High Metabolic Demand Research Articles

Identifying metabolic limitations in the tumor microenvironment.

Solid tumors are characterized by dysfunctional vasculature that limits perfusion and delivery of nutrients to the tumor microenvironment. Limited perfusion coupled with the high metabolic demand of growing tumors has led to the hypothesis that many tumors experience metabolic stress driven by limited availability of nutrients such as glucose, oxygen, and amino acids in the tumor. Such metabolic stress has important implications for the biology of cells in the microenvironment, affecting both disease progression and response to therapies. Recently, techniques have been developed to identify limiting nutrients and resulting metabolic stresses in solid tumors. These techniques have greatly expanded our understanding of the metabolic limitations in tumors. This review will discuss these experimental tools and the emerging picture of metabolic limitations in tumors arising from recent studies using these approaches.

Toxicity of extracellular cGAMP and its analogs to T cells is due to SLC7A1-mediated import.

STING agonists are promising innate immune therapies and can synergize with adaptive immune checkpoint blockade therapies for cancer treatment, but their effectiveness is limited by the toxicity to activated T cells. An important class of STING agonists are analogs of the endogenous STING agonist, cGAMP, and while transporters for these small molecules are known in some cell types, how they enter and kill T cells remains unknown. Here, we identify the cationic amino acid transporter SLC7A1 as the dominant transporter of cGAMP and its analogs in activated primary mouse and human T cells. T cells upregulate this transporter upon activation and rapid proliferation to meet their high metabolic demand, but this comes at the cost of enabling increased transport and toxicity of cGAMP. To circumvent the essentiality of SLC7A1 to proliferating T cells, we found that the residues responsible for cGAMP transport are separate from the arginine binding pocket allowing us to perturb cGAMP transport and STING-activation mediated killing without impacting arginine transport. These results suggest that SLC7A1 is a potential target for alleviating T cell toxicity associated with cGAMP and its analogs.

Recent Insights into Roles of Hypoxia-Inducible Factors in Retinal Diseases.

Hypoxia-inducible factors (HIFs) are transcriptional factors that function as strong regulators of oxygen homeostasis and cellular metabolisms. The maintenance of cellular oxygen levels is critical as either insufficient or excessive oxygen affects development and physiologic and pathologic conditions. In the eye, retinas have a high metabolic demand for oxygen. Retinal ischemia can cause visual impairment in various sight-threating disorders including age-related macular degeneration, diabetic retinopathy, and some types of glaucoma. Therefore, understanding the potential roles of HIFs in the retina is highly important for managing disease development and progression. This review focuses on the physiologic and pathologic roles of HIFs as regulators of oxygen homeostasis and cellular metabolism in the retina, drawing on recent evidence. Our summary will promote comprehensive approaches to targeting HIFs for therapeutic purposes in retinal diseases.

Increasing hexokinase 1 expression improves mitochondrial and glycolytic functional deficits seen in sporadic Alzheimer's disease astrocytes.

Abnormalities in cellular metabolism are seen early in Alzheimer's disease (AD). Astrocyte support for neuronal function has a high metabolic demand, and astrocyte glucose metabolism plays a key role in encoding memory. This indicates that astrocyte metabolic dysfunction might be an early event in the development of AD. In this paper we interrogate glycolytic and mitochondrial functional changes and mitochondrial structural alterations in patients' astrocytes derived with a highly efficient direct conversion protocol. In astrocytes derived from patients with sporadic (sAD) and familial AD (fAD) we identified reductions in extracellular lactate, total cellular ATP and an increase in mitochondrial reactive oxygen species. sAD and fAD astrocytes displayed significant reductions in mitochondrial spare respiratory capacity, have altered mitochondrial membrane potential and a stressed mitochondrial network. A reduction in glycolytic reserve and glycolytic capacity is seen. Interestingly, glycolytic reserve, mitochondrial spare respiratory capacity and extracellular lactate levels correlated positively with neuropsychological tests of episodic memory affected early in AD. We identified a deficit in the glycolytic enzyme hexokinase 1 (HK1), and correcting this deficit improved the metabolic phenotype in sAD not fAD astrocytes. Importantly, the amount of HK1 at the mitochondria was shown to be reduced in sAD astrocytes, and not in fAD astrocytes. Overexpression of HK1 in sAD astrocytes increases mitochondrial HK1 levels. In fAD astrocytes HK1 levels were unaltered at the mitochondria after overexpression. This study highlights a clear metabolic deficit in AD patient-derived astrocytes and indicates how HK1, with its roles in both oxidative phosphorylation and glycolysis, contributes to this.

Impact of anatomic variability and other vascular factors on lamina cribrosa hypoxia.

Insufficient oxygenation in the lamina cribrosa (LC) may contribute to axonal damage and glaucomatous vision loss. To understand the range of susceptibilities to glaucoma, we aimed to identify key factors influencing LC oxygenation and examine if these factors vary with anatomical differences between eyes. We reconstructed 3D, eye-specific LC vessel networks from histological sections of four healthy monkey eyes. For each network, we generated 125 models varying vessel radius, oxygen consumption rate, and arteriole perfusion pressure. Using hemodynamic and oxygen supply modeling, we predicted blood flow distribution and tissue oxygenation in the LC. ANOVA assessed the significance of each parameter. Our results showed that vessel radius had the greatest influence on LC oxygenation, followed by anatomical variations. Arteriole perfusion pressure and oxygen consumption rate were the third and fourth most influential factors, respectively. The LC regions are well perfused under baseline conditions. These findings highlight the importance of vessel radius and anatomical variation in LC oxygenation, providing insights into LC physiology and pathology. Pathologies affecting vessel radius may increase the risk of LC hypoxia, and anatomical variations could influence susceptibility. Conversely, increased oxygen consumption rates had minimal effects, suggesting that higher metabolic demands, such as those needed to maintain intracellular transport despite elevated intraocular pressure, have limited impact on LC oxygenation.

Influence of life-history traits on mitochondrial DNA substitution rates exceeds that of metabolic rates in teleost fishes

Abstract Understanding the molecular relevance of metabolic rate (MR) is crucial for unveiling the mechanisms driving the evolution of animals. In this study, we investigated the association between mitochondrial DNA characteristics and both resting and maximal MRs in conjunction with life-history traits among 139 species of teleost fish. We gathered fish MR data from various sources and procured sequences of 13 mitochondrial protein-encoding genes. We calculated the absolute substitution rate for entire nucleotide sequences and 4-fold degenerate sites of each gene, along with encoding amino acid sequences. Using the phylogenetic comparative method, we then explored the associations between MR and mitochondrial DNA absolute substitution rate. Additionally, we screened MR-associated single nucleotide variants in mitochondrial DNA. The findings indicate no positive correlation between MRs and any substitution rate values of both combined sequences and individual mitochondrial protein-coding genes, refuting the MR hypothesis. Instead, both maximum body size and longevity correlated negatively with molecular substitution rates, suggesting their influences on both mutation and fixation within mitochondrial genes in fish. Results also revealed significant correlations between base variation at ATP6_169 and both resting MR and maximum MR, identifying the unique ATP6_169G in Scombridae fish, which results in an extremely low isoelectric point (pI) value of the ATP6 protein. Considering its functional significance, the ATP6_169G in Scombridae fish might link to their lifestyle characterized by fast locomotion and high metabolic demands alongside a slower molecular evolutionary rate.

Endothelial β1 Integrins are Necessary for Microvascular Function and Glucose Uptake.

Microvascular insulin delivery to myocytes is rate limiting for the onset of insulin-stimulated muscle glucose uptake. The structural integrity of capillaries of the microvasculature is regulated, in part, by a family of transmembrane adhesion receptors known as integrins, which are composed of an α and β subunit. The integrin β1 (itgβ1) subunit is highly expressed in endothelial cells (EC). EC itgβ1 is necessary for the formation of capillary networks during embryonic during development and its knockdown in adult mice blunts the reactive hyperemia that manifests during ischemia reperfusion. In this study we investigated the contribution of skeletal muscle EC itgβ1 in microcirculatory function and glucose uptake. We hypothesized that loss of EC itgβ1 would impair microvascular hemodynamics and glucose uptake during insulin stimulation, creating 'delivery'-mediated insulin resistance. An itgβ1 knockdown mouse model was developed to avoid lethality of embryonic gene knockout and the deteriorating health resulting from early post-natal inducible gene deletion. We found that mice with (itgβ1fl/flSCLcre) and without (itgβ1fl/fl) inducible stem cell leukemia cre recombinase (SLCcre) expression at 10 days post cre induction have comparable exercise tolerance and pulmonary and cardiac functions. We quantified microcirculatory hemodynamics using intravital microscopy and the ability of mice to respond to the high metabolic demands of insulin-stimulated muscle using a hyperinsulinemic-euglycemia clamp. We show that itgβ1fl/flSCLcre mice compared to itgβ1fl/fl littermates have, i) deficits in capillary flow rate, flow heterogeneity, and capillary density; ii) impaired insulin-stimulated glucose uptake despite sufficient transcapillary insulin efflux; and iii) reduced insulin-stimulated glucose uptake due to perfusion-limited glucose delivery. Thus, EC itgβ1 is necessary for microcirculatory function and to meet the metabolic challenge of insulin stimulation.

Mitochondria in Retinal Ganglion Cells: Unraveling the Metabolic Nexus and Oxidative Stress.

This review explored the role of mitochondria in retinal ganglion cells (RGCs), which are essential for visual processing. Mitochondrial dysfunction is a key factor in the pathogenesis of various vision-related disorders, including glaucoma, hereditary optic neuropathy, and age-related macular degeneration. This review highlighted the critical role of mitochondria in RGCs, which provide metabolic support, regulate cellular health, and respond to cellular stress while also producing reactive oxygen species (ROS) that can damage cellular components. Maintaining mitochondrial function is essential for meeting RGCs' high metabolic demands and ensuring redox homeostasis, which is crucial for their proper function and visual health. Oxidative stress, exacerbated by factors like elevated intraocular pressure and environmental factors, contributes to diseases such as glaucoma and age-related vision loss by triggering cellular damage pathways. Strategies targeting mitochondrial function or bolstering antioxidant defenses include mitochondrial-based therapies, gene therapies, and mitochondrial transplantation. These advances can offer potential strategies for addressing mitochondrial dysfunction in the retina, with implications that extend beyond ocular diseases.

Harnessing Porphyrin Accumulation in Liver Cancer: Combining Genomic Data and Drug Targeting.

The liver, a pivotal organ in human metabolism, serves as a primary site for heme biosynthesis, alongside bone marrow. Maintaining precise control over heme production is paramount in healthy livers to meet high metabolic demands while averting potential toxicity from intermediate metabolites, notably protoporphyrin IX. Intriguingly, our recent research uncovers a disrupted heme biosynthesis process termed 'porphyrin overdrive' in cancers that fosters the accumulation of heme intermediates, potentially bolstering tumor survival. Here, we investigate heme and porphyrin metabolism in both healthy and oncogenic human livers, utilizing primary human liver transcriptomics and single-cell RNA sequencing (scRNAseq). Our investigations unveil robust gene expression patterns in heme biosynthesis in healthy livers, supporting electron transport chain (ETC) and cytochrome P450 function without intermediate accumulation. Conversely, liver cancers exhibit rewired heme biosynthesis and a massive downregulation of cytochrome P450 gene expression. Notably, despite diminished drug metabolism, gene expression analysis shows that heme supply to the ETC remains largely unaltered or even elevated with patient cancer progression, suggesting a metabolic priority shift. Liver cancers selectively accumulate intermediates, which are absent in normal tissues, implicating their role in disease advancement as inferred by expression analysis. Furthermore, our findings in genomics establish a link between the aberrant gene expression of porphyrin metabolism and inferior overall survival in aggressive cancers, indicating potential targets for clinical therapy development. We provide in vitro proof-of-concept data on targeting porphyrin overdrive with a drug synergy strategy.

Dynamic changes in structure and function of brain mural cells around chronically implanted microelectrodes.

Integration of neural interfaces with minimal tissue disruption in the brain is ideal to develop robust tools that can address essential neuroscience questions and combat neurological disorders. However, implantation of intracortical devices provokes severe tissue inflammation within the brain, which requires a high metabolic demand to support a complex series of cellular events mediating tissue degeneration and wound healing. Pericytes, peri-vascular cells involved in blood-brain barrier maintenance, vascular permeability, waste clearance, and angiogenesis, have recently been implicated as potential perpetuators of neurodegeneration in brain injury and disease. While the intimate relationship between pericytes and the cortical microvasculature have been explored in other disease states, their behavior following microelectrode implantation, which is responsible for direct blood vessel disruption and dysfunction, is currently unknown. Using two-photon microscopy we observed dynamic changes in the structure and function of pericytes during implantation of a microelectrode array over a 4-week implantation period. Pericytes respond to electrode insertion through transient increases in intracellular calcium and underlying constriction of capillary vessels. Within days following the initial insertion, we observed an influx of new, proliferating pericytes which contribute to new blood vessel formation. Additionally, we discovered a potentially novel population of reactive immune cells in close proximity to the electrode-tissue interface actively engaging in encapsulation of the microelectrode array. Finally, we determined that intracellular pericyte calcium can be modulated by intracortical microstimulation in an amplitude- and frequency-dependent manner. This study provides a new perspective on the complex biological sequelae occurring the electrode-tissue interface and will foster new avenues of potential research consideration and lead to development of more advanced therapeutic interventions towards improving the biocompatibility of neural electrode technology.

Illuminating mitochondrial translation through mouse models.

Mitochondria are hubs of metabolic activity with a major role in ATP conversion by oxidative phosphorylation (OXPHOS). The mammalian mitochondrial genome encodes 11 mRNAs encoding 13 OXPHOS proteins along with 2 rRNAs and 22 tRNAs, that facilitate their translation on mitoribosomes. Maintaining the internal production of core OXPHOS subunits requires modulation of the mitochondrial capacity to match the cellular requirements and correct insertion of particularly hydrophobic proteins into the inner mitochondrial membrane. The mitochondrial translation system is essential for energy production and defects result in severe, phenotypically diverse diseases, including mitochondrial diseases that typically affect postmitotic tissues with high metabolic demands. Understanding the complex mechanisms that underlie the pathologies of diseases involving impaired mitochondrial translation is key to tailoring specific treatments and effectively targeting the affected organs. Disease mutations have provided a fundamental, yet limited, understanding of mitochondrial protein synthesis, since effective modification of the mitochondrial genome has proven challenging. However, advances in next generation sequencing, cryoelectron microscopy, and multi-omic technologies have revealed unexpected and unusual features of the mitochondrial protein synthesis machinery in the last decade. Genome editing tools have generated unique models that have accelerated our mechanistic understanding of mitochondrial translation and its physiological importance. Here we review the most recent mouse models of disease pathogenesis caused by defects in mitochondrial protein synthesis and discuss their value for preclinical research and therapeutic development.

Neural Processing without O2 and Glucose Delivery: Lessons from the Pond to the Clinic.

Neuronal activity requires a large amount of ATP, leading to a rapid collapse of brain function when aerobic respiration fails. Here, we summarize how rhythmic motor circuits in the brain stem of adult frogs, which normally have high metabolic demands, transform to produce proper output during severe hypoxia associated with emergence from hibernation. We suggest that general principles underlying plasticity in brain bioenergetics may be uncovered by studying nonmammalian models that face extreme environments, yielding new insights to combat neurological disorders involving dysfunctional energy metabolism.

NRF2 activation by cysteine as a survival mechanism for triple-negative breast cancer cells

Triple-negative breast cancer (TNBC) is a very aggressive and heterogeneous group of tumors. In order to develop effective therapeutic strategies, it is therefore essential to identify the subtype-specific molecular mechanisms underlying disease progression and resistance to chemotherapy. TNBC cells are highly dependent on exogenous cystine, provided by overexpression of the cystine/glutamate antiporter SLC7A11/xCT, to fuel glutathione synthesis and promote an oxidative stress response consistent with their high metabolic demands. Here we show that TNBC cells of the mesenchymal stem-like subtype (MSL) utilize forced cystine uptake to induce activation of the transcription factor NRF2 and promote a glutathione-independent mechanism to defend against oxidative stress. Mechanistically, we demonstrate that NRF2 activation is mediated by direct cysteinylation of the inhibitor KEAP1. Furthermore, we show that cystine-mediated NRF2 activation induces the expression of important genes involved in oxidative stress response, but also in epithelial-to-mesenchymal transition and stem-like phenotype. Remarkably, in survival analysis, four upregulated genes (OSGIN1, RGS17, SRXN1, AKR1B10) are negative prognostic markers for TNBC. Finally, expression of exogenous OSGIN1, similarly to expression of exogenous NRF2, can prevent cystine depletion-dependent death of MSL TNBC cells. The results suggest that the cystine/NRF2/OSGIN1 axis is a potential target for effective treatment of MSL TNBCs.

The Importance of Intra-Islet Communication in the Function and Plasticity of the Islets of Langerhans during Health and Diabetes.

Islets of Langerhans are anatomically dispersed within the pancreas and exhibit regulatory coordination between islets in response to nutritional and inflammatory stimuli. However, within individual islets, there is also multi-faceted coordination of function between individual beta-cells, and between beta-cells and other endocrine and vascular cell types. This is mediated partly through circulatory feedback of the major secreted hormones, insulin and glucagon, but also by autocrine and paracrine actions within the islet by a range of other secreted products, including somatostatin, urocortin 3, serotonin, glucagon-like peptide-1, acetylcholine, and ghrelin. Their availability can be modulated within the islet by pericyte-mediated regulation of microvascular blood flow. Within the islet, both endocrine progenitor cells and the ability of endocrine cells to trans-differentiate between phenotypes can alter endocrine cell mass to adapt to changed metabolic circumstances, regulated by the within-islet trophic environment. Optimal islet function is precariously balanced due to the high metabolic rate required by beta-cells to synthesize and secrete insulin, and they are susceptible to oxidative and endoplasmic reticular stress in the face of high metabolic demand. Resulting changes in paracrine dynamics within the islets can contribute to the emergence of Types 1, 2 and gestational diabetes.

Abstract 3267: Understanding the role of dNTP pool on double-strand break and resistant to therapy in quiescent hypoxic glioblastoma

Abstract Glioblastoma (GBM) is the deadliest type of primary brain tumor. After surgical resection of GBM, chemotherapy and radiotherapy (RT) are used as treatment modalities to induce double-strand breaks (DSBs), the most cytotoxic and lethal form of DNA damage. However, an inadequate oxygen supply and the high metabolic demand in rapidly growing solid tumors like GBM result in severe hypoxic regions, contributing to altered cellular metabolism and resistance to these cancer therapies. These subpopulations of resistant and refractory hypoxic cancer cells are clinically significant as they lead to poor prognosis and adverse clinical outcomes in patients. Ionizing radiation (IR) and chemotherapeutics such as temozolomide induce DNA damage, such as base deletions, single-strand breaks (SSBs), and DSBs. Rapidly replicating cells are more sensitive to these therapeutic agents partly due to replication-induced conversion of SSB and other manageable damages to highly lethal DSB. A balanced deoxyribonucleoside triphosphate (dNTP) is crucial for DNA replication, repair, and maintenance of genomic stability. Cellular dNTP is mainly regulated by ribonucleotide reductase (RNR) and the SAM- and HD-domain-containing protein (SAMHD1). RNR boosts the cellular dNTP pool through the de novo synthesis, while SAMHD1 depletes the pool by hydrolyzing dNTPs. Also, previous studies have shown that hypoxic cancer cells have low dNTP levels due to oxygen dependency of its rate-limiting RNR enzyme, which could result in reduced DNA replication and cell cycle arrest. Here, we show the direct relationships between dNTP levels, DNA replication, and DSB induction in hypoxic GBM cancer cells, which is currently unknown. To bridge this knowledge gap, we exposed GBM cells to severe hypoxia and utilized cellular dNTP analysis to demonstrate that their dNTP pool can be elevated. Our results show that elevating the cellular dNTP pool relieved hypoxia-induced cell cycle arrest as determined by flow cytometry and increased their DNA replication. A single-cell microscopic analysis also revealed that increasing the dNTP pool significantly enhanced the induction of DSBs following exposure to IR and bleomycin. Furthermore, we detected an increased efficacy of DSB-inducing agents in hypoxic GBM, as demonstrated by apoptosis pathway activation. Our result suggests that increasing the intracellular dNTP pool in hypoxic GBM will stimulate DNA replication, increase genotoxin-induced DSB, and improve DSB-inducing therapeutic efficacy. In conclusion, this study provides a preclinical justification for using cellular dNTP modulation as a novel therapeutic strategy and an adjunct to classical DSB-inducing agents in hypoxic GBM. Citation Format: Edidiong R. Usoro, Dominique Monroe, Kristen Carver, Emily Steinbeck, Arilyn Williams, Shelby Aycox, Waaqo Daddacha. Understanding the role of dNTP pool on double-strand break and resistant to therapy in quiescent hypoxic glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3267.

Consensus of the Brazilian association of hematology, hemotherapy and cellular therapy on patient blood management: Anemia tolerance mechanisms

Understanding the physiological concepts of oxygen delivery is essential to discern the mechanisms that influence its increase, reduction or maintenance in the body. This text explores the different mechanisms that help maintain oxygen delivery even in the face of reduced hemoglobin levels. Adequate oxygen delivery ensures tissue and metabolic balance, which is crucial to avoid harmful consequences such as metabolic acidosis and cellular dysoxia. The complex interaction between variables such as cardiac output, hemoglobin and heart rate (HR) plays a fundamental role in maintaining oxygen delivery, allowing the body to temporarily adjust to situations of anemia or high metabolic demand. It is important to emphasize that blood transfusions should not be based on fixed values, but rather on individual metabolic needs. Strategies to reduce myocardial consumption and monitor macro and micro hemodynamics help in making rational decisions. Individualizing treatment and considering factors such as blood viscosity in relation to the benefits of transfusion are increasingly relevant to optimize therapy and minimize risks, especially in complex clinical scenarios, such as neurocritical patients and trauma victims.

Alterations in the gut microbiota and its metabolites contribute to metabolic maladaptation in dairy cows during the development of hyperketonemia.

Metabolic maladaptation in dairy cows after calving can lead to long-term elevation of ketones, such as β-hydroxybutyrate (BHB), representing the condition known as hyperketonemia, which greatly influences the health and production performance of cows during the lactation period. Although the gut microbiota is known to alter in dairy cows with hyperketonemia, the association of microbial metabolites with development of hyperketonemia remains unknown. In this study, we performed a multi-omics analysis to investigate the associations between fecal microbial community, fecal/plasma metabolites, and serum markers in hyperketonemic dairy cows during the transition period. Dynamic changes in the abundance of the phyla Verrucomicrobiota and Proteobacteria were detected in the gut microbiota of dairy cows, representing an adaptation to enhanced lipolysis and abnormal glucose metabolism after calving. Random forest and univariate analyses indicated that Frisingicoccus is a key bacterial genus in the gut of cows during the development of hyperketonemia, and its abundance was positively correlated with circulating branched-chain amino acid levels and the ketogenesis pathway. Taurodeoxycholic acid, belonging to the microbial metabolite, was strongly correlated with an increase in blood BHB level, and the levels of other secondary bile acid in the feces and plasma were altered in dairy cows prior to the diagnosis of hyperketonemia, which link the gut microbiota and hyperketonemia. Our results suggest that alterations in the gut microbiota and its metabolites contribute to excessive lipolysis and insulin insensitivity during the development of hyperketonemia, providing fundamental knowledge about manipulation of gut microbiome to improve metabolic adaptability in transition dairy cows.IMPORTANCEAccumulating evidence is pointing to an important association between gut microbiota-derived metabolites and metabolic disorders in humans and animals; however, this association in dairy cows from late gestation to early lactation is poorly understood. To address this gap, we integrated longitudinal gut microbial (feces) and metabolic (feces and plasma) profiles to characterize the phenotypic differences between healthy and hyperketonemic dairy cows from late gestation to early lactation. Our results demonstrate that cows underwent excessive lipid mobilization and insulin insensitivity before hyperketonemia was evident. The bile acids are functional readouts that link gut microbiota and host phenotypes in the development of hyperketonemia. Thus, this work provides new insight into the mechanisms involved in metabolic adaptation during the transition period to adjust to the high energy and metabolic demands after calving and during lactation, which can offer new strategies for livestock management involving intervention of the gut microbiome to facilitate metabolic adaptation.

Abstract B020: Understanding the role of dNTP pool on double-strand break and resistance to therapy in quiescent hypoxic glioblastoma

Abstract Glioblastoma (GBM) is the deadliest type of primary brain tumor. After surgical resection, chemotherapy, and radiotherapy (RT) are used as treatment modalities to induce double-strand breaks (DSBs), the most cytotoxic and lethal form of DNA damage. However, an inadequate oxygen supply and the high metabolic demand in rapidly growing solid tumors like GBM result in severe hypoxic regions contributing to altered cellular metabolism and resistance to these cancer therapies. These subpopulations of resistant and refractory hypoxic cancer cells are clinically significant as they lead to poor prognosis and adverse clinical outcomes in patients. Ionizing radiation (IR) and chemotherapeutics such as temozolomide induce DNA damage, such as base deletions, single-strand breaks (SSBs), and DSBs. Rapidly replicating cells are more sensitive to these therapeutic agents partly due to replication-induced conversion of SSB and other manageable damages to highly lethal DSB. A balanced deoxyribonucleoside triphosphate (dNTP) is crucial for DNA replication, repair, and maintenance of genomic stability. Cellular dNTP is mainly regulated by ribonucleotide reductase (RNR) and the SAM- and HD-domain-containing protein (SAMHD1). RNR boosts the cellular dNTP pool through the de novo synthesis while SAMHD1 depletes the pool by hydrolyzing dNTPs. Previous studies have shown that hypoxic cancer cells have low dNTP levels due to oxygen dependency of its rate limiting RNR enzyme, which could result in reduced DNA replication and cell cycle arrest. Here, we show the direct relationships between dNTP levels, DNA replication, and DSB induction in hypoxic GBM cancer cells, which is currently unknown. To bridge this knowledge gap, we exposed GBM cells to severe hypoxia and utilized cellular dNTP analysis to demonstrate that their dNTP pool can be elevated. Our result shows that elevating cellular dNTP pool relieved hypoxia-induced cell cycle arrest as determined by flow cytometry and increased their DNA replication. A single cell microscopic analysis also revealed that increasing the dNTP pool significantly enhanced the induction of DSBs following exposure to IR and bleomycin. Furthermore, we detected an increased efficacy of DSB-inducing agents in hypoxic GBM as demonstrated by apoptosis pathway activation. Our result suggest that increasing the intracellular dNTP pool in hypoxic GBM will stimulate DNA replication, increase genotoxin-induced DSB, and improve DSB-inducing therapeutics' efficacy. This study provides a preclinical justification for using cellular dNTP modulation as a novel therapeutic strategy and an adjunct to classical DSB-inducing agents in hypoxic GBM. Citation Format: Edidiong R. Usoro, Dominique Monroe, Kristen Carver, Maya Jenkins, Emily Steinbeck, Arilyn Williams, Shelby Aycox, Waaqo Daddacha. Understanding the role of dNTP pool on double-strand break and resistance to therapy in quiescent hypoxic glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B020.

Abstract B044: Elongation control of mRNA translation drives Group 3 medulloblastoma adaptation to nutrient deprivation

Abstract Background: Group 3 affiliation and MYC genetic amplification are associated with poor life expectancy and substantial morbidity in children suffering from medulloblastoma (MB). The high metabolic demand induced by MYC-driven transformation sensitizes MYC-overexpressing MB to cell death under conditions of nutrient deprivation (ND). Additionally, MYC-driven transformation is known to promote mitochondrial oxidative phosphorylation (OXPHOS). We previously reported that eukaryotic Elongation Factor Kinase 2 (eEF2K), the master regulator of mRNA translation elongation, promotes survival of MYC-overexpressing tumors under ND. Interestingly, eEF2K is overexpressed in MYC-driven MB and our preliminary proteomics data highlight large-scale alterations in OXPHOS components affecting eEF2K deficient MB cells. We therefore hypothesized that eEF2K activity is required for the selective translation of mRNAs needed for efficient OXPHOS, and for the progression of MYC-driven MB. METHODS: Multiplexed enhanced Protein Dynamic Mass Spectrometry performed in eEF2K knockdown MYC-overexpressing D425 MB cells to identify mRNAs selectively translated upon eEF2K activation. Time course experiments under ND were conducted in eEF2K knockout (KO) D425 cells to assess the presence of electron transport chain (ETC) complexes I-IV in their native state (via BN-PAGE), as well as transcript expression of individual ETC complex components (by qPCR). The effects of eEF2K inactivation on oxygen consumption, metabolic fluxes and mitochondrial membrane potential were studied with Seahorse technology and JC1/TMRE staining. The viability of eEF2K KO D425 cells was assessed by Incucyte system. Finally, MB orthotopic xenograft mouse models were used to confirm in vitro observations. RESULTS: Multiple (9 out of 10 detected) components of the mitochondrial OXPHOS pathway are selectively translated upon eEF2K activation. Inactivation of eEF2K by genetic KO leads to the disassembly of ETC complexes I-IV without affecting mRNA levels of their respective components. Consistently, eEF2K KO MB cells display decreased mitochondrial membrane potential and ~20% increased proton leak through the mitochondrial membrane. In addition, eEF2K inactivation results in increased D425 cell death under ND and doubles survival of MB bearing mice fed with calorie restricted diets (p<0.05). CONCLUSION: Control of mRNA translation elongation by eEF2K is critical for mitochondrial ETC complex assembly and efficient OXPHOS in MYC-overexpressing MB, likely representing an adaptive response by which MYC-driven MB cells cope with acute metabolic stress. Future therapeutic studies will aim to combine eEF2K inhibition with caloric restriction mimetic drugs as eEF2K activity appears critical under metabolic stress conditions. Citation Format: Alberto Delaidelli, Fares Burwag, Jessica Oliveira de Santis, Betty Yao, Haifeng Zhang, Yue Zhou Huang, Gian Luca Negri, Christopher Hughes, Gabriel Leprivier, Poul Sorensen. Elongation control of mRNA translation drives Group 3 medulloblastoma adaptation to nutrient deprivation [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B044.

Restriction of Glycolysis Increases Serial Killing Capacity of Natural Killer Cells.

Tumor cells rely heavily on glycolysis to meet their high metabolic demands. While this results in nutrient deprivation within the tumor microenvironment and has negative effects on infiltrating immune cells such as natural killer (NK) cells, it also creates a potential target for cancer therapies. Here we use Glupin, an inhibitor of glucose transporters, to study the effect of limited glucose uptake on NK cells and their anti-tumor functions. Glupin treatment effectively inhibited glucose uptake and restricted glycolysis in NK cells. However, acute treatment had no negative effect on NK cell cytotoxicity or cytokine production. Long-term restriction of glucose uptake via Glupin treatment only delayed NK cell proliferation, as they could switch to glutaminolysis as an alternative energy source. While IFN-γ production was partially impaired, long-term Glupin treatment had no negative effect on degranulation. Interestingly, the serial killing activity of NK cells was even slightly enhanced, possibly due to changes in NAD metabolism. This demonstrates that NK cell cytotoxicity is remarkably robust and insensitive to metabolic disturbances, which makes cellular metabolism an attractive target for immune-mediated tumor therapies.