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Mitochondrial Metabolism in Cell Decisions · Acedra
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Mitochondrial Metabolism in Cell Decisions
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0:08
Hi. My name is Jared Rutter, and I'm a Professor in the Department of Biochemistry and an Investigator of the Howard Hughes Medical Institute at the University of Utah. And I'm gonna tell you in this second part of my series about what I believe to be a very important role of mitochondria, and their metabolism, in controlling how cells make decisions. And in this talk, I'm going to allude to some data generated by Vettore Therapeutics, which is a company that I co-founded
0:39
and I'm quite involved in. So, as I alluded to in my first part, the first part of this series, my laboratory made it a goal to understand some of the uncharacterized mitochondrial proteins that are conserved across evolution. And that has led us into thinking about a lot of different mitochondrial processes and making what we believe are some interesting discoveries about how mitochondria work and how they communicate with the rest of the cell.
1:10
And what I'm going to tell you about today is a story about metabolite transport. So, when glucose is brought into the cell, it's converted through the actions of glycolysis to pyruvate. And that pyruvate, in most differentiated cells in our body, is taken into the mitochondria, where it's converted to acetyl-CoA, which then donates its carbons to the TCA cycle. And through this process, this enables highly efficient ATP production, as I alluded to in detail in the first part of my talk.
1:47
So, some cells in our body, however, don't do this quite so efficiently, and instead convert pyruvate and other glycolytic intermediates into building blocks that help to fuel cell growth and proliferation. And this is most famous in the context of cancer, where this is known as the Warburg effect. And again, this is thought to enable building blocks to be produced from carbon that's brought into the cell, rather than just the production of ATP. So, I want to also point out that in this context
2:22
some of that pyruvate can be converted to lactate, and that lactate be exported. And that will be very important toward the end of this talk. So, this is, arguably, the most well known, well studied metabolic pathway in all of biology. But surprisingly, one obligate component of this pathway was not molecularly identified until a few years ago, and that is the process by which pyruvate enters the mitochondria. Pyruvate is a charged molecule
2:56
and doesn't pass through membranes efficiently on its own. It needs a protein to enable that to happen. And that protein was, again, not molecularly identified until a few years ago, when it turns out that two of the proteins that we had been studying as being highly conserved but uncharacterized mitochondrial proteins turned out to form a dimeric complex known as the mitochondrial pyruvate carrier, or MPC. The MPC is an obligate heterodimer. There's an MPC1 protein and an MPC2 protein.
3:29
Those two proteins come together. Both of them are absolutely required for the function of this complex. And in the absence of one or the other, the other one just gets degraded. And I will allude to that later, when we talk about studies in mice. A graduate student, John Schell, was heavily involved in the discovery of the MPC and the early work at thinking about the roles of this complex, and he gives a great introduction to the discovery and the importance of this protein
4:01
in mediating some of the metabolic effects that we see in cancer. And it would be great to watch. But I just want to summarize that and tell you that one thing that John found, not surprisingly perhaps, is that many of those cells that do this so-called Warburg effect, where pyruvate is maintained in the cytosol, not imported into the mitochondria and oxidized... many of those cells actually have low expression of the MPC. Or, in the case of some cancers,
4:34
mutations or deletions that impair the activity of the MPC. And frequently, that is coupled with high expression of this MCT4 lactate exporter that removes lactate from the cytosol. But the question really is, does that matter? Does it matter that those cells have this MPC-low/MCT4-high situation, and as a result have a metabolic program that's characterized by aerobic glycolysis, so to sp... which is how it's known, as opposed to carbohydrate oxidation in the mitochondria?
5:14
Does that metabolism actually matter for the behavior of those cells? And the system in which we've studied that in greatest detail is depicted here, and this is the intestinal epithelium shown here. And the key feature of this is that these intestinal stem cells... those intestinal stem cells sit here at the base of the crypt, in a protected compartment, and proliferate and then differentiate as they move up the crypt and into the villus, eventually forming all the mature cell types
5:46
of the intestinal epithelium that perform the barrier function and all the other essential functions that this epithelium performs. There's also another great thing about studying the intestinal epithelium, and that is that it's very highly organized. Again, with the stem cells sitting at the base of this crypt, you know where they are, you know what they look like. And also, there are great ex-vivo systems for studying this system. And another great feature of the intestinal stem cell system
6:19
is the abil... the ability we have to make these so-called intestinal organoids, as shown here, and these are two examples shown. So, these organoids are essentially an intestinal epithelium that is folded back on itself to create an enclosed structure that is complete with intestinal crypts, as shown here, where the stem cells, again, sit at the base of this crypt, and as they proliferate and differentiate cause the extrusion of this crypt from what would otherwise be a spherical organoid.
6:53
So, one of the things that John wanted to do is ask the question of whether these stem cells, which normally have low expression of the MPC, actually require that low expression of the MPC to act like stem cells. So, what he did was rather simple: force those stem cells to express the MPC at a higher level and ask, what is the consequence of that? And what he found is that that essentially causes these stem cells to stop acting like stem cells.
7:22
They lose the ability to make new crypts, as shown here. Those cells don't die, but they stop acting like stem cells and even stop expressing many of the molecular markers of stem cells. And interestingly, one thing that he found is that this phenotype of MPC overexpression was completely reversed when he treated these organoids with an inhibitor of the MPC that had been... had been discovered almost 50 years ago now, and we now know to be a quite specific
7:52
and very useful inhibitor of the mitochondrial pyruvate carrier. And moreover, John did another experiment, which was to isolate stem cells from these wild type organoids, plate them again, and ask for their ability to make a new organoid. And what he found is that treatment with this MPC inhibitor in that experiment caused a rather dramatic increase in the ability of these stem cells to make a new organoid, to a similar or even greater level
8:25
than the effects caused by very canonical, well known drugs that are used to promote stemness: valproic acid and an inhibitor of the GSK3-beta protein, which causes activation of the Wnt/beta-catenin system. And I won't show you the data for this, but loss... genetic loss of the MPC in intestinal stem cells, in vivo in mice, not surprisingly leads to an expanded and hyperproliferative stem cell compartment in vivo. And I'll allude later to some of the consequences of that, we think.
8:59
So, the MPC sits here at this very critical juncture, between the metabolic programs operated by many stem cells and cancer cells, which require pyruvate metabolism in the cytosol, and those characterized by pyruvate oxidation in the mitochondria. It sits in this critical juncture. And we believe that this MPC activity -- the activity of this complex to promote mitochondrial pyruvate import -- has an active role in promoting differentiation and limiting stemness.
9:35
And I want to make one critical point. We've often thought about this, and people ask us all the time, well, does this mean these stem cells just don't have mitochondria? It turns out, as pointed to in... with yellow arrows here, these stem cells are chock full of mitochondria. They have more mitochondria than the differentiated cells around them, but it's just those mitochondria appear not to be focused
10:02
on doing mitochondrial pyruvate oxidation. It's really fascinating to think what they might be doing and how that mitochondrial function is controlled. So, the question is how this relates to the signaling that goes on in stem cells, because we all know about the signaling that tells a stem cell to be maintained a stem cell. And how does this metabolic program interface with that? And I want to just point to a couple of experiments done by colleagues of mine,
10:35
Roo Wisidagama, who was a graduate student in the lab of Carl Thummel in the Department of Human Genetics at the University of Utah. And they used the Drosophila system and have done really elegant work studying the impacts of the MPC there. And the system that they have employed is a system that enables the generation of clones in the Drosophila intestinal epithelium that, simultaneously to a genetic manipulation, also turn on the expression of GFP.
11:05
So, you can see a clone, here, that... in the control animals, that generates a clone of a certain number of cells. And when the APC gene... two genes in Drosophila... are deleted, that clone becomes much larger. And the APC gene is a tumor suppressor, the most commonly mutated gene in colon cancer, which causes hyperproliferation through constitutive activation of the Wnt/beta-catenin pathway. The same thing happens in flies, and as a result, you get hyperproliferation
11:36
of those stem cells and a large clone. And the experiment that they did, among many others, is now to force those stem cells to express the MPC, and ask, what is the effect of that? And the effect of that is that those stem cells essentially stop proliferating. And very interestingly, these stem cells don't die. They just stop proliferating, and this is quantified here. They just stop proliferating. So, even though the signaling is presumably telling these stem cells to proliferate...
12:12
APC is mutated, the... the... presumably the transcriptional program is driving proliferation. But when the metabolism doesn't cooperate, these stem cells don't proliferate. I think that puts this effect of the MPC controlling stemness and differentiation into a very interesting light. So, I alluded to data in mammals and in flies. There's data that I won't show you in fish, which shows, similarly, a very important role of the MPC. Others have shown this effect in other stem cell types.
12:50
So, does this actually have an impact on tumor formation? Does this effect of the MPC control oncogenesis, in vivo, in the intestine? So, Claire Bensard, a current MD-PhD student in the lab, did an experiment where she eliminated the MPC... again, specifically in intestinal stem cells, eliminated MPC1. It's interesting. This is a hetero... heterodimeric protein. So, we're deleting the MPC1 gene, and the mRNA for MPC1 is lost. MPC2 is not. But interestingly, this is an obligate heterodimer,
13:27
and as a result of that even though MPC2 presumably continues to be expressed, it's completely eliminated from the intestinal epithelium, presumably due to degradation because its partner, MPC1, is no longer being expressed. So, we end up in a situation where the MPC is absent from the intestinal epithelium. So, what effect does this have on tumorigenesis? So, Claire did a really nice experiment where she subjected these mice to an environmental, oncological stress in the intestine
14:00
and asked for their ability, or their propensity, to generate tumors in the intestine. And what she observed is a dose-dependent increase in tumorigenesis from the wild type to the heterozygote to the genetic loss animals, as shown by the height of these bars -- it's the number of lesions per animal. And the red colors indicate... indicate more aggressive tumors, again being generated in the... in those animals where in the stem cells lacked MP... the MPC.
14:31
So, more tumors, and those tumors were more aggressive. And again, all that's happening here is loss of this mitochondrial pyruvate carrier specifically in the stem cells. I think that's a very important consequence of loss of the MPC. So, not only does the MPC appear to limit stemness, directly, but also oncogenesis. Most likely an indirect effect of affecting stemness. And I haven't told you about this, but it's becoming clear from others in the field
15:04
that this process also plays a very important role in inflammation and fibrosis. So, based on this, we thought this would be a great idea for a way to maybe deal with some of the pathologies associated with these processes: oncogenesis, hyperinflammatory disease, fibrotic disease. And so, we decided to start a company along with my uncle, Bill Rutter, and decided to... can we find a way to activate the MPC? That seems to be what we need to do,
15:38
to activate this process, prevent oncogenesis or reverse it, and potentially also prevent inflammation and fibrosis. So, it... we started a company and hired a fantastic scientist to lead the scientific operations, Mark Parnell. And we figured out very quickly that activating the MPC was not going to be an easy task. And to date, we've completely failed. But what Mark did instead was to come upon a way to perform a related metabolic manipulation that seems to have many of the same consequences.
16:13
And that is through inhibition of this MCT4 protein. So, again, this is a lactate exporter that takes the lactate that's made from cytosolic pyruvate and exports it. And what appears to be the case.... when MCT4 is inhibited, presumably cytosolic lactate accumulates, cytosolic pyruvate accumulates, and that perhaps just drives, by mass action, mitochondrial pyruvate uptake and metabolism. And the net effect is similar to as if... to what we've seen when we overexpress the MPC genetically.
16:50
So, that's what we tried to do: inhibit the MCT4 protein. And Mark was able to develop some very potent and specific inhibitors of the M... of MCT4, and their statistics are shown here. The key features of this is that the MCT4 inhibitor that he found, this VB253 compound, is very potent for MCT4 and selective over the related MCT1 protein, inhibition of which seems to cause some toxicity. So, this protein... this VB253 molecule is also... has quite good pharmacological properties
17:27
and seems to be quite safe. So, I'm gonna show you some of the data that's been generated with this compound, again, with the idea that by manipulating these metabolic pathways we might be able to rewire metabolism, change cell behaviors in a way that would be beneficial therapeutically. One of the indications that we've been most interested in trying to treat with this VB253 compound is idiopathic pulmonary fibrosis. And there's still a lot to be understood
18:01
about the disease pathogenesis of IPF, but a few things that we do know... it's clear that fibroblasts... fibroblasts become activated and form this so-called myofibroblast cell type. And myofibroblasts, like cancer cells and like stem cells that we talked about previously, exhibit this highly glycolytic phenotype, characterized by low MPC expression, high MCT4 expression... again, characteristic of that metabolic phenotype. And this disease process is also contributed to
18:40
by pro-fibrotic macrophages, which also exhibit that same metabolic profile. So, this might be a scenario where, if we could inhibit this MCT4 protein in this context, this might reverse the pathogenic behaviors of these cells, limit the deposition of extracellular matrix and lung fibrosis. So, that's what we set out to test. So, just to show you some of the data behind what I just said... so, it turns out these pro-fibrotic myofibroblasts do express a large amount of this MCT4 protein,
19:17
as shown by staining here, as well as these activated macrophages. Both of them show this high MCT4 staining. And again, this is the target of this VB253 molecule. So, if this mol... if this protein is inhibited, does it have an effect? And it turns out that it does. So, what you're looking at here is pathological scoring, on the left, of a mouse model of idiopathic pulmonary fibrosis, where mice are given bleomycin to induce lung fibrosis, and then the fibrosis is scored as a function of time.
19:53
And interestingly, what was done here is to actually give bleomycin first, create injury, and then treat with this MCT4 inhibitor. And in spite of doing it in that order, which is a more challenging experimental paradigm, this VB253 molecule actually decreases the fibrosis score, a little bit better than what's the standard of care now in patients, which is a molecule called pirfenidone. And on the right, you see that smooth muscle actin, which again is a marker of fibrosis,
20:22
which is almost normalized by VB253. This are just examples of smooth muscle actin staining. Again, from... compared to the control, bleomycin causes a dramatic increase in staining with smooth muscle actin, coincident with fibrosis. This is partially reversed by pirfenidone, but seems to be almost completely reversed by inhibition of MCT4. And this seems to be cell autonomous. And this was a very important result for us. What's being done here is to take fibroblasts from IPF patients,
21:01
culture them in vitro, where they're the only cell type in the dish, and in that context inhibition of MCT4 leads to a decrease in the production of smooth muscle actin. So, that tells us that this effect on decreased smooth muscle actin at least can be partially explained by actions directly on these fibroblasts. It's not something complex going through the brain or the liver or the skeletal muscle. This seems to be happening locally in the lung. Finally, the last data slide I want to show you
21:36
is that this has an effect on the ability of the lung to contract in breathing. And this whole body plethysmography is a measure of bronchial obstruction. And you'll notice that when... upon bleomycin treatment, there is more bronchial obstruction, less breathing capacity. That is maybe decreased a little bit by these two molecules, which are, again, the standard of care approved for treatment in humans. But inhibition of MCT4 works a little bit better, even,
22:03
to decrease this bronchial obstruction and promote healthy lung function. So, we're really excited about the idea that rewiring metabolism in this way, by inhibition of MCT4, might change the behavior of these cells. Again, we have no evidence that these fibroblasts die or that these macrophages die. They just change their behavior. And that altered behavior decreases the production of the extracellular matrix that promotes fibrosis and leads to a decrease in fibrosis itself.
22:39
And we're really interested to try and understand not only the applications of this in human disease but also really fundamentally understand, how is it that by just altering the metabolism of these cells does that change their behavior? And again, this just reminds me to tell you that we think that this might be going on through the actions of the mitochondrial pyruvate carrier. Pyruvate that enters the mitochondria ends up being converted into very important signaling molecules,
23:10
like acetyl-CoA and other TCA cycle intermediates, that are known to have important signaling roles in the cytosol and the nucleus. And perhaps, one of those molecules plays an important role in changing cell behavior. There are also very important redox effects. So, I think it's critical for us to understand, how do our cells sense their metabolic state? And it's something that I believe we're just beginning to understand. How do they know what metabolites they have?
23:39
And I think if we could understand that, we might better understand how manipulations like inhibition of MCT4 change their behavior. And maybe we'd be able to make even better manipulations, build better drugs that would treat people better. So, I also think it's... you know, the MPC is not unique in being an important metabolic control point; there are many others. And if we can identify those metabolic control points and manipulate them, we might be able to make even better manipulations
24:08
to better change the behavior of cells to improve human health. And I told you a little bit about IPF. We think there are many manifestations -- cancer being one that's perhaps the most obvious -- where modulation of this metabolic program, the disposition of pyruvate, might have important consequences. And we're really anxious trying to understand the different ways that this can be used. So, I just want to thank the people that did the work. I alluded to many of them as we went through.
24:44
They've been fantastic collaborators, and thanks to those that paid for this work to be conducted, and thanks to you for listening.
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