 Thank you for the invitation to speak in the meeting. I received a particular letter from Tony and my Kim, so I want to thank them for the invitation. So I'm going to talk to you about the work that we're doing in the lab regarding HIF inhibition. And I modify, as you can see, the title. It's not called HIF Biology, but it is called HIF Target in the case of Rinalsel Carcinoma. The latest data from the TCGA that we're all aware about point to three broad categories of molecules that they are deregulated in the case of Rinalsel Carcinoma and they can be potentially targeted. So chromatin modulators is a very new and expanding space. And chromatin modulators are enzymes that they are losing their activity in the case of Rinalsel Carcinoma. So we're waiting for identification of critical targets downstream that are regulated in order to inhibit them. MTOR has been tried already in the clinic. It has some significant success, modest success, most probably not related to the inhibition of HIF. So what we have been focusing for a while in the lab is an effort to inhibit HIF in Rinalsel Carcinoma. The deletion of VHL, as we all are aware about, the deletion of VHL leads to a constitutive increase in HIF expression. And I would like to make few points here is that independently of the downstream modifiers of the chromatin or other secondary mutations that they're required for the development of Rinalsel Carcinoma, one of the background effects is the deletion of VHL in about 90% of the cases of RCC. Moreover, there are very intense preclinical data, and clear data that they say that if we reintroduce VHL or if we inhibit HIF in cells that are missing VHL, we're leading to tumor suppression. And again, this is preclinical data. One more caveat here before I present you the data about the HIF inhibitors that we have is that there are two paralogs of HIF, two family members of HIF, as you know, HIF1 alpha and HIF2 alpha. There are data that they're accumulating now for the last 10 years that they think they're quite compelling that HIF2 alpha is the oncogenic driver. There is evidence that the HIF1 alpha is a tumor suppressor, or if it is not a tumor suppressor, at least plays a secondary role in the case of development of Rinalsel Carcinoma. So I think that HIF2 alpha is a validated target, as we all know. And the last point I would like to make is that we heard so many of these meetings about inhibitors that they are in the clinic right now, but what they do is that they inhibit downstream targets of HIF. So we talk about the JF inhibition, PDGF inhibition, FGF inhibition, kind of a one-by-one inhibition of downstream targets of HIF. I will submit to you that when I hear the talks about potential resistance in treatment of Rinalsel Carcinoma to these inhibitors, the first thing that comes to my mind is that HIF has approximately 60 downstream targets. So inhibiting two or three or four of them leads me with approximately 57 reasons to tell you why these tumors develop resistance in the receptor tyrosine kinase that we currently hit. So an adventurous approach that we took for years in the lab was to go and directly attempt to inhibit HIF2 alpha. I'm not going to present data at presenting the previous meetings in which I show that we did develop small molecule inhibitors. We have done this by a cell-based screen. What we know about these inhibitors is that they can inhibit the HIF expression in every Rinalsel Carcinoma cell line, which is VHL negative. We know that they can inhibit HIF expression in every cell line that has been induced to hypoxia with very few exceptions. And we know a lot about the mechanism of action. And the mechanism of action is what they do. These inhibitors, like this is the red dot of the inhibitor, is that they inhibit HIF translation. This is a nem-tor independent translation of HIF. It occurs from an internal ribosomal entry site of the 5-UTR mRNA of HIF. And the way that they inhibit it is by precipitating on the mRNA of HIF, this protein which is called IRP1, iron-regulatory protein. One has been named this way because it has been identified initially through regulation of iron. And this IRP1 binds to the mRNA of HIF and inhibits the translation. So a VHL negative cell line, a VHL negative tumor, will have decreased availability of the HIF to alpha protein when it's treated with these inhibitors in principle because they inhibit the translation of HIF. What I'm going to present, you would like to present right now, is that unpublished data from our laboratory that has been quite gratifying and give us an enthusiasm about these inhibitors because what it says is that they do work in vivo. And the way we try them is that we didn't try them in the classic xenographed tumor model in the mouse because they have limited bioavailability and therefore they need the appropriate modification, but we went in an animal model that actually mimics VHL disease and this is a zebrafish animal model, this is a fish. I know this kind of a we are in a so clinical meeting to present the fish, but the fish has many actually phenotypes of the VHL disease. So this is a VHL negative fish and it develops increased angiogenesis. It develops erythrocytosis. It develops an increased size of the liver and the kidney, not frank kidney cancer. And also it has cardiomegaly and edema and it has a life, a short life span because this fish dies, the embryo of this fish dies approximately at 10 days due to all these physiologic changes. I put here the human cartoon because many of these features that I talked to you about the fish, they're actually encountered in the patients that they have VHL disease. So we tried, we took this fish and the other way to make it to mimic the VHL disease is to treat it with a chemical. This chemical is called DMOG. That poisons the prolihydroxylases that they deregulate here. So if we treat a wild type fish with this inhibitor, the DMOG, we make it appear if it has VHL. And we stain it here with a stain that stains red cells and therefore by staining red cells we can actually quantify the erythrocytosis and we can quantify also the development of the vessels. So we can train a computer algorithm to look at these dots into the fish, objectively quantify the intensity and then we can treat the fish with the HIF2-alpha inhibitor and so that we can significantly decrease the effect of HIF in erythrocytosis and in vascular development. We can do the same thing exactly with a VHL negative fish without treating with inhibitor. Here is the VHL negative fish, this is the VHL fish that's positive. You can see that the redness is more intense. This is exactly because they develop blood vessels and because they also develop erythrocytosis. Again, we can treat with inhibitor and we have a significant reduction of the staining which is the combined effect on the erythrocytosis and the vascular development. We can deconvolute this effect into an effect on the blood vessels or in an effect into the red cells and we deconvolute it this way, we cross this fish with another fish that has green vessels. It has been engineered genetically so that it has green vessels and what we see is that the VHL negative fish now we can mark the vessels, develops tumor lesions, antigenic lesions in the brain, in the retina and in the body. So we see the proliferation of the very intense vascular proliferation that we see with the loss of VHL in the tumors or with the antigenic lesions in the VHL patients and again when we treat with a drug we reduce the intensity of this vascular proliferation and we reduce the size of the tumors. Finally, we can bleed the fish and we can count the erythrocytes and we can count actually the mature forms and the immature forms of the erythrocytes what we can show is that by applying this HIF inhibitor we promote the maturation of the erythrocytes and we promote the maturation because it's a characteristic of the HIF expression that induces an immaturity in the blood cell development. So this combined effect on the muscularity on all the HIF targets that we looked translate into physiological changes in this fish. The VHL negative fish has a decreased cardiac contractility and when we treat with an inhibitor we actually improve the cardiac contractility and finally we do improve the overall survival of the fish that died significantly later when we treat them with HIF to alpha inhibitors. So I'm showing you this data to tell you that this is the first evidence that we have that we can actually treat with a pharmacologic HIF to alpha inhibitor an animal model that mimics the VHL disease and potentially mimics aspects of the human renal cell carcinoma and that we can actually improve this phenotype of the VHL disease. This is not, this is only a lead compound we are doing chemical synthesis of this compound right now, modifications and I can tell you that the first modified derivatives of this compound have a higher efficiency without having any toxicity. This study is well controlled, other drugs that they inhibit HIF on alpha they don't induce these effects and actually when we use chemical compounds that they're modified so that in the tissue culture do not inhibit HIF to alpha we do not see this amelioration in the effect in the fish. So I'm moving to the second target. This is one way that we think that it's possible to directly target the HIF. I'm moving the rest of the time in the second target and the second target is actually became clear to us when we started the effects of hypoxia and HIF on the metabolism. And there is a dramatic reprogramming of the tumor metabolism by HIF. So the normal cell that you can see here depends on glucose for very significant self-actions. The cell takes glucose, Amato presented some of this pathway through the glute one, takes it into the mitochondria, uses glucose as a fuel in this mitochondrial grinder and produces molecules that they are necessary for the cell to produce biomass, to increase the fatty acids, the DNA and the amino acids. And this combination gives the cell the ability to increase the mass and to divide. In the same time it produces energy. So when we expose cells to hypoxia, we see what has been well known to us as a Warbrook phenomenon. So the hypoxic cells do not put glucose into the mitochondria, but instead they shovel it over to the lactate, they divert the glucose. And therefore at the first glance, the question pops, how does a cell now that they cannot use the glucose as a fuel produces the nutrients to increase its mass, how does it produce its biomass and the same time how to produce the energy. So we visited this question and we visited as you understand very well because the VHL negative cells have a fixed phenotype in hypoxia because they expressed here. We were doing that with specific metabolic tracers. We can trace, we can label glucose, we can label other nutrients, we can label glutamine, we can put in the cell culture that we can crack the cell open and trace the pathway through which glucose or other nutrients enter what is called here the central carbon metabolism. So to make the story short, what we have shown is that the VHL negative cells, so the renal cell carcinoma cells have a very impressive phenotype. Instead of using glucose to create this biomass, they're using glutamine. And they're using glutamine through two pathways. One of them is that they enter glutamine to the crab cycle that we all remember from medical school and it goes through the oxidative pathway, but they also are using glutamine in a reverse way. It's the first time actually that we describe this in a mammalian cell system. It has been known and described in bacteria, but glutamine can go through this pathway that's called reductive carboxylation and then it's been used as a fuel for the biomass production. Why this happening? The hypoxic cells actually in here transactivate all these enzymes. One of them is PDK1 and PDK1 blocks the entrance of glucose into the cells. This is a fixed defect that the VHL negative cell has and therefore presents a potential opportunity to use it for therapy. This is not a subtle phenomenon. In blue you see the contribution of glucose and in red the contribution of glutamine into the biomass production inormoxane hypoxia. Every cell line that we test inormoxane hypoxia behaves this way and all the renal cell carcinoma cell lines behave this way. We went to show that this depends on HIF and that the HIF expression, the HIF12 expression specifically is necessary and it's efficient for the induction of this phenomenon. We went further into analyzing the mechanism and I'm not gonna spend a lot of time for the because of the time limitations but I think we understand why this is happening. This is happening because the metabolism of the cell has a plasticity. If you take the glucose, you block the entrance to the mitochondria, you throw it into the lactate, you create a deficiency inside the mitochondria and the deficiency that drives this reaction is citrate. So if you don't feed it, the citrate levels drop and because the citrate levels drop, they turn the reaction the other way from glutamine and they create this reductive carboxylation. This is not an in vitro only phenomenon. It happens only in vivo. We put human tumors in mice, we label the mice with glucose and with glutamine and we show that the renal cell carcinoma quickly take the glutamine out of the blood and they use it to feed the vessels and other amino acids. So it's not a surprise that DCDA data that they came actually very recently into publication show a significant deregulation of the glycolytic pathway and the pathway of the mitochondrial enzymes. I have two more slides. We can block this pathway in principle and this is the reasonable thing to do. Glutamine utilize an enzyme to go from glutamine to glutamate which is a necessary step to enter the mitochondria and perform this feed in the fueling of the mass and if we block this enzyme with a glutaminase inhibitor and there are several of them available pharmaceutically, we can show that the VHL deficient cells preferentially are killed over the cells or they express their VHL positive exactly because they have this fixed defect that does not allow them to replace the shortage of glutamine with glucose. It works in vivo, so if you inject mice with renal cell carcinoma, you can actually lead to tumor suppression by treating the cells with glutaminase inhibitor. This data with inhibitors that they're kind of in a quote, quote dirty, but there are clinical grade inhibitors that actually they're approaching the market right now and we are partnering with a pharmaceutical company to try some of these clinical grade inhibitors. So I present you two potential targets, one HIF to alpha itself and the other one, the glutaminase which is the consequence of the metabolic deregulation of the cell by HIF. These are the persons that they're working in my lab and these are many of my collaborators that they help with the studies. Paolo Gamero, let the metabolic studies Anna Metello is leading the HIF to inhibitor studies in the fish that I show you and I thank you very much for your attention.