Efficacy and safety of ivermectin in patients with mild COVID-19 in Japan and Thailand.

BACKGROUND: Ivermectin is an antiparasitic drug administered to hundreds of millions of people worldwide. Fundamental research suggests that ivermectin is effective against coronavirus disease 2019 (COVID-19); therefore, we investigated the efficacy and safety of ivermectin as a COVID-19 treatment option. METHODS: This multi-regional (Japan and Thailand), multicenter, placebo-controlled, randomized, double-blind, parallel-group, Phase III study evaluated the efficacy and safety of ivermectin in patients with mild COVID-19 (IVERMILCO Study). The participants took a specified number of the investigational product (ivermectin or placebo) tablets of, adjusted to a dose of 0.3-0.4 mg/kg, orally on an empty stomach once daily for three days. The primary efficacy endpoint was the time at which clinical symptoms first showed an improving trend by 168 h after investigational product administration. RESULTS: A total of 1030 eligible participants were assigned to receive the investigational product; 502 participants received ivermectin and 527 participants received a placebo. The primary efficacy endpoint was approximately 96 h (approximately four days) for both ivermectin and placebo groups, which did not show statistically significant difference (stratified log-rank test, p = 0.61). The incidence of adverse events and adverse drug reactions did not show statistically significant differences between the ivermectin and placebo groups (chi-square test, p = 0.97, p = 0.59). CONCLUSIONS: The results show that ivermectin (0.3-0.4 mg/kg), as a treatment for patients with mild COVID-19, is ineffective; however, its safety has been confirmed for participants, including minor participants of 12 years or older (IVERMILCO Study ClinicalTrials.gov number, NCT05056883.).

Genetic investigation of formaldehyde-induced DNA damage response in Schizosaccharomyces pombe.

Formaldehyde is a common environmental pollutant and is associated with adverse health effects. Formaldehyde is also considered to be a carcinogen because it can form DNA adducts, leading to genomic instability. How these adducts are prevented and removed is not fully understood. In this study, we used the fission yeast Schizosaccharomyces pombe as a model organism to investigate cellular tolerance pathways against formaldehyde exposure. We show that Fmd1 is a major formaldehyde dehydrogenase that functions to detoxify formaldehyde and that Fmd1 is critical to minimize formaldehyde-mediated DNA lesions. Our investigation revealed that nucleotide excision repair and homologous recombination have major roles in cellular tolerance to formaldehyde, while mutations in the Fanconi anemia, translesion synthesis, and base excision repair pathways also render cells sensitive to formaldehyde. We also demonstrate that loss of Wss1 or Wss2, proteases involved in the removal of DNA-protein crosslinks, sensitizes cells to formaldehyde and leads to replication defects. These results suggest that formaldehyde generates a variety of DNA lesions, including interstrand crosslinks, DNA-protein crosslinks, and base adducts. Thus, our genetic studies provide a framework for future investigation regarding health effects resulting from formaldehyde exposure.

Coordinated degradation of replisome components ensures genome stability upon replication stress in the absence of the replication fork protection complex.

The stabilization of the replisome complex is essential in order to achieve highly processive DNA replication and preserve genomic integrity. Conversely, it would also be advantageous for the cell to abrogate replisome functions to prevent inappropriate replication when fork progression is adversely perturbed. However, such mechanisms remain elusive. Here we report that replicative DNA polymerases and helicases, the major components of the replisome, are degraded in concert in the absence of Swi1, a subunit of the replication fork protection complex. In sharp contrast, ORC and PCNA, which are also required for DNA replication, were stably maintained. We demonstrate that this degradation of DNA polymerases and helicases is dependent on the ubiquitin-proteasome system, in which the SCF(Pof3) ubiquitin ligase is involved. Consistently, we show that Pof3 interacts with DNA polymerase ε. Remarkably, forced accumulation of replisome components leads to abnormal DNA replication and mitotic catastrophes in the absence of Swi1. Swi1 is known to prevent fork collapse at natural replication block sites throughout the genome. Therefore, our results suggest that the cell elicits a program to degrade replisomes upon replication stress in the absence of Swi1. We also suggest that this program prevents inappropriate duplication of the genome, which in turn contributes to the preservation of genomic integrity.

Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres.

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle-dependent recruitment of telomere-specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S-phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase epsilon (Polepsilon) arrived at telomeres earlier than the lagging strand DNA polymerases alpha (Polalpha) and delta (Poldelta). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polepsilon, whereas S-phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polalpha. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.

Comparative analysis of the catalytic components in the archaeal dye-linked L-proline dehydrogenase complexes.

Two types of hetero-oligomeric dye-linked L-proline dehydrogenases (α4ß4 and αßγδ types) are expressed in the hyperthermophilic archaea belonging to Thermococcales. In both enzymes, the ß subunit (PDHß) is responsible for catalyzing L-proline dehydrogenation. The genes encoding the two enzyme types form respective clusters that are completely conserved among Pyrococcus and Thermococcus strains. To compare the enzymatic properties of PDHßs from α4ß4- and αßγδ-type enzyme complexes, eight PDHßs (four of each type) from Pyrococcus furiosus DSM3638, Pyrococcus horikoshii OT-3, Thermococcus kodakaraensis KOD1 JCM12380 and Thermococcus profundus DSM9503 were expressed in Escherichia coli cells and purified to homogeneity using one-step Ni-chelating chromatography. The α4ß4-type PDHßs showed greater thermostability than most of the αßγδ-type PDHßs: the former retained more than 80 % of their activity after heating at 70 °C for 20 min, while the latter showed different thermostabilities under the same conditions. In addition, the α4ß4-type PDHßs utilized ferricyanide as the most preferable electron acceptor, whereas αßγδ-type PDHßs preferred 2, 6-dichloroindophenol, with one exception. These results indicate that the differences in the enzymatic properties of the PDHßs likely reflect whether they were from an αßγδ- or α4ß4-type complex, though the wider divergence observed within αßγδ-type PDHßs based on the phylogenetic analysis may also be responsible for their inconsistent enzymatic properties. By contrast, differences in the kinetic parameters among the PDHßs did not reflect the complex type. Interestingly, the k cat value for free α4ß4-type PDHß from P. horikoshii was much larger than the value for the same subunit within the α4ß4-complex. This indicates that the isolated PDHß could be a useful element for an electrochemical system for detection of L-proline.

The p53 DNA damage response and Fanconi anemia DNA repair pathway protect against acetaldehyde-induced replication stress in esophageal keratinocytes.

Alcohol contributes to cellular accumulation of acetaldehyde, a primary metabolite of alcohol and a major human carcinogen. Acetaldehyde can form DNA adducts and induce interstrand crosslinks (ICLs) that are repaired by the Fanconi anemia DNA repair pathway (FA pathway). Individuals with deficiency in acetaldehyde detoxification or in the FA pathway have an increased risk of squamous-cell carcinomas (SCCs) including those of the esophagus. In a recent report, we described the molecular basis of acetaldehyde-induced DNA damage in esophageal keratinocytes [1]. We demonstrated that, at physiologically relevant concentrations, acetaldehyde induces DNA damage at the DNA replication fork. This resulted in replication stress, leading to activation of the ATR-Chk1-dependent cell cycle checkpoints. We also reported that the p53 DNA damage response is elevated in response to acetaldehyde and that the FA pathway limits acetaldehyde-induced genomic instability. Here, we highlight these findings and present additional results to discuss the role of the FA pathway and p53 DNA damage response in the protection against genomic instability and esophageal carcinogenesis.

Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion.

The Timeless-Tipin protein complex has been reported to be important for replication checkpoint and normal DNA replication processes. However, the precise mechanisms by which Timeless-Tipin preserves genomic integrity are largely unclear. Here, we describe the roles of Timeless-Tipin in replication fork stabilization and sister chromatid cohesion. We show in human cells that Timeless is recruited to replication origin regions and dissociate from them as replication proceeds. Cdc45, which is known to be required for replication fork progression, shows similar patterns of origin association to those of Timeless. Depletion of Timeless-Tipin causes chromosome fragmentation and defects in damage repair in response to fork collapse, suggesting that it is required for replication fork maintenance under stress. We also demonstrate that depletion of Timeless-Tipin impairs sister chromatid cohesion and causes a defect in mitotic progression. Consistently, Timeless-Tipin co-purifies with cohesin subunits and is required for their stable association with chromatin during S phase. Timeless associates with the cohesion-promoting DNA helicase ChlR1, which, when overexpressed, partially alleviates the cohesion defect of cells depleted of Timeless-Tipin. These results suggest that Timeless-Tipin functions as a replication fork stabilizer that couples DNA replication with sister chromatid cohesion established at replication forks.

Maf1 limits RNA polymerase III-directed transcription to preserve genomic integrity and extend lifespan.

A key to longevity assurance is the nutrient-sensing mTOR pathway. Inhibition of mTOR extends lifespan in a variety of organisms. However, the downstream effectors of the mTOR pathway for lifespan regulation are elusive. In a recent report, we described the role of Maf1 as a critical lifespan regulator downstream of the mTOR pathway in fission yeast. Maf1 is the master negative regulator of RNA polymerase III-directed transcription (e.g. tRNAs and 5S rRNAs) and is regulated by mTOR-mediated phosphorylation. We demonstrated that Maf1 is required for lifespan extension under calorie restriction or when mTOR is inhibited. We also showed that Maf1 prevents DNA damage at tRNA genes, which appears to contribute to lifespan maintenance by Maf1. Here we highlight these observations and present additional results to discuss the role of the mTOR-Maf1-Pol III axis in promoting genomic integrity in the face of DNA replication-transcription conflicts in order to maintain normal lifespan.

FANCD2 limits acetaldehyde-induced genomic instability during DNA replication in esophageal keratinocytes.

Individuals with Fanconi anemia (FA), a rare genetic bone marrow failure syndrome, have an increased risk of young-onset head and neck squamous cell carcinomas (SCCs) and esophageal SCC. The FA DNA repair pathway is activated upon DNA damage induced by acetaldehyde, a chief alcohol metabolite and one of the major carcinogens in humans. However, the molecular basis of acetaldehyde-induced genomic instability in SCCs of the head and neck and of the esophagus in FA remains elusive. Here, we report the effects of acetaldehyde on replication stress response in esophageal epithelial cells (keratinocytes). Acetaldehyde-exposed esophageal keratinocytes displayed accumulation of DNA damage foci consisting of 53BP1 and BRCA1. At physiologically relevant concentrations, acetaldehyde activated the ATR-Chk1 pathway, leading to S- and G2/M-phase delay with accumulation of the FA complementation group D2 protein (FANCD2) at the sites of DNA synthesis, suggesting that acetaldehyde impedes replication fork progression. Consistently, depletion of the replication fork protection protein Timeless led to elevated DNA damage upon acetaldehyde exposure. Furthermore, FANCD2 depletion exacerbated replication abnormalities, elevated DNA damage, and led to apoptotic cell death, indicating that FANCD2 prevents acetaldehyde-induced genomic instability in esophageal keratinocytes. These observations contribute to our understanding of the mechanisms that drive genomic instability in FA patients and alcohol-related carcinogenesis, thereby providing a translational implication in the development of more effective therapies for SCCs.

Maf1-dependent transcriptional regulation of tRNAs prevents genomic instability and is associated with extended lifespan.

Maf1 is the master repressor of RNA polymerase III responsible for transcription of tRNAs and 5S rRNAs. Maf1 is negatively regulated via phosphorylation by the mTOR pathway, which governs protein synthesis, growth control, and lifespan regulation in response to nutrient availability. Inhibiting the mTOR pathway extends lifespan in various organisms. However, the downstream effectors for the regulation of cell homeostasis that are critical to lifespan extension remain elusive. Here we show that fission yeast Maf1 is required for lifespan extension. Maf1's function in tRNA repression is inhibited by mTOR-dependent phosphorylation, whereas Maf1 is activated via dephosphorylation by protein phosphatase complexes, PP4 and PP2A. Mutational analysis reveals that Maf1 phosphorylation status influences lifespan, which is correlated with elevated tRNA and protein synthesis levels in maf1∆ cells. However, mTOR downregulation, which negates protein synthesis, fails to rescue the short lifespan of maf1∆ cells, suggesting that elevated protein synthesis is not a cause of lifespan shortening in maf1∆ cells. Interestingly, maf1∆ cells accumulate DNA damage represented by formation of Rad52 DNA damage foci and Rad52 recruitment at tRNA genes. Loss of the Rad52 DNA repair protein further exacerbates the shortened lifespan of maf1∆ cells. Strikingly, PP4 deletion alleviates DNA damage and rescues the short lifespan of maf1∆ cells even though tRNA synthesis is increased in this condition, suggesting that elevated DNA damage is the major cause of lifespan shortening in maf1∆ cells. We propose that Maf1-dependent inhibition of tRNA synthesis controls fission yeast lifespan by preventing genomic instability that arises at tRNA genes.

Chromatin immunoprecipitation of replication factors moving with the replication fork.

Replication of chromosomes involves a variety of replication proteins including DNA polymerases, DNA helicases, and other accessory factors. Many of these proteins are known to localize at replication forks and travel with them as components of the replisome complex. Other proteins do not move with replication forks but still play an essential role in DNA replication. Therefore, in order to understand the mechanisms of DNA replication and its controls, it is important to examine localization of each replication factor. Here we describe a chromatin immunoprecipitation (ChIP) method to locate a replication factor at the replication fork. Defining the localization of replication proteins should provide important insight into mechanistic understanding of the regulation of the DNA replication process.

Assays used to study the DNA replication checkpoint in fission yeast.

The DNA replication checkpoint, also known as the intra-S or S-phase checkpoint, plays a central role in ensuring the accuracy of DNA replication. When replication is impeded by DNA damage or other conditions, this checkpoint delays cell cycle progression and coordinates resumption of replication with DNA repair pathways. One of its critical functions is to stabilize stalled replication forks in a replication-competent state, presumably by maintaining proper assembly of replisome components and preserving DNA structures. Here we describe a series of assays used to study the replication checkpoint. These assays allow us to investigate the specific functions of proteins involved in the replication checkpoint in fission yeast.

Rad22Rad52-dependent repair of ribosomal DNA repeats cleaved by Slx1-Slx4 endonuclease.

Slx1 and Slx4 are subunits of a structure-specific DNA endonuclease that is found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other eukaryotic species. It is thought to initiate recombination events or process recombination structures that occur during the replication of the tandem repeats of the ribosomal DNA (rDNA) locus. Here, we present evidence that fission yeast Slx1-Slx4 initiates homologous recombination events in the rDNA repeats that are processed by a mechanism that requires Rad22 (Rad52 homologue) but not Rhp51 (Rad51 homologue). Slx1 is required to generate approximately 50% of the spontaneous Rad22 DNA repair foci that occur in cycling cells. Most of these foci colocalize with the nucleolus, which contains the rDNA repeats. The increased fork pausing at the replication fork barriers in the rDNA repeats in a strain that lacks Rqh1 DNA helicase is further increased by expression of a dominant negative form of Slx1. These data suggest that Slx1-Slx4 cleaves paused replication forks in the rDNA, leading to Rad22-dependent homologous recombination that is used to maintain rDNA copy number.

The NuA4 acetyltransferase and histone H4 acetylation promote replication recovery after topoisomerase I-poisoning.

BACKGROUND: Histone acetylation plays an important role in DNA replication and repair because replicating chromatin is subject to dynamic changes in its structures. However, its precise mechanism remains elusive. In this report, we describe roles of the NuA4 acetyltransferase and histone H4 acetylation in replication fork protection in the fission yeast Schizosaccharomyces pombe. RESULTS: Downregulation of NuA4 subunits renders cells highly sensitive to camptothecin, a compound that induces replication fork breakage. Defects in NuA4 function or mutations in histone H4 acetylation sites lead to impaired recovery of collapsed replication forks and elevated levels of Rad52 DNA repair foci, indicating the role of histone H4 acetylation in DNA replication and fork repair. We also show that Vid21 interacts with the Swi1-Swi3 replication fork protection complex and that Swi1 stabilizes Vid21 and promotes efficient histone H4 acetylation. Furthermore, our genetic analysis demonstrates that loss of Swi1 further sensitizes NuA4 and histone H4 mutant cells to replication fork breakage. CONCLUSION: Considering that Swi1 plays a critical role in replication fork protection, our results indicate that NuA4 and histone H4 acetylation promote repair of broken DNA replication forks.

Sap1 promotes the association of the replication fork protection complex with chromatin and is involved in the replication checkpoint in Schizosaccharomyces pombe.

Sap1 is involved in replication fork pausing at rDNA repeats and functions during mating-type switching in Schizosaccharomyces pombe. These two roles are dependent on the ability of Sap1 to bind specific DNA sequences at the rDNA and mating-type loci, respectively. In S. pombe, Swi1 and Swi3 form the replication fork protection complex (FPC) and play important roles in the activation of the replication checkpoint and the stabilization of stalled replication forks. Here we describe the roles of Sap1 in the replication checkpoint. We show that Sap1 is involved in the activation of the replication checkpoint kinase Cds1 and that sap1 mutant cells accumulate spontaneous DNA damage during the S- and G2-phases, which is indicative of fork damage. We also show that sap1 mutants have a defect in the resumption of DNA replication after fork arrest. Sap1 is localized at the replication origin ori2004 and this localization is required for the association of the FPC with chromatin. We propose that Sap1 is required to recruit the FPC to chromatin, thereby contributing to the activation of the replication checkpoint and the stabilization of replication forks.

CDK phosphorylation of Drc1 regulates DNA replication in fission yeast.

Cyclin-dependent kinases (CDKs) are absolutely required for DNA replication in eukaryotic cells. CDKs are thought to activate one or more replication factors, but the identities of these proteins are unknown. Here we describe fission yeast Drc1, a protein required for DNA replication that is phosphorylated by Cdc2. Drc1 depletion leads to catastrophic mitotic divisions with incompletely replicated DNA, indicating that Drc1 is required for DNA synthesis and S-M replication checkpoint control. Drc1 associates with Cdc2 and is phosphorylated at the onset of S phase when Cdc2 is activated. Mutant Drc1 that lacks CDK phosphorylation sites is nonfunctional and fails to interact with Cut5 replication factor. These data suggest that Cdc2 promotes DNA replication by phosphorylating Drc1 and regulating its association with Cut5.

Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1.

The replication checkpoint is a dedicated sensor-response system activated by impeded replication forks. It stabilizes stalled forks and arrests division, thereby preserving genome integrity and promoting cell survival. In budding yeast, Tof1 is thought to act as a specific mediator of the replication checkpoint signal that activates the effector kinase Rad53. Here we report studies of fission yeast Swi1, a Tof1-related protein required for a programmed fork-pausing event necessary for mating type switching. Our studies have shown that Swi1 is vital for proficient activation of the Rad53-like checkpoint kinase Cds1. Together they are required to prevent fork collapse in the ribosomal DNA repeats, and they also prevent irreversible fork arrest at a newly identified hydroxyurea pause site. Swi1 also has Cds1-independent functions. Rad22 DNA repair foci form during S phase in swi1 mutants and to a lesser extent in cds1 mutants, indicative of fork collapse. Mus81, a DNA endonuclease required for recovery from collapsed forks, is vital in swi1 but not cds1 mutants. Swi1 is recruited to chromatin during S phase. We propose that Swi1 stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast.

Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+). The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.

Genetic controls of DNA damage avoidance in response to acetaldehyde in fission yeast.

Acetaldehyde, a primary metabolite of alcohol, forms DNA adducts and disrupts the DNA replication process, causing genomic instability, a hallmark of cancer. Indeed, chronic alcohol consumption accounts for approximately 3.6% of all cancers worldwide. However, how the adducts are prevented and repaired after acetaldehyde exposure is not well understood. In this report, we used the fission yeast Schizosaccharomyces pombe as a model organism to comprehensively understand the genetic controls of DNA damage avoidance in response to acetaldehyde. We demonstrate that Atd1 functions as a major acetaldehyde detoxification enzyme that prevents accumulation of Rad52-DNA repair foci, while Atd2 and Atd3 have minor roles in acetaldehyde detoxification. We found that acetaldehyde causes DNA damage at the replication fork and activates the cell cycle checkpoint to coordinate cell cycle arrest with DNA repair. Our investigation suggests that acetaldehyde-mediated DNA adducts include interstrand-crosslinks and DNA-protein crosslinks. We also demonstrate that acetaldehyde activates multiple DNA repair pathways. Nucleotide excision repair and homologous recombination, which are both epistatically linked to the Fanconi anemia pathway, have major roles in acetaldehyde tolerance, while base excision repair and translesion synthesis also contribute to the prevention of acetaldehyde-dependent genomic instability. We also show the involvement of Wss1-related metalloproteases, Wss1 and Wss2, in acetaldehyde tolerance. These results indicate that acetaldehyde causes cellular stresses that require cells to coordinate multiple cellular processes in order to prevent genomic instability. Considering that acetaldehyde is a human carcinogen, our genetic studies serve as a guiding investigation into the mechanisms of acetaldehyde-dependent genomic instability and carcinogenesis.

ALDH2 modulates autophagy flux to regulate acetaldehyde-mediated toxicity thresholds.

A polymorphic mutation in the acetaldehyde dehydrogenase 2 (ALDH2) gene has been epidemiologically linked to the high susceptibility to esophageal carcinogenesis for individuals with alcohol use disorders. Mice subjected to alcohol drinking show increased oxidative stress and DNA adduct formation in esophageal epithelia where Aldh2 loss augments alcohol-induced genotoxic effects; however, it remains elusive as to how esophageal epithelial cells with dysfunctional Aldh2 cope with oxidative stress related to alcohol metabolism. Here, we investigated the role of autophagy in murine esophageal epithelial cells (keratinocytes) exposed to ethanol and acetaldehyde. We find that ethanol and acetaldehyde trigger oxidative stress via mitochondrial superoxide in esophageal keratinocytes. Aldh2-deficient cells appeared to be highly susceptible to ethanol- or acetaldehyde-mediated toxicity. Alcohol dehydrogenase-mediated acetaldehyde production was implicated in ethanol-induced cell injury in Aldh2 deficient cells as ethanol-induced oxidative stress and cell death was partially inhibited by 4-methylpyrazole. Acetaldehyde activated autophagy flux in esophageal keratinocytes where Aldh2 deficiency increased dependence on autophagy to cope with ethanol-induced acetaldehyde-mediated oxidative stress. Pharmacological inhibition of autophagy flux by chloroquine stabilized p62/SQSTM1, and increased basal and acetaldehyde-mediate oxidative stress in Aldh2 deficient cells as documented in monolayer culture as well as single-cell derived three-dimensional esophageal organoids, recapitulating a physiological esophageal epithelial proliferation-differentiation gradient. Our innovative approach indicates, for the first time, that autophagy may provide cytoprotection to esophageal epithelial cells responding to oxidative stress that is induced by ethanol and its major metabolite acetaldehyde. Defining autophagymediated cytoprotection against alcohol-induced genotoxicity in the context of Aldh2 deficiency, our study provides mechanistic insights into the tumor suppressor functions of ALDH2 and autophagy in alcohol-related esophageal carcinogenesis.