Objective
We performed a large-scale genetic screen in which we used genetics to delete cell surface proteins called G protein coupled receptors (or GPCRs) from human islet cells to identify which GPCRs controlled beta cell replication. Somewhat unexpectedly, we also identified 30 GPCRs that when silenced resulted in complete beta cell death. We propose that using small molecule drugs that turn on these receptors in islets will help to promote their survival during isolation, dissociation, and transplantation.
This 1-year project has two objectives.
Objective 1 is to use commercially available drugs that turn on the 30 pro-survival GPCRs we uncovered in our screen to see if any of these drugs can promote survival of human beta cells and of beta cells derived from stem cells in the lab.
Objective 2 is to take the most promising pro-survival drug from the Objective 1 and test whether it can promote human beta cell survival after transplant into diabetic mice. While islet transplantation currently suffers from a lack of donor material, we have prioritized islet transplantation over stem cell transplantation as it is currently accepted clinical practice.
If we are successful and can demonstrate increased survival of islets cells after transplantation in mice, with a view to the future of using stem cells for transplantation, after the first year we will then evaluate the ability of the lead small molecule to promote survival of beta-like cells from stem cells.
Background Rationale
Islet Transplantation has brought us closer to a cure for T1D but is still limited by donor tissue availability, the need for lifelong immune suppression drugs, and ultimately, failure of transplanted islets. Thus, despite improving long-term outcomes, insulin independence is not permanent. Generating beta cells from stem cells (SCbeta cells) could provide a limitless source of insulin-producing cells for transplant. However, the promising outcomes of SCbeta cell transplants in mice are not necessarily predictive of how they will function in patients with T1D. A major reason for the failure of transplantation using islets or SCbeta cells is cell death, caused either by the immune system or loss of blood supply and survival signals. Therefore, new strategies aimed at protecting islets and SCbeta cells during this critical period may improve transplantation outcomes.
Beta cell death sits at the heart of the cause of T1D and is a central complication for current treatments. So far, strategies to prevent beta cell death in T1D have involved targeting proteins inside the cell that are turned on late in the cell death process. However, these strategies prolong the survival of poorly working cells that may no longer fulfill the desired role of mature beta cells. Recent exciting work has shown that beta cells are some of the longest living cells in mammals. From what we understand about long-lived cells, this suggests that they must have specific genes whose sole function it is to keep beta cells alive. Despite how vital beta cell survival is to prevent T1D and to current transplantation treatment approaches, the genes that direct beta cell survival in humans are poorly understood.
We have identified pro-survival GPCRs in human islets. Signals for survival come from outside the cell. We checked hundreds of cell surface receptors in human islets that receive these survival signals, called G-protein coupled receptors, or GPCRs, using our robotic screening system and identified 30 GPCRs that are essential for cell survival. This is exciting for several reasons. First, GPCRs are critical regulators of islet biology. Human islets have numerous GPCRs on their cell surface, and they control beta cell proliferation and function. Second, GPCRs are also the target of 1/3 of all drugs that are approved for use in humans by the Food and Drug Administration. Thus, GPCRs have a rich history of success as druggable targets with high therapeutic potential - the path to translating new results is clear. Third, despite the centrality of GPCRs to islet function, a comprehensive exploration of GPCR function in beta cell survival has not been performed. We hypothesize that pro-survival signaling pathways are directed by these 30 GPCRs and propose that turning on these GPCRs during cell dissociation and transplant will increase the surviving fraction of beta cells after transplantation. Given the importance of GPCRs to medicine, each of the 30 “pro-survival” GPCRs already has commercially available drugs that activate them. Thus, here we will test the ability of these drugs to promote cell survival of beta cells from human donors and SCbeta cells in the lab and in mice. Specifically, for this 1-year application, we will focus on the survival of human islets during the post-transplant period. We anticipate that our approach will quickly identify easily druggable pathways that can be used to promote survival of human beta cells. If successful, our findings will have applicability to all transplant approaches by increasing cell survival and function after transplantation. Promising results would prompt future drug use targeting a pro-survival GPCR as a potential therapy to prevent T1D.
Description of Project
Islet cell death is a limitation that lies at the heart of all current approaches to treating T1D. Islet transplantation has brought us closer to a cure for T1D but is still limited by donor tissue availability, the need for lifelong immune suppression, and the failure of the transplanted islets to survive. Islet survival is challenged by both the immune system of the host as well as the loss of blood supply and survival signals that are normally provided by the pancreas. An exciting alternative to transplanting islets is to transplant stem cell-derived beta (SCbeta) cells. SCbeta cells offer the promise of an unlimited supply of beta cells for transplant, these too are subject to loss of function and cell death, even when surrounded by devices designed to protect them from the immune system. As a result, unwanted cell death is a common feature of transplantation of both islets and SCbeta cells.
Our understanding of how beta cell death occurs in humans is still poor. For example, beta cells are very long lived, and as such, it stands to reason that they have genes that work to promote their survival throughout life. However, these genes and how they work to promote beta cell survival are largely unknown. This is important because we could harness the function of these genes to help promote beta cell survival, not only during transplant scenarios, but perhaps even to prevent the beta cell loss that triggers T1D.
Signals for survival come from outside the cell. We checked hundreds of cell surface receptors in human islets that receive these survival signals, called G-protein coupled receptors, or GPCRs, using our robotic screening system and identified 30 GPCRs that are essential for cell survival. This is exciting for several reasons. First, GPCRs are critical regulators of islet biology. Human islets have numerous GPCRs on their cell surface, and they control beta cell proliferation and function. Second, GPCRs are also the target of 1/3 of all drugs that are approved for use in humans by the Food and Drug Administration. Thus, GPCRs have a rich history of success as druggable targets with high therapeutic potential - the path to translating new results is clear. Third, despite the centrality of GPCRs to islet function, a comprehensive exploration of GPCR function in beta cell survival has not been performed.
We hypothesize that pro-survival signaling pathways are directed by these 30 GPCRs and propose that turning on these GPCRs during cell dissociation and transplant will increase the surviving fraction of beta cells after transplantation. Given the importance of GPCRs to medicine, each of the 30 “pro-survival” GPCRs already has commercially available drugs that activate them. We propose two specific aims to understand GPCRs identified in our screen might be leveraged to restore intrinsic survival GPCR signaling and minimize beta cell death:
Specific Aim 1. To identify if drugs that turn on GPCRs promote survival of human beta cells and stem cell-derived beta cells.
Specific Aim 2. To determine if drugs that turn on GPCRs that promote beta cell survival we identify in Specific Aim 1 also promote beta cell survival after transplantation in mice.
If successful, our findings will have applicability to all transplant approaches by increasing cell survival and function after transplantation. Promising results would prompt future drug use targeting a pro-survival GPCR as a potential therapy to prevent T1D.
Anticipated Outcome
The anticipated outcome for Objective 1 is the identification of a small molecule (drug) that can promote survival of human beta cell and stem cell-derived beta cells. We predict that we may find more than one drug that promotes beta cell survival, given that the receptors can act in a redundant, or “backup” fashion to one another. We will then bring the ‘best in class’ drug forward for evaluation in Objective 2.
In Objective 2, we predict that short term treatment of human islets with the pro-survival drug will reduce cell death in the few days after transplant. Furthermore, we expect that mice transplanted with human islets that were pretreated with the pro-survival drug to show lower blood sugar levels than in mice transplanted with human islets that did not receive the pro-survival drug treatment.
Knowledge gained in this work will not only further our understanding of how we might prevent beta cell death during islet isolation prior to transplant, but during the critical time after transplant, and possibly in the face of autoimmune attack. As such, with a view forward, identification of a pro-survival drug could be developed for use in prevention of T1D.
Relevance to T1D
The inability to produce enough insulin due to loss of pancreatic beta cells, the cells that make and secrete insulin after a meal, results in type 1 diabetes mellitus (T1D). A central challenge in treatments for T1D has been inappropriate loss of function and survival of transplanted islets. There is an initial wave of cell death after transplant and the reasons for this cell death and strategies to prevent it are largely unknown.
While it is widely appreciated that treating T1D will require increasing the number and health of beta cells, our current knowledge of the cell surface proteins, or “receptors”, that control human beta cell survival are lacking. Our vision is to establish a complete understanding of why human beta cells die after isolation and transplantation, and to determine how we can prevent this. The eventual goal is to get beta cells to survive during the early phase of transplantation to reduce the number of islets required for transplantation, thus increasing the number of potential recipients. If we are successful in finding a receptor that we can target with a drug and can demonstrate increased survival of islets cells after transplantation in mice, we will also evaluate the ability of the lead small molecule (or drug) to promote survival of beta-like cells from stem cells. Such a drug would also potentially be an exciting tool that could be developed to prevent T1D.