The Genomic and Epigenomic Landscape of Double-Negative Metastatic Prostate Cancer.

Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen signaling. Androgen deprivation therapy and second-generation androgen receptor (AR)-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by AR and neuroendocrine (NE) markers. Molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterized treatment-emergent mCRPC by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing from 210 tumors. AR-/NE- tumors were clinically and molecularly distinct from other mCRPC subtypes, with the shortest survival, amplification of the chromatin remodeler CHD7, and PTEN loss. Methylation changes in CHD7 candidate enhancers were linked to elevated CHD7 expression in AR-/NE+ tumors. Genome-wide methylation analysis nominated Krüppel-like factor 5 (KLF5) as a driver of the AR-/NE- phenotype, and KLF5 activity was linked to RB1 loss. These observations reveal the aggressiveness of AR-/NE- mCRPC and could facilitate the identification of therapeutic targets in this highly aggressive disease. SIGNIFICANCE: Comprehensive characterization of the five subtypes of metastatic castration-resistant prostate cancer identified transcription factors that drive each subtype and showed that the double-negative subtype has the worst prognosis.

Docking and Linking of Fragments To Discover Jumonji Histone Demethylase Inhibitors.

Development of tool molecules that inhibit Jumonji demethylases allows for the investigation of cancer-associated transcription. While scaffolds such as 2,4-pyridinedicarboxylic acid (2,4-PDCA) are potent inhibitors, they exhibit limited selectivity. To discover new inhibitors for the KDM4 demethylases, enzymes overexpressed in several cancers, we docked a library of 600,000 fragments into the high-resolution structure of KDM4A. Among the most interesting chemotypes were the 5-aminosalicylates, which docked in two distinct but overlapping orientations. Docking poses informed the design of covalently linked fragment compounds, which were further derivatized. This combined approach improved affinity by ∼ 3 log-orders to yield compound 35 (Ki = 43 nM). Several hybrid inhibitors were selective for KDM4C over the related enzymes FIH, KDM2A, and KDM6B while lacking selectivity against the KDM3 and KDM5 subfamilies. Cocrystal structures corroborated the docking predictions. This study extends the use of structure-based docking from fragment discovery to fragment linking optimization, yielding novel KDM4 inhibitors.

OCT1 is a high-capacity thiamine transporter that regulates hepatic steatosis and is a target of metformin.

Organic cation transporter 1, OCT1 (SLC22A1), is the major hepatic uptake transporter for metformin, the most prescribed antidiabetic drug. However, its endogenous role is poorly understood. Here we show that similar to metformin treatment, loss of Oct1 caused an increase in the ratio of AMP to ATP, activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride (TG) levels in livers from healthy and leptin-deficient mice. Conversely, livers of human OCT1 transgenic mice fed high-fat diets were enlarged with high TG levels. Metabolomic and isotopic uptake methods identified thiamine as a principal endogenous substrate of OCT1. Thiamine deficiency enhanced the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase. Metformin and the biguanide analog, phenformin, competitively inhibited OCT1-mediated thiamine uptake. Acute administration of metformin to wild-type mice reduced intestinal accumulation of thiamine. These findings suggest that OCT1 plays a role in hepatic steatosis through modulation of energy status. The studies implicate OCT1 as well as metformin in thiamine disposition, suggesting an intriguing and parallel mechanism for metformin and its major hepatic transporter in metabolic function.

Multiple targets of nitric oxide in the tricarboxylic acid cycle of Salmonella enterica serovar typhimurium.

Host nitric oxide (NO⋅) production is important for controlling intracellular bacterial pathogens, including Salmonella enterica serovar Typhimurium, but the underlying mechanisms are incompletely understood. S. Typhmurium 14028s is prototrophic for all amino acids but cannot synthesize methionine (M) or lysine (K) during nitrosative stress. Here, we show that NO⋅-induced MK auxotrophy results from reduced succinyl-CoA availability as a consequence of NO⋅ targeting of lipoamide-dependent lipoamide dehydrogenase (LpdA) activity. LpdA is an essential component of the pyruvate and α-ketoglutarate dehydrogenase complexes. Additional effects of NO⋅ on gene regulation prevent compensatory pathways of succinyl-CoA production. Microarray analysis indicates that over 50% of the transcriptional response of S. Typhimurium to nitrosative stress is attributable to LpdA inhibition. Bacterial methionine transport is essential for virulence in NO⋅-producing mice, demonstrating that NO⋅-induced MK auxotrophy occurs in vivo. These observations underscore the importance of metabolic targets for antimicrobial actions of NO⋅.

Incompatibility with formin Cdc12p prevents human profilin from substituting for fission yeast profilin: insights from crystal structures of fission yeast profilin.

Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in fission yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of fission yeast formin Cdc12p and poly-L-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161-1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in fission yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in fission yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider "wings" that interact with actin.