Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor, acting via the regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterized, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here we report a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B2) as a modulator of ferroptosis sensitivity. We demonstrate that riboflavin supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitizes cancer cells to ferroptosis. Our findings provide a rational strategy to modulate the FSP1-antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients, as well as their contributions to a cell's antioxidant capacity.

Gene activation and coregulation have been attributed to different mechanisms, such as enhancer-promoter interactions via chromatin looping or the accumulation of transcription factors into hubs or condensates. However, genome-wide studies exploring mechanistic differences in endogenous gene regulation in primary human cells are scarce. Here we dissect the proinflammatory gene expression programme induced by tumor necrosis factor (TNF) in human endothelial cells using sequencing- and imaging-based methods. Our findings, enabled by the co-accessibility analysis of deep-coverage single-cell chromatin accessibility data with our RWireX software, identified two distinct regulatory chromatin modules: autonomous links of co-accessibility (ACs) between separated sites and domains of contiguous co-accessibility (DCs) with increased local transcription factor binding. The TNF-dependent induction timing and strength as well as changes in transcriptional bursting kinetics differed for genes in the AC and DC modules, pointing to functionally distinct regulatory mechanisms. These findings provide a framework for understanding how cells achieve rapid and precise control of gene expression.

Cytosolic phospholipase A2 (cPLA2) controls some of the most powerful inflammatory lipids in vertebrates by releasing their metabolic precursor, arachidonic acid, from the inner nuclear membrane (INM). Ca2+ and INM tension (TINM) are thought to govern the interactions and activity of cPLA2 at the INM. However, as compensatory membrane flow from the contiguous endoplasmic reticulum (ER) may prevent TINM, the conditions permitting nuclear membrane mechanotransduction by cPLA2 or other mediators remain unclear. To test whether the ER buffers TINM, we created the genetically encoded, Ca²⁺-insensitive TINM biosensor amphipathic lipid-packing domain inside the nucleus (ALPIN). Confocal time-lapse imaging of ALPIN- or cPLA2-INM interactions, along with ER morphology, nuclear shape/volume and cell lysis revealed a link between TINM and disrupted ER-nuclear membrane contiguity in osmotically or ferroptotically stressed mammalian cells and at zebrafish wound margins in vivo. By combining ALPIN imaging with Ca2+-induced ER disruption, we reveal the causality of this correlation, which suggests that compensatory membrane flow from the ER buffers TINM without preventing it. Besides consolidating the biomechanical basis of cPLA2 activation by nuclear deformation, our results identify cell stress- and cell death-induced ER disruption as an additional nuclear membrane mechanotransduction trigger.

Therapeutic targeting of mutant KRAS pathways driving cancers is being actively investigated to identify feedback mechanisms responsible for the development of adaptive resistance to mutant KRAS inhibitors undergoing clinical trials. Here we report RASH3D19 as a mediator of RAS pathway activation through a positive feedback loop involving the KRAS-microRNA signalling axis. KRAS-induced miR-222 represses ETS1 expression and downstream transactivation of miR-301a leading to elevation of its target RASH3D19. RASH3D19 facilitates activation of RAS pathways by promoting dimerization and interaction of EGFR with the SOS2, GRB2, SHP2 and GAB1 complex. Genetic deletion of RASH3D19 in mutant KRAS-expressing cancer cells exhibits growth retardation in vitro, in vivo and sensitized pancreatic ductal adenocarcinoma and colorectal cancer cells, organoids and xenografts to mutant KRAS inhibitors, suppressing feedback reactivation of RAS pathways. Therapeutic targeting of RASH3D19 is expected to lead to tumour debulking and alleviating resistance to KRAS inhibitors in mutant KRAS-expressing cancers.