Citrate is a critical metabolite required to support both mitochondrial bioenergetics and cytosolic macromolecular synthesis. When cells proliferate under normoxic conditions, glucose provides the acetyl-CoA that condenses with oxaloacetate to support citrate production. Tricarboxylic acid (TCA) cycle anaplerosis is maintained primarily by glutamine. Here we report that some hypoxic cells are able to maintain cell proliferation despite a profound reduction in glucose-dependent citrate production. In these hypoxic cells, glutamine becomes a major source of citrate. Glutamine-derived α-ketoglutarate is reductively carboxylated by the NADPH-linked mitochondrial isocitrate dehydrogenase (IDH2) to form isocitrate, which can then be isomerized to citrate. The increased IDH2-dependent carboxylation of glutamine-derived α-ketoglutarate in hypoxia is associated with a concomitant increased synthesis of 2-hydroxyglutarate (2HG) in cells with wild-type IDH1 and IDH2. When either starved of glutamine or rendered IDH2-deficient by RNAi, hypoxic cells are unable to proliferate. The reductive carboxylation of glutamine is part of the metabolic reprogramming associated with hypoxia-inducible factor 1 (HIF1), as constitutive activation of HIF1 recapitulates the preferential reductive metabolism of glutaminederived α-ketoglutarate even in normoxic conditions. These data support a role for glutamine carboxylation in maintaining citrate synthesis and cell growth under hypoxic conditions. C itrate plays a critical role at the center of cancer cell metabolism. It provides the cell with a source of carbon for fatty acid and cholesterol synthesis (1). The breakdown of citrate by ATP-citrate lyase is a primary source of acetyl-CoA for protein acetylation (2). Metabolism of cytosolic citrate by aconitase and IDH1 can also provide the cell with a source of NADPH for redox regulation and anabolic synthesis. Mammalian cells depend on the catabolism of glucose and glutamine to fuel proliferation (3). In cancer cells cultured at atmospheric oxygen tension (21% O 2 ), glucose and glutamine have both been shown to contribute to the cellular citrate pool, with glutamine providing the major source of the four-carbon molecule oxaloacetate and glucose providing the major source of the two-carbon molecule acetyl-CoA (4, 5). The condensation of oxaloacetate and acetyl-CoA via citrate synthase generates the 6 carbon citrate molecule. However, both the conversion of glucose-derived pyruvate to acetyl-CoA by pyruvate dehydrogenase (PDH) and the conversion of glutamine to oxaloacetate through the TCA cycle depend on NAD + , which can be compromised under hypoxic conditions. This raises the question of how cells that can proliferate in hypoxia continue to synthesize the citrate required for macromolecular synthesis.This question is particularly important given that many cancers and stem/progenitor cells can continue proliferating in the setting of limited oxygen availability (6, 7). Louis Pasteur first highlighted the impact of hypoxia on nutrient metabol...

Intratumoral hypoxia and expression of Hypoxia Inducible Factor 1α (HIF1α) correlate with metastasis and poor survival in sarcoma patients. We demonstrate here that hypoxia controls sarcoma metastasis through a novel mechanism wherein HIF1α enhances expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2). We show that loss of HIF1α or PLOD2 expression disrupts collagen modification, cell migration and pulmonary metastasis (but not primary tumor growth) in allograft and autochthonous LSLKrasG12D/+; Trp53fl/fl murine sarcoma models. Furthermore, ectopic PLOD2 expression restores migration and metastatic potential in HIF1α-deficient tumors, and analysis of human sarcomas reveal elevated HIF1α and PLOD2 expression in metastatic primary lesions. Pharmacological inhibition of PLOD enzymatic activity suppresses metastases. Collectively, these data indicate that HIF1α controls sarcoma metastasis through PLOD2-dependent collagen modification and organization in primary tumors. We conclude that PLOD2 is a novel therapeutic target in sarcomas and successful inhibition of this enzyme may reduce tumor cell dissemination.

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Cancer cells are characterized by rapid proliferation and require adaptive metabolic responses to allow continued biosynthesis and cell growth in the setting of decreased oxygen (O 2 ) and nutrient availability. The hypoxia-inducible factors (HIFs) are a common link between adaptation to low O 2 , changes in cancer metabolism, and malignant progression. The HIF-a subunits differentially regulate metabolic enzymes and other key factors involved in glycolysis, changes in redox status, and oxidative phosphorylation. Importantly, metabolic changes can, in turn, regulate HIF activity. Finally, changes in metabolism under hypoxia lead to important crosstalk between cancer cells and the stromal compartment of the microenvironment.Keywords Hypoxia Á HIF Á Cancer metabolism Á Tumor microenvironment Hypoxia-inducible factors and their regulationA majority of healthy tissues experience 2-9 % O 2 , while hypoxia is defined as less than 2 % O 2 [1]. Low O 2 tensions are often exhibited by regions of intense inflammation such as within arthritic joints, regions of the bone marrow, and in highly proliferative cancer cells [2]. Hypoxia may occur secondary to necrosis or aberrant neovascularization resulting in poor perfusion. Additionally, cancer cells may also proliferate rapidly enough to outstrip their blood supply [3]. Cells adapt to changes in O 2 availability by altering gene expression of crucial metabolic enzymes in order to counter changes in nutrient availability and redox status. This response is mediated, in part, by O 2 -labile transcription factors hypoxia-inducible factors HIF-1a and HIF-2a, key regulators of cellular adaptation to hypoxic stress [3][4][5].Comprised of an O 2 -sensitive a subunit and constitutively expressed b subunit, HIFs are primarily regulated through post-translational modification and stabilization and are part of the basic helix-loop-helix-PAS (bHLH/ PAS) family of transcription factors [6]. Under normal O 2 tensions, prolyl hydroxylase domain enzymes (PHDs) hydroxylate two conserved proline residues (405 and 531 in HIF-1a) within the O 2 -dependent degradation (ODD) domain of the HIF-a subunit. After hydroxylation, the von Hippel-Lindau (VHL) tumor suppressor E3 ligase complex polyubiquitinates HIF-a and targets it for eventual degradation by the 26S proteasome [7][8][9]. Under low O 2 , HIFs are no longer modified by PHDs, but instead dimerize with ARNT/HIF-1b through HLH and PAS domain interactions, translocate to the nucleus, and recruit coactivators such as CBP/p300. HIF heterodimers bind and recognize hypoxiaresponse elements (HREs), with the consensus sequence G/ACGTG, within the promoter regions of target genes and drive adaptive gene transcription [10][11][12][13] (Fig. 1). While the HIF-1a subunit is expressed ubiquitously, HIF-2a is selectively expressed in a much more tissue-restricted manner but can be found at high levels in vascular endothelial cells and myeloid-derived cells [14].V. Mucaj and J.E.S. Shay contributed equally to this work.

Hypoxia-inducible factors (HIFs) accumulate in both neoplastic and inflammatory cells within the tumor microenvironment and impact the progression of a variety of diseases, including colorectal cancer. Pharmacological HIF inhibition represents a novel therapeutic strategy for cancer treatment. We show here that acriflavine (ACF), a naturally occurring compound known to repress HIF transcriptional activity, halts the progression of an autochthonous model of established colitis-associated colon cancer (CAC) in immunocompetent mice. ACF treatment resulted in decreased tumor number, size and advancement (based on histopathological scoring) of CAC. Moreover, ACF treatment corresponded with decreased macrophage infiltration and vascularity in colorectal tumors. Importantly, ACF treatment inhibited the hypoxic induction of M-CSFR, as well as the expression of the angiogenic factor (vascular endothelial growth factor), a canonical HIF target, with little to no impact on the Nuclear factor-kappa B pathway in bone marrow-derived macrophages. These effects probably explain the observed in vivo phenotypes. Finally, an allograft tumor model further confirmed that ACF treatment inhibits tumor growth through HIF-dependent mechanisms. These results suggest pharmacological HIF inhibition in multiple cell types, including epithelial and innate immune cells, significantly limits tumor growth and progression.

IMPORTANCE Alcohol-associated liver disease (ALD) is one of the most devastating complications of alcohol use disorder (AUD), an increasingly prevalent condition. Medical addiction therapy for AUD may play a role in protecting against the development and progression of ALD. OBJECTIVE To ascertain whether medical addiction therapy was associated with an altered risk of developing ALD in patients with AUD. DESIGN, SETTING, AND PARTICIPANTS This retrospective cohort study used the Mass General Brigham Biobank, an ongoing research initiative that had recruited 127 480 patients between its start in 2010 and August 17, 2021, when data for the present study were retrieved. The mean follow-up duration from AUD diagnosis was 9.2 years. International Statistical Classification of Diseases and Related Health Problems, Tenth Revision diagnosis codes were used to identify ALD and AUD diagnoses. EXPOSURES Medical addiction therapy was defined as the documented use of disulfiram, acamprosate, naltrexone, gabapentin, topiramate, or baclofen. Patients were considered to be treated if they initiated medical addiction therapy before the relevant outcome. MAIN OUTCOMES AND MEASURES Adjusted odds ratios (aORs) for the development of ALD and hepatic decompensation were calculated and adjusted for multiple risk factors. RESULTSThe cohort comprised 9635 patients with AUD, of whom 5821 were male individuals (60.4%), and the mean (SD) age was 54.8 (16.5) years. A total of 1135 patients (11.8%) had ALD and 3906 patients (40.5%) were treated with medical addiction therapy. In multivariable analyses, medical addiction therapy for AUD was associated with decreased incidence of ALD (aOR, 0.37; 95% CI, 0.31-0.43; P < .001). This association was evident for naltrexone (aOR, 0.67; 95% CI, 0.46-0.95; P = .03), gabapentin (aOR, 0.36; 95% CI, 0.30-0.43; P < .001), topiramate (aOR, 0.47; 95% CI, 0.32-0.66; P < .001), and baclofen (aOR, 0.57; 95% CI, 0.36-0.88; P = .01). In addition, pharmacotherapy for AUD was associated with lower incidence of hepatic decompensation in patients with cirrhosis (aOR, 0.35; 95% CI, 0.23-0.53, P < .001), including naltrexone (aOR, 0.27; 95% CI, 0.10-0.64; P = .005) and gabapentin (aOR, 0.36; 95% CI, 0.23-0.56; P < .001). This association persisted even when medical addiction therapy was initiated only after the diagnosis of cirrhosis (aOR, 0.41; 95% CI, 0.23-0.71; P = .002). CONCLUSIONS AND RELEVANCEResults of this study showed that receipt of medical addiction therapy for AUD was associated with reduced incidence and progression of ALD. The associations of individual pharmacotherapy with the outcomes of ALD and hepatic decompensation varied widely.

Glioblastomas are aggressive adult brain tumors, characterized by inadequately organized vasculature and consequent nutrient and oxygen (O2)-depleted areas. Adaptation to low nutrients and hypoxia supports glioblastoma cell survival, progression, and therapeutic resistance. However, specific mechanisms promoting cellular survival under nutrient and O2 deprivation remain incompletely understood. Here, we show that miR-124 expression is negatively correlated with a hypoxic gene signature in glioblastoma patient samples, suggesting that low miR-124 levels contribute to pro-survival adaptive pathways in this disease. Since miR-124 expression is repressed in various cancers (including glioblastoma), we quantified miR-124 abundance in normoxic and hypoxic regions in glioblastoma patient tissue, and investigated whether ectopic miR-124 expression compromises cell survival, during tumor ischemia. Our results indicate that miR-124 levels are further diminished in hypoxic/ischemic regions within individual glioblastoma patient samples, compared to regions replete in O2 and nutrients. Importantly, we also show that increased miR-124 expression affects the ability of tumor cells to survive under O2 and/or nutrient deprivation. Moreover, miR-124 re-expression increases cell death in vivo, and enhances the survival of mice bearing intracranial xenograft tumors. miR-124 exerts this phenotype in part by directly regulating TEAD1, MAPK14/p38α and SERP1, factors involved in cell proliferation and survival under stress. Simultaneous suppression of these miR-124 targets results in similar levels of cell death as caused by miR-124 restoration. Importantly, we further demonstrate that SERP1 re-introduction reverses the hypoxic cell death elicited by miR-124, indicating the importance of SERP1 in promoting tumor cell survival. In support of our experimental data, we observed a significant correlation between high SERP1 levels and poor patient outcome in glioblastoma patients. Collectively, among the many pro-tumorigeneic properties of miR-124 repression in glioblastoma, we delineated a novel role in promoting tumor cell survival under stressful microenvironments, thereby supporting tumor progression.

Key Points• ARNT promotes adult hematopoietic stem cell viability through regulation of BCL-2 and VEGF-A expression.• Fetal liver hematopoietic progenitors experience hypoxia and loss of hypoxiainduced transcription decreases their survival.Hypoxia-inducible factors (HIFs) are master regulators of the transcriptional response to low oxygen and play essential roles in embryonic development, tissue homeostasis, and disease. Recent studies have demonstrated that hematopoietic stem cells (HSCs) within the bone marrow localize to a hypoxic niche and that HIF-1a promotes HSC adaptation to stress. Because the related factor HIF-2a is also expressed in HSCs, the combined role of HIF-1a and HIF-2a in HSC maintenance is unclear. To this end, we have conditionally deleted the HIF-a dimerization partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) in the hematopoietic system to ablate activity of both HIF-1a and HIF-2a and assessed the functional consequence of ARNT deficiency on fetal liver and adult hematopoiesis. We determined that ARNT is essential for adult and fetal HSC viability and homeostasis. Importantly, conditional knockout of both Hif-1a and Hif-2a phenocopied key aspects of these HSC phenotypes, demonstrating that the impact of Arnt deletion is primarily HIF dependent. ARNT-deficient long-term HSCs underwent apoptosis, potentially because of reduced B-cell lymphoma 2 (BCL-2) and vascular endothelial growth factor A (VEGF-A) expression. Our results suggest that HIF activity may regulate HSC homeostasis through these prosurvival factors. (Blood. 2015;125(21):3263-3272) IntroductionHematopoietic stem cells (HSCs) reside in the bone marrow (BM), where they balance both cell-intrinsic and cell-extrinsic cues to achieve self-renewal and appropriate hematologic differentiation throughout the mammalian lifespan.1 HSCs are regulated by their microenvironment, which consists of endothelial, 2 perivascular, 2 adipocyte, 3 and osteoblast 4 support cells; secreted factors; and oxygen (O 2 ) availability. 5Hypoxia has become increasingly recognized as a critical regulator of stem cells, during both embryonic development and adulthood. 6 Importantly, HSCs reside in a poorly perfused hypoxic niche, [7][8][9][10] and recent data suggest that HSC oxygenation levels may be partially regulated by cell-specific mechanisms. 10 Although the biological importance of these observations is not entirely clear, hypoxia clearly imposes phenotypic consequences for HSCs. For instance, long-term HSCs (LT-HSCs) are highly quiescent, a cell cycle status often associated with O 2 -and nutrient-deprived cells. Although many pathways converge on metabolism, the primary transcriptional response to hypoxia is mediated by hypoxia-inducible factors (HIFs).HIFs are heterodimeric transcription factors composed of a HIF-a subunit ) and their common b subunit, HIF-1b or the aryl hydrocarbon receptor nuclear translocator (ARNT).13 HIFa/ARNT heterodimers stimulate the transcription of many genes, which promote survival and adaptation to ...