Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert cytotoxicity through DNA and DNA-protein crosslinking, likely contributing to the onset of the human DNA repair condition Fanconi Anaemia. Mutations in the genes coding for FA detoxifying enzymes underlie a human inherited bone marrow failure syndrome (IBMFS), even in the presence of functional DNA repair, raising the question of whether FA causes relevant cellular damage beyond genotoxicity. Here, we report that FA triggers cellular redox imbalance in human cells and in Caenorhabditis elegans. Mechanistically, FA reacts with the redox-active thiol group of glutathione (GSH), altering the GSH:GSSG ratio and causing oxidative stress. FA cytotoxicity is prevented by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which metabolizes FA-GSH products, lastly yielding reduced GSH. Furthermore, we show that GSH synthesis protects human cells from FA, indicating an active role of GSH in preventing FA toxicity. These findings might be relevant for patients carrying mutations in FA-detoxification systems and could suggest therapeutic benefits from thiol-rich antioxidants like N-acetyl-L-cysteine.

Blockade of vascular endothelial growth factor (VEGF) signaling with bevacizumab, a humanized anti-VEGF monoclonal antibody (mAb), or with receptor tyrosine kinase inhibitors, has improved progression-free survival and, in some indications, overall survival across several types of cancers by interrupting tumor angiogenesis. However, the clinical benefit conferred by these therapies is variable, and tumors from treated patients eventually reinitiate growth. Previously we demonstrated, in mouse tumor models, that galectin-1 (Gal1), an endogenous glycan-binding protein, preserves angiogenesis in anti-VEGF–resistant tumors by co-opting the VEGF receptor (VEGFR)2 signaling pathway in the absence of VEGF. However, the relevance of these findings in clinical settings is uncertain. Here, we explored, in a cohort of melanoma patients from AVAST-M, a multicenter, open-label, randomized controlled phase 3 trial of adjuvant bevacizumab versus standard surveillance, the role of circulating plasma Gal1 as part of a compensatory mechanism that orchestrates endothelial cell programs in bevacizumab-treated melanoma patients. We found that increasing Gal1 levels over time in patients in the bevacizumab arm, but not in the observation arm, significantly increased their risks of recurrence and death. Remarkably, plasma Gal1 was functionally active as it was able to reprogram endothelial cell biology, promoting migration, tubulogenesis, and VEGFR2 phosphorylation. These effects were prevented by blockade of Gal1 using a newly developed fully human anti-Gal1 neutralizing mAb. Thus, using samples from a large-scale clinical trial from stage II and III melanoma patients, we validated the clinical relevance of Gal1 as a potential mechanism of resistance to bevacizumab treatment.

25Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert 26 cytotoxicity through DNA and DNA-protein crosslinking. We show here that FA can cause cellular damage 27 beyond genotoxicity by triggering oxidative stress, which is prevented by the enzyme alcohol dehydrogenase 28 5 (ADH5/GSNOR). Mechanistically, we determine that endogenous FA reacts with the redox-active thiol group 29 of glutathione (GSH) forming S-hydroxymethyl-GSH, which is metabolized by ADH5 yielding reduced GSH thus 30 preventing redox disruption. We identify the ADH5-ortholog gene in Caenorhabditis elegans and show that 31 oxidative stress also underlies FA toxicity in nematodes. Moreover, we show that endogenous GSH can protect 32 cells lacking the Fanconi Anemia DNA repair pathway from FA, which might have broad implications for 33 Fanconi Anemia patients and for healthy BRCA2-mutation carriers. We thus establish a highly conserved 34 mechanism through which endogenous FA disrupts the GSH-regulated cellular redox homeostasis that is 35 critical during development and aging. 36 37 hydroxymethylglutathione, Cancer, Genotoxicity. 38 39 BMF, liver and kidney dysfunction and early cancer onset 9 , indicating that endogenous FA can drive cancer 50 initiation and Fanconi Anemia phenotypes. 51Genotoxicity has been widely indicated as the main consequence of FA reactivity in cells 4 . However, 52 the strong reactivity of the FA carbonyl group might also affect other molecules than DNA. In vitro, the 53 spontaneous electrophilic attack of the FA carbonyl group to the thiol-group of GSH leads to the formation of 54 the covalent product S-hydroxymethyl-GSH (HSMGSH) 12 . This reaction might be strongly favored inside cells, 55where GSH levels are in the millimolar range 13 . Accordingly, ADH5 metabolizes HSMGSH yielding formate, 56 which is directed to the one-carbon cycle for nucleotide synthesis 3 (Fig. 1A). 57Considering the electrophilicity of FA and the abundance of GSH we hypothesize that the reaction 58 between FA and GSH might affect the GSH pool having detrimental biological consequences. Indeed, 59 alterations in GSH homeostasis have been reported in multiple pathologies such as hemolytic anemia, 60 diabetes, liver diseases, cystic fibrosis, neurodegeneration and cancer [14][15][16][17] . GSH not only neutralizes reactive 61 oxygen species (ROS), but can also promote chemoresistance by forming GSH-xenobiotic conjugates that are 62 pumped out of the cell via multiple resistance-associated protein transporters (MRP) 18 . To replenish 63 intracellular GSH, cells synthesize GSH in a two-step metabolic pathway centered on the rate-limiting enzyme 64 glutamate cysteine ligase (GCL), which is composed of a catalytic (GCLC) and a regulatory (GCLM) subunit, and 65 the GSH synthetase (GS) ( Fig. 1a) 18 . Cells might thus also need to maintain the balance between GSH and the 66 oxidized GSH disulfide form (GSSG) -GSH:GSSG-to limit free FA and to prevent redox disruption. 67We report here that FA toxicity is inf...