Biology subjects

Lyssiotis, C. A.

Publications and source records attributed to Lyssiotis, C. A..

70 records · Page 4Linked to original sources

bioRxiv2020

Hyaluronic Acid Fuels Pancreatic Cancer Growth

Rewired metabolism is a hallmark of pancreatic ductal adenocarcinomas (PDA). Previously, we demonstrated that PDA cells enhance glycosylation precursor biogenesis through the hexosamine biosynthetic pathway (HBP) via activation of the rate limiting enzyme, glutamine-fructose 6-phosphate amidotransferase 1 (GFAT1). Here, we genetically ablated GFAT1 in PDA cell lines, which completely blocked proliferation in vitro and led to cell death. In contrast, GFAT1 knockout did not impair tumor growth, suggesting that cancer cells can maintain fidelity of glycosylation precursor pools by scavenging nutrients from the tumor microenvironment. Here, we show that hyaluronic acid (HA), an abundant carbohydrate polymer in pancreatic tumors composed of repeating N-acetyl-glucosamine (GlcNAc) and glucuronic acid sugars, can bypass GFAT1 to refuel the HBP via the GlcNAc salvage pathway. Furthermore, HA facilitates proliferation in nutrient-starved wild-type PDA. Together, these data show HA can serve as a nutrient fueling PDA metabolism beyond its previously appreciated structural and signaling roles.

cancer biology↗

bioRxiv2020

The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieves a durable remission. SIRT5 is a member of the sirtuin family of protein deacylases that regulate metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we show that SIRT5 is required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 is required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf;Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we find that SIRT5 is required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably include MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a novel, druggable genotype-independent addiction in melanoma.

cancer biology↗

bioRxiv2020

The Pancreatic Tumor Microenvironment Buffers Redox Imbalance Imposed by Disrupted Mitochondrial Metabolism

The tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDA) restricts vascularization and, consequently, access to blood-derived nutrients and oxygen, which impacts tumor growth. Intracellular redox imbalance is another restraint on cellular proliferation, yet it is unknown if the TME contributes to the maintenance of redox homeostasis in PDA cells. Here, we demonstrate that the loss of mitochondrial glutamate-oxaloacetate transaminase 2 (GOT2), a component in the malate-aspartate shuttle, disturbs redox homeostasis and halts proliferation of PDA cells in vitro. In contrast, GOT2 inhibition has no effect on in vivo tumor growth or tumorigenesis in an autochthonous model. We propose that this discrepancy is explained by heterocellular pyruvate exchange from the TME, including from cancer associated fibroblasts. More broadly, pyruvate similarly confers resistance to inhibitors of mitochondrial respiration. Genetic or pharmacologic inhibition of pyruvate uptake or metabolism abrogated pyruvate-mediated alleviation of reductive stress from NADH buildup. In sum, this work describes a potential resistance mechanism mediated by metabolic crosstalk within the pancreatic TME. These findings have important implications for metabolic treatment strategies since several mitochondrial inhibitors are currently in clinical trials for PDA and other cancers.

cancer biology↗

bioRxiv2020

Clonal Heterogeneity Supports Mitochondrial Metabolism in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDA) is characterized by a heterogenous and densely fibrotic microenvironment. This limits functional vasculature and diffusion of nutrients through the tumor1,2. Accordingly, pancreatic cancer cells develop numerous metabolic adaptations to survive and proliferate in nutrient austere conditions3-7. Subtypes of PDA have been characterized by transcriptional and functional differences8-12, which have been reported to exist within the same tumor13-15. However, it remains unclear if this diversity extends to metabolic programming. Here, using a combination of metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses within neoplastic populations isolated from a single pancreatic tumor. Furthermore, these populations are poised for metabolic crosstalk, and in examining this, we find an unexpected role for asparagine in maintaining cell proliferation following mitochondrial inhibition. Functionally, when challenged by mitochondrial inhibition, asparagine supplementation increases intracellular levels of asparagine and aspartate, a rate limiting biosynthetic precursor16-18. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes pancreatic tumors to mitochondrial targeting with phenformin. Together, these data extend the concept of metabolic diversity to neoplastic populations within individual tumors, while illustrating a new method of intratumoral communication that supports tumor fitness19,20. Finally, the combination of asparaginase with mitochondrial inhibition could provide a powerful new strategy for this difficult to treat disease.

cancer biology↗

bioRxiv2020

Purine metabolism regulates DNA repair and therapy resistance in glioblastoma

Intratumoral genomic heterogeneity in glioblastoma (GBM) is a barrier to overcoming therapy resistance, and new strategies that are effective independent of genotype are urgently needed. By correlating intracellular metabolite levels with radiation resistance across dozens of genomically-distinct models of GBM, we found that purine metabolites strongly correlated with radiation resistance. Inhibiting purine, but not pyrimidine, synthesis radiosensitized GBM cells and patient-derived neurospheres by impairing DNA repair in a nucleoside-dependent fashion. Likewise, administration of exogenous purine nucleosides protected sensitive GBM models from radiation by promoting DNA repair. Combining an FDA-approved inhibitor of de novo purine synthesis with radiation arrested growth in GBM xenograft models and depleted intratumoral guanylates. High expression of the rate-limiting enzyme of de novo GTP synthesis was associated with shorter survival in GBM patients. Together, these findings indicate that inhibiting de novo purine synthesis may be a promising strategy to overcome therapy resistance in this genomically heterogeneous disease.

neuroscience↗

bioRxiv2020

Asparagine signals mitochondrial respiration and can be targeted to impair tumour growth

Mitochondrial respiration is critical for cell proliferation. In addition to producing ATP via the electron transport chain (ETC), respiration is required for the generation of TCA cycle-derived biosynthetic precursors, such as aspartate, an essential substrate for nucleotide synthesis. Because mTORC1 coordinates availability of biosynthetic precursors with anabolic metabolism, including nucleotide synthesis, a link between respiration and mTORC1 is fitting. Here we show that in addition to depleting intracellular aspartate, ETC inhibition depletes aspartate-derived asparagine and impairs mTORC1 activity. Providing exogenous asparagine restores mTORC1 activity, nucleotide synthesis, and proliferation in the context of ETC inhibition without restoring intracellular aspartate in a panel of cancer cell lines. As a therapeutic strategy, the combination of ETC inhibitor metformin, which limits tumour asparagine synthesis, and either asparaginase or dietary asparagine restriction, which limit tumour asparagine consumption, effectively impairs tumour growth in several mouse models of cancer. Because environmental asparagine is sufficient to restore proliferation with respiration impairment, both in vitro and in vivo, our findings suggest that asparagine synthesis is a fundamental purpose of mitochondrial respiration. Moreover, the results suggest that asparagine signals active respiration to mTORC1 to communicate biosynthetic precursor sufficiency and promote anabolism.

cancer biology↗

bioRxiv2020

Mitochondrial Complex II In Intestinal Epithelial Cells Regulates T-cell Mediated Immunopathology

Intestinal epithelial cell (IEC) damage by T cells contributes to alloimmune, autoimmune and iatrogenic diseases such as graft-versus-host disease (GVHD), inflammatory bowel disease (IBD) and immune checkpoint blockade (ICB) mediated colitis, respectively. Despite significant advances in understanding the aberrant biology of T cells in these diseases, little is known about how the fundamental biological processes of the target IECs influence the disease severity. Here, through analyses of metabolic pathways of IECs, we identified disruption of oxidative phosphorylation without a concomitant change in glycolysis and an increase in succinate levels in several distinct in vivo models of T cell mediated gastrointestinal damage such as GVHD, IBD and ICB mediated colitis. Metabolic flux studies, complemented by imaging and protein analyses identified a critical role for IEC intrinsic succinate dehydrogenase A (SDHA), a component of mitochondrial complex II, in causing these metabolic alterations that contributed to the severity of intestinal damage. The critical mechanistic role of IEC intrinsic SDHA was confirmed by complementary chemical and genetic reduction of SDHA and with IEC specific deletion of SDHA. Further in vitro and in vivo mechanistic studies demonstrated that loss of SDHA in IECs was mediated by the perforin-granzyme from the T cells. The loss of SDHA was also validated in human clinical samples. These data identify a critical role for the alteration of the IEC specific mitochondrial complex II component SDHA in the regulation of the severity of T cell mediated intestinal diseases.

immunology↗

bioRxiv2020

GOT1 Inhibition Primes Pancreatic Cancer for Ferroptosis through the Autophagic Release of Labile Iron

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest solid malignancies, with a 5-year survival rate at ten percent. PDA have unique metabolic adaptations in response to cell-intrinsic and environmental stressors, and identifying new strategies to target these adaptions is an area of active research. We previously described a dependency on a cytosolic aspartate aminotransaminase (GOT1)-dependent pathway for NADPH generation. Here, we sought to identify metabolic dependencies induced by GOT1 inhibition that could be exploited to selectively kill PDA. Using pharmacological methods, we identified cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. Targeting any of these pathways was synthetic lethal in GOT1 knockdown cells and triggered ferroptosis, an oxidative, non-apoptotic, iron-dependent form of cell death. Mechanistically, GOT1 inhibition promoted the activation of autophagy in response to metabolic stress. This enhanced the availability of labile iron through ferritinophagy, the autolysosome-mediated degradation of ferritin. In sum, our study identifies a novel biochemical connection between GOT1, iron regulation, and ferroptosis, and suggests the rewired malate-aspartate shuttle plays a role in protecting PDA from severe oxidative challenge. HighlightsO_LIPDA exhibit varying dependence on GOT1 for in vitro and in vivo growth. C_LIO_LIExogenous cystine, glutathione synthesis, and lipid antioxidant fidelity are essential under GOT1 suppression. C_LIO_LIGOT1 inhibition sensitizes pancreatic cancer cell lines to ferroptosis. C_LIO_LIGOT1 inhibition represses anabolic metabolism and promotes the release of iron through autophagy. C_LI

cancer biology↗

bioRxiv2019

Induction of pancreatic tumor-selective ferroptosis through modulation of cystine import

Pancreatic ductal adenocarcinoma (PDA) is the third-leading cause of cancer mortality in the US and is highly resistant to classical, targeted, and immune therapies. We show that human PDA cells are dependent on the provision of exogenous cystine to avert a catastrophic accumulation of lipid reactive oxygen species (ROS) that, left unchecked, leads to ferroptotic cell death, both in vitro and in vivo. Using a dual-recombinase genetically engineered model, we found that acute deletion of Slc7a11 led to tumor-selective ferroptosis, tumor stabilizations/regressions, and extended overall survival. The mechanism of ferroptosis induction in PDA cells required the concerted depletion of both glutathione and coenzyme A, highlighting a novel branch of ferroptosis-relevant metabolism. Finally, we found that cystine depletion in vivo using the pre-IND agent cyst(e)inase phenocopied Slc7a11 deletion, inducing tumor-selective ferroptosis and disease stabilizations/regressions in the well-validated KPC model of PDA.\n\nOne Sentence SummaryGenetic and pharmacological targeting of cystine import induces pancreatic cancer-selective ferroptosis in vivo.

cancer biology↗

bioRxiv2019

Hypoxia inducible factor-2α increases sensitivity of colon cancer cells towards oxidative cell death

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US. Hypoxia is a hallmark of solid tumors which promotes tumor cell growth, survival, metastasis and confers resistance to chemo and radiotherapies. Targeting hypoxic cells has been difficult. Moreover, inhibitors for the major transcription factors, hypoxia inducible factor (HIF)-1 and HIF-2 have not shown long-term efficacy in most cancers. We have previously shown that HIF-2 is essential for colon tumorigenesis. Using an unbiased screen, we show a significant increase in synthetic lethality of HIF-2 overexpressing tumor enteroids to oxidative cell death activators. The treatment with hypoxia mimetic FG4592 (Roxadustat), led to a robust increase in erastin-, RSL3-, and dimethyl fumarate-induced cell death in a dose- and time-dependent manner. Further, our in-vitro data shows that HIF-2 knock-down cells are completely resistant to these drugs. HIF activation promotes upregulation of lipid synthesis genes in vitro and in vivo leading to oxidative stress. Taken together, our results suggest that this intrinsic sensitivity towards oxidative stress associated with hypoxia could be utilized as a persistent and dynamic form of cell death for colon cancer treatment.

cancer biology↗

bioRxiv2019

Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2, provides photoreceptor neuroprotection

Photoreceptor cell death is the ultimate cause of vision loss in many retinal disorders, and there is an unmet need for neuroprotective modalities to improve photoreceptor survival. Similar to cancer cells, photoreceptors maintain pyruvate kinase muscle isoform 2 (PKM2) expression, which is a critical regulator in aerobic glycolysis. Unlike PKM1, which has constitutively high catalytic activity, PKM2 is under complex regulation. Recently, we demonstrated that genetically reprogramming photoreceptor metabolism via PKM2-to-PKM1 substitution is a promising neuroprotective strategy. Here, we explored the neuroprotective effects of pharmacologically activating PKM2 via ML-265, a small molecule activator of PKM2, during acute outer retinal stress. We found that ML-265 increased PKM2 activity in 661W cells and in vivo in rat eyes without affecting the expression of genes involved in glucose metabolism. ML-265 treatment did, however, alter metabolic intermediates of glucose metabolism and those necessary for biosynthesis in cultured cells. Long-term exposure to ML-265 did not result in decreased photoreceptor function or survival under baseline conditions. Notably, though, ML-265-treatment did reduce entrance into the apoptotic cascade in in vitro and in vivo models of outer retinal stress. These data suggest that reprogramming metabolism via activation of PKM2 is a novel, and promising, therapeutic strategy for photoreceptor neuroprotection.

molecular biology↗

bioRxiv2019

Integration of high-content fluorescence imaging into the metabolic flux assay reveals insights into mitochondrial properties and functions

Metabolic flux technology with the Seahorse bioanalyzer has emerged as a standard technique in cellular metabolism studies, allowing for simultaneous kinetic measurements of respiration and glycolysis. Methods to extend the utility and versatility of the metabolic flux assay would undoubtedly have immediate and wide-reaching impacts. Herein, we describe a platform that couples the metabolic flux assay with high-content fluorescence imaging to simultaneously enhance normalization of respiration data with cell number; analyze cell cycle progression; quantify mitochondrial content, fragmentation state, membrane potential, and mitochondrial reactive oxygen species. Integration of fluorescent dyes directly into the metabolic flux assay generates a more complete data set of mitochondrial features in a single assay. Moreover, application of this integrated strategy revealed insights into mitochondrial function following PGC1a and PRC1 inhibition in pancreatic cancer and demonstrated how the Rho-GTPases impact mitochondrial dynamics in breast cancer.

molecular biology↗

bioRxiv2019

Tissue of Origin Dictates GOT1 Dependence and Confers Synthetic Lethality to Radiotherapy

Metabolic programs in cancer cells are influenced by genotype and the tissue of origin. We have previously shown that central carbon metabolism is rewired in pancreatic ductal adenocarcinoma (PDA) to support proliferation through a glutamate oxaloacetate transaminase 1 (GOT1)-dependent pathway. Here we tested if tissue type impacted GOT1 dependence by comparing PDA and colorectal cancer (CRC) cell lines and tumor models of similar genotype. We found CRC to be insensitive to GOT1 inhibition, contrasting markedly with PDA, which exhibit profound growth inhibition upon GOT1 knockdown. Utilizing a combination of metabolomics strategies and computational modeling, we found that GOT1 inhibition disrupted glycolysis, nucleotide metabolism, and redox homeostasis in PDA but not CRC. These insights were leveraged in PDA, where we demonstrate that radiotherapy potently enhanced the effect of GOT1 inhibition on tumor growth. Taken together, these results illustrate the role of tissue type in dictating metabolic dependencies and provide new insights for targeting metabolism to treat PDA.

cancer biology↗

bioRxiv2019

Meta-analysis of targeted metabolomics data from heterogeneous biological samples provides insights into metabolite dynamics

IntroductionMass spectrometry-based metabolomics coupled to liquid chromatography, or LC-MS metabolomics, has become the most popular tool for global metabolite abundance profiling to study metabolism. However, the physicochemical complexity of metabolites poses a major challenge for reliable measurements of metabolite abundance. One way to address the issue is to use multiple chromatographic methods to capture a greater range of molecular diversity. We previously developed a tandem mass spectrometry-based label-free targeted metabolomics analysis framework coupled to two distinct chromatographic methods, reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC), with dynamic multiple reaction monitoring (dMRM) for simultaneous detection of over 200 metabolites to study core metabolic pathways. ObjectivesWe aim to analyze a large-scale heterogeneous data compendium generated from our LC-MS/MS platform with both RPLC and HILIC methods to systematically assess measurement quality in biological replicate groups and to gain insights into metabolite dynamics across different biological conditions. MethodsOur metabolomics framework was applied in a wide range of experimental systems including cancer cell lines, tumors, extracellular media, primary cells, immune cells, organoids, organs (e.g. pancreata), tissues, and sera from human and mice. We also developed computational and statistical analysis pipelines, which include hierarchical clustering, replicate-group CV analysis, correlation analysis, and case-control paired analysis. ResultsWe generated a compendium of 42 heterogeneous deidentified datasets with 635 samples using both RPLC and HILIC methods. There exist signature metabolites that correspond to heterogeneous phenotypes, involved in several metabolic pathways. The RPLC method shows overall better reproducibility than the HILIC method for most metabolites including polar amino acids. Correlation analysis reveals high confidence metabolites irrespective of experimental systems such as methionine, phenylalanine, and taurine. We also identify homocystine, reduced glutathione, and phosphoenolpyruvic acid as highly dynamic metabolites across all case-control paired samples. ConclusionsOur study is expected to serve as a resource and a reference point for a systematic analysis of label-free LC-MS/MS targeted metabolomics data in both RPLC and HILIC methods with dMRM.

bioinformatics↗

bioRxiv2018

Macrophage Released Pyrimidines Inhibit Gemcitabine Therapy in Pancreatic Cancer

Pancreatic Ductal Adenocarcinoma (PDA) is characterized by abundant infiltration of tumor associated macrophages (TAMs). TAMs have been reported to drive resistance to gemcitabine, the front-line chemotherapy in PDA, though the mechanism of this resistance remains unclear. Profiling metabolite exchange, we demonstrate macrophages programmed by PDA cells release a spectrum of pyrimidine species. These include deoxycytidine, which inhibits gemcitabine through molecular competition at the level of drug uptake and metabolism. Accordingly, genetic or pharmacological depletion of TAMs in murine models of PDA sensitizes these tumors to gemcitabine. Consistent with this, patients with low macrophage burden demonstrate superior response to gemcitabine treatment. Additionally, we report pyrimidine release is a general function of anti-inflammatory myeloid cells, suggesting an unknown physiological role of pyrimidine exchange by immune cells.

cancer biology↗

bioRxiv2018

IDH1R132H acts as a tumor suppressor in glioma via epigenetic upregulation of the DNA damage response

One sentence summaryMutant IDH1 acts as a tumor suppressor when co-expressed together with TP53 and ATRX inactivating mutations in glioma, inducing genomic stability, DNA repair and resistance to genotoxic therapies.\n\nAbstractGlioma patients whose tumors carry a mutation in the Isocitrate Dehydrogenase 1 (IDH1R132H) gene are younger at the time of diagnosis and survive longer. The molecular glioma subtype which we modelled, harbors IDH1R132H, tumor protein 53 (TP53) and alpha-thalassemia/mental retardation syndrome X-linked (ATRX) loss. The impact of IDH1R132H on genomic stability, DNA damage response (DDR) and DNA repair in this molecular glioma subtype is unknown. We discovered that IDH1R132H expression in the genetic context of ATRX and TP53 inactivation: (i) increases median survival (MS), (ii) enhances DDR activity via epigenetic upregulation of Ataxia-telangiectasia mutated (ATM) signaling, and (iii) elicits tumor radioresistance. Pharmacological inhibition of ATM or checkpoint kinase 1 and 2 (CHK1/2), two essential kinases in the DDR pathways, restored tumors radiosensitivity. Translation of these findings for mlDH1 glioma patients could significantly improve the therapeutic efficacy of radiotherapy, and thus have a major impact on patient survival.

cancer biology↗