Pere Puigserver - US grants

DESCRIPTION (provided by applicant): Homeostatic mechanisms in mammals function to maintain blood glucose levels within a narrow range in response to hormones and nutrients. Glucose homeostasis is highly dysregulated in metabolic diseases such as obesity and diabetes as well as in dietary manipulations such as caloric restriction (CR). CR extends life span and causes many changes in glucose metabolism similar to fasting. However, how the metabolism of glucose might be connected to aging is largely unknown. One key component of glucose homeostasis in mammals is the transcriptional coactivator PGC-1alpha that controls glucose production in liver. Studies in yeast and worms have identified a Sir2 histone deacetylase protein as a possible link between caloric restriction and life span. Importantly, we have preliminary data showing that SIRT1 (mammalian Sir2 homolog) is regulated by insulin in hepatocytes. SIRT1 interacts with and deacetylates PGC-1alpha at specific lysine residues in an NAD+ dependent manner. In addition, in cultured hepatocytes SIRT1 regulates gluconeogenic/glycolytic genes and hepatic glucose output through PGC-lalpha. This application contains three large aims. First, we will identify the mechanisms of how insulinregulates SIRT1. Second, we will perform a detailed biochemical and cellular analysis of the physical and functional interaction between PGC-1alpha and SIRT1. We will focus on the regulatory mechanisms of SIRT1 on PGC-1alpha function on hepatic glucose metabolic genes. Finally, to bring the PGC-1alpha /SIRT1 mechanistic and cellular studies to the animal level we will perform a glucose-related gene expression and metabolic analysis using adenoviral delivery to the liver of mice. Taken together, the findings of these studies will allow us to identify the molecular mechanism by which two important regulated transcriptional components, PGC-1alpha and SIRT1 control glucose homeostasis in response to CR signals such as insulin. Understanding of these metabolically regulated molecular events could be used for anti-obesity, diabetes or aging drug development. These findings have strong implications for the basic pathways of energy homeostasis, diabetes and lifespan.

[unreadable] DESCRIPTION (provided by applicant): Several risks factors of the metabolic syndrome such as insulin resistance and obesity present mitochondrial dysfunction that is associated with increased intramyocellular lipid accumulation. In response to nutrients and cold stimuli, transcriptional complexes that contain PGC-1a control mitochondrial oxidative function to maintain energy homeostasis. mTOR is an important component that responds to nutrient and hormonal signals and regulates cell growth, size and survival. However, whether and how mTOR controls mitochondrial oxidative activities is unknown. We have preliminary experiments indicating that in skeletal muscle mTOR is necessary to maintain mitochondrial oxidative function. We have identified that the transcription factor YY1 and the coactivator PGC-1a are mediating mTOR mitochondrial effects through modulation of their physical interaction. However, the molecular mechanisms of the signal transduction from mTOR to YY1/PGC-1a are unknown. The major goal of this proposal is to identify the mechanisms by which mTOR pathway regulates mitochondrial gene expression through the YY1/PGC-1a and to test their functionality in [unreadable] in-vivo mouse models. To accomplish this goal, we will use a variety of biochemical, cellular and genetic approaches. We have three aims. Aim 1 is to perform molecular and functional analysis of how mTOR controls YY1 transcriptional function. Aim 2 is to carry out molecular and functional analysis of the mTOR activity-dependent interaction between YY1 and PGC-1a. Aim 3 will determine the effects of mTOR inhibition on the metabolic and bioenergetic function in mice. This investigation will allow us to identify the molecular mechanisms by which the nutrient sensor pathway regulates YY1/PGC-1a and how defects in this pathway results in dysregulated mitochondrial function and energy balance. PUBLIC HEALTH RELEVANCE: Since mitochondrial pathways are dysregulated metabolic diseases such as obesity and type 2 diabetes, studies in this grant proposal to understand how mTOR control mitochondrial function might translate into potential therapies for these diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Energy and nutrient homeostasis is maintained through a complex regulatory network formed by signaling and transcriptional components that control metabolic genes. In this regulatory network, metabolic flexibility in skeletal muscle is defined as the ability to undergo a nutrient switch of fuel substrate utilization between glucose and fatty acids. Clinically, the metabolic syndrome that include an array of risk factors such insulin resistance, obesity and dyslipidemia among others is associated with a loss of metabolic flexibility. Moreover, it is though that it is this lack of metabolic flexibility that correlates with an incomplete mitochondrial oxidation of fatty acids and increased accumulation of intramyocellular lipids, a putative cause of insulin resistance. We have identified an important component of the low nutrient/fasting switch from glucose to fatty acid oxidation that involves the SIRT1 deacetylase enzyme that targeting and activating the transcriptional coactivator PGC- 1a promotes this nutrient switch. Mimicking fasting metabolic response, active and deacetylated PGC-1a increases expression of genes encoding for regulatory proteins and enzymes linked to mitochondrial fatty acid oxidation. Interestingly, this pathway is altered in skeletal muscle of mice fed with high fat diet in which PGC- 1a is highly acetylated and correlates with a loss of metabolic flexibility and insulin resistance. Although activation of SIRT1 and PGC-1a are key regulators of this process, how low nutrient signals control SIRT1 enzymatic activity and how mechanistically deacetylated PGC-1a is a hyperactive protein is unknown. The major goal of this proposal is to identify the mechanisms by which fasting and low nutrient signals to SIRT1 enzymatic activity to induce PGC-1a deacetylation in skeletal muscle and to investigate their metabolic functionality in in-vivo mouse models. We have three aims: Aim 1 is to perform molecular and functional analysis of how fasting/low glucose controls SIRT1 deacetylase enzymatic activity in in-vitro as well in-vivo conditions. Aim 2 is to carry out molecular and functional analysis of PGC-1a acetylation and identify new proteins that account for the hyperactivity of deacetylated PGC-1. Aim 3 will determine the effects of fasting/PKA activation on glucose and lipid metabolism through generation of skeletal muscle transgenic mice expressing SIRT1 and PGC-1a mutant alleles in different dietary conditions. This investigation will allow us to identify the molecular mechanisms by which fasting and PKA activation in skeletal muscle controls glucose and lipid metabolism. Since dysregulation of these processes that involves a loss of metabolic flexibility is a major component of the metabolic syndrome including obesity and diabetes, our studies on the regulation of SIRT1 deacetylase activities and PGC-1a acetylation might translate into potential therapeutic interventions.

DESCRIPTION (provided by applicant): Exercise and caloric restriction exert very powerful protective effects against age-associated diseases including metabolic disorders such as obesity and diabetes. Among the key target tissues skeletal muscle is thought to be one of the mediators of these protective effects. A main regulatory component of these effects is the transcriptional coactivator PGC-11 which activity is upregulated upon exercise or under caloric restriction. Conversely, the activity of PGC-11 is suppressed in conditions of physical inactivity or after feeding with high fat calorie diets. Importantly, increases of PGC-11 activity in skeletal muscle are sufficient to prevent age- associated diseases and prolong life span. Deacetylation of PGC-11 through Sirt1 is one of the key chemical modifications that activate PGC-11 under exercise or nutrient deprivation conditions. In contrast, acetylation of PGC-11 by GCN5 results in an inactive protein found in non-exercise or high fat calorie diets situations. These results lead to the hypothesis that chemical compounds that induce PGC-11 deacetylation could mimic exercise or nutrient deprivation conditions. To address this hypothesis, this present proposal is aimed to identify these chemical compounds using a high throughput screening (HTS) that quantitatively measures the acetylation state of PGC-11. We have three Specific Aims: 1) Collaborate with MLPCN to implement a validated high-throughput screen to identify small molecules that decrease the acetylation level of PGC-11. We have developed and validated a physiological relevant cell-based assay using ELISA to screen the entire MLPCN library. 2) We will employ previously validated secondary assays to assess the biological relevance of identified hits on activation of PGC-11 target genes. We will focus on (i) to analyze the effects of the positive chemical compounds on counteracting pathways to eliminate possible side-effects of the drugs and, (ii) to identify the biologically relevant positive chemical compounds from the primary HTS assay by analyzing gene expression changes of PGC-11 targets. 3) Collaborate with MLPCN to develop and characterize chemical probes that affect PGC-11 acetylation using previously validated tertiary assays to test specificity and mechanism of action. We will focus on (i) we will confirm that increased expression of the PGC-11 target genes MCAD and CPT1b is dependent upon PGC-11 and, (ii) we will analyze the specific target mechanism by which positive chemical probes modulate PGC-11 acetylation through GCN5 and SIRT1. The outcomes of these studies will provide the identification of chemical compounds as well as the molecular mechanisms that control PGC-11 acetylation and as a consequence will modulate the metabolic activities regulated by this transcriptional coactivator. Since these metabolic activities mimic, at least to a large extend, exercise and calorie restriction it is very plausible that these chemical compounds might be used as potential therapies to treat metabolic disorders and age-associated diseases. PUBLIC HEALTH RELEVANCE: Exercise and calorie restriction have potent protective effects against age-associated diseases including metabolic diseases, thus studies in this grant proposal to identify chemical compounds that might mimic physical activity or nutrient deprivation through PGC-11 deacetylation might translate into potential therapies.

DESCRIPTION (provided by applicant): Nutrient homeostasis is maintained through a complex regulatory network formed by signaling and transcriptional components that control metabolic genes. The liver is one of the key tissues that depending of the physio/pathological conditions buffers whole body nutrient homeostasis. Insulin is one of the most powerful hormones that affect nutrient regulation and clinically, insulin resistance is a hallmark of the metabolic syndrome including type 2 diabetes. As a consequence of insulin resistance one of the metabolic processes that contribute to maintain the diabetic state is hepatic glucose production that is controlled, at least in part, at the transcriptional level. The PI3K/Akt pathway is one of the main effectors of insulin metabolic action. Akt controls expression of metabolic genes through direct phosphorylation and negative regulation of the forkhead transcription factor FoxO1 and coactivators such as PGC-11 and CRCT2, key components of the transcriptional gluconeogenic program. Although Akt can directly mediate this action, there are conditions such as late refeeding or diabetic states where active Akt does not entirely correlate with suppression hepatic glucose production. This indicates that additional key regulatory components, likely kinases, could mediate this repression. Along these lines, we have recently identified Cdc2-like kinase 2 (Clk2) as a novel component downstream of insulin/Akt and functions as part of the hepatic feeding response. Notably, Clk2 controls expression of gluconeogenic genes, hepatic glucose output and blood glucose levels. Moreover, obese/diabetic db/db mice have lower amounts of Clk2 protein and restoration of the levels corrects hyperglycemia. This result suggests that Clk2 might be dysregulated in conditions of obesity/diabetes and contributes to the clinical manifestations. Based on these findings, the major goal of this proposal is to identify the molecular mechanisms by which insulin controls Clk2 kinase activity and to test Clk2 metabolic functionality in-vitro and in in-vivo mouse models. We have three Specific Aims: Aim 1 is to perform molecular and functional analysis of how insulin controls Clk2 kinase activity focusing on regulation of Clk2 protein degradation through ubiquitin ligases. Aim 2 is devoted to carry out molecular and functional analysis of Clk2- mediated suppression of hepatic gluconeogenesis. We focus on the Clk2-induced phosphorylation and repression of PGC-11. Aim 3 will determine the effects of Clk2 on hepatic glucose and lipid metabolism in mice through loss-of-function of hepatic Clk2 in fasting/feeding cycles and genetic and diet-induced obesity. The outcomes of these studies will provide the identification of the molecular mechanisms by which insulin controls glucose and lipid metabolic effects through Clk2 kinase. Since insulin resistance and as a consequence increased and uncontrolled hepatic glucose output is a major defect that occurs in type 2 diabetes, our investigation on the regulation of Clk2 and PGC-11 might translate into potential therapies to treat this disease. PUBLIC HEALTH RELEVANCE: Insulin resistance and increased hepatic glucose production are hallmarks of metabolic diseases such as obesity and type 2 diabetes, thus studies in this grant proposal focusing on key regulators of these processes including a novel kinase Clk2 and PGC-11 might translate into potential therapies.

DESCRIPTION (provided by applicant): Energy expenditure (a major component of body weight) is regulated through a complex regulatory network formed by signaling and transcriptional components that control bioenergetic/metabolic function. Skeletal muscle and beige/brown adipose are key tissues that account for a large fraction of energy expenditure. Adrenergic/cAMP signaling is one of the powerful pathways that affect energy balance and cellular bioenergetics. Defects in components of the signaling/transcriptional and mitochondrial bioenergetic system is sufficient to promote obesity and associated disorders such as type 2 diabetes and atherosclerosis. Importantly, maintenance and activation of mitochondrial bioenergetic function strictly depend on basal and regulated transcription of nuclear genes encoding for mitochondrial proteins. Among the transcriptional regulators of these mitochondrial processes are the PGC1 family of coactivators and transcription factors including Nuclear Respiratory Factors, Hormone Nuclear Receptors and YY1. In the last years, our laboratory has identified the transcription factor YY1 as a key regulator of nuclear mitochondrial genes that has a major impact in mitochondrial bioenergetic capacity, both in cultured cells and in animals. Depending on YY1 phosphorylation at specific sites, phospho-YY1 forms an active complex on mitochondrial genes through recruitment of PGC1¿. In contrast, dephospho-YY1 forms a repressor complex through interacting with Polycomb Proteins that suppresses the expression of mitochondrial genes. Importantly, one of the signals that govern YY1-dependent phosphorylation interaction is the cAMP pathway. Based on these findings, the major goal of this proposal is to identify the regulatory mechanisms driving mitochondrial gene expression through YY1 transcriptional complex and to assess the functionality using in-vivo mouse models of obesity and diabetes. We have three Specific Aims: Aim 1 proposes to perform molecular mechanistic analysis of how the YY1 transcriptional complex controls mitochondrial function. Aim 2 is devoted to carry out cellular and functional mitochondrial bioenergetic and metabolic analysis mediated by the YY1 transcriptional protein complex in skeletal and adipose cultured cells. Aim 3 is focused to perform in-vivo metabolic and energetic analysis mediated by the YY1 transcription factor in skeletal muscle and adipose tissues. We will use genetic mouse models with gain and loss-of-function of YY1 in these tissues. The outcomes of these studies will provide the identification of the molecular mechanisms by which the YY1 transcriptional complex regulates mitochondrial bioenergetic capacities and how defects in this complex result in dysregulated mitochondrial function and energy balance. Based on the fact that these pathways are altered in metabolic diseases such as obesity and diabetes, studies proposed in this grant application might translate into potential therapies.

DESCRIPTION (provided by applicant): Melanoma tumors are characterized by their aggressiveness as well as by their resistance to therapeutic treatments. In the last years, major breakthroughs have been achieved in melanoma treatment as exemplified by the successful development and use of BRAF mutant inhibitors, a mutation that occurs in approximately 50% of human melanomas. However, the high degree of heterogeneity of melanoma tumors allows them to rapidly develop resistant mechanisms after clinical treatment. Thus, there are fundamental questions in melanoma biology that need to be addressed. Among them are the metabolic vulnerabilities and mechanisms of metastasis that will be investigated in this grant. Cancer cells use metabolic/energetic pathways that are critical to maintain survival and promote tumor growth and metastasis, however the specific role of these pathways and the effect on tumor progression are not completely understood. We have recently identified a subset of human melanomas (8-10%) that aberrantly overexpress the transcriptional coactivator PGC1? (OXPHOS high) that induces mitochondrial and ROS detoxification genes. PGC1? maintains survival and protect these tumors against oxidative stress and ROS-inducing drugs, but are vulnerable to mitochondrial OXPHOS inhibition. However, PGC1? suppression in melanoma tumors allows metabolic/energetic compensation through HIF1?-dependent glycolysis and metastatic progression. These results underscore the metabolic plasticity of melanomas and show that a rational combinatorial therapy will be required to treat tumors using metabolic targets. Here, we propose to continue our studies on the identification of mechanisms and targets by which human melanoma tumors reprogram metabolism conferring different vulnerabilities on cell growth and survival as well as the impact on metastasis. The goals are centered in three central aims: 1) defining the mechanisms and targets by which PGC1? controls melanoma tumor progression (Specific Aim 1), 2) identifying the metabolic compensatory mechanisms that allow survival in melanoma tumors via the HIF1? pathway (Specific Aim 2) and, 3) defining the impact and mechanisms of PGC1? on melanoma metastatic processes (Specific Aim 3). The outcomes of this proposal will advance our understanding of tumor melanoma and metabolic/energetic vulnerabilities and inform us on the identification of novel therapeutic targets in melanoma progression and metastasis.

Abstract Obesity and associated metabolic diseases including type 2 diabetes is a current epidemic in the US and worldwide. There are no current safe anti-obesity drugs available and bariatric surgery, in combination with dietary and physical activity regimens, is considered the best option to treat obese patients. Thus, there is an urgent medical need to identify new therapeutic targets and develop new and safer drugs to treat obesity. Defects in energy expenditure in response to diet or lower temperatures cause obesity. A major site of energy expenditure is the brown or beige adipose tissue that contains thermogenic mitochondria equipped to uncouple respiration and produce heat. The presence in humans of these thermogenic adipocytes opens up the possibility to activate them and protect against obesity. In the previous funded period we have found that deficiency of the protein Clk2, a kinase downstream of feeding signals and insulin, in adipose tissue decreases energy expenditure and exacerbates body weight upon high fat diet feeding. Clk2-deficient brown adipocytes exhibit a failure in thermogenic function associated with low levels of Uncoupling protein 1. However, the regulatory components and mechanisms of how the feeding-regulated Clk2 kinase activity affects cold- and diet-induced thermogenic activity in brown and beige adipose cells and control whole body energy expenditure are unknown. The major goal of this grant renewal is to identify and analyze the molecular and regulatory mechanism whereby the protein kinase Clk2 activates thermogenic function in brown/beige adipose tissue in response to cold and diet and increases energy expenditure to protect against obesity and diabetes. The research strategy is focused on three central aims: 1) Molecular and functional analysis of how Clk2 kinase controls thermogenic gene expression programs in brown/beige adipose cells (Specific Aim 1), 2) Cellular, metabolic and bioenergetic analysis mediated by Clk2 kinase action in adipose cells and fat tissues ex vivo (Specific Aim 2) and, 3) In vivo metabolic and energetic analysis driven by Clk2 kinase in cold- and diet- induced thermogenesis (Specific Aim 3). The outcomes from these studies will identify novel molecular mechanisms and regulatory components by which brown and beige adipocytes control rates of energy expenditure and protect against diet-induced obesity. Since insufficient energy expenditure is a hallmark of obesity and associated pathologies, our studies may translate into potential therapies.

Project Summary Mitochondrial diseases comprise a heterogeneous group of genetic inherited disorders resulting from mutations in mitochondrial or nuclear DNA that cause failures in mitochondrial energetic and metabolic function. As a consequence of this mitochondrial failure, high energy demanding tissues such as brain, skeletal muscle, liver, kidney, endocrine and respiratory systems are severely affected. Current available therapies remain supportive but an effective cure is still missing. Therefore, there is an urgent medical need to identify new therapeutic targets to treat mitochondrial diseases. Identification of specific targets and drugs that increase and rescue mitochondrial bioenergetics through different complexes of the electron transport chain can be of therapeutic value to treat mitochondrial diseases. An example is activation of the transcriptional coactivator PGC-1?, a major component of mitochondrial biogenesis, that rescue bioenergetic defects caused by mutations or mouse models of mitochondrial diseases and ameliorates clinical symptoms. Using a chemical and genome-wide CRISPR editing screens in trans-mitochondrial cybrids cell carrying a mutation in a mitochondrial encoded complex I subunit, we have identified Brd4 (Bromodomain protein 4) as potential target to treat mitochondrial diseases. Bromodomain inhibition or loss-of-Brd4 enhances oxidative phosphorylation activity and rescues the bioenergetic defects caused by genetic inhibition of mitochondrial complex I and promotes cell survival under high energetic demands. However, the precise mechanisms of how bromodomain inhibition controls mitochondrial bioenergetics and the effects on mitochondrial disease in in vivo models are unknown. The major goal of this grant application is to identify and analyze the molecular mechanisms whereby bromodomain inhibition activates mitochondrial energetics function and whether it rescues mitochondrial disease symptoms in in vivo mouse models. The research strategy is focused on three central aims: 1) Molecular and functional analysis of how Brd4 controls mitochondrial gene expression programs in trans-mitochondrial cybrid cells. (Specific Aim 1), 2) Cellular, metabolic and bioenergetic analysis mediated by bromodomain inhibition in cybrid cell lines and mitochondrial disease patient derived fibroblasts (Specific Aim 2) and, 3) In vivo and Ex vivo metabolic, energetic and survival analysis by bromodomain inhibitors in mitochondrial disease mouse models (Specific Aim 3). The outcomes from these studies will identify novel molecular mechanisms and regulatory components by which bromodomain inhibition and loss of Brd4 rescue bioenergetic defects caused by mitochondrial electron transfer chain defects. Since mitochondrial bioenergetic failure is a hallmark of mitochondrial diseases, our studies may translate into potential therapies.

Summary Obesity is a current major health problem associated with life threatening complications such as cardiovascular disease, type 2 diabetes and cancer. Obesity is managed with dietary regimens that selectively reduce energy intake and/or exercise programs focused on energy expenditure. These treatments have high failure and relapse rates urging new strategies focused on drug-targeted therapies. Recent findings in humans showing that activation of brown and beige thermogenic function can increase energy expenditure open a new route to target the molecular components in these tissues that control body temperature and weight. However, the regulatory mechanisms of the components that control thermogenic gene expression and whole body energy balance are not completely understood. The transcription factor YY1 controls the thermogenic function in brown and beige fat through transcriptional and epigenetic changes in genes encoding for this metabolic and energetic program. Our previous studies found that mice deficient in the transcription factor YY1 in adipose tissue are strongly protected against diet-induced obesity. Mechanistically, this protection is caused by the fact that YY1 can repress brown fat secreted proteins that activate beige fat thermogenic activity. In this grant renewal, we propose studies that will focus on two main processes in thermogenic adipocytes. (1) Define the specific YY1 activation function through the INO80 complex that controls mitochondrial bioenergetic gene expression, and (2) define the YY1 repression function that controls brown fat secreted proteins that activate beige adipose thermogenic function. Because both regulatory processes are directly linked to energy expenditure they have strong significance towards potential treatments for metabolic diseases. The major goal of this application is to identify the transcriptional and epigenetic mechanisms, focusing on the YY1/INO80 chromatin remodeling complex and secreted proteins, underlying the brown and beige adipose thermogenic function which promotes energy expenditure and protects against obesity. Three different aims are proposed, 1) Transcriptional and epigenetic regulatory analysis of how the YY1/INO80 chromatin remodeling complex controls mitochondrial/thermogenic and secreted proteins gene expression programs in brown adipose tissue (Specific Aim 1), 2) Metabolic and bioenergetic analysis mediated by the YY1/INO80-dependent thermogenic and secreted gene expression programs in brown and beige adipose cells (Specific Aim 2) and, 3) Energy and metabolic analysis in response to cold- and diet-induced thermogenesis mediated through the YY1/INO80 transcriptional complex and GDF15 secreted protein (Specific Aim 3). The outcomes from this application will identify the transcriptional and epigenetic mechanisms that control adjustable thermogenesis in response to cold and overnutrition driven through the YY1/INO80 chromatin remodeling complex. Since obesity is linked to insufficient energy expenditure that is unable to counteract increased dietary intake, our studies have therapeutic implications for obesity treatment.

Abstract Type 2 diabetes (T2D), particularly associated with obesity, is an epidemic in the US and worldwide and it is the leading cause of renal diseases, non-traumatic loss of limb and blindness. Despite the fact that there are current antidiabetic drugs, such as insulin secretagogues and metformin, used in the treatment of T2D, there is an urgent medical need of additional targeted therapies for an improved management of this disease in patients in which these current drugs have moderate efficacy. As T2D progresses, there is exacerbated and uncontrolled hepatic glucose production that strongly contributes to chronic hyperglycemia, a major cause of the various diabetic pathologies. Acetylation of the transcriptional coactivator PGC-1? selectively suppresses hepatic glucose production and ameliorates T2D. We have used a series of chemical high throughput and secondary assays to identify a small molecule, SR-18292, that increases PGC-1? acetylation, suppressing its gluconeogenic activity. SR-18292 inhibits glucagon and PGC-1?-dependent gluconeogenic gene expression through increased binding between PGC-1? and the GCN5 Acetyl Transferase, displacing the transcription factor HNF4? from gluconeogenic promoters and reducing epigenetic histone activation marks. In diabetic mice, SR-18292 decreases hepatic glucose output, hyperglycemia and increases liver insulin sensitivity. Combined, these studies support targeting this pathway for potential therapeutic intervention for T2D. Thus, the primary goal of this application is to characterize SR-18292 and optimize analogs that could have the potential to become a new anti-diabetic drug therapy in T2D. We will perform a complete SAR (structure and activity relationship), DMPK (drug metabolism and pharmacokinetics), toxicity and a series of in vitro and in vivo metabolic studies to validate the pathway and to identify a SR-18292 analog with robust anti-diabetic activities. The experimental design is focused on two aims: 1) SAR and DMPK studies based on the SR-18292 molecule scaffold using in-vitro and in-vivo assays and target identification (Specific Aim 1) and, 2) toxicology and in-vivo metabolic studies using the SR-18292 analog (Specific Aim 2). The outcomes of this proposal will provide significant contribution to the early-stage preclinical validation for the SR-18292 analogs as therapeutic leads for management of T2D and insulin resistance.

Project Summary Metastatic melanoma was until recently considered an untreatable disease, but the discovery of small molecules that inhibit oncogenic BRAF(V600E) and approaches that unleash the immune system against tumors have brought hope to melanoma patients. Not every patient will have meaningful therapeutic benefit from these treatments and durable disease remission remains elusive for most. Among the causes of the failure to respond or early relapse is a dynamic cancer cell heterogeneity that facilitates outgrowth of therapy resistant tumors with enhanced malignancy traits. In order to extend the use of current therapies, we propose to identify alternative molecular targets that could be harnessed for combinatorial treatment exploit and might hold promise for sustainable therapeutic benefit. Specifically, whether metabolic and epigenetic processes provide collateral dependencies within highly metastatic and chronic BRAF-targeted drug-adapted melanomas is largely unknown. To this end, a third of melanomas display heightened expression of the transcriptional coactivator PGC1? that integrates mitochondrial biogenesis and bioenergetic activity to ensure cellular survival. Previously we found an inverse functional relationship between PGC1? expression and vertical growth phase within primary melanoma that associates with poor patient prognosis, and genetic targeting of PGC1? provoked enhanced metastatic traits in cell line models. Consistent with a functional role for adaptive expression of PGC1? and enhanced malignancy traits, our current preliminary data supports that chronic adaptation to BRAF-targeted drugs silences PGC1? expression through altered histone marks across its promoter region. We now propose to seek the molecular mechanisms that attenuate PGC1? expression that links enhanced metastatic spread and chronic adaptation to BRAF-targeted drugs. In an integrated study plan that includes clinical melanoma specimens, established cell lines and in vivo tumor modeling, the experimental design is focused on two aims: 1) to determine epigenetic mechanisms that silence PGC1? expression during chronic adaptation to targeted BRAF(V600E) treatment; and 2) to identify collateral metabolic and epigenetic vulnerabilities arising from chronic adaptation to targeted BRAF(V600E) treatment. Outcomes from these studies will identify metabolites and epigenetic regulators that provoke vulnerabilities within alternate PGC1?- dependent epigenetic states. Successful completion of the proposed study plan may help predict patients at heightened clinical risk as well as provide means to break chronic adaptation to BRAF-targeted drugs.