okay. So here's the problem in trying to target rats. You would think that the most frequent oncogene associated with human cancers would be intensely studied, would be the intense target to develop new therapies. But here's the problem with RAS. If you take a look at it, that's sort of a high end CGI generated structure of it, is RAS has been turned an undruggable protein.

And that was a term that was done by the head of the NIH. And chemists took exception to it because what do you mean it's undruggable? We can come up with a drum for it. Well, the challenge is this, grass is it's been described as a greasy little ball. It looks like a billiard ball.

There's no nooks. There's no crannies. There's nothing you've got on RASP that you could really very easily find a small molecule to bind to. That's been the challenge, is that in order to find a drug that can target a specific protein, you need to have a hook. You gotta have something that your drug could latch on to.

Well, fortunately, here's what's interesting. There's one amino acid that allows you to do that because of its chemical reactivity, and that is this mutant form up here, g 12 c. Now so it turns out you've got other possible alleles of RAS besides that one, but RAS g 12 c is prevalent enough that that's the target of opportunity that you could use to find a small molecule that will bind to that cysteine. You're taking advantage of the reactivity of that survival group. K?

And here's where things are headed. And they're called molecular glues. Okay? Not a big buzzword. At least it's not militaristic.

But it's glue. And it's kinda like making a sticky bomb, I guess, if you wanna make it militaristic. And it's interesting how 2 of them work. These are 2 of the little molecular glues that are being developed, and they're in in clinical trials to target RAS. Now, here's what's interesting.

Okay? The problem with RAS is that it hydrolyzes it loses the ability to hydrolyze PTP, but it doesn't lose it entirely. So even the RAS, g 12 c, hydrolyzes GTP at a very, very low rate. Okay? So that means within a given tumor cell, you are going to have an opportunity.

You've got an opportunity at the point where RAS has hydrolyzed GTP to GDP. You can target that. And that's what these two drugs do. So what it is, is it's a taurabrasim or adagrasim. Okay.

You see they work RAS into the name. And anytime you see -ib, it means it's an inhibitor. This is what's interesting. Is that what these two little glues do is they bind to the inactive form of RAS. So what that means is, obviously, that's gonna be the rarest species in the cell at any one time.

So it means you're gonna have to deliver the drug for a long enough period of time, ultimately, to capture a lot of the RAS as it cycles through. And what it does is that once these drugs bind to the inactive form of RAF, they prevent it from being reactivated. Okay? So in essence, what you're doing is you're trying to k? So in essence, what you're doing is you're trying to shunt the pool of brass from its active form where it can cycle back on and off to get it over here to where it is permanently shut off.

So that's one approach. The other idea of using a molecular glue is shown over here. And that is we take a small molecule. You guys don't need to worry about the structure. And the small molecule binds and forms a bond with cysteine at cysteine 12.

So again, all these drugs take advantage of binding. Now, they do it non covalently. It doesn't make a covalent bond, but they bind to cysteine, and they can stick. In this case, the small molecule is basically a glue. One region of it binds to RAS, cysteine 12.

The other binds to this protein over here called Prolyl Isomerase CYP8. You go, why the heck would we care about a Prolyl Isomerase? It just turns out to be a protein that serendipitously can bind to RAS. There was no real reason to find it. They just noticed there were certain proteins that RAS could bind to in addition to RAS.

So, what you do is the following. We just make a little jigsaw puzzle. And in essence, if we take the drug, the molecular glue binds to RAS, it will then recruit this protein CYPA. That complex acts as a competitive inhibitor. So what it means is is when RAS is locked and it's bound to this complex, it can't bind to RAF, which is a signaling target that we're going through what we're talking about.

But actually, RAS activates a dual signaling pathway that we're not getting into, and that is a signaling pathway that goes through a different kinase called PI 3 k. We're not dealing with that one. But the point is, RAS normally activates 2 pathways that are in parallel. And that's why RAS RAS is particularly effective as an oncogene because it's one effector that basically turns on 2 pro proliferative pathways. And one of the common problems that you have is that if you target a component in the RAF dependent pathway, you can pick up advantageous mutations in the PI 3 k pathway.

So that's another reason it gets complicated. But the point is, what you're seeing right here in this little slide from a paper that just came out a little more than a year ago, those are the first successful attempts after all these years to be able to target RAS. K? So what is required, as you can see, you're just having to be more clever because most drugs inhibit the biochemical activity of its target. Well, you can't inhibit a biological activity if it doesn't work.

So if you've got a protein that lacks a GTPase activity, you can't inhibit the GTPase activity, obviously. So it's not like when you try and target a kinase, for example, where you can inhibit the kinase activity. Okay? Alright. So here's next in line.

Okay? So now we've gone from the receptor, the g protein. Now we're going into the realm of kinases. Because as you guys know, most of the rest of the RTK pathway are protein kinases. Okay?

Now, the thing that you run into here that's why we're gonna call these oncokinases because, in fact, the vast majority of oncogenes that are out there are protein kinases. Okay? Now, as you guys know, kinases have a very defined biological activity. They phosphorylate target proteins. They generally have a fair amount of substrate specificity, but that depends on the particular kinase.

The challenge is this, if you're trying to design a competitive inhibitor of a protein kinase, you're kind of restricted in your chemical diversity. Because in essence, you've got to design a drug that's probably gonna have something on it that kinda looks like ATP, because ATP is the cofactor. That's the source of the the phosphate to be donated. So most kinase inhibitors, if you take a look at them, yeah, they might have something that sort of looks like that. The other thing that you can look for, though, is that the protein kinases recognize these are serine, threonine, or tyrosine, and flanked by particular amino acids.

But the problem is, as you guys remember from the structure of serine, threonine, and tyrosine kinases, they're all pretty similar. The only thing to differ in is really the depth of the clot if there are tyrosine kinase relative to serine training. But, structurally, one of the downsides of kinase inhibitors, they have collateral damage. Okay? It's next to impossible to design a kinase inhibitor that won't also have effects on other similar types of serine threagentryltransfer kinases unless you have a unique protein structure that exists on the oncogene that doesn't exist on the normal kinase.

And that's what we're gonna have here with the protein kinase RAF. Okay? So here's a very, very successful drug that was developed. You see the structure over here. It's kind of a beast.

And it goes by the name Vimerafenib. Vimerafenib is the standard of care treatment to go after most RAF addicted cancers. 1st and foremost, the most common RAF driven cancer is melanoma, skin cancer. And part of the reason is is that the particular region we're looking at here, the gene is sensitive to UV light. Here's another thing that's kind of interesting.

Okay? If you think about your skin, when it's exposed to sunlight, those are the cells that are gonna be most probably to develop melanoma. Cool with that? Well, it turns out that the UV damage occurs in the nucleus. Right?

We're gonna get binding dimers, cytosine dimers that occur in our DNA sequences in the nucleus. Cool with that? The arrangement of the nucleus. Cool with that. The arrangement of our genes within the nucleus of our cells is not random.

Okay? And that is our chromosomes aren't just floating around arranged freely within the nucleus of our cells. The nucleus has a three-dimensional architecture where our chromosomes are arranged in every cell type with a particular structure. And if it it'd be like if you imagine taking the human genome on our chromosomes and putting it in this auditorium, some of them might be attached up here near the ceiling, some of them down here by the door, some of them on the floor, some of them in the seats. The point is, it's a three-dimensional structure.

The raft gene of all the genes that you can think of, the raft gene is located and attached to the nuclear membrane closest to where the nuclear membrane is exposed to the exterior of the cell. So when you take a look down at a cell and you look within the nucleus, the RAF gene is right at the top of the nuclear membrane with, like, bringing on, hitting it. And it does. So RAF itself is intrinsically more sensitive to ultraviolet light mutagenesis than in any, many other genes just because of where it happens to be located in the chromosomes and in the nuclear structure of our cells. So it's just a bad luck type of situation.

Okay? But here's RAF. Okay? Remember RAF? This is probably all I should have shown for that first slide when we were looking at RAF.

K? Remember that RAF is this little folded over dumbbell structure because it has this internal regulatory domain that keeps it from being active until it can bind with RAS. Okay? So RAS activity, normal RAS, not an oncogene, normal RAF is contingent on RAS for its activity. So that's why if you have an oncogenic form of RAS, RAF is always going to be on.

Okay? So and remember, it forms these dimers, and it hooks up, and that's what then activates MAP Kinase Kinase, and it activates MAP Kinase, and off we go. Okay. That's normal RAF. But here's the most frequent oncogenic mutation that you find in RAF.

And that is, there's a single amino acid located right here that's called v it's v 600e. It's a veiling that's been altered to glutamic acid. So it's v 600e. Okay? You guys are gonna start acquiring this little jargon.

When we talk about brass, you go, oh, yeah. G 12 c. For this one, for RAS, it's b 600 e. K? This mutation is right here where you've got this interface between the nuclear, the nuclear the nuke I'm sorry.

In terminal regulatory domain, that thing there, and the kinase domain. That single amino acid substitution allows RAFT to be constituently active. It's still flopped over like it is here, but now that single amino acid substitution enables the kinase to be constituently active. Okay? It means it no longer needs an activated RAS.

So that means if you had a drug that targeted the receptor, who cares? You're resistant. It means that if you had a drug that targeted RAS, doesn't matter. You're still gonna be resistant. But here's what's unique that we have with this RAF mutant that we don't have for a lot of kinases, is that that interaction there is driving the activity of the mutant kinase.

That means we have unique protein structure there because we've got amino acid sequences contributed by the kinase domain, and they're contributed by the regulatory domain. We've got a neat platform here, and that's how the Mirafinib is discovered. So the Mirafinib, what gives it its specificity and reduces its side effects, vomerifenib cannot bind to normal RAF. It can only bind to the mutant RAF that has that same nonconcerned amino acid substitution. Okay?

So the beauty of the merifenib here is it will only inhibit the oncogenic form of RAT, but not normal RAT. That's what you're looking for. You're looking for that type of specificity if you're going to target a particular oncogene. Okay? So pomerafenib is very, very, very effective at stopping melanoma when it's first diagnosed.

Same with colon cancer, where RAF is the culprit instead of rest. But as you guys know the story of that, well, it's just a matter of time before you acquire resistance. And here's the most common way that cells acquire resistance to the mRNA. Okay? And that is the rapv600e mutation undergoes a deletion.

And what the deletion does, it chops off the entire regulatory domain. It kind of resembles that one receptor mutation that we talked about at the beginning, the truncated, I think it was Erb b. Okay? The one that basically you take the Erb 1 receptor, and let's just chop off the exercise of the domain, and it constitutively dimerizes. It's kinda taking a page out of that book.

So the way that you acquire the meripinib resistance here is that we just lose that region, the catalytic domains dimerize without the regulatory domain. But what you can see is, in essence, what the cell did, it just deleted the region of the protein that's essential to bind to the drug. So in this particular instance, you now become resistance to resistant to murofenab. K? So again, guys, can you see that the types of adaptive mutations that cells undergo is pretty diverse?

We can undergo gene amplification. We can undergo missense mutations. We can undergo deletions. So the point is, all these genetic alterations, all of them are available to a cell to select for anything that will cause resistance. The thing to appreciate is that these are incredibly rare events.

The rates of mutation in a cancer cell are like 1 out of a1000000 each cell division. But the point is that one cell that acquires the mutation will not be killed by the drug, and now you're selecting for it. It's very, very much similar to how antibiotic resistant bacteria evolve. Okay? The frequency of the mutations are relatively low, but the point is everybody who didn't get the mutation dies.

So that's pretty strong evolutionary selection to basically generate resistance alleles in cancer cells. Okay? Alright. So here's what I want to finish up with, just as kind of a lead in to what we'll get into next Monday when you guys are back from break. And that is another mechanism to generate Ompan kinases.

Okay? And this particularly is applicable to immune cells. Lymphomas and leukemias. And these are chromosomal rearrangements now. So we're not just confined to looking at, oh, here's a point mutation and a misnace mutation.

Oh, here's a little small deletion. Okay. Or over replication. This is going to involve the generation of chimeric proteins. So we've been tortured all semester looking at chimeric proteins, where we can look at targeting the different places in the cells.

Different little chimeric receptors to screw up signal pathways. These are the real deals. And these are generating novel fusion kinases that are basically due to the fact we've got 2 genes that are going to be found on different chromosomes that are going to undergo a rearrangement, and they're going to be fused together. So we're taking 1 gene on 1 chromosome, another gene on a separate chromosome. Each gene is normally very well behaved.

We put them together and we create this little chimeric monster. The reason that you notice that all of these types of gene fusions, you notice they're all immune cells. And the reason is the immune cells are the ones that are normally in the business of chromosomal rearrangements to generate immune diversity. That's how we get that's how we basically get immune diversity generation, because that's how we shuffle the antibody genes to come up with different combinations of chemo, chimneys, and light chains. So in essence, they are susceptible to these types of mutations.

So the one that we'll take a look at next Monday, what we're back in, is this one up here, which is the BCR ABL oncogene. And what it is is that we're gonna take a normal BCR gene, fuse it with a normal Abel gene, which is a tyrosine kinase, and we generate this fusion gene that underlies the 2 most prevalent forms of leukemia that are out there, CML, which is chronic myelogenous leukemia, and AML, which is acute myelogenous leukemia. They're the most prevalent forms out there. But they provided the opportunity to develop the first kinasepecific chemotherapeutic drug, and it was one of the first blockbuster drugs to be discovered, and it goes by the name of Gleevec or imitoneal. And so we're gonna go through the development of that particular drug.

And, again, what we're gonna take advantage of here is we have a unique situation that where we have a fusion of 2 different proteins into a single polypeptide. We've got a little region there that's not gonna be found in any other cell. And that's gonna prove to be the target site for BCR aBL that will allow them to develop in the cleavage. Okay? Alright.

So have a good break, rest up, eat a lot, chill, and, see you guys back here on Monday. There will be a home there is some homework assignment for today, but you won't be doing till next Monday. Okay?