For several well-documented reasons, it has been challenging to develop artificial small molecule inhibitors of protein/protein complexes. Such reagents are of particular interest for transcription factor complexes given links between their misregulation and disease. Here we report parallel approaches to identify regulators of a hypoxia signaling transcription factor complex, involving the ARNT subunit of the HIF (Hypoxia Inducible Factor) activator and the TACC3 (Transforming Acidic Coiled Coil Containing Protein 3) coactivator. In one route, we used in vitro NMR and biochemical screening to identify small molecules that selectively bind within the ARNT PAS (Per-ARNT-Sim) domain that recruits TACC3, identifying KG-548 as an ARNT/TACC3 disruptor. A parallel, cell-based screening approach previously implicated the small molecule KHS101 as an inhibitor of TACC3 signaling. Here, we show that KHS101 works indirectly on HIF complex formation by destabilizing both TACC3 and the HIF component HIF-1α. Overall, our data identify small molecule regulators for this important complex and highlight the utility of pursuing parallel strategies to develop protein/protein inhibitors.

G protein-coupled receptor (GPCR) pathways control glucose and fatty acid metabolism and the onset of obesity and diabetes. Regulators of G protein signaling (RGS) are GTPase-activating proteins (GAPs) for G i and G q ␣-subunits that control the intensity and duration of GPCR signaling. Herein we determined the role of Rgs16 in GPCR regulation of liver metabolism. Rgs16 is expressed during the last few hours of the daily fast in periportal hepatocytes, the oxygen-rich zone of the liver where lipolysis and gluconeogenesis predominate. Rgs16 knock-out mice had elevated expression of fatty acid oxidation genes in liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma ␤-ketone levels compared with wild type mice. By contrast, transgenic mice that overexpressed RGS16 protein specifically in liver exhibited reciprocal phenotypes as well as low blood glucose levels compared with wild type littermates and fatty liver after overnight fasting. The transcription factor carbohydrate response element-binding protein (ChREBP), which induces fatty acid synthesis genes in response to high carbohydrate feeding, was unexpectedly required during fasting for maximal Rgs16 transcription in liver and in cultured primary hepatocytes during gluconeogenesis. Thus, RGS16 provides a signaling mechanism for glucose production to inhibit GPCRstimulated fatty acid oxidation in hepatocytes.Body weight homeostasis is maintained, in part, by complex communication between G protein-coupled receptors (GPCRs) 5 localized in the brain and in the periphery. Long term and short term satiety signals are integrated to create a dynamic equilibrium between energy expenditure and food intake.The activation cycle of heterotrimeric G proteins revolves around receptor-catalyzed guanine nucleotide exchange on the G␣ subunit; the G␣ GDP ␤␥ heterotrimer is inactive, whereas hormone binding to receptor catalyzes formation of active G␣ GTP (1). RGS proteins are GTPase-activating proteins (GAPs) for G i -and G q/11 -class ␣-subunits and can terminate signaling by restoring the inactive G␣ GDP ␤␥ heterotrimer, thereby uncoupling hormone-bound receptor from effector protein activation (2-4). An important complexity of G protein signaling is that both G␣ GTP and free G␤␥ subunits can independently regulate the production of second messengers by effector proteins. RGS proteins can integrate and coordinate responses to separate G␣ and G␤␥ signals to generate an emergent property, such as RGS-mediated Ca 2ϩ oscillations evoked by G␣ q/11 -coupled agonists (5-8).Given that Rgs mRNAs were up-regulated by GPCR agonists controlling mating responses and nutrient sensing in fungi (9 -11), we hypothesized that Rgs expression could be utilized as a marker for unknown G i -or G q/11 -mediated signal transduction in mammalian physiology. To explore novel G protein function in liver, we surveyed differential regulation of Rgs genes in liver of fasted and refed wild type mice (12). Interestingly, of the 21 Rgs genes, only Rgs16 mRNA was diurnally express...

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related deaths in the United States, and is projected to be second by 2025. It has the worst survival rate among all major cancers. Two pressing needs for extending life expectancy of affected individuals are the development of new approaches to identify improved therapeutics, addressed herein, and the identification of early markers. PDA advances through a complex series of intercellular and physiological interactions that drive cancer progression in response to organ stress, organ failure, malnutrition, and infiltrating immune and stromal cells. Candidate drugs identified in organ culture or cell-based screens must be validated in preclinical models such as KIC (p48Cre;LSL-KrasG12D;Cdkn2af/f) mice, a genetically engineered model of PDA in which large aggressive tumors develop by 4 weeks of age. We report a rapid, systematic and robust in vivo screen for effective drug combinations to treat Kras-dependent PDA. Kras mutations occur early in tumor progression in over 90% of human PDA cases. Protein kinase and G-protein coupled receptor (GPCR) signaling activates Kras. Regulators of G-protein signaling (RGS) proteins are coincidence detectors that can be induced by multiple inputs to feedback-regulate GPCR signaling. We crossed Rgs16::GFP bacterial artificial chromosome (BAC) transgenic mice with KIC mice and show that the Rgs16::GFP transgene is a KrasG12D-dependent marker of all stages of PDA, and increases proportionally to tumor burden in KIC mice. RNA sequencing (RNA-Seq) analysis of cultured primary PDA cells reveals characteristics of embryonic progenitors of pancreatic ducts and endocrine cells, and extraordinarily high expression of the receptor tyrosine kinase Axl, an emerging cancer drug target. In proof-of-principle drug screens, we find that weanling KIC mice with PDA treated for 2 weeks with gemcitabine (with or without Abraxane) plus inhibitors of Axl signaling (warfarin and BGB324) have fewer tumor initiation sites and reduced tumor size compared with the standard-of-care treatment. Rgs16::GFP is therefore an in vivo reporter of PDA progression and sensitivity to new chemotherapeutic drug regimens such as Axl-targeted agents. This screening strategy can potentially be applied to identify improved therapeutics for other cancers.

Background: Heterotrimeric G protein signaling in liver helps maintain carbohydrate and lipid homeostasis. G protein signaling is activated by binding of extracellular ligands to G protein coupled receptors and inhibited inside cells by regulators of G protein signaling (RGS) proteins. RGS proteins are GTPase activating proteins, and thereby regulate Gi and/or Gq class G proteins. RGS gene expression can be induced by the ligands they feedback regulate, and RGS gene expression can be used to mark tissues and cell-types when and where Gi/q signaling occurs. We characterized the expression of mouse RGS genes in liver during fasting and refeeding to identify novel signaling pathways controlling changes in liver metabolism.

Pancreatic ductal adenocarcinoma (PDA) is the 4th leading cause of cancer related deaths. Progress towards effective therapy for PDA has been very limited. We are developing a systematic and robust in vivo screen for effective drug combinations. Kras mutations (e.g. KrasG12D) are found in over 90% of human PDA and occur early in tumor progression. Protein kinase and G‐Protein Coupled Receptor (GPCR) signaling can initiate Ras activation. Regulators of G‐protein Signaling (RGS) proteins are coincidence detectors that can be induced by multiple inputs to feedback regulate GPCR signaling. We previously described Rgs16 expression during embryonic and postnatal pancreas development in pancreatic progenitor and endocrine cells (DMM3, 567). Here, we show that the Rgs16::GFP transgene is a KrasG12D dependent marker of all neoplastic stages in the LSL‐KrasG12D; Cdkn2af/f; p48Cre (KIC) mice. Rgs16::GFP expression first emerges in ductal Pancreatic Intraepithelial Neoplasia two weeks after birth. The distribution and intensity of Rgs16::GFP increase proportional to tumor burden and extend to acinar cells of distal lobes after occlusion of proximal ducts. RNA‐seq gene expression analysis of primary PDA cell culture shows characteristics of embryonic progenitors of pancreatic ducts and endocrine cells. The receptor tyrosine kinase Axl is a new target for drug development and overexpressed in PDA cells. In a proof‐of‐principle for drug screens, we find PDA weanling mice treated with gemcitabine and Axl inhibitors for 2 weeks have significantly lower quantitative Rgs16::GFP expression and reduced tumor size and occurrence than gemcitabine alone. Rgs16::GFP is hence an in vivo reporter of PDA progression and sensitivity to new chemotherapeutic drug regimens. Supported by NCI CA161624.