ABSTRACT: Introduction: Altered metabolism is a key hallmark of tumour cells (Pavlova & Thompson, 2016) and has long been associated with relapse and resistance to treatment (Gonçalves et al., 2021; Lv et al., 2022). Recently, we identified a new role for histone deacetylase 6 (HDAC6) in the regulation of glycolytic metabolism (Dowling et al., 2021). HDAC6 is an atypical member of the HDAC family as it is localised in the cytosol, has two deacetylase domains, and contains a ubiquitin-binding domain (Gallinari et al., 2007). HDAC6 has been shown to deacetylate proteins involved in processes including microtubule remodelling and stress responses (Hubbert et al., 2002; Kawaguchi et al., 2003). In a recent publication, we showed that HDAC6 knockdown (K/D) or inhibition, with a compound we discovered called BAS-2, altered the acetylation of glycolytic enzymes and reduced glycolysis in triple-negative breast cancer (TNBC) cells (Dowling et al., 2021). However, we did not investigate whether HDAC6 inhibition altered other metabolic pathways at the time. The mitochondrial tricarboxylic acid (TCA) cycle plays an important role in tumour biology, not alone for energy production through the respiratory chain, but also for metabolic intermediates that serve as building blocks for lipid and nucleic acid synthesis (Eniafe & Jiang, 2021). Some of these intermediates of the TCA cycle have dual roles as ‘oncometabolites’ (Frezza, 2017). Both fumarate hydratase (FH) and succinate dehydrogenase (SDH), two key enzymes in the TCA cycle, are known tumour suppressors (Rustin et al., 2002; Schmidt et al., 2020). Loss of FH is associated with an aggressive form of kidney cancer called hereditary leiomyomatosis and renal cell carcinoma (HLRCC) (Tomlinson et al., 2002). Deficiency of FH causes an increase in fumarate, which has various signalling properties including the non-enzymatic formation of 2-(succino)cysteine (2-SC) adducts that can alter protein activity, a process termed succination (Guberovic & Frezza, 2024; Kinch et al., 2011; Tyrakis et al., 2017). Two recent publications identified that macrophages with FH deficiency show an increase of intracellular fumarate and that this alters mitochondrial structure, causing the release of mitochondrial DNA or RNA to activate inflammatory responses (Hooftman et al., 2023; Zecchini et al., 2023). To date, the majority of studies on FH have been carried out using knockout mice to show the role of FH in tumourigenesis or macrophage inflammation (Valcarcel-Jimenez & Frezza, 2023; Zecchini et al., 2023). However, there are limited studies in tumour cells on the role of FH or on how FH activity is regulated. In this study, we identified that HDAC6 can alter mitochondrial structure, causing cristae damage and the release of mtDNA into the cytosol. We then show by mass spectrometry, immunoprecipitation, and super-resolution imaging that HDAC6 and FH directly interact. Inhibition of HDAC6 with BAS-2 reduces FH activity, resulting in an increase of fumarate, with a downstream increase in protein succination. Utilising stochastic optical reconstruction microscopy (STORM), we pinpoint sites of interaction to the mitochondria, thereby demonstrating a novel regulation of FH in tumour cells.
Kieran Wynne 
