Faculty Bibliography

Oxygen is critical for a multitude of metabolic processes that are essential for human life. Biological processes can be identified by treating cells with ¹⁸O₂ or other isotopically labelled gases and systematically identifying biomolecules incorporating labeled atoms. Here we labelled cell lines of distinct tissue origins with ¹⁸O₂ to identify the polar oxy-metabolome, defined as polar metabolites labelled with ¹⁸O under different physiological O₂ tensions. The most highly ¹⁸O-labelled feature was 4-hydroxymandelate (4-HMA). We demonstrate that 4-HMA is produced by hydroxyphenylpyruvate dioxygenase-like (HPDL), a protein of previously unknown function in human cells. We identify 4-HMA as an intermediate involved in the biosynthesis of the coenzyme Q10 (CoQ10) headgroup in human cells. The connection of HPDL to CoQ10 biosynthesis provides crucial insights into the mechanisms underlying recently described neurological diseases related to HPDL deficiencies^1-4 and cancers with HPDL overexpression⁵.

Pancreatic ductal adenocarcinoma (PDAC) tumors have a nutrient-poor, desmoplastic, and highly innervated tumor microenvironment. Although neurons can release stimulatory factors to accelerate PDAC tumorigenesis, the metabolic contribution of peripheral axons has not been explored. We found that peripheral axons release serine (Ser) to support the growth of exogenous Ser (exSer)-dependent PDAC cells during Ser/Gly (glycine) deprivation. Ser deprivation resulted in ribosomal stalling on two of the six Ser codons, TCC and TCT, and allowed the selective translation and secretion of nerve growth factor (NGF) by PDAC cells to promote tumor innervation. Consistent with this, exSer-dependent PDAC tumors grew slower and displayed enhanced innervation in mice on a Ser/Gly-free diet. Blockade of compensatory neuronal innervation using LOXO-101, a Trk-NGF inhibitor, further decreased PDAC tumor growth. Our data indicate that axonal-cancer metabolic crosstalk is a critical adaptation to support PDAC growth in nutrient poor environments.

Neurons communicate by the activity-dependent release of small-molecule neurotransmitters packaged into synaptic vesicles (SVs). Although many molecules have been identified as neurotransmitters, technical limitations have precluded a full metabolomic analysis of synaptic vesicle content. Here, we present a workflow to rapidly isolate SVs and to interrogate their metabolic contents at high-resolution using mass spectrometry. We validated the enrichment of glutamate in SVs of primary cortical neurons using targeted polar metabolomics. Unbiased and extensive global profiling of SVs isolated from these neurons revealed that the only detectable polar metabolites they contain are the established neurotransmitters glutamate and GABA. In addition, we adapted the approach to enable quick capture of SVs directly from brain tissue and determined the neurotransmitter profiles of diverse brain regions in a cell-type specific manner. The speed, robustness, and precision of this method to interrogate SV contents will facilitate novel insights into the chemical basis of neurotransmission.

A hallmark of metastasis is the adaptation of tumor cells to new environments. Metabolic constraints imposed by the serine and glycine-limited brain environment restrict metastatic tumor growth. How brain metastases overcome these growth-prohibitive conditions is poorly understood. Here, we demonstrate that 3-phosphoglycerate dehydrogenase (PHGDH), which catalyzes the rate-limiting step of glucose-derived serine synthesis, is a major determinant of brain metastasis in multiple human cancer types and preclinical models. Enhanced serine synthesis proved important for nucleotide production and cell proliferation in highly aggressive brain metastatic cells. In vivo, genetic suppression and pharmacological inhibition of PHGDH attenuated brain metastasis, but not extracranial tumor growth, and improved overall survival in mice. These results reveal that extracellular amino acid availability determines serine synthesis pathway dependence, and suggests that PHGDH inhibitors may be useful in the treatment of brain metastasis.

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

OBJECTIVE:WWOX, a well-established tumor suppressor, is frequently lost in cancer and plays important roles in DNA damage response and cellular metabolism. METHODS:We re-analyzed several genome-wide association studies (GWAS) using the Type 2 Diabetes Knowledge Portal website to uncover WWOX's association with metabolic syndrome (MetS). Using several engineered mouse models, we studied the effect of somatic WWOX loss on glucose homeostasis. RESULTS:mice display reduced amounts of slow-twitch fibers, decreased mitochondrial quantity and activity, and lower glucose oxidation levels. Mechanistically, we found that WWOX physically interacts with the cellular energy sensor AMP-activated protein kinase (AMPK) and that its loss is associated with impaired activation of AMPK, and with significant accumulation of the hypoxia inducible factor 1 alpha (HIF1α) in SKM. CONCLUSIONS:Our studies uncover an unforeseen role of the tumor suppressor WWOX in whole-body glucose homeostasis and highlight the intimate relationship between cancer progression and metabolic disorders, particularly obesity and type-2 diabetes. SUBJECT AREAS/UNASSIGNED:Genetics, Metabolic Syndrome, Diabetes.

One-carbon metabolism generates the one-carbon units required to synthesize many critical metabolites, including nucleotides. The pathway has cytosolic and mitochondrial branches, and a key step is the entry, through an unknown mechanism, of serine into mitochondria, where it is converted into glycine and formate. In a CRISPR-based genetic screen in human cells for genes of the mitochondrial pathway, we found sideroflexin 1 (SFXN1), a multipass inner mitochondrial membrane protein of unclear function. Like cells missing mitochondrial components of one-carbon metabolism, those null for SFXN1 are defective in glycine and purine synthesis. Cells lacking SFXN1 and one of its four homologs, SFXN3, have more severe defects, including being auxotrophic for glycine. Purified SFXN1 transports serine in vitro. Thus, SFXN1 functions as a mitochondrial serine transporter in one-carbon metabolism.

Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016).

Background: Antimetabolites (e.g. methotrexate and gemcitabine) are not frequently used in the treatment of most solid tumors, but are effective in the treatment of UBC. Rapid cancer cell proliferation relies on an abundance of serine-derived one carbon units to support macromolecule synthesis. Specifically, PHGDH, which encodes a key enzyme of de novo serine synthesis, is amplified in breast cancer and in melanoma, and small molecule inhibitors of enzymes in this pathway are in early clinical development. However, the enzymes of serine and one carbon metabolism have not been widely investigated in UBC.
Method(s): We conducted an observational analysis of The Cancer Genome Atlas UBC cohort, focusing on gene expression data from a targeted panel indicated by Yang, et al. to be involved in serine and one carbon metabolism. Univariate Cox proportional hazard models were utilized to identify genes impacting OS and RFS, and a subsequent multivariate model was employed to control for inter-gene associations.
Result(s): Expression data from 14 genes were analyzed among 436 UBC patients, of whom complete data were available for 422. At a median follow-up of 17 months, 188 of 422 patients had died. On univariate analysis, 7 of 14 genes were significantly associated with OS: PHGDH, PSPH, MTHFD1, MTHFD2, MTHFD1L, MTHFD2L, and ALDH1L2 (all P < 0.05). Interestingly, overexpression was associated with worse OS for all but one gene, MTHFD2L (HR 0.74), which is known to be underexpressed by cancer cells in favor of MTHFD2 (HR 1.21). In multivariate analysis, overexpression of PHGDH (HR 1.19, P = 0.008), MTHFD1 (HR 1.33, P = 0.041), and ALDH1L2 (HR 1.21, P < 0.001) were independent predictors of poor survival. RFS analysis was limited by missing data; nevertheless, univariate analyses found MTHFD1, MTHFD2, MTHFD1L, MTHFD2L, and ALDH1L2 to be associated with RFS (all P < 0.05).
Conclusion(s): Within the limits of this observational study, these data suggest that serine and one carbon metabolism is important in the progression and prognosis of muscle-invasive bladder cancer. Subsequent in vitro analyses are needed to validate the prognostic and therapeutic significance of these findings