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Biology subjects

Fets, L.

Publications and source records attributed to Fets, L..

5 recordsLinked to original sources

Multimodal imaging reveals a lysosomal drug reservoir that drives heterogeneous distribution of PARP inhibitors

For all drugs, effective target engagement requires sufficient intracellular concentrations of drug to be reached, but whether tumour heterogeneity impacts drug distribution and efficacy is poorly studied. PARP inhibitors have transformed treatment of high-grade serous ovarian carcinoma (HGSOC), but resistance remains a clinical hurdle in this highly heterogeneous tumour type. We developed a patient-derived explant multi-modal imaging pipeline, which demonstrated that cell-intrinsic PARP inhibitor accumulation is highly variable, both between patients and within tumours. Spatial transcriptomics revealed enrichment of apoptotic and lysosomal signatures in high-drug regions. Rucaparib, an intrinsically fluorescent PARP inhibitor, accumulates heterogeneously at the single-cell level, with rucaparib-high cells demonstrating increased drug response relative to rucaparib low. Mechanistically, lysosomal sequestration creates a rucaparib reservoir that determines drug levels in the nucleus. Perturbation of lysosomal content altered intracellular levels of weak base PARP inhibitors rucaparib and niraparib, but not olaparib. Together these data suggest that lysosomes act as a reservoir for a subset of PARP inhibitor drugs to improve drug response.

cancer biology↗

Mitochondrial dysfunction underlies monocyte immune deficiency in patients with severe alcohol-related hepatitis

Severe alcohol-related hepatitis (sAH) is a life-threatening form of alcohol-related liver disease (ARLD) associated with a significant short-term mortality. Opportunistic infections due to impaired immune function are a major cause of patient mortality. Mitochondrial dysfunction within the liver is a well-recognised feature of ARLD and sAH. However, whether these hepatic mitochondrial defects extend to the immune system of sAH patients, underlying their immune dysfunction, remains unclear. Here, we demonstrate that sAH monocytes exhibit an increased content of inefficient, dysfunctional mitochondria. These changes were underpinned by abnormal mitochondrial cristae ultrastructure, which were associated with depletion of cristae structural proteins and alterations in cardiolipin profiles. Overall, our study uncovers novel structural and functional mitochondrial defects, which likely contribute to impaired monocyte immune function in sAH.

immunology↗

GOT1 primes the cellular response to hypoxia by supporting glycolysis and HIF1α stabilisation

Adaptation to chronic hypoxia occurs through changes in protein expression, which are controlled by hypoxia inducible factor 1a (HIF1) and are necessary for cancer cell survival. However, the mechanisms that enable cancer cells to adapt in early hypoxia, prior to full activation of HIF1, remain poorly understood. Here we show that aspartate transaminase 1 (GOT1), which supports NAD+ production by malate dehydrogenase 1 (MDH1), is required, in addition to reserve lactate dehydrogenase (LDH) capacity, for the HIF1-independent increase in glycolysis we observe early upon exposure of cells to hypoxia. Additionally, GOT1 maintains low -ketoglutarate levels, thereby limiting prolyl hydroxylase activity to promote HIF1 stabilisation in early hypoxia and robust HIF1 target gene expression in later hypoxia. Our findings reveal that, in normoxia, GOT1 maintains cells in a primed state and ready to support increased glycolysis and HIF1 stabilisation upon oxygen limitation, until other adaptive processes that require more time, are fully established.

cancer biology↗

Development of novel MOG analogues with increased stability to explore MCT2 and α-ketoglutarate biology in vivo

-ketoglutarate (KG) is a central metabolic node with a broad influence on cellular physiology. The KG analogue N-oxalylglycine (NOG) and its membrane-permeable pro-drug derivative dimethyl-oxalylglycine (DMOG) have been extensively used as tools to study prolyl hydroxylases (PHDs) and other KG-dependent processes. In cell culture media, DMOG is rapidly converted to MOG, which enters cells through monocarboxylate transporter MCT2, leading to intracellular NOG concentrations that are sufficiently high to inhibit glutaminolysis enzymes and cause cytotoxicity. Therefore, the degree of (D)MOG instability together with MCT2 expression levels determine the intracellular targets NOG engages with and, ultimately, its effects on cell viability. Here we designed and characterised a series of MOG analogues with the aims of improving stability and exploring the functional requirements for interaction with MCT2, a relatively understudied member of the SLC16 family. We report MOG analogues that maintain ability to enter cells via MCT2, and identify compounds that do not inhibit glutaminolysis or cause cytotoxicity but can still inhibit PHDs. We use these analogues to show that glutaminolysis-induced activation of mTORC1 can be uncoupled from PHD activity. Therefore, these new compounds can help deconvolute cellular effects that result from the polypharmacological action of NOG.

biochemistry↗

Functional cross-talk between allosteric effects of activating and inhibiting ligands underlies PKM2 regulation

Allosteric regulation is central to the role of the glycolytic enzyme pyruvate kinase M2 (PKM2) in cellular metabolism. Multiple activating and inhibitory allosteric ligands regulate PKM2 activity by controlling the equilibrium between high activity tetramers and low activity dimers and monomers. However, it remains elusive how allosteric inputs upon simultaneous binding of different ligands are integrated to regulate PKM2 activity. Here, we show that, in the presence of the allosteric inhibitor L-phenylalanine (Phe), the activator fructose 1,6-bisphosphate (FBP) can induce PKM2 tetramerisation, but fails to maximally increase enzymatic activity. Guided by a new computational framework we developed to identify residues that mediate FBP-induced allostery, we generated two PKM2 mutants, A327S and C358A, in which activation by FBP remains intact but cannot be attenuated by Phe. Our findings demonstrate a role for residues involved in FBP-induced allostery in enabling the integration of allosteric input from Phe and reveal a mechanism that underlies the co-ordinate regulation of PKM2 activity by multiple allosteric ligands.

biochemistry↗