Biology subjects

Mathews, T. P.

Publications and source records attributed to Mathews, T. P..

23 records · Page 2Linked to original sources

bioRxiv2022

Comprehensive isotopomer analysis of glutamate and aspartate in small tissue samples

Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance spectroscopy (NMRS) or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMRS is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring 1% of the sample used for NMRS. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase deficiency in human renal cancers, and investigate the contributions of TCA cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMRS or standard MS to provide outstanding sensitivity in isotope labeling experiments, particularly in vivo.

biochemistry↗

bioRxiv2021

De Novo Pyrimidine Synthesis is a Targetable Vulnerability in IDH Mutant Glioma

Mutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they appear less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1 mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH mutant gliomas. Mechanistically, this vulnerability selectively applies to de novo pyrimidine, but not purine, synthesis because glioma cells engage disparate programs to produce these nucleotide species and because IDH oncogenes increase DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

cancer biology↗

bioRxiv2021

G6PD deficiency sensitizes metastasizing melanoma cells to oxidative stress and glutaminolysis

The pentose phosphate pathway is a major source of NADPH for oxidative stress resistance in cancer cells but there is limited insight into its role in metastasis, when some cancer cells experience high levels of oxidative stress. To test this, we mutated the substrate binding site of Glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the first step of the pentose phosphate pathway, in patient-derived melanomas. G6PD mutant melanomas had significantly decreased G6PD enzymatic activity and depletion of intermediates in the oxidative branch of the pentose phosphate pathway. Reduced G6PD function had little effect on the formation of primary subcutaneous tumors but when these tumors spontaneously metastasized the frequency of circulating melanoma cells in the blood and metastatic disease burden were significantly reduced. G6PD mutant melanomas exhibited increased levels of reactive oxygen species (ROS), decreased NADPH levels, and depleted glutathione as compared to control melanomas. G6PD mutant melanomas compensated for this increase in oxidative stress by increasing the production of NADPH through glutaminolysis. This generated a new metabolic vulnerability as G6PD mutant melanomas were more dependent upon glutamine as compared to control melanomas. The oxidative pentose phosphate pathway and compensatory glutaminolysis thus confer layered protection against oxidative stress during metastasis. SignificanceMelanoma metastasis is limited by oxidative stress. Cells that enter the blood experience high levels of ROS and usually die of ferroptosis. We found that melanoma cells become more dependent upon the oxidative branch of the pentose phosphate pathway to manage oxidative stress during metastasis. When pentose phosphate pathway function was disabled by G6PD mutation, the melanoma cells increased their utilization of malic enzyme, fueled by increased consumption of glutamine in the tricarboxylic acid cycle. Melanoma cells thus have redundant and layered protection against oxidative stress.

cancer biology↗

bioRxiv2021

Cell-type specific requirement for pyruvate dehydrogenase in hematopoiesis and leukemia development.

Cancer cells have different metabolic requirements as compared to their corresponding normal tissues. This is thought to reflect metabolic reprogramming during transformation. An alternative possibility is that some metabolic requirements of cancer cells reflect a maintenance of the metabolism of the specific normal cell type from which cancer cells originate. Here, we investigate this hypothesis by comparing glucose use in normal hematopoiesis and in leukemia. T cell progenitors in the thymus were glucose avid and oxidized more glucose in the tricarboxylic acid (TCA) cycle through pyruvate dehydrogenase (PDH) as compared to hematopoietic stem cells (HSCs) or other hematopoietic cells. PDH deletion reduced the number of double positive (DP) T cell progenitors but had no effect on HSCs, myeloid progenitors and other hematopoietic cells we examined. PDH deletion blocked the development of T cell leukemia from Pten-deficient DP progenitors, but not the development of a myeloid neoplasm from Pten-deficient HSCs or myeloid progenitors. Therefore, the requirement of glucose oxidation for leukemia development is inherited from the normal cell of origin and occurs independently of the driver genetic lesion. PDH was not required in vivo to generate acetyl-CoA or maintain levels of TCA cycle metabolites but to prevent pyruvate accumulation and to maintain glutathione levels and redox homeostasis.

cancer biology↗

bioRxiv2020

Metabolomic profiling of rare cell populations isolated by flow cytometry from tissues

Little is known about the metabolic regulation of rare cell populations because most metabolites are hard to detect in small numbers of cells. We previously described a method for metabolomic profiling of flow cytometrically-isolated hematopoietic stem cells (HSCs) that detects approximately 60 metabolites in 10,000 cells (Agathocleous et al., 2017). Here we describe a new method involving hydrophilic liquid interaction chromatography (HILIC) and high-sensitivity orbitrap mass spectrometry that detected approximately 160 metabolites in 10,000 HSCs, including many more glycolytic and lipid intermediates. We improved chromatographic separation, increased mass resolution, minimized ion suppression, extracted with acetonitrile, and eliminated sample drying. Most metabolites did not significantly change during cell preparation and sorting. We used this method to profile HSCs and circulating melanoma cells. HSCs exhibited increased glycerophospholipid metabolites relative to unfractionated bone marrow cells and altered purine biosynthesis after methotrexate treatment in vivo. Circulating melanoma cells were depleted for purine intermediates relative to subcutaneous tumors, suggesting they decrease purine synthesis during metastasis. These methods facilitate the routine metabolomic analysis of rare cell populations from tissues. Impact statementWe developed a method for metabolomic analysis of small numbers of flow cytometrically isolated cells from rare cell populations such as hematopoietic stem cells and circulating cancer cells.

cell biology↗