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Summary: Inhibition of oxidative phosphorylation (OXPHOS) by 1-cyclopropyl-4-(4-[(5-methyl-3-(3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl]pyridin-2-yl)piperazine (BAY87-2243, abbreviated as B87), a complex I inhibitor, fails to kill human cancer cells in vitro. Driven by this consideration, we attempted to identify agents that engage in synthetically lethal interactions with B87. Here, we report that dimethyl α-ketoglutarate (DMKG), a cell-permeable precursor of α-ketoglutarate that lacks toxicity on its own, kills cancer cells when combined with B87 or other inhibitors of OXPHOS. DMKG improved the antineoplastic effect of B87, both in vitro and in vivo. This combination caused MDM2-dependent, tumor suppressor protein p53 (TP53)-independent transcriptional reprogramming and alternative exon usage affecting multiple glycolytic enzymes, completely blocking glycolysis. Simultaneous inhibition of OXPHOS and glycolysis provoked a bioenergetic catastrophe culminating in the activation of a cell death program that involved disruption of the mitochondrial network and activation of PARP1, AIFM1, and APEX1. These results unveil a metabolic liability of human cancer cells that may be harnessed for the development of therapeutic regimens. : Sica et al. show that respiratory chain inhibition by 1-cyclopropyl-4-(4-[(5-methyl-3-(3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl)-1H-pyrazol-1-yl)methyl]pyridin-2-yl)piperazine (BAY87-2243, abbreviated as B87) becomes lethal for cancer cells when glycolysis is simultaneously suppressed. When combined with B87, dimethyl α-ketoglutarate acquires the capacity to suppress glycolysis, thus lethally poisoning bioenergetics metabolism. This therapeutic combination effect relies on transcriptional reprogramming that can be reverted by pharmacological inhibition of MDM2. Keywords: MDM2, Krebs cycle, glycolysis, mitochondrial fragmentation, regulated cell death, parthanatos, cancer metabolism

Acellular fraction of ascites might play an active role in tumor development. Nevertheless the mechanisms involved in the tumor modulating properties are still controversial. Here we demonstrate that malignant ascites from 8 patients with epithelial ovarian cancer did not influence proliferative or invasive properties of ovarian cancer cells but promoted H2O2 induced apoptosis and increased sensitivity to paclitaxel. Malignant ascites induced BRCA1 Fas and FasL expression and phosphorylation of JNK but not the activation of caspase pathway. Ascites induced apoptosis of ovarian cancer cells was strongly inhibited by a JNK inhibitor suggesting a critical role of JNK pathway in ascite induced apoptosis. The use of siRNA JNK confirmed the importance of JNK in ascites induced Fas and FasL expression. These results demonstrate that malignant ascites induce apoptosis of ovarian cancer cells and encourage us to think about the clinical management of ovarian cancer patients with malignant ascites.

Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate driven respiration as well as perturbed metabolic profiles and both defects could be restored by reexpression of MPC1. Labeling experiments using 13C labeled glucose and glutamine demonstrated that MPC deficiency causes increased glutaminolysis and reduced contribution of glucose derived pyruvate to the TCA cycle. Morphological defects were observed in mutant embryonic brains together with major alterations of their metabolome including lactic acidosis diminished TCA cycle intermediates energy deficit and a perturbed balance of neurotransmitters. Strikingly these changes were reversed when the pregnant dams were fed a ketogenic diet which provides acetyl CoA directly to the TCA cycle and bypasses the need for a functional MPC. This allowed the normal gestation and development of MPC deficient pups even though they all died within a few minutes post delivery. This study establishes the MPC as a key player in regulating the metabolic state necessary for embryonic development neurotransmitter balance and post natal survival.

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNA(Met), and mt-tRNA(Phe), and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.