Basal Glycolysis Research Articles

Energy Deficit Phenotype of Cerebral Microvascular Network in the Aged Brains

ObjectiveAging is associated with an increased risk of neurodegenerative diseases and stroke. Impaired microvascular function was proposed to be the connecting link, but the underlying mechanisms are unclear. Microvasculature is critical for cerebrovascular blood flow, neurovascular coupling, and nutrient transport, and blood‐brain‐barrier. Maintaining these energy‐demanding functions by cerebral microvasculature needs optimal functioning of energy pathways like glycolysis and mitochondrial respiration. Characterization of cellular energy metabolism in the young and aged microvasculature was essential to understand the aging‐induced cerebrovascular pathologies.MethodsWe isolated the microvessels from the brains from young (3 months) and aged mice (24 months). Microvessels were isolated by a combination of filtration (40μm – 300μm) and gradient centrifugation. Microvessels were plated on Seahorse XFe24 culture plates. Basal and compensatory glycolysis, and proton efflux rate (PER) were measured from extracellular acidification rates (ECAR) using glycolysis rate kit. Similarly, mitochondrial respiratory parameters were determined by the measurements from the oxygen consumption rate (OCR), and the fuel dependency of the microvasculature was measured using mitofuel kit.ResultsAged brain microvasculature showed 26% (73.87±7.6 vs. 100.1±2.9 picomoles of H+/min/μg protein, p<0.01, n=7–9 mice/phenotype) decrease in basal glycolysis when compared with the microvessels from the young mouse brain. Compensatory glycolysis is decreased by 28.3% (121.0±7.0 vs 168.9±13.4 picomoles of H+/min/μg protein, p<0.004, n=7–9 mice/phenotype) in the aged mouse brain microvessels compared to the young mouse brain vessels, whereas the basal proton efflux is decreased by 35.3% (105.2±6.3 vs 162.8±5.9 picomoles of H+/min/μg protein, p<0.0001, n=7–9 mice/phenotype). Microvessels from the aged mouse brain had 36% lower mitochondrial respiration compared to the young mouse brain microvasculature (64.0±10.4 vs 100.0±7.6 picomoles of O2/min/μg protein, p<0.01, n=7–9 mice/phenotype). Glucose (65.6±0.2%, n=4 mice) is the primary fuel preference by the mitochondria in the young mouse brain microvessels followed by glutamine (32.9±0.5%) and fatty acids (27.9±3.0%). Aged microvasculature exhibited unaltered fuel preferences concerning glucose and fatty acids, whereas glutamine utilization is increased by 58% (54.1±2.9% vs 34.3% vs 3.57, n=3 mice) when compared to the young mouse brain microvessels.ConclusionsWe conclude that aged brain microvasculature exhibits compromised energy metabolism including both glycolysis and the mitochondrial respiration. We further conclude that mitochondrial utilization of glutamine as energy fuel is increased in the aged brain microvasculature. The energy deficit phenotype of the brain microcirculatory network may contribute to the aging‐associated cerebrovascular complications.Support or Funding InformationNational Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834) and National Institute on Aging (Mostany: AG047296).

Paradoxical Effect of TNFα on Mitochondrial Function and Metabolic Activity in the Retinal Pigment Epithelium (RPE)

The retinal pigment epithelium (RPE) acts as a metabolic gatekeeper between photoreceptors and the choroidal vasculature to maintain healthy retinal function. RPE dysfunction is a key feature of age‐related macular degeneration (AMD), the leading cause of blindness in developed countries. Tumor necrosis factor‐alpha (TNFα), a potent pro‐inflammatory cytokine, has been implicated in the pathogenesis of AMD. Growing evidence supports metabolic dysfunction as another key mechanism driving AMD. To date, there is no literature on the metabolic effects of TNFα on RPE and thus, this study sheds light on the impact of TNFα on mitochondrial morphology and metabolic function in RPE. Matured ARPE‐19 (human RPE cell line) were treated with TNFα (10 ng/ml) for up to 72h. TNFα induced ARPE‐19 to elongate into spindle‐shaped cells, reminiscent of epithelial‐mesenchymal transition (EMT). However, qPCR showed that TNFα reduced the expression of EMT genes (α‐SMA, Col1A1, fibronectin, MMP2, CTGF) indicating that the elongated cells were not mesenchymal in nature. To explore the effects of TNFα on metabolism, two major energy‐generating pathways, oxidative phosphorylation (OXPHOS) and glycolysis were assessed by Seahorse high‐resolution respirometry and qPCR. The Seahorse Mito Stress Test revealed an increase in basal respiration but reduced spare respiratory capacity following TNFα treatment. TNFα increased glycolytic reserve and capacity. Despite the increased OXPHOS and glycolysis, qPCR showed a significant reduction in expression of OXPHOS (ATP5O, COX4I1, COX5B, NDUFB5) and glycolysis (G6P, PFKL, PFKFB3, PGK1, LDHA) genes with TNFα treatment. ARPE‐19 were transfected with mitochondria‐tagged GFP and imaged using confocal microscopy. Parameters on mitochondrial morphology were extracted from the confocal images using automated image segmentation and revealed that TNFα disrupted mitochondrial network integrity. This was supported by qPCR data showing significantly reduced expression of genes regulating mitochondrial function (PGC‐1α, TFAM, POLG, MFN1, MFN2, FIS1, OPA1). TNFα significantly elevated SOD2, a mitochondrial antioxidant enzyme, but not SOD1. Taken together, we find that TNFα robustly disrupts mitochondrial function and morphology in RPE, although shifting the bioenergetic profile in a paradoxical manner, i.e. TNFα raised the levels of basal respiration and glycolysis despite the suppression of genes regulating OXPHOS and glycolysis. These findings highlight the potential of targeting metabolic pathways in RPE as a promising therapeutic avenue for AMD. Further research is required to elucidate the mechanisms underlying these intriguing TNFα‐driven metabolic changes.Support or Funding InformationGrimshaw‐Gudewicz Charitable Foundation; National Council of Scientific and Technological Development—Brazil (PDE 210474/2014‐9); The Iraty Award; NEI Core Grant P30EYE003790; Department of Defense, Spinal Vision Research Program under Award no. VR180132

Sigma‐1 Receptors Promote Lymphatic Endothelial Barrier Function

The lymphatic endothelium plays important roles in lymphatic contractile activity, lymph transport and maintaining a barrier between the lymph and interstitial compartments and its dysfunction is a key factor in the pathogenesis of lymphatic diseases such as lymphedema. Sigma‐1 (σ1) receptors are chaperone proteins located in the endoplasmic reticulum were recently identified to promote collecting lymphatic dilation through endothelial production of nitric oxide. However, the role of σ1 in other functions of lymphatic endothelial cells are not clear. The aim of the current study was to test the hypothesis that σ1 promotes barrier function of lymphatic endothelium. We confirmed expression of σ1 in adult and juvenile human dermal lymphatic endothelial cells (HDLEC) using the ProteinSimple Western (WES) system and immunofluorescence microscopy. To test whether activation of σ1 promotes lymphatic endothelial barrier function, we applied the selective agonist PRE‐084 to cultured monolayers of HDLEC and determined the transendothelial electrical resistance (TER) as an index of barrier function using electrical cell‐substrate impedance sensing. The contribution of basal σ1 activity to HDLEC TER was also assessed either with the σ1 antagonist BD1047 (200 nM) or by silencing σ1 expression with siRNA. Because σ1 has been previously described to be located in the ER‐mitochondrial membrane interface, we also evaluated its role in endothelial bioenergetics using the Agilent Seahorse system glycolytic rate assay. Lastly, we tested the ability of PRE‐084 to counteract IL‐1β‐induced barrier dysfunction in HDLEC monolayers. The results show that the activation of σ1 receptor with PRE‐084 increases HDLEC TER in a concentration‐dependent manner, with 50, 100, 150, and 200 μM PRE‐084 eliciting significant elevations in TER (100 μM was chosen for subsequent experiments). Blockade of σ1 either with the antagonist BD‐1047 or reducing σ1 expression with siRNA resulted in decreased HDLEC TER. PRE‐084 also enhanced basal and compensatory glycolysis as well as proton efflux rate in HDLEC. Moreover, PRE‐084 partially counteracted the IL‐1β‐induced TER drop indicating its potential anti‐inflammatory impact on lymphatic endothelial cells. Collectively our results suggest that σ1 contributes to basal lymphatic endothelial barrier function, potentially through its ability to enhance glycolytic ATP production. Our work also highlights the therapeutic potential of σ1 agonists for preventing lymphatic barrier dysfunction caused by inflammatory mediators.Support or Funding InformationThis work was supported by NIH Grant R01GM120774.

Dose-dependent regulation of mitochondrial function and cell death pathway by sorafenib in liver cancer cells

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the fourth most frequent cause of cancer-related death worldwide. Sorafenib is the first line recommended therapy for patients with locally advanced/metastatic HCC. The low response rate is attributed to intrinsic resistance of HCC cells to Sorafenib. The potential resistance to Sorafenib-induced cell death is multifactorial and involves all hallmarks of cancer. However, the presence of sub-therapeutic dose can negatively influence the antitumoral properties of the drug. In this sense, the present study showed that the sub-optimal Sorafenib concentration (10 nM) was associated with activation of caspase-9, AMP-activated protein kinase (AMPK), sustained autophagy, peroxisome proliferator-activated receptor-coactivator 1α (PGC-1α) and mitochondrial function in HepG2 cells. The increased mitochondrial respiration by Sorafenib (10 nM) was also observed in permeabilized HepG2 cells, but not in isolated rat mitochondria, which suggests the involvement of an upstream component in this regulatory mechanism. The basal glycolysis was dose dependently increased at early time point studied (6 h). Interestingly, Sorafenib increased nitric oxide (NO) generation that played an inhibitory role in mitochondrial respiration in sub-therapeutic dose of Sorafenib. The administration of sustained therapeutic dose of Sorafenib (10 µM, 24 h) induced mitochondrial dysfunction and dropped basal glycolysis derived acidification, as well as increased oxidative stress and apoptosis in HepG2. In conclusion, the accurate control of the administered dose of Sorafenib is relevant for the potential prosurvival or proapoptotic properties induced by the drug in liver cancer cells.

Metabolic Reprograming Due to Deletion of <i>CISH</i> in Human Natural Killer Cells Promotes <i>in vivo</i> Persistence and Enhances Anti-Tumor Activity

Cytokine-inducible SH2-containing protein (CIS, encoded by the gene CISH) is a key negative regulator of IL-15 signaling in natural killer (NK) cells. Here, we developed human CISH knockout (CISH-/-) NK cells using an induced pluripotent stem cell-derived NK cell (iPSC-NK cell) platform. CISH-/- iPSC-derived NK cells demonstrate increased IL-15 mediated JAK-STAT pathway signaling activity. Consequently CISH-/- iPSC-NK cells exhibit improved expansion and increased cytotoxic activity against multiple tumor cell lines when maintained at low cytokine concentration. CISH-/- iPSC-NK cells display significantly increased in vivo persistence and inhibition of tumor progression in a leukemia xenograft model. Mechanistically, CISH-/- iPSC-NK cells display improved metabolic fitness characterized by a significant increase in basal glycolysis, glycolytic capacity, maximal mitochondrial respiration, ATP-linked respiration and spare capacity respiration which are mediated by mTOR signaling and directly contribute to enhanced NK cell function. In addition, we demonstrate that deletion of CISH in human NK cells increases single-cell polyfunctionality by secreting multiple effector cytokines, a profile has been proven to be positively associated with clinical outcome of CAR-T cells. Together, these studies demonstrate CIS plays a key role to regulate human NK cell metabolic activity and deletion of CIS leads to enhanced anti-tumor activity.

Platelet glycolytic metabolism correlates with hemodynamic severity in pulmonary arterial hypertension.

Group 1 pulmonary hypertension (PH), i.e., pulmonary arterial hypertension (PAH), is associated with a metabolic shift favoring glycolysis in cells comprising the lung vasculature as well as skeletal muscle and right heart. We sought to determine whether this metabolic switch is also detectable in circulating platelets from PAH patients. We used Seahorse Extracellular Flux to measure bioenergetics in platelets isolated from group 1 PH (PAH), group 2 PH, patients with dyspnea and normal pulmonary artery pressures, and healthy controls. We show that platelets from group 1 PH patients exhibit enhanced basal glycolysis and lower glycolytic reserve compared with platelets from healthy controls but do not differ from platelets of group 2 PH or dyspnea patients without PH. Although we were unable to identify a glycolytic phenotype unique to platelets from PAH patients, we found that platelet glycolytic metabolism correlated with hemodynamic severity only in group 1 PH patients, supporting the known link between PAH pathology and altered glycolytic metabolism and extending this association to ex vivo platelets. Pulmonary artery pressure and pulmonary vascular resistance in patients with group 1 PH were directly associated with basal platelet glycolysis and inversely associated with maximal and reserve glycolysis, suggesting that PAH progression reduces the capacity for glycolysis even while demanding an increase in glycolytic metabolism. Therefore, platelets may provide an easy-to-harvest, real-time window into the metabolic shift occurring in the lung vasculature and represent a useful surrogate for interrogating the glycolytic shift central to PAH pathology.

Mitochondrial transfer from MSCs to T cells induces Treg differentiation and restricts inflammatory response.

Mesenchymal stem cells (MSCs) have fueled ample translation for the treatment of immune-mediated diseases. They exert immunoregulatory and tissue-restoring effects. MSC-mediated transfer of mitochondria (MitoT) has been demonstrated to rescue target organs from tissue damage, yet the mechanism remains to be fully resolved. Therefore, we explored the effect of MitoT on lymphoid cells. Here, we describe dose-dependent MitoT from mitochondria-labeled MSCs mainly to CD4+ T cells, rather than CD8+ T cells or CD19+ B cells. Artificial transfer of isolated MSC-derived mitochondria increases the expression of mRNA transcripts involved in T-cell activation and T regulatory cell differentiation including FOXP3, IL2RA, CTLA4, and TGFβ1, leading to an increase in a highly suppressive CD25+ FoxP3+ population. In a GVHD mouse model, transplantation of MitoT-induced human T cells leads to significant improvement in survival and reduction in tissue damage and organ T CD4+ , CD8+ , and IFN-γ+ expressing cell infiltration. These findings point to a unique CD4+ T-cell reprogramming mechanism with pre-clinical proof-of-concept data that pave the way for the exploration of organelle-based therapies in immune diseases.

Abstract B081: Regulation and function of monocarboxylate transporters in osteosarcoma

Abstract Background: Osteosarcoma (OS), the most common malignant bone tumor in humans and dogs, shares several features in both species including clinical presentation and molecular alterations. Despite numerous efforts there have been no improvements in outcome: 30% of people and 90% of dogs still die of metastasis. In both species, macroscopic metastasis exhibits inherent resistance to multiple agents (kinase inhibitors, chemotherapy, immunotherapy, among others). In both dogs and humans, the OS genomes exhibit low somatic mutational load and no clear molecular drivers. These data, along with a history of failed clinical trials suggest that contemporary approaches to therapeutic advancement (kinase inhibitors, immune checkpoint blockade) will likely have limited clinical impact, necessitating therapeutic innovation. Monocarboxylate transporters (MCTs) move lactic acid across the plasma membrane, providing a mechanism for tumor cells to meet their bioenergetic needs in a variety of limiting microenvironments. The objective of this work is credential MCTs (MCT1/MCT4) as therapeutic targets in OS and investigate mechanisms of synthetic lethality. Methods: STAT3, MCT1 and MCT4 expression and function were modulated in canine and human OS cell lines using a combination of CRISPR/cas9 techniques and pharmacologic agents (AZD3965, AstraZeneca; syrosingopine, Sigma Aldrich). Western blotting, luciferase reporter assays and chromatin immunoprecipitation (ChIP) were used to investigate the transcriptional regulation of MCT1/MCT4. The metabolic effects of STAT3 and MCT inhibition were investigated through measurement of lactate (Lactate-Glo, Promega) and respirometry (Seahorse XFe96 extracellular flux analyzer), while cell proliferation and invasion were evaluated using sulforhodamine B and Matrigel invasion assays. Results: MCT1/MCT4 and were found to be expressed in all OS cell lines, although at variable levels. Following knockdown of STAT3, MCT4 expression was downregulated in several OS cell lines. ChIP assays confirm enrichment of STAT3 and FOXM1 at the MCT4 promoter, and this was confirmed in STAT3 deficient OS lines that exhibited decreased MCT4-dependent luciferase reporter activity. The absence of MCT1 or MCT4 was associated with decreased basal and compensatory glycolysis and increased intracellular lactate. In addition, decreased proliferation and invasive capacity were noted concurrent with MCT loss. Dual MCT1/MCT4 inhibition exhibited synergy with doxorubicin, as determined by a combination index < 1. Conclusions: Loss of MCT1 or MCT4 function in OS cells decreases basal and compensatory glycolysis, cellular proliferation and invasive capacity. MCT4 is a direct transcriptional target of STAT3 and FOXM1, both of which exhibit constitutive activation in OS, supporting a link between MCT4/STAT3/FOXM1 and aerobic glycolysis. Despite promising results in vitro, rational drug combinations may be needed to maximize the effect of MCT1/4 inhibition. Together these studies will create a blueprint for clinical translational with the goal of optimizing strategies in dogs with OS prior to human trials. Citation Format: Heather L. Gardner, Joelle M. Fenger, Cheryl A. London. Regulation and function of monocarboxylate transporters in osteosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B081. doi:10.1158/1535-7163.TARG-19-B081

8-Chloroadenosine Alters the Metabolic Profile and Downregulates Antioxidant and DNA Damage Repair Pathways in Macrophages.

The exposure of RNA and DNA nucleobases to the oxidant hypochlorous acid (HOCl) results in the generation of different stable chlorinated products. These chlorinated nucleobases are formed in vivo, particularly in chronic inflammatory pathologies, which are characterized by the overproduction of HOCl by myeloperoxidase. As such, chlorinated nucleosides are used as biomarkers of inflammation. However, these compounds have also attracted attention as potential chemotherapeutic agents with 8-chloro-adenosine (8ClA), for example, currently in clinical trials for the treatment of hematological cancers, including chronic lymphocytic leukemia. 8ClA has mainly RNA-directed effects in malignant cells, with exposure resulting in ATP depletion and apoptotic cell death. Whether 8ClA has significant reactivity with nonmalignant cells has not been widely studied. Here we show that prolonged incubation of J774A.1 macrophage-like cells with 8ClA results in the perturbation of cellular metabolism and apoptotic cell death. These effects are associated with an accumulation of 8-chloroadenosine triphosphate (8Cl-ATP), an effect not seen in experiments utilizing other chlorinated nucleosides. Exposure of the macrophages to 8ClA did not significantly change basal mitochondrial respiration or glycolysis but resulted in an increase in maximal mitochondrial respiration as well as spare respiratory capacity within these cells. Additionally, 8ClA exposure also altered the mRNA expression of a range of antioxidant and DNA damage repair genes in the macrophages in a manner consistent with a reduction in the capacity of the cells to cope with oxidative stress and repair DNA damage. Taken together, these results provide new insight into pathways by which the production of HOCl during chronic inflammation could perturb immune cell function and may also have implications for the use of 8ClA as a chemotherapeutic drug.

Panobinostat (LBH589) in Combination with the β-Catenin Inhibitor Tegavivint (BC2059) Exerts Significant Anti-Myeloma Activity Both in Vitro and In Vivo

Introduction: Panobinostat (LBH589) is approved for the treatment of relapsed multiple myeloma (MM). The Wnt canonical pathway and its key player, β-catenin are dysregulated in advanced MM supporting the evaluation of β-catenin inhibition as a potential therapy. We evaluated the anti-MM effect of Tegavivint in combination with Panobinostat in vitro and in vivo. Results and Methods : In vitro combination of low doses (BC2059≤100nM; LBH589≤10nM) for 48h was synergistic against OCI-My1 and U266 MM cells, with combination indices from 0.569 to 0.883 (CI<1: synergism). The combination demonstrated synergistic killing of primary MM tumour cells in an autologous co-culture assay with synergism quotients from 1.2 to 2 (SQ>1: synergism). The combination rapidly (<24h) decreased the expression of downstream β-catenin targets myc, cyclin D1 and cyclin D2 as shown by immunoblotting. Furthermore, by 20h, the combination decreased both oxidative phosphorylation and aerobic glycolysis as measured by oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively (Seahorse XFe96 analyser). Basal, maximal and ATP coupled OCR were significantly reduced by the combination when compared to vehicle (for OCI-My1 from 203.93 to 56.20, 105.81 to 33.29 and 156.29 to 34.67 pmol/min/50K cells OCR respectively). Similarly, basal glycolysis and glycolytic capacity were both reduced by the combination (from 48.88 to 9.77 and 61.68 to 8.58 mpH/min/50K cells ECAR, respectively). In vivo, the combination was superior to either single drug treatment in a murine xenograft of U266-luciferase cells. Disease burden was reduced in the combination arm compared to single drug and vehicle arms as early as day 14 after inoculation (p=0.02). This difference increased further until the last bioluminescence imaging (day 49, p<0.001) and translated into significantly prolonged OS for the combination (p=0.006). Potential on-target toxicities with BC2059 are a concern as the Wnt canonical pathway is essential for stem cell maintenance in several organs and has a role in bone homeostasis, but the combination did not result in cytopenias nor body weight loss. After euthanasia, µCT demonstrated that neither BC2059 nor the combination negatively affected bone morphometric indices (bone volume fraction, trabecular thickness, connectivity density). Likewise, osteoblastic activity as measured by serum osteocalcin was unaffected, whereas osteoclastic activity (as indicated by serum CTX1) was reduced when compared to healthy mice (p=0.008 and p=0.013, respectively). In conclusion Tegavivint in combination with Panobinostat may be a useful therapeutic modality for MM patients with advanced/refractory disease and warrants further evaluation. Disclosures Khong: Novartis Oncology: Research Funding. Spencer:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding; Specialised Therapeutics Australia: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Servier: Consultancy, Honoraria; Secura Bio: Consultancy, Honoraria.

Deletion of CISH in Human Pluripotent Stem Cell-Derived Natural Killer Cells Enhances Anti-Tumor Activity Via Metabolic Reprogramming

Cellular metabolism is now recognized to play a key role in regulating function and differentiation of immune cells. Immune cells undergo dynamic metabolic shifts to support their activity. Recent studies have shown the importance of metabolism to regulate NK cell function under different conditions. IL-15 can regulate NK cell metabolism in a dose- and time- dependent manner. However, whether improving metabolic fitness of NK cells can lead to improved anti-tumor activity is largely unknown. Here, we deleted CIS (encoded by the CISH gene in human induced pluripotent stem cells (iPSC)-derived NK cells (CISH-/- iPSC-NK) using CRISPR/Cas9 technology. CIS is known to function as a critical negative regulator of IL-15 signaling and a potent inhibitory checkpoint in NK cell-mediated anti-tumor activity. Surprisingly, more than 3000 differentially expressed genes were identified in CISH-/- iPSC-NK cells through RNA sequencing analysis. Gene ontology (GO) enrichment analysis showed that the top 2 up-regulated signaling pathways were associated with leukocyte differentiation and activation. Notably, more than 50 genes that involved in positive regulation of lymphocyte activation including genes associated with NK cell effector functions were significantly up-regulated in CISH-/- iPSC-NK cells compared with unmodified iPSC-NK cells. As expected, CISH-/- iPSC-NK cells exhibited increased sensitivity to IL-15 stimulation. Specifically, IL-15 stimulated JAK1 tyrosine phosphorylation, STAT3 and STAT5 phosphorylation were all dramatically increased in CISH-/- iPSC-NK cells, compared with unmodified iPSC-NK cells. In addition, when incubated with low concentrations of IL-15 (5 ng/ml), a concentration that does not support the growth and functions of unmodified iPSC-NK cells, CISH-/- iPSC-NK cells maintained significantly better expansion with >10 fold expansion for CISH-/- iPSC-NK cells in 3 weeks, whereas most of WT iPSC-NK cells died under these conditions (p<0.001). These CISH-/- iPSC-NK cells exhibited improved cytotoxic against acute myeloid leukemia (AML) cell lines K562 and Molm-13. More importantly, in an AML (MOLM-13) xenograft model, CISH-/- iPSC-NK cells displayed significantly increased in vivo persistence thus lead to significantly better control of tumor progression. To further understand the underling mechanisms contributing to the increased functionality in CISH-/- iPSC-NK cells, we then analyzed the metabolic profiles of these cells using Agilent Seahorse assays. After treatment with low concentration IL-15 for 7 days, CISH-/- iPSC-NK cells displayed both increased glucose metabolism and increased mitochondrial respiration profiles when compared with unmodified iPSC-NK cells. Increased glucose metabolism include a significant increase in basal glycolysis (p=0.0003, 84.5 ± 1.0 vs 61.1 ± 1.8 mpH/min, n=6), and glycolytic capacity (p=0.0049, 15.2±0.4 vs 11.7±0.5 mpH/min, n=6). And increased mitochondrial respiration profile is characterized by an increase in the spare respiratory capacity (p=0.0009, 16.3±1.9 vs 6.9±0.5 pmol/min, n=6), maximal respiration (p<0.0001, 31.6±1.9 vs 15.7±0.8 pmol/min, n=6) and ATP-linked respiration (p<0.0001, 11.5 ± 0.4 vs 7.0±0.3 pmol/min, n=6). Interestingly, limiting the increase of glucose metabolism and mitochondrial respiration via utilization of an mTOR inhibitor (rapamycin) attenuated the increased functionality in CISH-/- iPSC-NK cells. The findings presented here show that deletion of CISH in iPSC-NK cells enhance anti-tumor activity, at least in part via metabolic reprogramming. These findings suggest that improving NK cell functions for cancer immunotherapy through metabolic reprogramming provides a promising therapeutic strategy. Disclosures Guan: OncoImmune: Equity Ownership. Kaufman:FATE Therapeutics: Consultancy, Research Funding.

RUNX2 Regulates Cell Migration in T-Cell Lineage Acute Lymphoblastic Leukemia

T-lineage acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy accounting for 10-15% of pediatric ALLs. While cure rates have recently improved, event free survival is <10% in patients with relapsed disease. We recently showed that Runt related transcription factor 2 (RUNX2) is upregulated in high-risk T-ALL including patients harboring KMT2A-R (p< 0.0001), ETP phenotype (p< 0.0001) and those who failed induction therapy (p <0.005) (Sharma et al., AACR Annual Meeting 2018; #4129). We also demonstrated that RUNX2 positively regulates T-ALL survival via Akt/β-catenin/survivin axis and observed a significant increase in survival of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice engrafted with shRNA-RUNX2 depleted T-ALL cells (median survival 52 days) compared to animals engrafted with negative control cells (median survival 38 days; p< 0.0001). Because RUNX2 is involved in invasiveness and metastasis of many solid tumors, we hypothesized that RUNX2 might modulate the migration and dissemination of T-ALL. To test the regulatory effects of RUNX2 on T-ALL migration we used lentiviral transduction to induce or knock down RUNX2 expression in T-ALL cell lines (n = 4) and/or primary patient samples (n = 3), and/or BaF3 cells expressing KMT2A-R (KMT2A-MLLT4 or KMT2A-MLLT1). While induced expression of RUNX2 had no effect on T-ALL cell growth and cell cycle, we discovered that RUNX2 positively regulated the expression of CXCR4 receptor and migration of T-ALL cells towards CXCL12 chemokine (p< 0.0005). Because cell migration is overall an energy-demanding process, we used the Agilent Seahorse XF Analyzer to study leukemic cell metabolism. We found that RUNX2 upregulation leads to increased oxygen consumption rate and ATP production (p< 0.0001) as well as the increase in basal and compensatory glycolysis (p< 0.005), suggesting that RUNX2 potentiates metabolic activity of T-ALL cells. We next utilized flow-based Mito-Tracker Red CMXRos and Reactive Oxygen Species (ROS) assays to examine the impact of RUNX2 on mitochondrial activity. Forced expression of RUNX2 increased mitochondrial membrane potential corresponding to decreased ROS levels. RUNX2 positively regulated the expression of critical regulators of mitochondrial fission (P-DRP1 Ser616, DRP1, MFF and FIS1), fusion (OPA1 and Mfn1, Mfn2) and mitochondrial biogenesis (PGC1α and NRF1). In converse experiments, RUNX2 depletion reduced the oxidative phosphorylation, glycolytic flux and mitochondrial activities in the tested T-ALL cells suggesting that RUNX2 displays unique metabolic signatures that might enhance leukemia aggressiveness. Disruption of mitochondrial fission with Midivi-1 inhibitor (1 μΜ, 16 hr) reduced RUNX2-mediated T-ALL migration towards CXCL12 (P< 0.0001) suggesting that RUNX2 regulates mitochondrial dynamics to fosters T-ALL cell migration in vitro. Similarly, treatment with 2-deoxy-D-glucose (2DG), a glucose analog reduced the levels of migrating T-ALL cells (p< 0.005) indicating the roles of metabolic reprograming in RUNX2-mediated T-ALL cell migration. Our results suggest that RUNX2 regulates mitochondrial dynamics and cell metabolism to promote T-ALL migration. We propose deregulation of RUNX2 expression as a novel mechanism governing T-ALL migration and dissemination. Ongoing in vivo studies will reveal the roles of RUNX2 in T-ALL infiltration to extramedullary tissue. Disclosures Loh: Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Hunger:Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Jazz: Honoraria; Novartis: Consultancy.

Abstract 280: The Effect of YAP/TAZ on Glycolysis and Mitochondrial Oxidative Phosphorylation in Cardiomyocytes

The heart requires a high rate of adenosine triphosphate (ATP) production to maintain healthy cardiac function and viability. Anaerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the main metabolic pathways by which ATP is generated in mammalian cells, including cardiomyocytes. Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), which are major downstream effectors of the Hippo signaling pathway, play an important role in the regulation of heart size and cellular homeostasis of cardiomyocytes. However, interplay between YAP/TAZ and cardiac energy metabolism has been poorly understood. Here we examined the effect of YAP/TAZ on glycolysis and OXPHOS in isolated neonatal rat ventricular cardiomyocytes (NRVMs). Extracellular acidification rate (ECAR; an index of glycolysis) and oxygen consumption rate (OCR; an index of OXPHOS) of NRVMs transduced with adenoviruses harboring YAP, TAZ, or LacZ (control) for 6 days were assessed by Seahorse XFe96 analyzer. Both YAP and TAZ significantly increased not only the ECAR of basal glycolysis (1.5-fold and 1.3-fold, respectively, p<0.05), glycolytic maximal capacity (1.4-fold and 1.3-fold, respectively, p<0.05), and glycolytic reserve capacity (1.3-fold and 1.2-fold, respectively, P<0.05) (n=12-15) but also basal OCR (1.3-fold and 1.3-fold, respectively, p<0.05) (n=6-10) compared to control LacZ. Quantitative PCR analysis showed that mRNA levels of Pfkm, a key glycolytic enzyme, and subunits of protein complexes I though V in the electron transport chain are significantly increased (1.3-1.9-fold, n=5-6, p<0.05) by both YAP and TAZ compared with control. These results suggest that YAP/TAZ increase ATP production by accelerating glycolysis and OXPHOS in cardiomyocytes.

Dynamic bioenergetic alterations in colorectal adenomatous polyps and adenocarcinomas

BackgroundEnergy metabolism in carcinogenesis is poorly understood. It is widely accepted the majority of colorectal cancers (CRCs) arise from adenomatous polyps (APs). We aimed to characterize the bioenergetic alterations in APs and CRCs. MethodsFifty-six APs, 93 CRCs and adjacent normal mucosae were tested. Oxygen consumption rate (OCR) was measured representing mitochondrial oxidative phosphorylation (OxPhos), and extracellular acidification rate (ECAR)was measured representing glycolysis. Mitochondrial DNA (mtDNA) variants and mutations were studied. Over-expressed metabolic genes in APs were identified by microarray and validated by qRT-PCR, Western blots and immunohistochemistry. Identified genes were knocked down in WiDr and colo205 CRC cell lines, and their expression was analyzed in APs/CRCs with enhanced glycolysis. FindingsECAR, not OCR, was significantly increased in APs. While no difference of ECAR was found between CRCs and normal mucosae, OCR was significantly reduced in CRCs. OCR/ECAR ratio was decreased in APs over 1 cm, APs with a villous component and CRCs, indicating their glycolytic tendencies. The number of mtDNA mutations was increased in APs and CRCs, but not correlated with metabolic profiles. Two metabolic genes ALDOB and SLC16A4 were up-regulated in APs. Both ALDOB-knockdown and SLC16A4-knockdown CRC cell lines showed increased basal motichondrial OxPhos and decreased basal glycolysis. Moreover, the increase of mitochondrial ATP-linked respiration and the decrease of glycolytic capacity were showed in SLC16A4-knockdown cells. Finally, APs/CRCs with enhanced glycolysis had increased SLC16A4 expression. InterpretationATP production shifts from OxPhos to glycolysis in the process of AP enlargement and villous transformation. OxPhos defects are present in CRCs but not in APs. APs and CRCs tend to accumulate mtDNA mutations, but these are not correlated with bioenergetic profiles. Finally, the ALDOB and SLC16A4 may contribute to the glycolytic shift in APs/CRCs.

Attenuation of Endoplasmic Reticulum Stress, Impaired Calcium Homeostasis, and Altered Bioenergetic Functions in MPP+-Exposed SH-SY5Y Cells Pretreated with Rutin.

Parkinson's disease (PD) is a common neurodegenerative disorder that affects approximately 1% of the population over the age of 65years. While treatment options for PD are limited, reports show that plant-derived bioactive compounds such as rutin possess numerous pharmacological benefits, including antioxidant and antiapoptotic activities. This study aimed to investigate the potential role of rutin in MPP+-treated SH-SY5Y neuroblastoma cells, an established cell model of PD. Our findings reveal increased concentrations of Ca2+ and endoplasmic reticulum (ER) stress as well as impaired mitochondrial membrane potential and bioenergetic status in SH-SY5Y cells treated with MPP+ only. This is demonstrated by a significant reduction in the expression levels of BiP, significantly reduced basal respiration, maximal respiration, and spare respiratory capacity as well as a significant increase in the expression levels of CHOP; however, these effects were significantly attenuated following pretreatment with rutin. Also, rutin significantly improved basal and compensatory glycolysis as a response to an impaired oxidative phosphorylation system triggered by MPP+, characterized by deficient ATP production. In conclusion, our findings provide the first evidence on the ability of rutin to maintain Ca2+ homeostasis, inhibit ER stress, and protect the mitochondria in MPP+-treated SH-SY5Y cells.

Abstract WP337: The Ischemic Neuroprotectant Protein Kinase C Epsilon, Phosphorylates Enzymes in Neuronal NADH Shuttle and Regulates Mitochondrial Respiration and Glycolysis

Background: Among PKCε-mediated potential protective mechanisms in ischemic preconditioning, one target that we surmise is crucial for neuroprotection is the malate-aspartate shuttle (MAS). MAS is the most important NAD + /NADH shuttle importing reducing equivalents (NADH) generated by glycolysis from the cytosol into the mitochondria, and thus, tightly controls neuronal respiration. We hypothesized that PKCε regulates MAS in the brain and mediates neuronal mitochondrial respiration. Method: Phosphoproteomics analysis and immunoprecipitation (IP) were used to determine PKCε-induced phosphorylation of MAS components. Synaptosomes from cortices of naïve SD rats were treated with Tat-conjugated ΨεRACK (selective PKCε activator) or control Tat peptide and sent for phosphoproteomics analysis. To confirm the predicted phosphorylation, IP was performed. SD rats were treated with ΨεRACK (i.p.) or Tat and sacrificed 48hrs later. Proteins from rat cortical synaptosomes were immunoprecipitated with phosphoserine/threonine/tyrosine and immunoblotted for predicted phosphorylated proteins. Once phosphorylation was determined, we evaluated energy metabolism in primary neurons. Neuronal cultures were obtained from embryonic pups cortices and treated with ΨεRACK or Tat on DIV 7-10. Cellular respiration and glycolytic function were evaluated 48hrs after treatment using Seahorse XF Analyzer. Result: Phosphoproteomics analysis and IP results revealed increased phosphorylation of key MAS components: the mitochondrial aspartate aminotransferase (GOT2) and the oxoglutarate carrier (OGC). After PKCε activation, GOT2 and OGC exhibited serine phosphorylation increased by 2.14 fold (P < 0.05, n=4-5), and 1.74 fold (P < 0.01, n=4-5), respectively. In addition, in primary neurons, ΨεRACK treatment increased the basal and maximum cellular respiration by 0.13 (P < 0.05, n=4), and 0.22 fold (P < 0.01, n=4), respectively. The basal and maximum glycolysis were increased by 0.14, and 0.21 fold (P<0.05, n=4), respectively. Conclusion: Our data strongly suggest that PKCε regulates MAS, and increases neuronal metabolism by promoting cellular respiration and glycolysis. These changes may be linked to our observed PKCε-induced ischemic tolerance in the brain.

Abstract A089: The effect of lactate dehydrogenase-A (LDH-A) knockdown and human prostate-specific membrane antigen (hPSMA) directed CAR T-cell treatment on hPSMA(+) Myc-CaP tumors

Abstract Objective: Determine whether LDH-A knockdown enhances CAR T-cell tumor-targeting and treatment response.Background: It has been shown that T-cells are restricted from entering many human and murine solid tumors, including both murine and human prostate tumors. CD3(+) T-cells are restricted from Myc-CaP murine prostate tumors and localize around the periphery of the tumor nodules (1). Human prostate specific membrane antigen (hPSMA) targeted CAR T-cells are also restricted from hPSMA(+) Myc-CaP tumors and this is only partially reversed by anti-PD1 treatment (1). This report describes the benefit of LDH-A depletion and a greater response of CAR T-cells targeting Myc-CaP tumors with low expression of LDH-A. Methods: LDH-A depletion in hPSMA(+) Myc-CaP cells bearing a bioluminescence reporter (Renilla Luciferase), was achieved by shRNA knockdown (KD) (1). A scrambled IgG shRNA was used as a control (NC). LDH-A expression was quantified by digital droplet PCR (ddPCR), Western blotting and LDH enzyme activity. The glycolytic activity of KD and NC cells was measured using Seahorse XF96 and XFp analyzers. Intratumoral lactate levels were monitored by magnetic resonance spectroscopy (MRS). The preparation and characterization of “second generation” hPSMA-directed CAR T-cells and the hPSMA(+) Myc-CaP tumor models in SCID mice have been described (1). To monitor CAR T-cell trafficking, T-cells were transduced with a Click Beetle Red luciferase reporter to enable efficient visualization by bioluminescence imaging (BLI) of CAR T-cell trafficking, persistence and viability within the hPSMA(+)Myc-CaP tumor mass. Tumor volume was calculated from caliper measurements. CD31 and PD-L1 expression was quantified with immunofluorescent staining and Metamorph Offline image analysis. Results and Discussion: These studies demonstrated that LDH-A KD was the dominant factor in reducing tumor growth. The difference in tumor doubling time (DT) between KD and NC tumors in the presence of CAR T-cell therapy was significant (p<0.0001): DT= 5.0±0.3 vs 2.9±0.5 days, respectively. A significant difference was also found in the absence of CAR T-cell therapy. Although LDH-A KD produced the larger single-treatment effect, the addition of CAR T-cells was additive. To explain these observations and study the effect of LDH-A KD on tumor metabolism and microenvironment, Seahorse and MRS experiments were performed. LDH-A depletion (KD) resulted in: lower rates of basal (NC: 24.0±1.9 vs KD: 6.9±1.9 pmol H+/min/µg protein; p<0.0001) and compensatory glycolysis (NC: 43.0±2.3 vs KD: 18.3±3.9 pmol H+/min/µg protein, p<0.0001), and lactate production. In contrast, basal and maximal respiration increased following LDH-A KD (NC: 4.0±0.8 vs KD: 8.3±0.6, p=0.0011, and NC: 6.7±1.4 vs KD: 13.8±0.6 pmol O2/min/µg protein, p=0.0003, respectively). However, total tumor lactate, measured in vivo by MRS, showed only marginal differences between KD and NC tumors. Nevertheless, there were significant differences in CAR T-cell trafficking-to and persistence-in KD compared to NC tumors, and the increase in tumor doubling time was directly related to the CAR T-cell expansion ratio. Interestingly, tumor vascularity (CD31), but not PD-L1, was significantly lower in KD compared to NC tumors. Necrosis was minimal in both KD and NC tumors, with or without CAR T-cell treatment. Conclusions: LDHA knockdown (KD) in the Myc-CaP murine prostate tumor model has a significant effect on tumor cell metabolism resulting in reduced glycolysis, reduced lactate production and increased oxidative phosphorylation that impacts the tumor microenvironment. The LDH-A KD induced changes in tumor metabolism and the microenvironment diminish Myc-CaP tumor growth rate and impact CAR T-cell trafficking and persistence in the tumor. Future studies will compare the effects of anti-PD1 and anti-CTLA4 therapy in combination with LDH-A knockdown. Reference: 1. Serganova I, et al. Molecular Therapy: Oncolytics 2017;4:41. Citation Format: Mayuresh M. Mane, Khalid Shalaby, Ivan Cohen, Avi Albeg, Jenny Ijoma, Myat Ko, Masatomo Maeda, Kiranmayi Vemuri, Jaya Satagopan, Anna Moroz, Juan Zurita, Larissa Shenker, Ellen Ackerstaff, Masahiro Shindo, Ekaterina Moroz, Maxim A. Moroz, Inna Serganova, Jason Koutcher, Vladimir Ponomarev, Ronald G. Blasberg. The effect of lactate dehydrogenase-A (LDH-A) knockdown and human prostate-specific membrane antigen (hPSMA) directed CAR T-cell treatment on hPSMA(+) Myc-CaP tumors [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A089.

Classical monocytes maintain ex vivo glycolytic metabolism and early but not later inflammatory responses in older adults

BackgroundInflammaging is a condition of chronic low-grade inflammation due to the aging process and is associated with a variety of chronic diseases. Monocytes are innate immune cells which contribute to inflammation and are dysregulated during aging, demonstrated reduced phagocytosis, increased inflammation, and alterations in subset proportions. Metabolism is known to determine immune cell function, with quiescent and anti-inflammatory cells primarily relying on fatty acid oxidation, while activated and inflammatory cells primarily rely on glycolysis. We have previously shown an age-related decrease in mitochondrial respiratory capacity in monocytes, so we hypothesized here that a compensatory shift toward glycolysis would occur which would also exacerbate inflammation.ResultsUsing Seahorse assays, we profiled glycolysis in classical monocytes isolated from older (60–80 yr) and younger (18–35 yr) adults. Aging did not affect parameters of basal glycolysis in the glycolysis stress test, nor did it alter glycolytic activation early (2 h) or later (24 h) post-LPS stimulation. Cytokine gene expression was unchanged between aged and young subjects at 2 h post-LPS but was reduced in older subjects at 24 h post-LPS either significantly (IL1B) or near-significantly (IL6, IL10).ConclusionsAging appears not to affect glycolytic metabolism ex vivo in classical monocytes, but may reduce cytokine expression at later timepoints. Studies examining monocytes stimulated with age-altered circulating factors or with other pattern recognition receptor agonists may shed further light on monocyte metabolism as a determinant of immunosenescence and inflammaging.

The age-related changes and differences in energy metabolism and glutamate-glutamine recycling in the d-gal-induced and naturally occurring senescent astrocytes in vitro.

Previously, we successfully established a d-galactose (d-gal)-induced astrocyte aging model in vitro. However, whether the changes in the aged astrocytes induced by d-gal are similar to those occurred in naturally are unknown. Therefore, in current study, we simultaneously established d-gal-induced and naturally aged astrocyte aging models in vitro to explore the age-related changes and to compare the differences in these two astrocyte aging models. The Seahorse Extracellular Flux Analyzer was used to examine the mitochondrial metabolism and glycolysis activities of the young and senescent astrocytes. The results showed that the mitochondrial ATP-linked oxygen consumption rates (OCRs) were decreased markedly both in the d-gal-induced and naturally occurring senescent astrocytes. The basal glycolysis activity was increased in the naturally occurring senescent astrocytes, whereas it was decreased in the d-gal-induced senescent astrocytes. Western blot analysis showed that isocitrate dehydrogenase 3 (IDH3), succinate dehydrogenase (SDH) and malate dehydrogenase 2 (MDH2) were markedly decreased both in these two aging models, whereas the iron‑sulfur cluster assembly enzyme (ISCU) was up-regulated in the naturally occurring senescent astrocytes but was down-regulated in the d-gal-induced senescent astrocytes. The expression levels of glial glutamate transporter-1 (GLT-1), glutamine synthetase (GS) and γ-aminobutyric acid type B receptor subunit 2 (GABABR2) were also markedly decreased in these two aging models. In addition, the PI3K/AKT signaling pathway was to be inactivated both in the d-gal-induced and naturally occurring senescent astrocytes. These results indicate that the age-related changes in d-gal-induced senescent astrocytes are not fully consistent with those in naturally occurring senescent astrocytes, and it may be not suitable to use d-gal-induced senescent astrocytes to replace the naturally occurring senescent astrocytes to explore the aging mechanisms under some circumstances.

Ovarian Cancer Relies on Glucose Transporter 1 to Fuel Glycolysis and Growth: Anti-Tumor Activity of BAY-876.

The recent progresses in understanding of cancer glycolytic phenotype have offered new strategies to manage ovarian cancer and other malignancies. However, therapeutic targeting of glycolysis to treat cancer remains unsuccessful due to complex mechanisms of tumor glycolysis and the lack of selective, potent and safe glycolytic inhibitors. Recently, BAY-876 was identified as a new-generation inhibitor of glucose transporter 1 (GLUT1), a GLUT isoform commonly overexpressed but functionally poorly defined in ovarian cancer. Notably, BAY-876 has not been evaluated in any cell or preclinical animal models since its discovery. We herein took advantage of BAY-876 and molecular approaches to study GLUT1 regulation, targetability, and functional relevance to cancer glycolysis. The anti-tumor activity of BAY-876 was evaluated with ovarian cancer cell line- and patient-derived xenograft (PDX) models. Our results show that inhibition of GLUT1 is sufficient to block basal and stress-regulated glycolysis, and anchorage-dependent and independent growth of ovarian cancer cells. BAY-876 dramatically inhibits tumorigenicity of both cell line-derived xenografts and PDXs. These studies provide direct evidence that GLUT1 is causally linked to the glycolytic phenotype in ovarian cancer. BAY-876 is a potent blocker of GLUT1 activity, glycolytic metabolism and ovarian cancer growth, holding promise as a novel glycolysis-targeted anti-cancer agent.