Bright Untargeted Metabolomics Approaches and Flux Analysis to Decipher Cellular Metabolism

Deciphering the Role of Second Messenger in Mycobacterial Drug Tolerance Using Extracellular Flux Analysis and 13C Stable-isotope Tracing

Antimicrobial tolerance is the gateway to the development of antimicrobial resistance (AMR) and is therefore a major issue that needs to be addressed. Bioenergetics and metabolism are intimately connected and can be read out of bacterial physiology. Indeed, Seahorse bioenergetics combined to flux analysis care unevaluable tools to decipher the mechanism by which mycobacteria become tolerant and resistant to antibiotics. The second messenger 3’,5’-cyclic adenosine monophosphate (cAMP), which is conserved across all taxa, is involved in propagating signals from environmental stimuli and converting these signals into a response. However, cAMP signalling in mycobacteria is particularly complex, making the investigation of cAMP signalling and its involvement in antimicrobial tolerance difficult.

To address this pressing need, we identified a new cyclic nucleotides degrading phosphodiesterase enzyme (Rv1339) and used it as a tool to significantly decrease cAMP levels in mycobacteria. This analysis revealed that in Mycobacterium smegmatis mc2155, the expression of Rv1339 reduced cAMP levels, altered genes expression leading to impaired bioenergetics, increased peptidoglycan turnover and led to decreased tolerance to antimicrobials that target cell wall synthesis. By combining Seahorse bioenergetics and LC/MS based 13C stable isotope tracing, this work represents an important milestone by showing that targeting nucleotide signalling is a promising new avenue for antimicrobial development and expands our understanding of nucleotide signalling in mycobacteria.

Presenter: Gerald Larrouy-Maumus, PhD (Senior Lecturer, Imperial College of London)

Gerald Larrouy-Maumus did a Ph.D, in Toulouse (France), to determine the biogenesis of the mycobacterial cell wall, especially the identification of the glycosyltransferases potentially involved in the biosynthesis of lipopolysaccharides which constitute the Achilles’ hill of the mycobacterial cell-envelope. Then, after a Post-doc in National Institute for Medical Research in London, UK, focusing on annotation of orphan enzymes using metabolomics approaches, he was recruited at Imperial College London in 2014 as lecturer. Now, as a senior lecturer in Molecular Microbiology at the MRC-CMBI, Imperial College, his laboratory explores deciphering the environmental adaptation of Mycobacterium tuberculosis within the host. Mainly Metabolomics, and Transcriptomics and Lipidomics are used as tools for the read-out of the first steps in this adaptation. The findings will potentially lead to the discovery of new drug targets and have a better understanding on resistant bacteria in context of the host.

Isocitrate dehydrogenase 1 Sustains a Hybrid Cytoplasmic–mitochondrial Tricarboxylic Acid Cycle in Prostate Cancer

The androgen receptor (AR) is an established orchestrator of cell metabolism in prostate cancer (PCa), notably by inducing an oxidative mitochondrial program. Intriguingly, AR also specifically regulates cytoplasmic isocitrate dehydrogenase 1 (IDH1), but not its mitochondrial counterparts, IDH2 and IDH3. Here, we aimed at understanding the functional role of IDH1 in PCa. IDH1 was found to be responsible of >90% of the total IDH activity in prostate and PCa models and its expression to be increased in tumors. Pharmacological and genetic inhibition of IDH1 impaired mitochondrial respiration, as assessed using real time cellular bioenergetics (Seahorse), suggesting that this cytoplasmic enzyme contributes to the mitochondrial tricarboxylic acid (TCA) cycle in PCa. Furthermore, mass spectrometry (MS)-based metabolomics analyses confirmed this hypothesis, showing that inhibition of IDH1 impairs carbon flux into the TCA cycle. Consequently, inhibition of IDH1 decreased PCa proliferation. Taken together, these results demonstrate that PCa cells exhibit a hybrid cytoplasmic-mitochondrial TCA cycle that depends on IDH1. This metabolic enzyme thus represents a metabolic vulnerability of PCa cells and a potential new therapeutic target.

Presenter: Étienne Audet-Walsh, PhD (Assistant Professor - Department of Molecular Medicine Université Laval)

Dr. Étienne Audet-Walsh is an Assistant Professor at the Department of Molecular Medicine at Université Laval since 2017. He completed his Ph.D. in 2012 in molecular endocrinology and performed his postdoctoral fellowship at McGill University, where he studied the regulation of cell metabolism by nuclear receptors. Since his establishment as an independent investigator at Université Laval, he works on the regulation of metabolism by members of the nuclear receptor NR3 subfamily, which include the androgen and the estrogen receptors.He uses metabolomic toolsin combination with functional genomic approaches such as ChIP-seq and RNA-seq to demonstrate that hormone receptors are major orchestrators of cell metabolism.His research program aims at understanding the metabolic functions of sex-steroid hormone receptors in a cell-specific manner by combining molecular endocrinology, functional genomics, and metabolomics.

Metabolic Vulnerabilities in Breast Cancer Progression

The main focus of this talk will be on metabolic adaptations fueling cancer progression. During the last decade, our team has contributed to understanding the role of the metabolic regulators PGC-1s in cancer, with a particular focus on poor outcome breast cancers. We showed that PGC-1alpha plays a key role in setting the metabolic state of poor outcome breast cancers, and that it promotes breast cancer growth and metastasis. Currently, we are pursuing research projects on metabolic adaptations promoting therapeutic resistance. In this seminar, we will highlight how combining mass spectrometry metabolomics and real-time cell bioenergetics uncovered metabolic vulnerabilities of breast cancers during disease progression and how we can use this knowledge to design potential combinatorial therapeutic interventions

Presenter: Julie St. Pierre, PhD (Professor, Director - Metabolomics Core Facility University of Ottawa)

The central research focus of the St-Pierre laboratory is the understanding of metabolic adaptations in cancer. Dr. St-Pierre received her PhD at the University of Cambridge and was Postdoctoral Fellow at the University of Cambridge, Harvard Medical School, and University of Montréal. From 2008-2017, she was Assistant and Associate Professor at McGill University as well as co-director of the Goodman Cancer Research Centre metabolomics core facility. Currently, she is a Full Professor and the University of Ottawa and Director of the metabolomics core facility. She holds a CRC tier 1 in Cancer Metabolism and she is co-leading a Terry Fox Program Project Grant on oncometabolism.