Reversal of mitochondrial malate dehydrogenase 2 enables anaplerosis via redox rescue in respiration-deficient cells

Inhibition of the electron transport chain (ETC) prevents the regeneration of mitochondrial NAD+, resulting in cessation of the oxidative tricarboxylic acid (TCA) cycle and a consequent dependence upon reductive carboxylation for aspartate synthesis. NAD+ regeneration alone in the cytosol can rescue the viability of ETC-deficient cells. Yet, how this occurs and whether transfer of oxidative equivalents to the mitochondrion is required remain unknown. Here, we show that inhibition of the ETC drives reversal of the mitochondrial aspartate transaminase (GOT2) as well as malate and succinate dehydrogenases (MDH2 and SDH) to transfer oxidative NAD+ equivalents into the mitochondrion. This supports the NAD+-dependent activity of the mitochondrial glutamate dehydrogenase (GDH) and thereby enables anaplerosis—the entry of glutamine-derived carbon into the TCA cycle and connected biosynthetic pathways. Thus, under impaired ETC function, the cytosolic redox state is communicated into the mitochondrion and acts as a rheostat to support GDH activity and cell viability.P.A.-M was supported by a Marie Skłodowska-Curie Actions individual fellowship and the Beug Foundation. A.V. was supported by Fonds Wetenschappelijk Onderzoek (FWO Vlaanderen). J.E.-H. was supported by an MRC studentship. J.C.A was supported by a Cancer Research UK Career Development Fellowship (C47559/A16243). S.-M.F. acknowledges funding from the European Research Council under the ERC Consolidator grant agreement no. 771486–MetaRegulation, FWO Projects, Fonds Baillet Latour, KU Leuven-FTBO/Internal Funding, Stichting Tegen Kanker and the King Baudouin Foundation. Work in the A.J.F. group was supported by a Wellcome Trust-ISSF grant, funding from Barts Charity (MGU0404), and by a Cancer Research UK Centre Grant to Barts Cancer Institute (C355/A25137). The illustrations in the graphical abstract and Figure 5F were created using BioRender.com

A roadmap for patient-public involvement and engagement (PPIE) : recounting the untold stories of breast cancer patient experiences

Introduction Breast cancer remains a prevalent disease in women worldwide. Though significant advancements in the standard of care for breast cancer have contributed to improved patient survival and quality of life, a breast cancer diagnosis and subsequent treatment interventions have a long-lasting impact on patients’ lived experiences. A high-quality healthcare system uses a patient-centred approach to healthcare, with patient engagement being a central pillar in the delivery of patient-centred care. However, the disconnect between patients and researchers can translate into research lacking real-world relevance to patient health needs. Here, we report a patient and stakeholder engagement workshop series that was conceptualized with the goal of promoting dialogue between patients with breast cancer, breast cancer researchers and the clinician involved in their care. We present the collaborative learning process and emerging opportunities from this patient engagement workshop series as a community-academic partnership. Method We report on a three-part storytelling workshop, with the scope of the workshops including topics related to raising awareness of the patient lived experience following a breast cancer diagnosis, breast cancer research activities undertaken by researchers, and the approach used by multidisciplinary healthcare teams in the management of breast cancer using storytelling as a tool. We used an iterative approach to cohort trust and relationship building, narrative development, and the use of multiple media formats to capture patient stories. This included the use of object memories, storytelling prompt cards and open-mic audio format to capture patient stories from diagnosis to treatment, and remission. Results 20 patients shared their stories with key themes emerging from the qualitative analysis of audio recordings. For many, this was the first time they had spoken about their breast cancer experience beyond family and friends. Emerging themes included common public misconceptions about a breast cancer diagnosis, the importance of self-advocacy in patient decision making about treatment, and the complex emotional journey experienced by patients diagnosed with breast cancer. The group-based storytelling approach provided collective empowerment to share personal experiences and connect meaningfully across the peer community. Conclusion While a breast cancer diagnosis can be overwhelming from a physical, social, emotional and cognitive perspective, storytelling as a patient engagement approach can build patient trust in researchers, ensuring that as key stakeholders they are involved in the process of research. Understanding the patient perspective of a breast cancer diagnosis and subsequent experiences can support healthcare professionals in developing an empathetic approach to sharing information, and involving patients in shared decision making about their healthcare

Nab-paclitaxel/gemcitabine combination is more effective than gemcitabine alone in locally advanced, unresectable pancreatic cancer - A GISCAD phase II randomized trial

The role of combination chemotherapy has not yet been established in unresectable locally advanced pancreatic cancer (LAPC) lacking dedicated randomized trials

Mitochondrial hyperfusion via metabolic sensing of regulatory amino acids

The relationship between nutrient starvation and mitochondrial dynamics is poorly understood. We find that cells facing amino acid starvation display clear mitochondrial fusion as a means to evade mitophagy. Surprisingly, further supplementation of glutamine (Q), leucine (L), and arginine (R) did not reverse, but produced stronger mitochondrial hyperfusion. Interestingly, the hyperfusion response to Q + L + R was dependent upon mitochondrial fusion proteins Mfn1 and Opa1 but was independent of MTORC1. Metabolite profiling indicates that Q + L + R addback replenishes amino acid and nucleotide pools. Inhibition of fumarate hydratase, glutaminolysis, or inosine monophosphate dehydrogenase all block Q + L + R-dependent mitochondrial hyperfusion, which suggests critical roles for the tricarboxylic acid (TCA) cycle and purine biosynthesis in this response. Metabolic tracer analyses further support the idea that supplemented Q promotes purine biosynthesis by serving as a donor of amine groups. We thus describe a metabolic mechanism for direct sensing of cellular amino acids to control mitochondrial fusion and cell fate

The Differential Metabolic Signature of Breast Cancer Cellular Response to Olaparib Treatment

Metabolic reprogramming and genomic instability are key hallmarks of cancer, the combined analysis of which has gained recent popularity. Given the emerging evidence indicating the role of oncometabolites in DNA damage repair and its routine use in breast cancer treatment, it is timely to fingerprint the impact of olaparib treatment in cellular metabolism. Here, we report the biomolecular response of breast cancer cell lines with DNA damage repair defects to olaparib exposure. Following evaluation of olaparib sensitivity in breast cancer cell lines, we immunoprobed DNA double strand break foci and evaluated changes in cellular metabolism at various olaparib treatment doses using untargeted mass spectrometry-based metabolomics analysis. Following identification of altered features, we performed pathway enrichment analysis to measure key metabolic changes occurring in response to olaparib treatment. We show a cell-line-dependent response to olaparib exposure, and an increased susceptibility to DNA damage foci accumulation in triple-negative breast cancer cell lines. Metabolic changes in response to olaparib treatment were cell-line and dose-dependent, where we predominantly observed metabolic reprogramming of glutamine-derived amino acids and lipids metabolism. Our work demonstrates the effectiveness of combining molecular biology and metabolomics studies for the comprehensive characterisation of cell lines with different genetic profiles. Follow-on studies are needed to map the baseline metabolism of breast cancer cells and their unique response to drug treatment. Fused with genomic and transcriptomics data, such readout can be used to identify key oncometabolites and inform the rationale for the design of novel drugs or chemotherapy combinations

Untargeted metabolomics and proteomics application of mass spectrometry: from biomarker identification within cells to their spatial localization on tissues

Metabolomics is the newest omics science which studies the chemical changes of small molecules (metabolites) within cells and tissues of a living organism. Thanks to the implementation of liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry imaging (MSI) analytical technologies, and the integration of multiple omics tools, metabolomics provides insight into the mechanisms underlying physiological and pathological conditions including ageing and cancer. Time-dependent accumulation of DNA damage has been observed during senescence, the state of cell cycle arrest and resistance to death which has been recognised as a driver of the ageing process. Similarly, genomic instability can initiate cancer and influence the overall prognosis of affected patients. Metabolomics has shown that metabolic reprogramming is another key characteristic of both ageing and cancer, necessary to sustain their survival in adverse conditions. Genomic instability and metabolic reprogramming contribute to the highly heterogeneous and dynamic phenotype of ageing and cancer, therefore predisposing patients to inferior clinical outcomes and resistance to treatments. Due to their heterogeneous and dynamic nature, isolating and effectively analysing the different phenotypes of both ageing and cancer is still a challenge. Owing to the implementation of mass spectrometry and its combination with microscopy technologies it is now possible to identify and spatially localise the distribution of new reliable and specific biomarkers for each individual phenotype. In this thesis, molecular assays coupled to mass spectrometry-based global metabolomics and proteomics techniques were employed to examine the changes occurring during cellular senescence upon induction of DNA damage, brain ageing in mice, and different breast cancer subtypes in response to DDR inhibition. The results presented show that at the cellular level senescence can be induced through replication stress, irradiation and DNA damage-inducing chemicals (hydroxyurea and etoposide), which share similar molecular features (growth arrest, flattened shape, expression of ß-galactosidase, DNA damage foci and cell cycle alteration), but different intra and extracellular metabolic components specific for each phenotype. At the tissue level, integration of global metabolomics and proteomics analysis allowed to design a putative metabolic map of the changes in the metabolites and proteins that were altered in the aged brain of mice. Moreover, the employment of mass spectrometry imaging (MSI) enabled the spatial localization of metabolites within specific regions of the brain. Finally, changes in cellular metabolism (glutamine and lipids metabolism) were observed in different breast cancer sub-phenotypes in response to DDR inhibition through Olaparib treatment. Overall, this thesis presents metabolomics – combined with molecular, proteomics studies and the high-resolution spatial determination of metabolites – as a powerful tool to reveal novel therapeutic targets for the treatment of ageing and age-related diseases (including cancer) and to comprehensively stratify different phenotypes relative to their tissue localization and based on their altered genetic alterations. When transferred into clinical diagnostics, this approach has future potential design personalised therapeutic approaches.Metabolomics is the newest omics science which studies the chemical changes of small molecules (metabolites) within cells and tissues of a living organism. Thanks to the implementation of liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry imaging (MSI) analytical technologies, and the integration of multiple omics tools, metabolomics provides insight into the mechanisms underlying physiological and pathological conditions including ageing and cancer. Time-dependent accumulation of DNA damage has been observed during senescence, the state of cell cycle arrest and resistance to death which has been recognised as a driver of the ageing process. Similarly, genomic instability can initiate cancer and influence the overall prognosis of affected patients. Metabolomics has shown that metabolic reprogramming is another key characteristic of both ageing and cancer, necessary to sustain their survival in adverse conditions. Genomic instability and metabolic reprogramming contribute to the highly heterogeneous and dynamic phenotype of ageing and cancer, therefore predisposing patients to inferior clinical outcomes and resistance to treatments. Due to their heterogeneous and dynamic nature, isolating and effectively analysing the different phenotypes of both ageing and cancer is still a challenge. Owing to the implementation of mass spectrometry and its combination with microscopy technologies it is now possible to identify and spatially localise the distribution of new reliable and specific biomarkers for each individual phenotype. In this thesis, molecular assays coupled to mass spectrometry-based global metabolomics and proteomics techniques were employed to examine the changes occurring during cellular senescence upon induction of DNA damage, brain ageing in mice, and different breast cancer subtypes in response to DDR inhibition. The results presented show that at the cellular level senescence can be induced through replication stress, irradiation and DNA damage-inducing chemicals (hydroxyurea and etoposide), which share similar molecular features (growth arrest, flattened shape, expression of ß-galactosidase, DNA damage foci and cell cycle alteration), but different intra and extracellular metabolic components specific for each phenotype. At the tissue level, integration of global metabolomics and proteomics analysis allowed to design a putative metabolic map of the changes in the metabolites and proteins that were altered in the aged brain of mice. Moreover, the employment of mass spectrometry imaging (MSI) enabled the spatial localization of metabolites within specific regions of the brain. Finally, changes in cellular metabolism (glutamine and lipids metabolism) were observed in different breast cancer sub-phenotypes in response to DDR inhibition through Olaparib treatment. Overall, this thesis presents metabolomics – combined with molecular, proteomics studies and the high-resolution spatial determination of metabolites – as a powerful tool to reveal novel therapeutic targets for the treatment of ageing and age-related diseases (including cancer) and to comprehensively stratify different phenotypes relative to their tissue localization and based on their altered genetic alterations. When transferred into clinical diagnostics, this approach has future potential design personalised therapeutic approaches

The differential metabolic signature of breast cancer cellular response to olaparib treatment

Metabolic reprogramming and genomic instability are key hallmarks of cancer, the combined analysis of which has gained recent popularity. Given the emerging evidence indicating the role of oncometabolites in DNA damage repair and its routine use in breast cancer treatment, it is timely to fingerprint the impact of olaparib treatment in cellular metabolism. Here, we report the biomolecular response of breast cancer cell lines with DNA damage repair defects to olaparib exposure. Following evaluation of olaparib sensitivity in breast cancer cell lines, we immunoprobed DNA double strand break foci and evaluated changes in cellular metabolism at various olaparib treatment doses using untargeted mass spectrometry-based metabolomics analysis. Following identification of altered features, we performed pathway enrichment analysis to measure key metabolic changes occurring in response to olaparib treatment. We show a cell-line dependent response to olaparib exposure, and an increased susceptibility to DNA damage foci accumulation in triple-negative breast cancer cell lines. Metabolic changes in response to olaparib treatment were cell-line and dose- dependent, where we predominantly observed metabolic reprogramming of glutamine-derived amino acids and lipids metabolism. Our work demonstrates the effectiveness of combining molecular biology and metabolomics studies for the comprehensive characterisation of cell lines with different genetic profiles. Follow-on studies are needed to map the baseline metabolism of breast cancer cells and their unique response to drug treatment. Fused with genomic and transcriptomics data, such readout can be used to identify key oncometabolites and inform the rationale for the design of novel drugs or chemotherapy combinations

The differential metabolic signature of breast cancer cellular response to olaparib treatment

Metabolic reprogramming and genomic instability are key hallmarks of cancer, the combined analysis of which has gained recent popularity. Given the emerging evidence indicating the role of oncometabolites in DNA damage repair and its routine use in breast cancer treatment, it is timely to fingerprint the impact of olaparib treatment in cellular metabolism. Here, we report the biomolecular response of breast cancer cell lines with DNA damage repair defects to olaparib exposure. Following evaluation of olaparib sensitivity in breast cancer cell lines, we immunoprobed DNA double strand break foci and evaluated changes in cellular metabolism at various olaparib treatment doses using untargeted mass spectrometry-based metabolomics analysis. Following identification of altered features, we performed pathway enrichment analysis to measure key metabolic changes occurring in response to olaparib treatment. We show a cell-line-dependent response to olaparib exposure, and an increased susceptibility to DNA damage foci accumulation in triple-negative breast cancer cell lines. Metabolic changes in response to olaparib treatment were cell-line and dose-dependent, where we predominantly observed metabolic reprogramming of glutamine-derived amino acids and lipids metabolism. Our work demonstrates the effectiveness of combining molecular biology and metabolomics studies for the comprehensive characterisation of cell lines with different genetic profiles. Follow-on studies are needed to map the baseline metabolism of breast cancer cells and their unique response to drug treatment. Fused with genomic and transcriptomics data, such readout can be used to identify key oncometabolites and inform the rationale for the design of novel drugs or chemotherapy combinations