A Mitochondrial RNAi Screen Defines Cellular Bioenergetic Determinants and Identifies an Adenylate Kinase as a Key Regulator of ATP Levels

Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels. We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC) perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity. Additionally, we identified a mitochondrial adenylate kinase (AK4) that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome

Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine

Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by 13C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis

Transmembrane Protease TMPRSS11B Promotes Lung Cancer Growth by Enhancing Lactate Export and Glycolytic Metabolism

Summary: Pathways underlying metabolic reprogramming in cancer remain incompletely understood. We identify the transmembrane serine protease TMPRSS11B as a gene that promotes transformation of immortalized human bronchial epithelial cells (HBECs). TMPRSS11B is upregulated in human lung squamous cell carcinomas (LSCCs), and high expression is associated with poor survival of non-small cell lung cancer patients. TMPRSS11B inhibition in human LSCCs reduces transformation and tumor growth. Given that TMPRSS11B harbors an extracellular (EC) protease domain, we hypothesized that catalysis of a membrane-bound substrate modulates tumor progression. Interrogation of a set of soluble receptors revealed that TMPRSS11B promotes solubilization of Basigin, an obligate chaperone of the lactate monocarboxylate transporter MCT4. Basigin release mediated by TMPRSS11B enhances lactate export and glycolytic metabolism, thereby promoting tumorigenesis. These findings establish an oncogenic role for TMPRSS11B and provide support for the development of therapies that target this enzyme at the surface of cancer cells. : Updegraff et al. show that transmembrane protease TMPRSS11B is upregulated in lung squamous cell carcinoma, where it interacts with MCT4 and its obligate chaperone Basigin. TMPRSS11B catalytic activity promotes Basigin solubilization, which enhances lactate export and glycolytic metabolism, thereby promoting tumorigenesis. Keywords: TMPRSS11B, lung squamous cell carcinoma, MCT4, Basigin, lactate export, lung cancer, transmembrane serine protease, glycolytic metabolism, transposon mutagenesis, CRISPR-mediated genome editin

Cytochrome c Oxidase Activity Is a Metabolic Checkpoint that Regulates Cell Fate Decisions During T Cell Activation and Differentiation

T cells undergo metabolic reprogramming with major changes in cellular energy metabolism during activation. In patients with mitochondrial disease, clinical data were marked by frequent infections and immunodeficiency, prompting us to explore the consequences of oxidative phosphorylation dysfunction in T cells. Since cytochrome c oxidase (COX) is a critical regulator of OXPHOS, we created a mouse model with isolated dysfunction in T cells by targeting a gene, COX10, that produces mitochondrial disease in humans. COX dysfunction resulted in increased apoptosis following activation in vitro and immunodeficiency in vivo. Select T cell effector subsets were particularly affected; this could be traced to their bioenergetic requirements. In summary, the findings presented herein emphasize the role of COX particularly in T cells as a metabolic checkpoint for cell fate decisions following T cell activation, with heterogeneous effects in T cell subsets. In addition, our studies highlight the utility of translational models that recapitulate human mitochondrial disease for understanding immunometabolism. [Display omitted] •Patients with mitochondrial disease have an underappreciated immune phenotype•COX regulates activation and proliferation in T cells through apoptosis•Effector T cell subsets are differentially affected by COX deficiency•Mouse T cell COX deficiency produces immunodeficiency in vivo Mitochondrial diseases are disorders of oxidative phosphorylation. Using mitochondrial disease as a model system, Tarasenko et al. demonstrate that cytochrome c oxidase deficiency differentially affects T cell effector subsets based on their bioenergetic requirements. Mouse T cell COX deficiency produces an immunodeficiency similar to that of patients with mitochondrial disease

Biomarker Accessible and Chemically Addressable Mechanistic Subtypes of BRAF Melanoma

Abstract Genomic diversity among melanoma tumors limits durable control with conventional and targeted therapies. Nevertheless, pathologic activation of the ERK1/2 pathway is a linchpin tumorigenic mechanism associated with the majority of primary and recurrent disease. Therefore, we sought to identify therapeutic targets that are selectively required for tumorigenicity in the presence of pathologic ERK1/2 signaling. By integration of multigenome chemical and genetic screens, recurrent architectural variants in melanoma tumor genomes, and patient outcome data, we identified two mechanistic subtypes of BRAFV600 melanoma that inform new cancer cell biology and offer new therapeutic opportunities. Subtype membership defines sensitivity to clinical MEK inhibitors versus TBK1/IKBKϵ inhibitors. Importantly, subtype membership can be predicted using a robust quantitative five-feature genetic biomarker. This biomarker, and the mechanistic relationships linked to it, can identify a cohort of best responders to clinical MEK inhibitors and identify a cohort of TBK1/IKBKϵ inhibitor–sensitive disease among nonresponders to current targeted therapy. Significance: This study identified two mechanistic subtypes of melanoma: (1) the best responders to clinical BRAF/MEK inhibitors (25%) and (2) nonresponders due to primary resistance mechanisms (9.9%). We identified robust biomarkers that can detect these subtypes in patient samples and predict clinical outcome. TBK1/IKBKϵ inhibitors were selectively toxic to drug-resistant melanoma. Cancer Discov; 7(8); 832–51. ©2017 AACR. See related commentary by Jenkins and Barbie, p. 799. This article is highlighted in the In This Issue feature, p. 783</jats:p

Phosphoglycerate Mutase 1 Coordinates Glycolysis and Biosynthesis to Promote Tumor Growth

It remains unclear how cancer cells coordinate glycolysis and biosynthesis to support rapidly growing tumors. We found that glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, contributes to biosynthesis regulation in part by controlling intracellular levels of its substrate 3-phosphoglycerate (3-PG) and product 2-phosphoglycerate (2-PG). 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP), while 2-PG activates 3-phosphoglycerate dehydrogenase to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA or a small molecule inhibitor PGMI-004A results in increased 3-PG and decreased 2-PG levels in cancer cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth