Ras-dependent activation of BMAL2 regulates hypoxic metabolism in pancreatic cancer.

To identify novel drivers of malignancy in pancreatic ductal adenocarcinoma (PDAC), we employed regulatory network analysis, which calculates the activity of transcription factors and other regulatory proteins based on the integrated expression of their positive and negative target genes. We generated a regulatory network for the malignant epithelial cells of human PDAC using gene expression data from a set of 197 laser capture microdissected human PDAC samples and 45 low-grade precursors, for which we had matched histopathological, clinical, and epidemiological annotation. We then identified the most highly activated and repressed regulatory proteins (e.g. master regulators or MRs) associated with four malignancy phenotypes: precursors vs. PDAC (initiation), low-grade vs. high grade histopathology (progression), survival post resection, and association with KRAS activity. Integrating across these phenotypes, the top MR of PDAC malignancy was found to be BMAL2, a member of the PAS family of bHLH transcription factors. Although the canonical function of BMAL2 is linked to the circadian rhythm protein CLOCK, annotation of BMAL2 target genes highlighted a potential role in hypoxia response. We previously demonstrated that PDAC is hypovascularized and hypoperfused, and here show that PDAC from the genetically engineered KPC model exists in a state of extreme hypoxia, with a partial oxygen pressure of <1mmHg. Given the close homology of BMAL2 to HIF1ß (ARNT) and its potential to heterodimerize with HIF1A and HIF2A, we investigated whether BMAL2 plays a role in the hypoxic response of PDAC. Indeed, BMAL2 controlled numerous hypoxia response genes and could be inhibited following treatment with multiple RAF, MEK, and ERK inhibitors, validating its association with RAS activity. Knockout of BMAL2 in four human PDAC cell lines led to defects in growth and invasion in the setting of hypoxia. Strikingly, BMAL2 null cells failed to induce glycolysis upon exposure to severe hypoxia and this was associated with a loss of expression of the glycolytic enzyme LDHA. Moreover, HIF1A was no longer stabilized under hypoxia in BMAL2 knockout cells. By contrast, HIF2A was hyper-stabilized under hypoxia, indicating a dysregulation of hypoxia metabolism in response to BMAL2 loss. We conclude that BMAL2 is a master regulator of hypoxic metabolism in PDAC, serving as a molecular switch between the disparate metabolic roles of HIF1A- and HIF2A-dependent hypoxia responses.

BRCA1 and TP53 codeficiency causes a PARP inhibitor-sensitive erythroproliferative neoplasm.

Mutations in the BRCA1 tumor suppressor gene, such as 5382insC (BRCA1insC), give carriers an increased risk for breast, ovarian, prostate, and pancreatic cancers. We have previously reported that, in mice, Brca1 deficiency in the hematopoietic system leads to pancytopenia and, as a result, early lethality. We explored the cellular consequences of Brca1-null and BRCA1insC alleles in combination with Trp53 deficiency in the murine hematopoietic system. We found that Brca1 and Trp53 codeficiency led to a highly penetrant erythroproliferative disorder that is characterized by hepatosplenomegaly and by expanded megakaryocyte erythroid progenitor (MEP) and immature erythroid blast populations. The expanded erythroid progenitor populations in both BM and spleen had the capacity to transmit the disease into secondary mouse recipients, suggesting that Brca1 and Trp53 codeficiency provides a murine model of hematopoietic neoplasia. This Brca1/Trp53 model replicated Poly (ADP-ribose) polymerase (PARP) inhibitor olaparib sensitivity seen in existing Brca1/Trp53 breast cancer models and had the benefits of monitoring disease progression and drug responses via peripheral blood analyses without sacrificing experimental animals. In addition, this erythroid neoplasia developed much faster than murine breast cancer, allowing for increased efficiency of future preclinical studies.

Functional Interaction of BRCA1 and CREBBP in Murine Hematopoiesis.

Both BRCA1 and CREBBP are tumor suppressor genes that are important for hematopoiesis. We have previously shown that mouse Brca1 is essential for hematopoietic stem cell (HSC) viability. In contrast to Brca1 deficiency, which results in pancytopenia, we report here that Crebbp deficiency results in myeloproliferation associated with an increase of splenic HSCs as well as a lethal systemic inflammatory disorder (LD50 = 86 days). To investigate the interaction of these two proteins in hematopoiesis, we generated double Crebbp/Brca1 knockout mice (DKOs). To our surprise, DKOs had accelerated bone marrow failure compared with Brca1-deficient mice and this was associated with an even shorter lifespan (LD50 = 88.5 versus 33 days). Furthermore, Crebbp or Brca1 heterozygosity influenced the hematopoietic phenotype associated with complete deficiency of Brca1 or Crebbp, respectively. We also observed lower BRCA1 protein levels in hematopoietic tissues when CREBBP is absent. Collectively, these data suggest Crebbp and Brca1 functionally interact to maintain normal hematopoiesis.

Experimental microdissection enables functional harmonisation of pancreatic cancer subtypes.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDA) has among the highest stromal fractions of any cancer and this has complicated attempts at expression-based molecular classification. The goal of this work is to profile purified samples of human PDA epithelium and stroma and examine their respective contributions to gene expression in bulk PDA samples. DESIGN: We used laser capture microdissection (LCM) and RNA sequencing to profile the expression of 60 matched pairs of human PDA malignant epithelium and stroma samples. We then used these data to train a computational model that allowed us to infer tissue composition and generate virtual compartment-specific expression profiles from bulk gene expression cohorts. RESULTS: Our analysis found significant variation in the tissue composition of pancreatic tumours from different public cohorts. Computational removal of stromal gene expression resulted in the reclassification of some tumours, reconciling functional differences between different cohorts. Furthermore, we established a novel classification signature from a total of 110 purified human PDA stroma samples, finding two groups that differ in the extracellular matrix-associated and immune-associated processes. Lastly, a systematic evaluation of cross-compartment subtypes spanning four patient cohorts indicated partial dependence between epithelial and stromal molecular subtypes. CONCLUSION: Our findings add clarity to the nature and number of molecular subtypes in PDA, expand our understanding of global transcriptional programmes in the stroma and harmonise the results of molecular subtyping efforts across independent cohorts.

Deficiency of the Endocytic Protein Hip1 Leads to Decreased Gdpd3 Expression, Low Phosphocholine, and Kypholordosis.

Deficiency of huntingtin-interacting protein 1 (Hip1) results in degenerative phenotypes. Here we generated a Hip1 deficiency allele where a floxed transcriptional stop cassette and a human HIP1 cDNA were knocked into intron 1 of the mouse Hip1 locus. CMV-Cre-mediated germ line excision of the stop cassette resulted in expression of HIP1 and rescue of the Hip1 knockout phenotype. Mx1-Cre-mediated excision led to HIP1 expression in spleen, kidney and liver, and also rescued the phenotype. In contrast, hGFAP-Cre-mediated, brain-specific HIP1 expression did not rescue the phenotype. Metabolomics and microarrays of several Hip1 knockout tissues identified low phosphocholine (PC) levels and low glycerophosphodiester phosphodiesterase domain containing 3 (Gdpd3) gene expression. Since Gdpd3 has lysophospholipase D activity that results in the formation of choline, a precursor of PC, Gdpd3 downregulation could lead to the low PC levels. To test whether Gdpd3 contributes to the Hip1 deficiency phenotype, we generated Gdpd3 knockout mice. Double knockout of Gdpd3 and Hip1 worsened the Hip1 phenotype. This suggests that Gdpd3 compensates for Hip1 loss. More-detailed knowledge of how Hip1 deficiency leads to low PC will improve our understanding of HIP1 in choline metabolism in normal and disease states.

Liver LXRα expression is crucial for whole body cholesterol homeostasis and reverse cholesterol transport in mice.

Liver X receptors (LXRα and LXRß) are important regulators of cholesterol and lipid metabolism, and their activation has been shown to inhibit cardiovascular disease and reduce atherosclerosis in animal models. Small molecule agonists of LXR activity are therefore of great therapeutic interest. However, the finding that such agonists also promote hepatic lipogenesis has led to the idea that hepatic LXR activity is undesirable from a therapeutic perspective. To investigate whether this might be true, we performed gene targeting to selectively delete LXRα in hepatocytes. Liver-specific deletion of LXRα in mice substantially decreased reverse cholesterol transport, cholesterol catabolism, and cholesterol excretion, revealing the essential importance of hepatic LXRα for whole body cholesterol homeostasis. Additionally, in a pro-atherogenic background, liver-specific deletion of LXRα increased atherosclerosis, uncovering an important function for hepatic LXR activity in limiting cardiovascular disease. Nevertheless, synthetic LXR agonists still elicited anti-atherogenic activity in the absence of hepatic LXRα, indicating that the ability of agonists to reduce cardiovascular disease did not require an increase in cholesterol excretion. Furthermore, when non-atherogenic mice were treated with synthetic LXR agonists, liver-specific deletion of LXRα eliminated the detrimental effect of increased plasma triglycerides, while the beneficial effect of increased plasma HDL was unaltered. In sum, these observations suggest that therapeutic strategies that bypass the liver or limit the activation of hepatic LXRs should still be beneficial for the treatment of cardiovascular disease.

LRH-1 and PTF1-L coregulate an exocrine pancreas-specific transcriptional network for digestive function.

We have determined the cistrome and transcriptome for the nuclear receptor liver receptor homolog-1 (LRH-1) in exocrine pancreas. Chromatin immunoprecipitation (ChIP)-seq and RNA-seq analyses reveal that LRH-1 directly induces expression of genes encoding digestive enzymes and secretory and mitochondrial proteins. LRH-1 cooperates with the pancreas transcription factor 1-L complex (PTF1-L) in regulating exocrine pancreas-specific gene expression. Elimination of LRH-1 in adult mice reduced the concentration of several lipases and proteases in pancreatic fluid and impaired pancreatic fluid secretion in response to cholecystokinin. Thus, LRH-1 is a key regulator of the exocrine pancreas-specific transcriptional network required for the production and secretion of pancreatic fluid.

The G protein-coupled bile acid receptor, TGR5, stimulates gallbladder filling.

TGR5 is a G protein-coupled bile acid receptor present in brown adipose tissue and intestine, where its agonism increases energy expenditure and lowers blood glucose. Thus, it is an attractive drug target for treating human metabolic disease. However, TGR5 is also highly expressed in gallbladder, where its functions are less well characterized. Here, we demonstrate that TGR5 stimulates the filling of the gallbladder with bile. Gallbladder volume was increased in wild-type but not Tgr5(-/-) mice by administration of either the naturally occurring TGR5 agonist, lithocholic acid, or the synthetic TGR5 agonist, INT-777. These effects were independent of fibroblast growth factor 15, an enteric hormone previously shown to stimulate gallbladder filling. Ex vivo analyses using gallbladder tissue showed that TGR5 activation increased cAMP concentrations and caused smooth muscle relaxation in a TGR5-dependent manner. These data reveal a novel, gallbladder-intrinsic mechanism for regulating gallbladder contractility. They further suggest that TGR5 agonists should be assessed for effects on human gallbladder as they are developed for treating metabolic disease.

AKR1B7 is induced by the farnesoid X receptor and metabolizes bile acids.

Although bile acids are crucial for the absorption of lipophilic nutrients in the intestine, they are cytotoxic at high concentrations and can cause liver damage and promote colorectal carcinogenesis. The farnesoid X receptor (FXR), which is activated by bile acids and abundantly expressed in enterohepatic tissues, plays a crucial role in maintaining bile acids at safe concentrations. Here, we show that FXR induces expression of Akr1b7 (aldo-keto reductase 1b7) in murine small intestine, colon, and liver by binding directly to a response element in the Akr1b7 promoter. We further show that AKR1B7 metabolizes 3-keto bile acids to 3ß-hydroxy bile acids that are less toxic to cultured cells than their 3α-hydroxy precursors. These findings reveal a feed-forward, protective pathway operative in murine enterohepatic tissues wherein FXR induces AKR1B7 to detoxify bile acids.

Regulation of bile acid synthesis by fat-soluble vitamins A and D.

Bile acids are required for proper absorption of dietary lipids, including fat-soluble vitamins. Here, we show that the dietary vitamins A and D inhibit bile acid synthesis by repressing hepatic expression of the rate-limiting enzyme CYP7A1. Receptors for vitamin A and D induced expression of Fgf15, an intestine-derived hormone that acts on liver to inhibit Cyp7a1. These effects were mediated through distinct cis-acting response elements in the promoter and intron of Fgf15. Interestingly, transactivation of both response elements appears to be required to maintain basal Fgf15 expression levels in vivo. Furthermore, whereas induction of Fgf15 by vitamin D is mediated through its receptor, the induction of Fgf15 by vitamin A is mediated through the retinoid X receptor/farnesoid X receptor heterodimer and is independent of bile acids, suggesting that this heterodimer functions as a distinct dietary vitamin A sensor. Notably, vitamin A treatment reversed the effects of the bile acid sequestrant cholestyramine on Fgf15, Shp, and Cyp7a1 expression, suggesting a potential therapeutic benefit of vitamin A under conditions of bile acid malabsorption. These results reveal an unexpected link between the intake of fat-soluble vitamins A and D and bile acid metabolism, which may have evolved as a means for these dietary vitamins to regulate their own absorption.

SUMO-mediated inhibition of glucocorticoid receptor synergistic activity depends on stable assembly at the promoter but not on DAXX.

Multiple transcription factors, including members of the nuclear receptor family, harbor one or more copies of a short regulatory motif that limits synergistic transactivation in a context-dependent manner. These synergy control (SC) motifs exert their effects by serving as sites for posttranslational modification by small ubiquitin-like modifier (SUMO) proteins. By analyzing the requirements for both synergy control and SUMOylation in the glucocorticoid receptor (GR), we find that an intact ligand-binding domain and an engaged DNA- binding domain dimerization interface are necessary for effective synergy control. However, these features, which promote stable assembly of GR-DNA complexes, are required downstream of SUMOylation because their disruption or deletion does not interfere with SUMO modification. Remarkably, in the absence of these features, sensitivity to the effects of SUMOylation can be restored simply by stabilization of DNA interactions through a heterologous DNA binding domain. The data indicate that stable interaction with DNA is an important prerequisite for SUMO-dependent transcriptional inhibition. Analysis of genomic regions occupied by GR indicates that the effects of SC motif SUMOylation are most evident at multiple, near-ideal GR binding sites and that SUMOylation selectively affects the induction of linked endogenous genes. Although the SUMO-binding protein DAXX has been proposed to mediate the inhibitory effects of GR SUMOylation, we find that inhibition by DAXX is independent of GR SUMOylation. Furthermore, neither expression nor knockdown of DAXX influences SUMO effects on GR. We therefore propose that stable binding of GR to multiple sites on DNA allows for the SUMO-dependent recruitment of inhibitory factors distinct from DAXX.

Identification of a hormonal basis for gallbladder filling.

The cycle of gallbladder filling and emptying controls the flow of bile into the intestine for digestion. Here we show that fibroblast growth factor-15, a hormone made by the distal small intestine in response to bile acids, is required for gallbladder filling. These studies demonstrate that gallbladder filling is actively regulated by an endocrine pathway and suggest a postprandial timing mechanism that controls gallbladder motility.

A small conserved surface in SUMO is the critical structural determinant of its transcriptional inhibitory properties.

Small ubiquitin-like modifier (SUMO) modification of sequence-specific transcription factors has profound regulatory consequences. By providing an intrinsic inhibitory function, SUMO isoforms can suppress transcriptional activation, particularly at promoters harboring multiple response elements. Through a comprehensive structure-function analysis, we have identified a single critical sector along the second beta sheet and the following alpha helix of SUMO2. This distinct surface is defined by four basic residues (K33, K35, K42, R50) that surround a shallow pocket lined by aliphatic (V30, I34) and polar (T38) residues. Substitutions within this area specifically and dramatically affected the ability of both SUMO2 and SUMO1 to inhibit transcription and revealed that the positively charged nature of the key basic residues is the main feature responsible for their functional role. This highly conserved surface accounts for the inhibitory properties of SUMO on multiple transcription factors and promoter contexts and likely defines the interaction surface for the corepressors that mediate the inhibitory properties of SUMO.

Direct and distinguishable inhibitory roles for SUMO isoforms in the control of transcriptional synergy.

Functional interactions between factors bound at multiple sites on DNA often lead to a synergistic or more-than-additive transcriptional response. We previously defined a class of peptide sequences termed synergy control motifs (SC motifs) that function in multiple regulators by selectively inhibiting synergistic activity driven from multiple but not single response elements. By studying the prototypic SC motifs of the glucocorticoid receptor, we show that SC motifs inhibit transcription per se both in cis and in trans, and that a requirement for multiple contacts with DNA renders them selective for compound response elements. Notably, SC motifs are sites for SUMOylation, and the degree of modification correlates strongly with the extent of synergy control. Recruiting SUMO to the promoter either independently or as a fusion to the glucocorticoid receptor is sufficient to recapitulate the in trans and in cis inhibition by SC motifs without apparent changes in subcellular localization. Moreover, we find that the core ubiquitin fold domain of SUMO is sufficient for inhibition and that, independently of their potential for polySUMO chain formation, SUMO-2 and SUMO-3 are more effective inhibitors than SUMO-1.