PEGPH20, a PEGylated Human Hyaluronidase, Induces Radiosensitization by Reoxygenation In Pancreatic Cancer Xenografts. A Molecular Imaging Study

PEGylated human hyaluronidase (PEGPH20) enzymatically depletes hyaluronan, an important component of the extracellular matrix, in tumors. The resultant improvement in vascular patency and perfusion has been shown to increase the delivery of therapeutic molecules. We show that PEGPH20 also improves the efficacy of radiation therapy in a human pancreatic adenocarcinoma BxPC3 mouse model overexpressing hyaluronan synthase 3 (BxPC3-HAS3) while exerting little effect on the corresponding wild type tumors. Mice overexpressing HAS3 developed fast growing, radiation resistant tumors that became rapidly more hypoxic as time progressed. Treatment with PEGPH20 increased survival times when used in combination with radiation therapy, significantly more than either radiation therapy or PEGPH20 alone. Radiosensitization in BxPC3-HAS3 tumors was attributed to an increase in local pO2 as studied by by EPR imaging. No effect on survival, radiation treatment, or pO2 was seen in wild type tumors after PEGPH20 treatment. Dynamic contrast enhanced (DCE) MRI and MRI based blood volume imaging showed improved perfusion/permeability and local blood volume, respectively, in BxPC3-HAS3 tumors after PEGPH20 treatment, accounting for the increase in tumor oxygenation. Photoacoustic imaging indicated immediate changes in tumor oxygenation after treatment. Metabolic MRI using hyperpolarized [1-13C] pyruvate suggested a metabolic shift towards decreased glycolytic flux after PEGPH20 treatment. In summary, the results showed that PEGPH20 may be useful for radiosensitization of pancreatic cancer but only in the subset of tumors with substantial hyaluronan accumulation and the response of the treatment may potentially be monitored non-invasive imaging of the hemodynamic and metabolic changes in the tumor microenvironment.

Real-Time Monitoring of the Yeast Intracellular State During Bioprocesses With a Toolbox of Biosensors

Industrial fermentation processes strive for high robustness to ensure optimal and consistent performance. Medium components, fermentation products, and physical perturbations may cause stress and lower performance. Cellular stress elicits a range of responses, whose extracellular manifestations have been extensively studied; whereas intracellular aspects remain poorly known due to lack of tools for real-time monitoring. Genetically encoded biosensors have emerged as promising tools and have been used to improve microbial productivity and tolerance toward industrially relevant stresses. Here, fluorescent biosensors able to sense the yeast intracellular environment (pH, ATP levels, oxidative stress, glycolytic flux, and ribosome production) were implemented into a versatile and easy-to-use toolbox. Marker-free and efficient genome integration at a conserved site on chromosome X of Saccharomyces cerevisiae strains and a commercial Saccharomyces boulardii strain was developed. Moreover, multiple biosensors were used to simultaneously monitor different intracellular parameters in a single cell. Even when combined together, the biosensors did not significantly affect key physiological parameters, such as specific growth rate and product yields. Activation and response of each biosensor and their interconnection were assessed using an advanced micro-cultivation system. Finally, the toolbox was used to screen cell behavior in a synthetic lignocellulosic hydrolysate that mimicked harsh industrial substrates, revealing differences in the oxidative stress response between laboratory (CEN.PK113-7D) and industrial (Ethanol Red) S. cerevisiae strains. In summary, the toolbox will allow both the exploration of yeast diversity and physiological responses in natural and complex industrial conditions, as well as the possibility to monitor production processes.

Glycolytic flux-signaling controls mouse embryo mesoderm development

How cellular metabolic state impacts cellular programs is a fundamental, unresolved question. Here we investigated how glycolytic flux impacts embryonic development, using presomitic mesoderm (PSM) patterning as the experimental model. First, we identified fructose 1,6-bisphosphate (FBP) as an in vivo sentinel metabolite that mirrors glycolytic flux within PSM cells of post-implantation mouse embryos. We found that medium-supplementation with FBP, but not with other glycolytic metabolites, such as fructose 6-phosphate and 3-phosphoglycerate, impaired mesoderm segmentation. To genetically manipulate glycolytic flux and FBP levels, we generated a mouse model enabling the conditional overexpression of dominant active, cytoplasmic Pfkfb3 (cytoPfkfb3). Overexpression of cytoPfkfb3 indeed led to increased glycolytic flux/FBP levels and caused an impairment of mesoderm segmentation, paralleled by the downregulation of Wnt-signaling, reminiscent of the effects seen upon FBP-supplementation. To probe for mechanisms underlying glycolytic flux-signaling, we performed subcellular proteome analysis and revealed that cytoPfkfb3 overexpression altered subcellular localization of certain proteins, including glycolytic enzymes, in PSM cells. Specifically, we revealed that FBP supplementation caused depletion of Pfkl and Aldoa from the nuclear-soluble fraction. Combined, we propose that FBP functions as a flux-signaling metabolite connecting glycolysis and PSM patterning, potentially through modulating subcellular protein localization.

STIM1 is a core trigger of airway smooth muscle remodeling and hyperresponsiveness in asthma

Airway remodeling and airway hyperresponsiveness are central drivers of asthma severity. Airway remodeling is a structural change involving the dedifferentiation of airway smooth muscle (ASM) cells from a quiescent to a proliferative and secretory phenotype. Here, we show up-regulation of the endoplasmic reticulum Ca2+ sensor stromal-interacting molecule 1 (STIM1) in ASM of asthmatic mice. STIM1 is required for metabolic and transcriptional reprogramming that supports airway remodeling, including ASM proliferation, migration, secretion of cytokines and extracellular matrix, enhanced mitochondrial mass, and increased oxidative phosphorylation and glycolytic flux. Mechanistically, STIM1-mediated Ca2+ influx is critical for the activation of nuclear factor of activated T cells 4 and subsequent interleukin-6 secretion and transcription of pro-remodeling transcription factors, growth factors, surface receptors, and asthma-associated proteins. STIM1 drives airway hyperresponsiveness in asthmatic mice through enhanced frequency and amplitude of ASM cytosolic Ca2+ oscillations. Our data advocates for ASM STIM1 as a target for asthma therapy.

Molecular mechanism of glycolytic flux control intrinsic to human phosphoglycerate kinase

Glycolysis plays a fundamental role in energy production and metabolic homeostasis. The intracellular [adenosine triphosphate]/[adenosine diphosphate] ([ATP]/[ADP]) ratio controls glycolytic flux; however, the regulatory mechanism underlying reactions catalyzed by individual glycolytic enzymes enabling flux adaptation remains incompletely understood. Phosphoglycerate kinase (PGK) catalyzes the reversible phosphotransfer reaction, which directly produces ATP in a near-equilibrium step of glycolysis. Despite extensive studies on the transcriptional regulation of PGK expression, the mechanism in response to changes in the [ATP]/[ADP] ratio remains obscure. Here, we report a protein-level regulation of human PGK (hPGK) by utilizing the switching ligand-binding cooperativities between adenine nucleotides and 3-phosphoglycerate (3PG). This was revealed by nuclear magnetic resonance (NMR) spectroscopy at physiological salt concentrations. MgADP and 3PG bind to hPGK with negative cooperativity, whereas MgAMPPNP (a nonhydrolyzable ATP analog) and 3PG bind to hPGK with positive cooperativity. These opposite cooperativities enable a shift between different ligand-bound states depending on the intracellular [ATP]/[ADP] ratio. Based on these findings, we present an atomic-scale description of the reaction scheme for hPGK under physiological conditions. Our results indicate that hPGK intrinsically modulates its function via ligand-binding cooperativities that are finely tuned to respond to changes in the [ATP]/[ADP] ratio. The alteration of ligand-binding cooperativities could be one of the self-regulatory mechanisms for enzymes in bidirectional pathways, which enables rapid adaptation to changes in the intracellular environment.

Metabolic dysregulation induces impaired lymphocyte memory formation during severe SARS-CoV-2 infection

Cellular metabolic dysregulation is a consequence of COVID-19 infection that is a key determinant of disease severity. To understand the mechanisms underlying these cellular changes, we performed high-dimensional immune cell profiling of PBMCs from COVID-19-infected patients, in combination with single cell transcriptomic analysis of COVID-19 BALFs. Hypoxia, a hallmark of COVID-19 ARDS, was found to elicit a global metabolic reprogramming in effector lymphocytes. In response to oxygen and nutrient-deprived microenvironments, these cells shift from aerobic respiration to increase their dependence on anaerobic processes including glycolysis, mitophagy, and glutaminolysis to fulfill their bioenergetic demands. We also demonstrate metabolic dysregulation of ciliated lung epithelial cells is linked to significant increase of proinflammatory cytokine secretion and upregulation of HLA class 1 machinery. Augmented HLA class-1 antigen stimulation by epithelial cells leads to cellular exhaustion of metabolically dysregulated CD8 and NK cells, impairing their memory cell differentiation. Unsupervised clustering techniques revealed multiple distinct, differentially abundant CD8 and NK memory cell states that are marked by high glycolytic flux, mitochondrial dysfunction, and cellular exhaustion, further highlighting the connection between disrupted metabolism and impaired memory cell function in COVID-19. Our findings provide novel insight on how SARS-CoV-2 infection affects host immunometabolism and anti-viral response during COVID-19.

Extracellular Lactate Acts as a Metabolic Checkpoint and Shapes Monocyte Function Time Dependently

Elevated blood lactate levels are frequently found in critically ill patients and thought to result from tissue hypoperfusion and cellular oxygen shortage. Considering the close relationship between immune cell function and intracellular metabolism, lactate is more than a glycolytic waste molecule but able to regulate the immune response. Our aim was to elucidate the temporal and mechanistic effect of extracellular lactate on monocytes. To this end, primary human monocytes and the human monocytic cell line MonoMac6 were stimulated with various toll-like-receptor agonists after priming with Na-L-lactate under constant pH conditions. As readout, cytokine production was measured, real-time assessment of intracellular energy pathways was performed, and intracellular metabolite concentrations were determined. Irrespective of the immunogenic stimulus, short-term Na-lactate-priming strongly reduced cytokine production capacity. Lactate and hexoses accumulated intracellularly and, together with a decreased glycolytic flux, indicate a lactate-triggered impairment of glycolysis. To counteract intracellular hyperglycemia, glucose is shunted into the branching polyol pathway, leading to sorbitol accumulation. In contrast, long-term priming with Na-L-lactate induced cellular adaption and abolished the suppressive effect. This lactate tolerance is characterized by a decreased cellular respiration due to a reduced complex-I activity. Our results indicate that exogenous lactate shapes monocyte function by altering the intracellular energy metabolism and acts as a metabolic checkpoint of monocyte activation.

Cannabinoids Induce Cell Death in Leukemic Cells through Parthanatos and PARP-Related Metabolic Disruptions

BACKGROUND: We have previously described the antitumor effect of the cannabinoid WIN-55,212-2 (WIN-55) and a set of cannabinoid derivatives (CNB) specific for CB2 in multiple myeloma (Barbado et al, 2018). In AML, we also observed a potent and selective antileukemic effect, affecting signaling and metabolic pathways essential for the viability of tumor cells. Among them, we found an increased stress on the endoplasmic reticulum, mitochondrial damage, and alteration of the metabolism of ceramides, although none of these events turned out to be the main trigger of cell death, since the inhibition of each of them did not prevent the antileukemic effect of CNB. On the other hand, disruption of the mecanisms of DNA repair have been identified as a key oncogeneic event in different solid tumors, and some studies have also suggested that it might be involved in leukemogenesis. More specifically, PARP1 is involved in DNA damage repair. Other functions include the regulation of glycolysis enzymes through the addition of Poly ADP-Ribose (PAR) and the execution of Parthanatos, that occurs whenever PARP-1 becomes over-activated in response to extreme damage inducing nuclear translocation of AIF and depletion of NAD +. OBJECTIVES: In this study we set out to identify the ultimate mechanism that justifies the aforementioned pleiotropic effect of CNB on the metabolism of leukemic cells and their viability. METHODS: Cell viability was determined by MTT and flow cytometry. The mRNA and / or protein expression profile of AML samples or healthy progenitor cells were studied by qPCR and / or Western blot. Glycolytic flux was studied with the XF Glycolytic Rate Assay (Seahorse Biosciences). NAD + levels and glycolytic enzyme activity were measured using quantification kits. Parylation of different enzymes were confirmed by Co-IP using the corresponding antibodies. Finally, NOD/scid/IL-2R gammae null (NSG) mice were xenotransplanted with HL60-Luciferase cell line. Once the presence of leukemic cells was confirmed, treatment with vehicle, WIN-55 cannabinoid at a dose of 5 mg/kg/day or citarabine (ARA-C) at 50 mg/kg during 5 days was administered. Also we tested the effect of these compounds on normal hematopoiesis by treating healthy BALB-C mice. RESULTS: Pretreatment of leukemic cells with Olaparib, a PARP1 inhibitor, reversed WIN-55 induced apoptosis by almost 100%. WIN-55 affected the activity of most glycolysis enzymes, with a marked drop of the activity of GAPDH and pyruvate kinase which was reversed by Olaparib pretreatment. Also G6PDH activity was markedly affected upon culture with WIN-55. Co-IP confirmed parylation of these enzymes which was reversed with Olaparib. ECAR data detected by Seahorse also confirmed the drop in glycolytic capacity produced by WIN-55 in leukemic cells which again was reversed upon culture with Olaparib. In addition, the addition of nicotinamide mononucleotide (NAM), a precursor of NAD +, reversed the loss of viability produced by WIN-55. Next, we confirmed that PARP1 levels were significantly higher in leukemic cell lines and in a series of 40 AML patients as compared to healthy hematopoietic stem cells (HSC). Finally, we observed a translocation of AIF to the nucleus, confirming that WIN-55 produces PARTHANATOS. In a murine model we confirmed treatment with WIN-55,212-2 significantly prolonged survival in AML xenograft mice, with disappearance of the leukemic clone in a significant proportion of cases. By contrast, cannabinoids did not affect the viability of hematopoietic stem cells (HSC) in vivo, resulting in a lack of myeloid toxicity in healthy treated mice. CONCLUSIONS: WIN-55 exerts a selective antileukemic effect through the overactivation of PARP1, affecting the levels of parylation in enzymes involved in glycolysis and pentose phosphate pathways, leading to the translocation of AIF to the nucleus and to depletion of NAD +, which were reversed through PARP1 inhibition. These effects are not observed in normal HSC. These data are confirmed in murine models in which we confirmed the antileukemic effect of WIN-55 withouth hampering normal hematopoiesis. Figure 1 Figure 1. Disclosures Pérez-Simón: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Fbxo7 promotes Cdk6 activity to inhibit PFKP and glycolysis in T cells.

Deregulated Fbxo7 expression is associated with many pathologies, including anaemia, male sterility, cancer, and Parkinson's disease, demonstrating its critical role in a variety of cell types. Although Fbxo7 is an F-box protein that recruits substrates for SCF-type E3 ubiquitin ligases, it also promotes the formation of cyclin D/Cdk6/p27 complexes in an E3-ligase independent fashion. We discovered PFKP, the major gatekeeper of glycolysis, in a screen for Fbxo7 substrates. PFKP has been previously shown to be a critical substrate of Cdk6 for the viability of T-ALL cells. We investigated the molecular relationships between Fbxo7, Cdk6 and PFKP, and the functional effect Fbxo7 has on T cell metabolism, viability, and activation. Fbxo7 promotes Cdk6-independent ubiquitination and Cdk6-dependent phosphorylation of PFKP. Importantly Fbxo7-deficient cells have reduced Cdk6 activity, and haematopoietic and lymphocytic cell lines show a significant dependency on Fbxo7. Compared to WT cells, CD4+ T cells with reduced Fbxo7 expression show increased glycolysis, despite lower cell viability and activation levels. Metabolomic studies of activated CD4+ T cells confirm increased glycolytic flux in Fbxo7-deficient cells, as well as altered nucleotide biosynthesis and arginine metabolism. We show Fbxo7 expression is glucose-responsive at the mRNA and protein level, and we propose Fbxo7 inhibits PFKP and glycolysis via its activation of Cdk6.

NIMG-50. DEUTERIUM METABOLIC IMAGING OF THE ALTERNATIVE LENGTHENING OF TELOMERES PATHWAY REPORTS ON TUMOR BURDEN AND PSEUDOPROGRESSION IN LOW-GRADE GLIOMAS

Glioma patient management relies heavily on magnetic resonance imaging (MRI). However, MRI is often inadequate for assessment of tumor burden and pseudoprogression. Non-invasive methods that report on molecular pathways such as telomere maintenance that drive tumor proliferation are needed. Among brain tumors, low-grade astrocytomas (LGAs) use the alternative lengthening of telomeres (ALT) pathway for telomere maintenance. The goal of this study was to identify ALT-linked metabolic alterations that can be exploited for non-invasive magnetic resonance spectroscopy (MRS)-based imaging of LGAs. We examined the patient-derived BT257 model and compared neurospheres that are ALT-dependent (BT257 ALT+) with those in which the ALT pathway has been silenced (BT257 ALT-). Our studies suggest that expression and activity of the rate-limiting glycolytic enzyme phosphofructokinase-1 are significantly higher in BT257 ALT+ neurospheres relative to ALT-, an effect that is associated with elevated glucose flux to lactate. Studies indicate that poly(ADP-ribose) polymerase inhibitors such as niraparib selectively induce telomeric fusion and cell death in ALT-dependent cells. We find that the telomeric fusion-mediated cytotoxicity of niraparib is associated with significantly reduced glycolytic flux in BT257 ALT+ neurospheres. We then examined whether 2H-MRS using [6,6’-2H]-glucose, which is a clinically translatable method of imaging glycolytic flux, can be used to monitor the ALT pathway in vivo. [6,6’-2H]-glucose flux to lactate is elevated in tumor relative to normal brain in mice bearing orthotopic BT257 tumors. Importantly, following treatment of BT257 tumor-bearing mice with niraparib, lactate production from [6,6’-2H]-glucose is significantly reduced at early timepoints when alterations in tumor volume cannot be observed by MRI, pointing to the ability of [6,6’-2H]-glucose to report on pseudoprogression in vivo. Collectively, our studies mechanistically link the ALT pathway with elevated glycolytic flux via phosphofructokinase-1 and identify deuterium metabolic imaging as a novel, non-invasive method of imaging tumor burden and treatment response in LGAs in vivo.