TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during Development and in Aging.

Aging is a major risk factor for both genetic and sporadic neurodegenerative disorders. However, it is unclear how aging interacts with genetic predispositions to promote neurodegeneration. Here, we investigate how partial loss of function of TBK1, a major genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) comorbidity, leads to age-dependent neurodegeneration. We show that TBK1 is an endogenous inhibitor of RIPK1 and the embryonic lethality of Tbk1-/- mice is dependent on RIPK1 kinase activity. In aging human brains, another endogenous RIPK1 inhibitor, TAK1, exhibits a marked decrease in expression. We show that in Tbk1+/- mice, the reduced myeloid TAK1 expression promotes all the key hallmarks of ALS/FTD, including neuroinflammation, TDP-43 aggregation, axonal degeneration, neuronal loss, and behavior deficits, which are blocked upon inhibition of RIPK1. Thus, aging facilitates RIPK1 activation by reducing TAK1 expression, which cooperates with genetic risk factors to promote the onset of ALS/FTD.

TAK1 is an essential kinase for STING trafficking.

The translocation of stimulator of interferon genes (STING) from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC) enables its activation. However, the mechanism underlying the regulation of STING exit from the ER remains elusive. Here, we found that STING induces the activation of transforming growth factor beta-activated kinase 1 (TAK1) prior to STING trafficking in a TAK1 binding protein 1 (TAB1)-dependent manner. Intriguingly, activated TAK1 directly mediates STING phosphorylation on serine 355, which facilitates its interaction with STING ER exit protein (STEEP) and thereby promotes its oligomerization and translocation to the ERGIC for subsequent activation. Importantly, activation of TAK1 by monophosphoryl lipid A, a TLR4 agonist, boosts cGAMP-induced antitumor immunity dependent on STING phosphorylation in a mouse allograft tumor model. Taken together, TAK1 was identified as a checkpoint for STING activation by promoting its trafficking, providing a basis for combinatory tumor immunotherapy and intervention in STING-related diseases.

AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System.

AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis.

Tyrosine Kinase SYK Licenses MyD88 Adaptor Protein to Instigate IL-1α-Mediated Inflammatory Disease.

Mice carrying a hypomorphic point mutation in the Ptpn6 gene (Ptpn6spin mice) develop an inflammatory skin disease that resembles neutrophilic dermatosis in humans. Here, we demonstrated that interleukin-1α (IL-1α) signaling through IL-1R and MyD88 in both stromal and immune cells drive inflammation in Ptpn6spin mice. We further identified SYK as a critical kinase that phosphorylates MyD88, promoted MyD88-dependent signaling and mediates dermatosis in Ptpn6spin mice. Our studies further demonstrated that SHP1 encoded by Ptpn6 binds and suppresses SYK activation to inhibit MyD88 phosphorylation. Downstream of SHP1 and SYK-dependent counterregulation of MyD88 tyrosine phosphorylation, we have demonstrated that the scaffolding function of receptor interacting protein kinase 1 (RIPK1) and tumor growth factor-ß activated kinase 1 (TAK1)-mediating signaling were required to spur inflammatory disease. Overall, these studies identify SHP1 and SYK crosstalk as a critical regulator of MyD88 post-translational modifications and IL-1-driven inflammation.

Galectins Control mTOR in Response to Endomembrane Damage.

The Ser/Thr protein kinase mTOR controls metabolic pathways, including the catabolic process of autophagy. Autophagy plays additional, catabolism-independent roles in homeostasis of cytoplasmic endomembranes and whole organelles. How signals from endomembrane damage are transmitted to mTOR to orchestrate autophagic responses is not known. Here we show that mTOR is inhibited by lysosomal damage. Lysosomal damage, recognized by galectins, leads to association of galectin-8 (Gal8) with the mTOR apparatus on the lysosome. Gal8 inhibits mTOR activity through its Ragulator-Rag signaling machinery, whereas galectin-9 activates AMPK in response to lysosomal injury. Both systems converge upon downstream effectors including autophagy and defense against Mycobacterium tuberculosis. Thus, a novel galectin-based signal-transduction system, termed here GALTOR, intersects with the known regulators of mTOR on the lysosome and controls them in response to lysosomal damage. VIDEO ABSTRACT.

Small molecule activators of TAK1 promotes its activity-dependent ubiquitination and TRAIL-mediated tumor cell death.

TAK1 is a key modulator of both NF-κB signaling and RIPK1. In TNF signaling pathway, activation of TAK1 directly mediates the phosphorylation of IKK complex and RIPK1. In a search for small molecule activators of RIPK1-mediated necroptosis, we found R406/R788, two small molecule analogs that could promote sustained activation of TAK1. Treatment with R406 sensitized cells to TNF-mediated necroptosis and RIPK1-dependent apoptosis by promoting sustained RIPK1 activation. Using click chemistry and multiple biochemical binding assays, we showed that treatment with R406 promotes the activation of TAK1 by directly binding to TAK1, independent of its original target Syk kinase. Treatment with R406 promoted the ubiquitination of TAK1 and the interaction of activated TAK1 with ubiquitinated RIPK1. Finally, we showed that R406/R788 could promote the cancer-killing activities of TRAIL in vitro and in mouse models. Our studies demonstrate the possibility of developing small molecule TAK1 activators to potentiate the effect of TRAIL as anticancer therapies.

Aberrantly activated TAK1 links neuroinflammation and neuronal loss in Alzheimer's disease mouse models.

Neuroinflammation is causally associated with Alzheimer's disease (AD) pathology. Reactive glia cells secrete various neurotoxic factors that impair neuronal homeostasis eventually leading to neuronal loss. Although the glial activation mechanism in AD has been relatively well studied, how it perturbs intraneuronal signaling, which ultimately leads to neuronal cell death, remains poorly understood. Here, we report that compound stimulation with the neurotoxic factors TNF and glutamate aberrantly activates neuronal TAK1 (also known as MAP3K7), which promotes the pathogenesis of AD in mouse models. Glutamate-induced Ca2+ influx shifts TNF signaling to hyper-activate TAK1 enzymatic activity through Ca2+/calmodulin-dependent protein kinase II, which leads to necroptotic cellular damage. Genetic ablation and pharmacological inhibition of TAK1 ameliorated AD-associated neuronal loss and cognitive impairment in the AD model mice. Our findings provide a molecular mechanism linking cytokines, Ca2+ signaling and neuronal necroptosis in AD.

Humans pIKK-up NLRP3 to skip NEK7.

NLRP3 inflammasome regulation is essential for controlling cell death and inflammation. Mechanistic studies in murine cells suggest a two-step model of priming and activation with an indispensable role for NEK7. However, in a recent article in Immunity, Schmacke et al. report that, in humans, transcription-independent NLRP3 activation occurs by circumventing NEK7 via IKKß.

Deubiquitinase USP18 inhibits hepatic stellate cells activation and alleviates liver fibrosis via regulation of TAK1 activity.

BACKGROUND & AIMS: Activation of hepatic stellate cells (HSCs) is the key process underlying liver fibrosis. Unveiling its molecular mechanism may provide an effective target for inhibiting liver fibrosis. Protein ubiquitination is a dynamic and reversible process. Deubiquitinases (DUBs) catalyze the removal of ubiquitin chains from substrate proteins, thereby inhibiting the biological processes regulated by ubiquitination signals. However, there are few studies revealing the role of deubiquitination in the activation of HSCs. METHODS & RESULTS: Single-cell RNA sequencing (scRNA-seq) revealed significantly decreased USP18 expression in activated HSCs when compared to quiescent HSCs. In mouse primary HSCs, continuous activation of HSCs led to a gradual decrease in USP18 expression whilst restoration of USP18 expression significantly inhibited HSC activation. Injection of USP18 lentivirus into the portal vein of a CCl4-induced liver fibrosis mouse model confirmed that overexpression of USP18 can significantly reduce the degree of liver fibrosis. In terms of mechanism, we screened some targets of USP18 in mouse primary HSCs and found that USP18 could directly bind to TAK1. Furthermore, we demonstrated that USP18 can inhibit TAK1 activity by interfering with the K63 ubiquitination of TAK1. CONCLUSIONS: Our study demonstrated that USP18 inhibited HSC activation and alleviated liver fibrosis via modulation of TAK1 activity; this may prove to be an effective target for inhibiting liver fibrosis.

Emerging role of TAK1 in the regulation of skeletal muscle mass.

Maintenance of skeletal muscle mass and strength throughout life is crucial for heathy living and longevity. Several signaling pathways have been implicated in the regulation of skeletal muscle mass in adults. TGF-ß-activated kinase 1 (TAK1) is a key protein, which coordinates the activation of multiple signaling pathways. Recently, it was discovered that TAK1 is essential for the maintenance of skeletal muscle mass and myofiber hypertrophy following mechanical overload. Forced activation of TAK1 in skeletal muscle causes hypertrophy and attenuates denervation-induced muscle atrophy. TAK1-mediated signaling in skeletal muscle promotes protein synthesis, redox homeostasis, mitochondrial health, and integrity of neuromuscular junctions. In this article, we have reviewed the role and potential mechanisms through which TAK1 regulates skeletal muscle mass and growth. We have also proposed future areas of research that could be instrumental in exploring TAK1 as therapeutic target for improving muscle mass in various catabolic conditions and diseases.

Inhibition of phosphodiesterase 10A mitigates neuronal injury by modulating apoptotic pathways in cold-induced traumatic brain injury.

Brain injury develops from a complex series of pathophysiological phases, resulting in acute necrotic or delayed apoptotic cell death after traumatic brain injury (TBI). Inhibition of apoptotic cell death is critical for the treatment of acute neurodegenerative disorders, such as TBI. Here, we investigated the role of phosphodiesterase 10A (PDE10A) in the development of neuronal injury, particularly in apoptotic cell death. Using the PDE10A inhibitor TAK-063, we found that PDE10A inhibition is associated with decreased brain injury, brain swelling, and blood brain barrier disruption 48 h after cold-induced TBI. Furthermore, a particularly notable result was observed with 3 mg/kg TAK-063, which reduced disseminated neuronal injury. Protein abundance analysis revealed that PDE10A inhibition activates survival kinases AKT and ERK-1/-2, which were associated with the decreased activation of MMP-9 and PTEN. Additionally, iNOS and nNOS levels significantly reduced in the TAK-063 group, playing roles in inflammation and apoptosis. A planar surface immunoassay was performed for in-depth analyses of the apoptotic signaling pathways. We observed that inhibition of PDE10A resulted in the decreased expression of TNFRSF1A, TNFRSF10B, and TNFRSF6 receptors, particularly inducing apoptotic cell death. Moreover, these findings correlated with reduced levels of pro-apoptotic proteins, including PTEN, p27, Cytochrome-c, cleaved Caspase-3, Bad, and p53. Interestingly, TAK-063 treatment reduced levels of anti-apoptotic proteins or enzymes, including XIAP, Claspin, and HIF1α, without affecting Bcl-x, MCL-1, SMAC, HO-1, HO-2, HSP27, HSP60, and HSP70. The findings suggest that PDE10A regulates cellular signaling predominantly pro-apoptotic pathways, and inhibition of this protein is a promising approach for the treatment of acute brain injury.

The protein phosphatase PP6 promotes RIPK1-dependent PANoptosis.

BACKGROUND: The innate immune system serves as the first line of host defense. Transforming growth factor-ß-activated kinase 1 (TAK1) is a key regulator of innate immunity, cell survival, and cellular homeostasis. Because of its importance in immunity, several pathogens have evolved to carry TAK1 inhibitors. In response, hosts have evolved to sense TAK1 inhibition and induce robust lytic cell death, PANoptosis, mediated by the RIPK1-PANoptosome. PANoptosis is a unique innate immune inflammatory lytic cell death pathway initiated by an innate immune sensor and driven by caspases and RIPKs. While PANoptosis can be beneficial to clear pathogens, excess activation is linked to pathology. Therefore, understanding the molecular mechanisms regulating TAK1 inhibitor (TAK1i)-induced PANoptosis is central to our understanding of RIPK1 in health and disease. RESULTS: In this study, by analyzing results from a cell death-based CRISPR screen, we identified protein phosphatase 6 (PP6) holoenzyme components as regulators of TAK1i-induced PANoptosis. Loss of the PP6 enzymatic component, PPP6C, significantly reduced TAK1i-induced PANoptosis. Additionally, the PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 had redundant roles in regulating TAK1i-induced PANoptosis, and their combined depletion was required to block TAK1i-induced cell death. Mechanistically, PPP6C and its regulatory subunits promoted the pro-death S166 auto-phosphorylation of RIPK1 and led to a reduction in the pro-survival S321 phosphorylation. CONCLUSIONS: Overall, our findings demonstrate a key requirement for the phosphatase PP6 complex in the activation of TAK1i-induced, RIPK1-dependent PANoptosis, suggesting this complex could be therapeutically targeted in inflammatory conditions.

Efficacy and Safety of a Tetravalent Dengue Vaccine (TAK-003) in Children With Prior Japanese Encephalitis or Yellow Fever Vaccination.

BACKGROUND: We explored the impact of prior Yellow fever (YF) or Japanese encephalitis (JE) vaccination on the efficacy of Takeda's dengue vaccine candidate, TAK-003 (NCT02747927). METHODS: Children 4-16 years of age were randomized 2:1 to receive TAK-003 or placebo and were under active febrile surveillance. Symptomatic dengue was confirmed by serotype-specific RT-PCR. YF and JE vaccination history was recorded. RESULTS: Of the 20,071 children who received TAK-003 or placebo, 21.1% had a YF and 23.9% had a JE vaccination history at randomization. Fifty-seven months after vaccination, vaccine efficacy was 55.7% (95% CI, 39.7%-67.5%) in those with YF vaccination, 77.8% (70.8%-83.1%) for JE vaccination, and 53.5% (45.4%-60.4%) for no prior YF/JE vaccination. Regional differences in serotype distribution confound these results. The apparent higher vaccine efficacy in the JE vaccination subgroup could be largely explained by serotype-specific efficacy of TAK-003. Within 28 days of any vaccination, the proportions of participants with serious adverse events in the YF/JE prior vaccination population were comparable between the TAK-003 and placebo groups. CONCLUSIONS: The available data do not suggest a clinically relevant impact of prior JE or YF vaccination on TAK-003 performance. Overall, TAK-003 was well-tolerated and efficacious in different epidemiological settings.

An AP-3-dependent pathway directs phagosome fusion with Rab8 and Rab11 vesicles involved in TLR2 signaling.

Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.

The host antiviral protein SAMHD1 suppresses NF-κB activation by interacting with the IKK complex during inflammatory responses and viral infection.

Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) inhibits HIV-1 replication in nondividing cells by reducing the intracellular dNTP pool. SAMHD1 also suppresses NF-κB activation induced by inflammatory stimuli and viral infections. Specifically, SAMHD1-mediated reduction of NF-κB inhibitory protein (IκBα) phosphorylation is important for the suppression of NF-κB activation. However, while the inhibitors of NF-κB kinase subunit alpha and beta (IKKα and IKKß) regulate IκBα phosphorylation, the mechanism by which SAMHD1 regulates phosphorylation of IκBα remains unclear. Here, we report that SAMHD1 suppresses phosphorylation of IKKα/ß/γ via interaction with IKKα and IKKß, thus inhibiting subsequent phosphorylation of IκBα in monocytic THP-1 cells and differentiated nondividing THP-1 cells. We show that knockout of SAMHD1 enhanced phosphorylation of IKKα, IKKß, and IKKγ in THP-1 cells treated with the NF-κB activator lipopolysaccharide or infected with Sendai virus and SAMHD1 reconstitution inhibited phosphorylation of IKKα/ß/γ in Sendai virus-infected THP-1 cells. We demonstrate that endogenous SAMHD1 interacted with IKKα and IKKß in THP-1 cells and recombinant SAMHD1 bound to purified IKKα or IKKß directly in vitro. Mapping of these protein interactions showed that the HD domain of SAMHD1 interacts with both IKKα and IKKß and that the kinase domain of IKKα and the ubiquitin-like domain of IKKß are required for their interactions with SAMHD1, respectively. Moreover, we found that SAMHD1 disrupts the interaction between upstream kinase TAK1 and IKKα or IKKß. Our findings identify a new regulatory mechanism by which SAMHD1 inhibits phosphorylation of IκBα and NF-κB activation.

Immunogenicity, safety, and efficacy of a tetravalent dengue vaccine in children and adolescents: an analysis by age group.

BACKGROUND: Dengue is an increasing threat to global health. This exploratory analysis evaluated the immunogenicity, safety, and vaccine efficacy (VE) of a live-attenuated tetravalent dengue vaccine (TAK-003) in participants enrolled in the phase 3 DEN-301 trial (NCT02747927), stratified by baseline age (4-5 years; 6-11 years; or 12-16 years). METHODS: Participants were randomized 2:1 to receive 2 doses of TAK-003, administered 3 months apart, or placebo. Dengue serostatus was evaluated at enrolment. VE against virologically-confirmed dengue (VCD) and hospitalized VCD; immunogenicity (geometric mean titers; GMTs); and safety were evaluated per age group through ∼4 years post-vaccination. RESULTS: VE against VCD across serotypes was 43.5% (95% confidence interval: 25.3%, 57.3%) for 4-5 year-olds; 63.5% (56.9%, 69.1%) for 6-11 year-olds, and 67.7% (57.8%, 75.2%) for 12-16 year-olds. VE against hospitalized VCD was 63.8% (21.1%, 83.4%), 85.1% (77.1%, 90.3%), and 89.7% (77.9%, 95.2%), for the three age groups, respectively. GMTs remained elevated against all four serotypes for ∼4 years post-vaccination, with no evident differences across age groups. No clear differences in safety by age were identified. CONCLUSIONS: This exploratory analysis shows TAK-003 was efficacious in dengue prevention across age groups in children and adolescents 4-16 years of age living in dengue endemic areas. Relatively lower VE in 4-5 year-olds was potentially confounded by causative serotype distribution, small sample size, and VE by serotype, and should be considered in benefit-risk evaluations in this age group.

Smyd3 negatively regulates the anti-viral pathway by promoting TAK1 degradation in teleost fish.

IMPORTANCE: In this study, we have found that the existence of Smyd3 promoted the replication of SCRV. Additionally, we report that Smyd3 negatively regulates the NF-κB and IRF3 signaling pathway by facilitating the degradation of TAK1 in fish. Our findings suggest that Smyd3 interacts with TAK1. Further investigations have revealed that Smyd3 specifically mediates K48-linked ubiquitination of TAK1 and enhances TAK1 degradation, resulting in a significant inhibition of the NF-κB and IRF3 signaling pathway. These results not only contribute to the advancement of fish anti-viral immunity but also provide new evidence for understanding the mechanism of TAK1 in mammals.

Toll-Like Receptor 2 Attenuates the Formation and Progression of Angiotensin II-Induced Abdominal Aortic Aneurysm in ApoE-/- Mice.

INTRODUCTION: We demonstrated Toll-like receptor (TLR) 4 in the pathogenesis of angiotensin II (AngII)-mediated abdominal aortic aneurysm (AAA) formation. Here, we study TLR2 in the AAA formation. METHODS: Male ApoE-/- and ApoE-/-TLR2-/- mice were treated with AngII. Mice were injected with the TLR2 agonist Pam3CSK4. The incidence and severity of AAA were determined. MCP-1, MCP-5, RANTES, CXCL10, CCR5, and CXCR3 were analyzed. M1 and M2 macrophages in the aorta were detected by flow cytometry. RESULTS: These studies demonstrated an increase in AAA formation in TLR2-/- mice and a decrease by Pam3CSK4. Pam3CSK4 decreased the ratio of M1/M2 and the levels of RANTES, CXCL10, CCR5, and CXCR3. Furthermore, Pam3CSK4 treatment 1 week following AngII retarded the progression of AAA. CONCLUSION: These data demonstrated a protective effect of TLR2 signaling on AAA in association with a decrease in the ratio of M1 to M2 macrophages and the expression of chemokines and their receptors. Furthermore, the treatment of Pam3CSK4 after AngII demonstrated a marked retardation of lesion progression. Given the fact that most AAA patients are detected late in the disease process, these findings suggest that TLR2 stimulation may play a therapeutic role in retarding disease progression.

Dual-specificity phosphatase 26 protects against kidney injury caused by ischaemia-reperfusion through restraint of TAK1-JNK/p38-mediated apoptosis and inflammation of renal tubular epithelial cells.

Dual-specificity phosphatase 26 (DUSP26) acts as a pivotal player in the transduction of signalling cascades with its dephosphorylating activity. Currently, DUSP26 attracts extensive attention due to its particular function in several pathological conditions. However, whether DUSP26 plays a role in kidney ischaemia-reperfusion (IR) injury is unknown. Aims of the current work were to explore the relevance of DUSP26 in kidney IR damage. DUSP26 levels were found to be decreased in renal tubular epithelial cells following hypoxia-reoxygenation (HR) and kidney samples subjected to IR treatments. DUSP26-overexpressed renal tubular epithelial cells exhibited protection against HR-caused apoptosis and inflammation, while DUSP26-depleted renal tubular epithelial cells were more sensitive to HR damage. Upregulation of DUSP26 in rat kidneys by infecting adenovirus expressing DUSP26 markedly ameliorated kidney injury caused by IR, while also effectively reducing apoptosis and inflammation. The mechanistic studies showed that the activation of transforming growth factor-ß-activated kinase 1 (TAK1)-JNK/p38 MAPK, contributing to kidney injury under HR or IR conditions, was restrained by increasing DUSP26 expression. Pharmacological restraint of TAK1 markedly diminished DUSP26-depletion-exacebated effects on JNK/p38 activation and HR injury of renal tubular cells. The work reported a renal-protective function of DUSP26, which protects against IR-related kidney damage via the intervention effects on the TAK1-JNK/p38 axis. The findings laid a foundation for understanding the molecular pathogenesis of kidney IR injury and provide a prospective target for treating this condition.

Xanthine oxidoreductase mediates genotoxic drug-induced autophagy and apoptosis resistance by uric acid accumulation and TGF-ß-activated kinase 1 (TAK1) activation.

Autophagy is a highly conserved cellular process that profoundly impacts the efficacy of genotoxic chemotherapeutic drugs. TGF-ß-activated kinase 1 (TAK1) is a serine/threonine kinase that activates several signaling pathways involved in inducing autophagy and suppressing cell death. Xanthine oxidoreductase (XOR) is a rate-limiting enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid and hydrogen peroxide in the purine catabolism pathway. Recent studies showed that uric acid can bind to TAK1 and prolong its activation. We hypothesized that genotoxic drugs may induce autophagy and apoptosis resistance by activating TAK1 through XOR-generated uric acid. Here, we report that gemcitabine and 5-fluorouracil (5-FU), two genotoxic drugs, induced autophagy in HeLa and HT-29 cells by activating TAK1 and its two downstream kinases, AMP-activated kinase (AMPK) and c-Jun terminal kinase (JNK). XOR knockdown and the XOR inhibitor allopurinol blocked gemcitabine-induced TAK1, JNK, AMPK, and Unc51-like kinase 1 (ULK1)S555 phosphorylation and gemcitabine-induced autophagy. Inhibition of the ATM-Chk pathway, which inhibits genotoxic drug-induced uric acid production, blocked gemcitabine-induced autophagy by inhibiting TAK1 activation. Exogenous uric acid in its salt form, monosodium urate (MSU), induced autophagy by activating TAK1 and its downstream kinases JNK and AMPK. Gene knockdown or the inhibitors of these kinases blocked gemcitabine- and MSU-induced autophagy. Inhibition of autophagy by allopurinol, chloroquine, and 5Z-7-oxozeaenol (5Z), a TAK1-specific inhibitor, enhanced gemcitabine-induced apoptosis. Our study uncovers a previously unrecognized role of XOR in regulating genotoxic drug-induced autophagy and apoptosis and has implications for designing novel therapeutic strategies for cancer treatment.