SUMMARY: Cyclin-Dependent Kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50=5nM) that has broad anti-cancer activity in-vitro and is effective in in-vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

SUMMARY Mitochondrial Ca2+ Uniporter (MCU)-dependent mitochondrial Ca2+ uptake is the primary mechanism for increasing matrix Ca2+ in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1, MCUR1, have severely impaired [Ca2+]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation and migration but elicited autophagy. These studies establish the existence of a MCU complex which assembles at the mitochondrial integral membrane and regulates Ca2+-dependent mitochondrial metabolism.

Background: TRPM2 channels play an essential role in cell death following oxidative stress. Results: Dominant negative TRPM2-S decreases growth of neuroblastoma xenografts and increases doxorubicin sensitivity through modulation of HIF-1/2␣ expression, mitophagy, and mitochondrial function. Conclusion: TRPM2 is important for neuroblastoma growth and viability through modulation of HIF-1/2␣. Significance: Modulation of TRPM2 may be a novel approach in cancer therapeutics.

Obesity-linked insulin resistance greatly increases the risk for type 2 diabetes, hypertension, dyslipidemia, and non-alcoholic fatty liver disease, together known as the metabolic or insulin resistance syndrome. How obesity promotes insulin resistance remains incompletely understood. Plasma concentrations of free fatty acids and proinflammatory cytokines, endoplasmic reticulum (ER) stress, and oxidative stress are all elevated in obesity and have been shown to induce insulin resistance. However, they may be late events that only develop after chronic excessive nutrient intake. The nature of the initial event that produces insulin resistance at the beginning of excess caloric intake and weight gain remains unknown. We show that feeding healthy men with ~6000 kcal/day of the common U.S. diet [~50% carbohydrate (CHO), ~ 35% fat, and ~15% protein] for 1 week produced a rapid weight gain of 3.5 kg and the rapid onset (after 2 to 3 days) of systemic and adipose tissue insulin resistance and oxidative stress but no inflammatory or ER stress. In adipose tissue, the oxidative stress resulted in extensive oxidation and carbonylation of numerous proteins, including carbonylation of GLUT4 near the glucose transport channel, which likely resulted in loss of GLUT4 activity. These results suggest that the initial event caused by overnutrition may be oxidative stress, which produces insulin resistance, at least in part, via carbonylation and oxidation-induced inactivation of GLUT4.

The acetylating enzyme, spermidine/spermine N 1 -acetyltransferase, participates in polyamine homeostasis by regulating polyamine export and catabolism. Previously, we reported that overexpression of the enzyme in cultured tumor cells and mice activates metabolic flux through the polyamine pathway and depletes the N 1 -acetyltransferase coenzyme and fatty acid precursor, acetyl-CoA. Here, we investigate this possibility in spermidine/spermine N 1 -acetyltransferase transgenic mice in which the enzyme is systemically overexpressed and in spermidine/spermine N 1 -acetyltransferase knock-out mice. Tissues of the former were characterized by increased N 1 -acetyltransferase activity, a marked elevation in tissue and urinary acetylated polyamines, a compensatory increase in polyamine biosynthetic enzyme activity, and an increase in metabolic flux through the polyamine pathway. These polyamine effects were accompanied by a decrease in white adipose acetyl-and malonyl-CoA pools, a major (20-fold) increase in glucose and palmitate oxidation, and a distinctly lean phenotype. In SSAT-ko mice, the opposite relationship between polyamine and fat metabolism was observed. In the absence of N 1 -acetylation of polyamines, there was a shift in urinary and tissue polyamines indicative of a decline in metabolic flux. This was accompanied by an increase in white adipose acetyl-and malonyl-CoA pools, a decrease in adipose palmitate and glucose oxidation, and an accumulation of body fat. The latter was further exaggerated under a high fat diet, where knock-out mice gained twice as much weight as wild-type mice. A model is proposed whereby the expression status of spermidine/spermine N 1 -acetyltransferase alters body fat accumulation by metabolically modulating tissue acetyl-and malonyl-CoA levels, thereby influencing fatty acid biosynthesis and oxidation.The polyamines putrescine (Put), 3 spermidine (Spd), and spermine (Spm) are known for their critical role in supporting cell proliferation, albeit in ways that have not yet been clearly defined. For the most part, polyamines do not incorporate into macromolecules but rather bind electrostatically to negatively charged molecules, such as DNA, RNA, and phospholipids. Thus, as metabolically distinct entities, homeostatic control of intracellular polyamines is critical to their role in supporting cell proliferation. This is achieved by effector systems that regulate biosynthesis, catabolism, uptake, and export of these molecules. The enzyme, spermidine/spermine N 1 -acetyltransferase (SSAT), catalyzes the transfer of acetyl groups from acetyl-CoA to the terminal amines of polyamines and, thus, readies the molecule for export or catabolism via polyamine oxidase. The enzyme is short lived, sensitively regulated by intracellular polyamine pools, and highly inducible by polyamine analogues and various cytotoxic agents (1, 2).Although most antiproliferative strategies targeting the polyamine pathway seek to deplete intracellular pools by inhibiting biosynthesis, we have been investigating t...