Oncogene-induced senescence (OIS), a cellular programme initiated by activation of oncogenic signalling, provides a barrier to transformation and is accompanied by major reprogramming of cellular metabolism. We show here that induction of OIS by BRAFV600E expression in human diploid fibroblasts led to global changes in the cellular lipidome, characterised by a strong increase in triglycerides (TG) and a marked reduction in membrane phosphoglycerides carrying polyunsaturated fatty acids (PUFA) in their acyl-chains. Induction of BRAFV600E OIS resulted in a marked resistance towards lipid peroxidation and ferroptosis. Inhibition of TG synthesis by blocking diacylglycerol O-acyltransferase 1 (DGAT1) resulted in PUFA re-distribution to membrane lipids and increased ferroptosis sensitivity of senescent cells. Inhibition of DGAT also altered the senescence-associated secretory phenotype (SASP) and enhanced the secretion of oxylipins by BRAFV600E OIS cells. Combined blockade of DGAT1-dependent TG and COX2-dependent oxylipin synthesis fully restored ferroptosis sensitivity in BRAFV600E OIS cells. Together, these findings indicate that channelling of PUFA towards TG synthesis confers protection from oxidative stress and ferroptosis during BRAFV600E OIS but also limits the production of pro-inflammatory lipid mediators, a key feature of the senescent phenotype.

Abstract The MYC oncoprotein promotes immune evasion of pancreatic ductal adenocarcinoma (PDAC), but the underlying molecular mechanisms are not fully understood. Here we show that MYC protects PDAC tumors from CD4 + T cell-dependent elimination. Single cell sequencing shows that MYC suppression in tumor cells increases amino acid availability and broadly activates amino acid-responsive gene expression programs in immune cell populations. This occurs because MYC-driven uptake depletes free amino acids from tumor interstitial fluid and plasma, while MYC compromises macropinocytosis and autophagy, both of which depend on lysosomal protein degradation. MYC engages the POZ/BTB transcription factor MIZ1 to suppress lysosomal genes regulated by the TFE3/TFEB/MITF network or by free MIZ1, thereby inhibiting lysosomal protein degradation. An orthogonal genetic model enabling transient, selective inhibition of amino acid uptake in tumor cells recapitulates the effects of MYC depletion on amino acid levels in the tumor microenvironment and induces complete, CD4 + T cell-dependent tumor eradication with long-term survival. We propose that MYC-mediated, cell-autonomous disruption of lysosome function coupled to non-cell-autonomous protection from immune clearance allows MYC-low cells to benefit from MYC-high neighbors, such that intratumoral heterogeneity in MYC expression confers a selective advantage to the entire tumor. Abstract Figure

Genomic instability is a hallmark of cancer, driving oncogenic mutations that enhance tumor aggressiveness and drug resistance. MYC, a master transcription factor that is deregulated in nearly all human tumors, paradoxically induces replication stress and associated DNA damage while also increasing expression of DNA repair factors and mediating resistance to DNA-damaging therapies. Emerging evidence supports a nontranscriptional role for MYC in preserving genomic integrity at sites of active transcription and protecting stalled replication forks under stress. Understanding how MYC's genotoxic and genoprotective functions diverge may reveal new therapeutic strategies for MYC-driven cancers. Here, we identify a noncanonical role of MYC in DNA damage response (DDR) through its association with DNA breaks. We show that phosphorylation at serine 62 (pS62-MYC) is crucial for the efficient recruitment of MYC to damage sites, its interaction with repair factors BRCA1 and RAD51, and effective DNA repair to support cell survival under stress. Proteomic profiling of the MYC interactome confirms a conserved interaction with components of the DDR pathway. These findings establish pS62-MYC as a key regulator of genomic stability and a potential therapeutic target in cancers.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is almost inevitably fatal and largely resistant to current chemotherapy regimens. Senolysis, i. e. the selective elimination of senescent cells, and the targeting of cancer specific cellular metabolism are two novel treatment approaches that have proven to be effective for a variety of cancer types in pre-clinical studies. We now demonstrate that pharmacological depletion of arginine with pegylated recombinant human arginase 1 (PEG-rhARG1) induced senescence and activated the integrated stress response (IRS) in PDAC cells. Cells treated in such a manner were strikingly susceptible towards senolysis with ABT-263 (navitoclax) and also sensitive towards inhibition of the IRS. These results demonstrate a novel mechanism for an induced sensitivity of PDAC cells that could be exploited for cancer therapy.

In response to perturbed transcription elongation, the MYC oncoprotein multimerizes and undergoes a phase transition. Here, we demonstrate that MYC globally relocalizes from its canonical positions on DNA to nascent RNA upon accumulation of intronic RNA. Upon binding to RNA, MYC forms multimers that concentrate the nuclear exosome, an RNA exonuclease, and its targeting complexes around double-stranded RNA and R-loops. MYC harbors four RNA-binding regions (RBRI-IV). RBRIII promotes MYC multimerization and is necessary for recruiting the exosome to R-loops. RBRIII is dispensable for transcriptional activation and pancreatic tumor cell proliferation in culture, but it is indispensable for sustaining tumor growth in vivo. Via RBRIII, MYC suppresses the accumulation of R-loop-derived RNA-DNA hybrids and prevents them from activating the innate immune kinase TBK1 via the TLR3 pattern recognition receptor. Our data demonstrate that the phase transition of MYC is an RNA-driven stress response that suppresses the accumulation of immunogenic RNA-DNA hybrids.

MYC proteins are classically viewed as oncoproteins because they act as DNA-bound transcription factors that drive characteristic gene expression programs. Building on this view, recent work has shown that MYC proteins engage in multiple protein complexes that resolve transcription-associated stress and that they function as both DNA- and RNA-binding proteins. These findings suggest that, through these activities, MYC proteins enhance the stress resilience of proliferating cells and enable tumor cells to sustain nonphysiological, oncogenic gene expression programs.

The androgen receptor (AR) is a key driver of prostate cancer progression, making androgen deprivation therapy and AR signaling inhibitors the standard of care therapies for treating prostate cancer. Although these treatments are effective initially, they often lead to the development of resistance, limiting their long-term efficacy. This acquired resistance is frequently linked to the MYC oncogene family, which, together with reduced AR signaling, is associated with poor clinical prognosis. Among the resistant phenotypes, treatment-induced neuroendocrine prostate cancer (tNEPC) is a particularly aggressive AR-independent subtype characterized by loss of AR signaling and epigenetic reprogramming. These molecular changes promote cell lineage plasticity, creating a permissive environment for MYCN activation, which further drives tumor progression and neuroendocrine differentiation. In summary, MYC proteins pose a major biological challenge, but also offer opportunities to develop new therapeutic approaches. To address these, we generated LNCaP-derived prostate cancer cell lines that constitutively express c-MYC or MYCN. Their sustained proliferation under enzalutamide treatment confirmed the development of a resistant phenotype. In addition, RNA sequencing of MYCN-overexpressing cells revealed transcriptional reprogramming, including the activation of neural lineage markers and epithelial-mesenchymal transition programs consistent with a neuroendocrine-like phenotype. To identify dependency factors, we performed a genome-wide shRNA screen to uncover genes whose knockdown selectively impaired growth of LNCaP cells expressing c-MYC or MYCN under enzalutamide. In c-MYC-expressing cells approximately 80 significantly downregulated genes were identified, many of which had previously been associated with therapeutic resistance. Interestingly, a substantial proportion of these factors were also significantly enriched in RNA processing and metabolic pathways. Both c-MYC and MYCN bind directly to DNA as well as RNA, and recent studies have revealed that MYC proteins play distinct mechanistic roles in transcriptional regulation and RNA metabolism. The newly described RNA-related functions of MYC highlight the importance of RNA regulatory mechanisms as key drivers of MYC-dependent oncogenesis in prostate cancer. Approximately 20 genes were significantly downregulated in MYCN-expressing cells, most of which are involved in RNA splicing. This is an intriguing observation given that MYCN is a well-characterized transcription factor that orchestrates transcriptional reprogramming. The data suggest that MYCN can influence oncogenic processes by modulating RNA splicing mechanisms, revealing an additional level of regulatory complexity in tNEPC. Ongoing validation and integrative analyses, including bulk mRNA-sequencing of patient data, aim to define the roles of these genes, delineate pathways, and identify novel therapeutic targets to overcome MYC-mediated and AR-targeted therapy resistance. Saskia Elena. Haarmann, Martin Eilers, Steffi Herold. Global shRNA screen to identify factors that are involved in MYC/MYCN-dependent growth of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr B028.

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.

Clonal expansion and effector differentiation of T cells require extensive metabolic reprogramming. This includes the restructuring of the inner mitochondrial membrane (IMM) to enhance respiration by increasing chemiosmotic coupling efficiency. Cardiolipin, a unique phospholipid that is exclusively synthesized and localized in the IMM, modulates the biophysical properties of the electron transport chain (ETC) in tissues with high energy demands, such as cardiomyocytes and skeletal muscle. However, it remains unclear whether cardiolipin is also important for metabolic remodeling during T helper (Th) cell differentiation. In this study, we show that cardiolipin transacylation, catalyzed by the enzyme Tafazzin, supports the clonal expansion and effector function of inflammatory Th1 and Th17 cells in vitro and in models of autoimmune colitis and encephalomyelitis. At the molecular level, we demonstrate that loss of Tafazzin-mediated cardiolipin maturation induces a metabolic and transcriptional stress response in Th cells to compensate for impaired coupling efficiency of the ETC complexes and disrupted cellular redox homeostasis. However, the genetic program that restores cellular homeostasis and mitigates oxidative stress concurrently impairs the effector functions of inflammatory T cells, such as cytokine production. Our findings also provide insights into the complex clinical manifestation of patients with Barth syndrome (BTHS) caused by mutations in the human TAFAZZIN gene. BTHS is characterized by cardiac and skeletal myopathy as well as neutropenia and an increased susceptibility to infections. Although the molecular basis of the immunodeficiency remains poorly understood, our findings suggest that impaired Th cell function contributes to the immunopathology observed in BTHS patients. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, SFB-TR 12 (“FungiNet”), project number: 210879364, SFB 1526 (“PANTAU”), project number: 454193335, SFB-TR 338 (“LETSimmun”), project number: 452881907, SFB 1583 (“DECIDE”), project number: 49262049, VA882/2-1, VA882/3-2

High-risk neuroblastoma is a poor prognosis cancer of the sympathetic nervous system that accounts for a disproportionate number of childhood cancer deaths. Many viable biological targets have been identified, and the number of potential combinations is even larger. Several products have attained marketing authorization for treatment of patients with neuroblastoma. Patient outcomes remain poor, with approximately 50% of children with newly diagnosed high-risk neuroblastoma cured of their disease. International, multistakeholder Neuroblastoma Drug Development Strategy (NDDS) meetings were established more than a decade ago. This third NDDS meeting included academia, industry, regulatory, and patient advocacy representatives to prioritize agents and to address key challenges in drug development in this disease. Given the central role that anti-GD2 therapy plays, novel GD2-directed combinations were a key focus, including epigenetic enzymes such as EZH2 and immunologic targets such as IL15 and TIGIT as potential combination partners. GD2-directed chimeric antigen receptor (CAR)-T cells were a top priority, along with emerging CAR-T targets such as B7-H3 and GPC2. Recognizing that combination therapies are likely to be most impactful for patients and for advancing therapies to frontline, another key focus was on high priority combinations of targeted therapies, including Aurora A kinase plus BCL2 or ATR inhibitors. Additional targets and agents were prioritized or deprioritized based upon current data. Access to drugs for clinical trials was viewed as a major barrier to progress. Strategies to overcome this challenge focused on united efforts by the international scientific and advocacy community and early engagement by industry with regulatory authorities.

Aurora A kinase (AURKA) is an oncogene frequently overexpressed in adult solid tumors, hematologic malignancies, and pediatric cancers. AURKA plays an important role in mitosis, and cancer cells sensitive to loss of AURKA include lines derived from MYCN amplified tumors such as neuroblastoma, as well as those with RB1 loss such as neuroendocrine small cell cancers and CDK4/6-resistant breast cancer (Mou, et al., 2021). Several AURKA inhibitors are effective in preclinical tumor models, but this activity has failed to translate into clinical efficacy. Recent studies have found that AURKA has kinase-independent scaffolding functions that are not effectively blocked through enzymatic inhibition. For example, AURKA limits the proteasomal degradation of MYCN through direct protein-protein interactions and prevents transcription/replication conflicts during S phase (Otto, et al, Cancer Cell, 2009; Buchel, et al., Cell Reports, 2017). To address the limitations of inhibitors, we have designed bifunctional targeted protein degraders of AURKA that enable removal of both enzymatic and scaffolding functions. Combinatorial chemical libraries of AURKA binders, CRBN binders, and linkers were generated by high-throughput parallel synthesis and screened for optimal properties in vitro and in vivo. These degraders form stable ternary complexes between AURKA and the E3 ligase CRBN, leading to efficient ubiquitylation and proteosome-dependent elimination of AURKA. Optimized AURKA degraders potently and selectively degraded AURKA in the MYCN amplified neuroblastoma cell line IMR32. Compounds with oral bioavailability and CNS exposure in mice were prioritized for further development. Our lead AURKA degrader, NRX-4972, has 58% oral bioavailability in C57BL/6 mice and moderate clearance (17.5 mL/min/kg). Three days of daily oral administration at 30 mg/kg to athymic nude mice bearing subcutaneous IMR32 tumors resulted in strong AURKA degradation (84% degraded relative to vehicle controls 6 hours after the final administration), high exposure in plasma (6.94 µM) and tumors (6,939 ng/g) and brain exposure of 178 ng/g. The closely related kinase AURKB was not degraded. AURKA degradation also promoted rapid regression of IMR32 neuroblastoma tumors, while inhibitors only achieved stasis. Analysis of tumors isolated from treated mice revealed that AURKA degraders, but not inhibitors, rapidly induced DNA damage and apoptosis. The differentiated activity of AURKA degraders compared to enzymatic inhibitors in these preclinical studies suggests the potential for improved safety and efficacy in cancer patients. The ability of AURKA degraders to penetrate the CNS not only broadens their therapeutic potential to include pediatric brain cancers such as medulloblastoma but also offers a promising avenue for treating adult cancers with brain metastases, addressing an urgent unmet medical need. Hua Tian, Eric Wegrzyniak, Ya-Wen Lu, Jeffrey T. Mihalic, Ryan Rountree, Tina Acholla, Bikash Adhikari, Karthik Arumugam, Paul Auger, Graham Carlson, Robert Cass, Coleen E. Casey, Abhinav Chaterjee, Lorenz Eing, James Iuliano, Adrienne Le, Yifan Li, Victoria Louie, Filippo Marchioni, Daniel Medina-Cleghorn, Isabel Morgado, Michael Mormino, Madeleine Nemchek, Erick Palomares, Rusha Sardhara, Julie Sheung, Sangita Sridharan, Gintvile Valinciute, Dipna Venkatachalam, Simon Vezina-Dawod, Derek Wodka, Stephanie Yung, Martin Eilers, John M. Maris, Yael P. Mossé, Martine F. Roussel, Elmar Wolf, Gwenn M. Hansen. Identification of selective, orally bioavailable aurora A degraders for treatment of pediatric and adult cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6379.

Deregulated MYCN is a driver of aggressive pediatric and adult neuroendocrine tumors, but critical oncogenic processes downstream of MYCN remain poorly defined. In neuroblastoma, MYCN interacts with and activates the Aurora-A kinase. Here we show that Aurora-A is a CDK-activating kinase for CDK12 by phosphorylating T893 in the T-loop, thereby enhancing its kinase activity. Aurora-A-dependent activation of CDK12 controls phosphorylation of T4 of RNA polymerase and recruits transcription termination complexes, thereby preventing transcription-replication conflicts. Enhanced crosslinking and immunoprecipitation sequencing reveals that Aurora-A associates with splice sites on nascent RNA. RNA-bound Aurora-A is catalytically inactive. MYCN competes with RNA for binding to Aurora-A and displaces Aurora-A from RNA in cells, promoting its CDK12 kinase activity. Combining Aurora-A and CDK12 inhibition potently suppresses the growth of MYCN-amplified neuroblastoma cells and patient-derived xenografts. Our data demonstrate that an Aurora-A/CDK12-dependent transcription termination pathway is a critical and targetable dependency of MYCN-driven tumors. ### Competing Interest Statement M.B. is a shareholder and employee of Proxygen. V.N.-P. is an employee of Proxygen.

Unchecked inflammation can lead to tissue damage. Liu and colleagues show that the transcription factor Miz1 recruits the histone deacetylase HDAC1 to restore repression of Cebpd transcription, which is necessary for the termination of LPS-induced inflammatory responses. Inflammation is essential for host defense but can cause tissue damage and organ failure if unchecked. How the inflammation is resolved remains elusive. Here we report that the transcription factor Miz1 was required for terminating lipopolysaccharide (LPS)-induced inflammation. Genetic disruption of the Miz1 POZ domain, which is essential for the transactivation or repression activity of Miz1, resulted in hyperinflammation, lung injury and greater mortality in LPS-treated mice but a lower bacterial load and mortality in mice with Pseudomonas aeruginosa pneumonia. Loss of the Miz1 POZ domain prolonged the expression of proinflammatory cytokines. After stimulation, Miz1 was phosphorylated at Ser178, which was required for recruitment of the histone deacetylase HDAC1 to repress transcription of the gene encoding C/EBP-δ, an amplifier of inflammation. Our data provide a long-sought mechanism underlying the resolution of LPS-induced inflammation.

Current treatment protocols have limited success against MYCN-amplified neuroblastoma. Adoptive T cell therapy presents an innovative strategy to improve cure rates. However, L1CAM-targeting CAR T cells achieved only limited response against refractory/relapsed neuroblastoma so far. We investigated how oncogenic MYCN levels influence tumor cell response to CAR T cells, as one possible factor limiting clinical success. A MYCN-inducible neuroblastoma cell model was created. L1CAM-CAR T cell effector function was assessed (activation markers, cytokine release, tumor cytotoxicity) after coculture with the model or MYCN-amplified neuroblastoma cell lines. RNA sequencing datasets characterizing the model were compared to publicly available RNA/proteomic datasets. MYCN-directed L1CAM regulation was explored using public ChIP-sequencing datasets. Synergism between CAR T cells and the indirect MYCN inhibitor, MLN8237, was assessed in vitro using the Bliss model and in vivo in an immunocompromised mouse model. Inducing high MYCN levels in the neuroblastoma cell model reduced L1CAM expression and, consequently, L1CAM-CAR T cell effector function in vitro. Primary neuroblastomas possessing high MYCN levels expressed lower levels of both the L1CAM transcript and L1CAM tumor antigen. MLN8237 treatment restored L1CAM tumor expression and L1CAM-CAR T cell effector function. Combining MLN8237 and L1CAM-CAR T cell treatment synergistically enhanced MYCN-overexpressing tumor cytotoxicity in vitro and in vivo concomitant with severe in vivo toxicity. We identify target antigen downregulation as source of resistance against L1CAM-CAR T cells in MYCN-driven neuroblastoma cells. These data suggest that L1CAM-CAR T cell therapy combined with pharmacological MYCN inhibition may benefit patients with MYCN-amplified neuroblastoma.

Protein synthesis is an essential process, deregulated in multiple tumor types showing differential dependence on translation factors compared to untransformed tissue. We show that colorectal cancer (CRC) with loss-of-function mutation in the APC tumor suppressor depends on an oncogenic translation program regulated by the ability to sense phosphorylated eIF2α (p-eIF2α). Despite increased protein synthesis rates following APC loss, eIF2α phosphorylation, typically associated with translation inhibition, is enhanced in CRC. Elevated p-eIF2α, and its proper sensing by the decameric eIF2B complex, are essential to balance translation. Knockdown or mutation of eIF2Bα and eIF2Bδ, two eIF2B subunits responsible for sensing p-eIF2α, impairs CRC viability, demonstrating that the eIF2B/p-eIF2α nexus is vital for CRC. Specifically, the decameric eIF2B linked by two eIF2Bα subunits is critical for translating growth-promoting mRNAs which are induced upon APC loss. Depletion of eIF2Bα in APC-deficient murine and patient-derived organoids establishes a therapeutic window, validating eIF2Bα as a target for clinical intervention. In conclusion, we demonstrate how the expression of the oncogenic signature in CRC is crucially controlled at the translational level.

The contribution of deubiquitylating enzymes (DUBs) to β-Catenin stabilization in intestinal stem cells and colorectal cancer (CRC) is poorly understood. Here, and by using an unbiassed screen, we discovered that the DUB USP10 stabilizes β-Catenin specifically in APC-truncated CRC in vitro and in vivo. Mechanistic studies, including in vitro binding together with computational modelling, revealed that USP10 binding to β-Catenin is mediated via the unstructured N-terminus of USP10 and is outcompeted by intact APC, favouring β-catenin degradation. However, in APC-truncated cancer cells USP10 binds to β-catenin, increasing its stability which is critical for maintaining an undifferentiated tumour identity. Elimination of USP10 reduces the expression of WNT and stem cell signatures and induces the expression of differentiation genes. Remarkably, silencing of USP10 in murine and patient-derived CRC organoids established that it is essential for NOTUM signalling and the APC super competitor-phenotype, reducing tumorigenic properties of APC-truncated CRC. These findings are clinically relevant as patient-derived organoids are highly dependent on USP10, and abundance of USP10 correlates with poorer prognosis of CRC patients. Our findings reveal, therefore, a role for USP10 in CRC cell identity, stemness, and tumorigenic growth by stabilising β-Catenin, leading to aberrant WNT signalling and degradation resistant tumours. Thus, USP10 emerges as a unique therapeutic target in APC truncated CRC.

During early transcription, RNA polymerase II (RNAPII) undergoes a series of structural transitions controlled by cyclin-dependent kinases. How protein ubiquitylation and proteasomal degradation control the function of RNAPII is less well understood. Here we show that the deubiquitinating enzyme USP11 forms a complex with TCEAL1, a member of the TFIIS (TCEA)-like protein family. TCEAL1 shares sequence homology with the RNAPII interaction domain of the elongation factor TFIIS (which controls the fate of backtracked RNAPII) and competes with TFIIS for binding to core promoters. USP11 protects TCEAL1 from proteasomal degradation, and TCEAL1 recruits USP11 to RNAPII. Both USP11 and TCEAL1 promote transcription elongation and maintain expression of RPB8, an essential subunit of all three nuclear RNA polymerases. In neuroblastoma, USP11- and TCEAL1-dependent genes define a gene expression program that is characteristic for mesenchymal tumors, which are described as able to escape from many treatments, suggesting that the USP11/TCEAL1 complex promotes transcription elongation to support a critical oncogenic gene expression program.

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. Deficiencies in these protective systems lead to an increased propensity to phospholipid peroxidation and ferroptosis. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor acting via regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterised, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here, we conducted a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B₂) as a new modulator of ferroptosis sensitivity. We demonstrate that riboflavin, unlike other vitamins that act as radical-trapping antioxidants, 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 sensitises cancer cells to ferroptosis. Thus, we uncover a direct and actionable role for riboflavin in maintaining membrane integrity by promoting membrane tolerance to lipid peroxidation. 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 and their contributions to a cell’s antioxidant capacity. ### Competing Interest Statement M.C. and B.P. are co-founders and shareholders of ROSCUE Therapeutics GmbH. Deutsche Forschungsgemeinschaft, FR 3746/3-1, FR 3746/6-1, FR 3746/5-1, INST 269/886-1, 514894665 European Research Council, https://ror.org/0472cxd90, ERC-Consolidator, DeciFERR Deutsche José Carreras Leukämie-Stiftung, DJCLS 01 R/2022 São Paulo Research Foundation, 2023/04397-4

Replication stress is a driver of genomic instability, contributing to carcinogenesis by causing DNA damage and mutations. While YAP, the downstream co-activator of the Hippo signaling pathway, plays a crucial role in regulating cell growth and differentiation, it is unclear whether it generates replication stress exploitable for therapy. Here, we report that oncogenic YAP shortens the G1 phase through increased CDK4/6 activity, leading to early S-phase entry. This causes origin underlicensing, an overall reduced rate of DNA replication, and, unusually, an accelerated speed of individual replication forks. CHK1 inhibition in cells expressing oncogenic YAP results in DNA damage during S-phase, which is not due to premature CDK1 activation or mitotic entry. Sensitivity to CHK1 inhibition depends on the YAP-TEAD interaction and involves a global increase in transcription and an increase in transcription-replication conflicts (TRCs). Replication stress from oncogenic YAP can be mitigated by restoring G1 length through partial CDK4/6 inhibition or by reducing YAP-induced hypertranscription. Our findings suggest a potential therapeutic strategy for targeting YAP-dependent cancers by exploiting their vulnerability to replication stress.