Drug conjugates for the treatment of lung cancer: from drug discovery to clinical practice.

A drug conjugate consists of a cytotoxic drug bound via a linker to a targeted ligand, allowing the targeted delivery of the drug to one or more tumor sites. This approach simultaneously reduces drug toxicity and increases efficacy, with a powerful combination of efficient killing and precise targeting. Antibody‒drug conjugates (ADCs) are the best-known type of drug conjugate, combining the specificity of antibodies with the cytotoxicity of chemotherapeutic drugs to reduce adverse reactions by preferentially targeting the payload to the tumor. The structure of ADCs has also provided inspiration for the development of additional drug conjugates. In recent years, drug conjugates such as ADCs, peptide‒drug conjugates (PDCs) and radionuclide drug conjugates (RDCs) have been approved by the Food and Drug Administration (FDA). The scope and application of drug conjugates have been expanding, including combination therapy and precise drug delivery, and a variety of new conjugation technology concepts have emerged. Additionally, new conjugation technology-based drugs have been developed in industry. In addition to chemotherapy, targeted therapy and immunotherapy, drug conjugate therapy has undergone continuous development and made significant progress in treating lung cancer in recent years, offering a promising strategy for the treatment of this disease. In this review, we discuss recent advances in the use of drug conjugates for lung cancer treatment, including structure-based drug design, mechanisms of action, clinical trials, and side effects. Furthermore, challenges, potential approaches and future prospects are presented.

Clinical and genetic interpretation of uncertain DMD missense variants: evidence from mRNA and protein studies.

BACKGROUND: Pathogenic missense variants in the dystrophin (DMD) gene are rarely reported in dystrophinopathies. Most DMD missense variants are of uncertain significance and their pathogenicity interpretation remains complicated. We aimed to investigate whether DMD missense variants would cause aberrant splicing and re-interpret their pathogenicity based on mRNA and protein studies. METHODS: Nine unrelated patients who had an elevated serum creatine kinase level with or without muscle weakness were enrolled. They underwent a detailed clinical, imaging, and pathological assessment. Routine genetic testing and muscle-derived mRNA and protein studies of dystrophin and sarcoglycan genes were performed in them. RESULTS: Three of the 9 patients presented with a Duchenne muscular dystrophy (DMD) phenotype and the remaining 6 patients had a suspected diagnosis of Becker muscular dystrophy (BMD) or sarcoglycanopathy based on their clinical and pathological characteristics. Routine genetic testing detected only 9 predicted DMD missense variants in them, of which 6 were novel and interpreted as uncertain significance. Muscle-derived mRNA studies of sarcoglycan genes didn't reveal any aberrant transcripts in them. Dystrophin mRNA studies confirmed that 3 predicted DMD missense variants (c.2380G > C, c.4977C > G, and c.5444A > G) were in fact splicing and frameshift variants due to aberrant splicing. The 9 DMD variants were re-interpreted as pathogenic or likely pathogenic based on mRNA and protein studies. Therefore, 3 patients with DMD splicing variants and 6 patients with confirmed DMD missense variants were diagnosed with DMD and BMD, respectively. CONCLUSION: Our study highlights the importance of muscle biopsy and aberrant splicing for clinical and genetic interpretation of uncertain DMD missense variants.

Simultaneous Activation of Immunogenic Cell Death and cGAS-STING Pathway by Liver- and Mitochondria-Targeted Gold(I) Complexes for Chemoimmunotherapy of Hepatocellular Carcinoma.

Induction of immunogenic cell death (ICD) and activation of the cyclic GMP-AMP synthase stimulator of interferon gene (cGAS-STING) pathway are two potent anticancer immunotherapeutic strategies in hepatocellular carcinoma (HCC). Herein, 12 liver- and mitochondria-targeting gold(I) complexes (9a-9l) were designed and synthesized. The superior complex 9b produced a considerable amount of reactive oxygen species (ROS) and facilitated DNA excretion, the ROS-induced ICD and DNA activated the cGAS-STING pathway, both of which evoked an intense anticancer immune response in vitro and in vivo. Importantly, 9b strongly inhibited tumor growth in a patient-derived xenograft model of HCC. Overall, we present the first case of simultaneous ICD induction and cGAS-STING pathway activation within the same gold-based small molecule, which may provide an innovative strategy for designing chemoimmunotherapies for HCC.

NSAID-Au(I) Complexes Induce ROS-Driven DAMPs and Interpose Inflammation to Stimulate the Immune Response against Ovarian Cancer.

Inflammation contributes to the development of ovarian cancer, and chemoresistance is a principal obstacle in ovarian cancer treatment. Herein, we designed and synthesized a series of gold(I) complexes derived from NSAIDs or their analogues. Among them, complex B3 (Npx-Au) displayed higher antitumor activity than cisplatin and other gold(I) complexes. Npx-Au could induce oxidative stress and the damage-associated molecular patterns (DAMPs) process by the inhibition of TrxR activity. Mechanistic studies revealed that simultaneous downregulation of COX-2 and PD-L1 was observed after Npx-Au treatment. Interestingly, in vivo experiments demonstrated that Npx-Au treatment could stimulate the immune response via reducing the expression of PD-L1, inducing DC maturation and increasing the infiltration of T (CD4+ and CD8+) cells. Collectively, our studies found that the gold(I) complex Npx-Au could elicit immunogenic cell death (ICD) and provide a promising strategy for chemotherapy combined with immunotherapy in the treatment of ovarian cancer.

Association of smoking with brain gray and white matter volume: a Mendelian randomization study.

BACKGROUND: Observational studies have found a significant association between smoking and smaller gray matter volume, but this finding was limited by the reverse causality bias and possible confounding factors. Therefore, we conducted a Mendelian randomization (MR) study to explore the causal association of smoking with brain gray and white matter volume from a genetic perspective, and to investigate the possible mediators influencing the association. METHODS: Smoking initiation (ever being a regular smoker) was used as the primary exposure from the GWAS & Sequencing Consortium of Alcohol and Nicotine use in up to 1,232,091 individuals of European descent. Their associations with brain volume were acquired from a recent genome-wide association study of brain imaging phenotypes conducted among 34,298 individuals of the UK Biobank. The random-effects inverse-variance weighted method was applied as the main analysis. Multivariable MR analysis was performed to assess the potential interference of confounding factors on causal effect. RESULTS: Genetic liability to smoking initiation was significantly associated with lower gray matter volume (beta, -0.100; 95% CI, -0.156 to -0.043; P=5.23×10-4) but not with white matter volume. Multivariable MR results suggested that the association with lower gray matter volume might be mediated by alcohol drinking. Regarding localized gray matter volume, genetic liability to smoking initiation was associated with lower gray matter volume in left superior temporal gyrus, anterior division and right superior temporal gyrus, posterior division. CONCLUSIONS: This MR study supports the association between smoking and lower gray matter volume, and highlights the importance of never smoking.

Design, synthesis and biological evaluation of fluorinated selective estrogen receptor degraders (FSERDs) --- A promising strategy for advanced ER positive breast cancer.

Although endocrine therapies involving pharmaceuticals, such as tamoxifen and aromatase inhibitors, had initially demonstrated good responses in patients with estrogen receptor-positive (ER+) breast cancer, they often led to drug resistance. ER plays a vital role in the progression of metastatic diseases. Fulvestrant, a first generation selective estrogen receptor degrader (SERD), can effectively downregulate the ER protein and inhibit its downstream signaling pathways. However, as the drug needs to be intramuscularly injected, its widespread use is limited owing to poor patient compliance. Herein, we described a novel class of orally bioavailable fluorine-substituted SERDs that exhibit improved pharmacokinetic profiles. We substituted the hydroxyl group of clinical SERD candidate 6 with a fluorine atom to diminish phase II metabolism. The subsequent structure-activity relationship (SAR) investigation identified 22h and 27b, which can effectively degrade ER in a dose-dependent manner and exhibit considerable antiproliferative potency and efficacy in vitro and in vivo. The excellent pharmacokinetic profiles of 27b render it promising candidate of clinically useful oral SERD.

SERD-NHC-Au(I) complexes for dual targeting ER and TrxR to induce ICD in breast cancer.

The development of selective estrogen receptor degraders (SERDs) has brought new ideas for the clinical treatment of ER-positive advanced breast cancer. The successful application of combinational therapy inspired the exploration of other targets to prevent breast cancer progression. Thioredoxin reductase (TrxR) is an important enzyme that can regulate redox balance in cells and it was considered as a potential target for anticancer treatment. In this study, we firstly combine a clinical SERD candidate--G1T48 (NCT03455270), with a TrxR inhibitor--N-heterocyclic carbene gold(I) [NHC-Au(I)] to form dual targeting complexes that can regulate both signaling pathways. The most efficacious complex 23 exhibited significant antiproliferative profile through degrading ER and inhibiting TrxR activity. Interestingly, it can induce immunogenic cell death (ICD) caused by ROS. This is the first evidence to elucidate the role of ER/TrxR-ROS-ICD axis in ER positive breast cancer and this research may inspire new drug development with novel mechanisms. The in vivo xenograft study demonstrated that complex 23 had excellent antiproliferative activity toward MCF-7 cells in mice model.

Research Progress on Bioactive Metal Complexes against ER-Positive Advanced Breast Cancer.

Breast cancer is the most prevalent cancer in women and represents a serious disease that is harmful to life and health. In 1977, with the approval of tamoxifen, endocrine therapy has become the main clinical treatment for ER-positive (ER+) breast cancer. Although patients initially respond well to endocrine therapies, drug resistance often emerges and side effects can be challenging. To overcome drug resistance, the exploration for new drugs is a priority. Metal complexes have demonstrated significant antitumor activities, and platinum complexes are widely used in the clinic against various cancers, including breast cancer. In this Perspective, the first section describes the classification and mechanism of endocrine therapy drugs for ER+ breast cancer, and the second section summarizes research since 2000 into metal complexes with activity toward ER+ breast cancer. Finally, we discuss the opportunities, challenges, and future directions for metal complexes in the treatment of ER+ breast cancer.

Tumor-Targeting NHC-Au(I) Complex Induces Immunogenic Cell Death in Hepatocellular Carcinoma.

Immunogenic cell death (ICD) is a promising direction of cancer immunotherapy in hepatocellular carcinoma (HCC). A series of novel NHC-Au(I) complexes derived from 4,5-diarylimidazole, containing glycyrrhetinic acid (GA) as an efficient targeting ligand for HCC, were herein designed and synthesized. Among these, complex 4C exhibited excellent effectiveness for tumor targeting and antitumor activity, which induced the occurrence of ICD in HCC cells. Additionally, 4C can effectively inhibit TrxR enzyme activity, increase reactive oxygen species (ROS) expression, lead to redox homeostasis disorder, mediate mitochondrial dysfunction and endoplasmic reticulum stress (ERS), and cause the characteristic discharge of damage-associated molecular patterns (DAMPs) in HCC cells. More importantly, 4C showed a great ICD-inducing effect in a vaccination mouse model and activated antitumor immunity in a tumor-bearing C57BL/6 mouse model, which is consistent with the in vitro results. In conclusion, we found the potential of Au(I) complex with HCC-targeted capability for effective tumor immunotherapy.

Induction of immunogenic cell death by novel platinum-based anticancer agents.

Traditional platinum-based anticancer drugs, led by cisplatin, play an important role in chemotherapy. However, the development of platinum compounds is limited due to serious toxicity and side effects. In recent years, studies have showed that immunogenic cell death (ICD) may be one of the potential action mechanisms of classical platinum drugs, such as oxaliplatin. This strategy combining chemotherapy and immunotherapy can effectively utilize the body's immune system to help platinum compounds to fight against tumors, and the dose can be appropriately reduced to limit toxic side effects. The induction of ICD by platinum compounds has become a research hotspot and one of the future development directions of metal drugs. Here, the progress of platinum compounds were collected and comprehensively summarized, their capacity of ICD induction and mechanism of action are exposed, providing reference for the design and synthesis of new anticancer platinum ICD inducers.

CCL24/CCR3 axis plays a central role in angiotensin II-induced heart failure by stimulating M2 macrophage polarization and fibroblast activation.

AIMS: We aimed to investigate the effect and mechanism of pleiotropic chemokine CCL24 in heart failure. METHODS AND RESULTS: Compared with normal donators, the expression of CCL24 and number of cardiac M2 macrophages in heart were higher in heart failure patients, the same as plasma CCL24. Treatment with CCL24 antibody hindered Ang II (1500 ng/kg/min)-induced cardiac adverse remodeling through preventing cardiac hypertrophy and fibrosis. RNA-seq showed that CCL24/CCR3 axis was involved in immune and inflammatory responses. Single-cell analysis of cytometry by time of flight (CyTOF) revealed that CCL24 antibody decreased the M2 macrophage and monocyte polarization during Ang II stimulation. Immunofluorescence co-localization analysis confirmed the expression of CCR3 in macrophage and fibroblasts. Then, in vitro experiments confirmed that CCL24/CCR3 axis was also involved in cardiac primary fibroblast activation through its G protein-coupled receptor function. CONCLUSION: CCL24/CCR3 axis plays a crucial part in cardiac remodeling by stimulating M2 macrophage polarization and cardiac fibroblast activation. Cardiac M2 macrophages, CCL24 and circulation CCL24 increased in heart failure patients. Treatment with CCL24 Ab hindered Ang II induced cardiac structural dysfunction and electrical remodeling. In CCL24 Ab group RNA-seq found that it was related to immune responses and hypertrophic cardiomyopathy, CytoF revealed M2 macrophages and monocytes decreased obviously. In vitro,CCL24 promoted activation and migration of cardiac fibroblast.

Association of cannabis use disorder with cardiovascular diseases: A two-sample Mendelian randomization study.

Background: The use of cannabis has increased globally due to more regions decriminalizing marijuana use for therapeutic and recreational aims. Several observational studies have revealed that cannabis use is associated with an increased risk of adverse cardiovascular pathologies and diseases. Nevertheless, the causal associations between cannabis use and cardiovascular diseases remain unclear. Hence, we performed single-variable and multivariable Mendelian randomization (MR) to evaluate the association between cannabis use disorder and various cardiovascular diseases. Materials and methods: Summary statistics were collected from the largest-to-date genome-wide association studies (GWAS) of cannabis use disorder. The 12 SNPs for cannabis use disorder were used as instrumental variables in this study. MR estimates were pooled using a random-effects inverse-variance weighted (IVW) method. Simple median and weighted median methods were conducted as sensitivity analyses. Results: The genetic liability to cannabis use disorder was associated with an augmented risk of coronary artery disease, myocardial infarction, atrial fibrillation, heart failure, deep venous thrombosis, pulmonary embolism, and stroke. Except for stroke, the results were inconsistent in the sensitivity analyses. The overall patterns for the associations of cannabis use disorder with atrial fibrillation, heart failure, pulmonary embolism and stroke remained in multivariable MR analyses adjusting for potential mediators, including smoking, alcohol, body mass index, blood lipid, type 2 diabetes, hypertension, and depression. However, the association with coronary artery disease, myocardial infarction, and deep venous thrombosis did not persist in multivariable MR analyses. Mediation analysis demonstrated that smoking, body mass index, low-density lipoprotein, hypertension, and depression have more significant mediation effects, which suggests that these factors partly mediate the link from cannabis use disorder to coronary artery disease, myocardial infarction, and deep venous thrombosis. Conclusion: The genetic liability to cannabis use disorder was associated with a higher risk of atrial fibrillation, heart failure, pulmonary embolism, and stroke. The evidence for the association between cannabis use disorder, coronary artery disease, myocardial infarction, and deep venous thrombosis was weak. Hence, future use of cannabis for therapeutic and recreational aims should consider its potential impact on cardiovascular diseases.

Biotin-Targeted Au(I) Radiosensitizer for Cancer Synergistic Therapy by Intervening with Redox Homeostasis and Inducing Ferroptosis.

The search for highly selective sensitizers with a novel mechanism for tumor targeting therapy is of considerable interest. In this work, we have developed a series of new biotin-targeted Au(I) complexes. Through systematic biological evaluation and comparison, biotinylated Au(I) complex 3a containing a triphenylphosphine ligand was screened, as it realized both prominent efficient inhibition and selective cytotoxicity to cancer cells, and the effect was better than that of popularly used auranofin. Meanwhile, complex 3a, as a potent radiosensitizer, enhances anticancer effects in vitro and in vivo and has sensitization selectivity. From the action mechanism study, we provide evidence that complex 3a could intervene in redox homeostasis through targeted binding and strong suppression of thioredoxin reductase (TrxR) and induce the ferroptosis death process, enabling it to sensitize tumor cells to radiotherapy. Thus, complex 3a has enormous potential as an efficient and specific radiosensitizing agent in cancer therapy.

Mecheliolide elicits ROS-mediated ERS driven immunogenic cell death in hepatocellular carcinoma.

The nonnegligible reason for the poor prognosis of hepatocellular carcinoma (HCC) is resistance to conventional chemotherapy. Immunogenic cell death (ICD) is a rare immunostimulatory form of cell death that can reengage the tumor-specific immune system. ICD can improve the clinical outcomes of chemotherapeutics by promoting a long-term cancer immunity. The discovery of potential ICD inducers is emerging as a promising direction. In the present study, micheliolide (MCL), a natural guaianolide sesquiterpene lactone, was screened out by the virtual screening strategies, identified as an inhibitor of thioredoxin reductase (TrxR) and was evaluated to have high potential to induce ICD. Here, we showed that MCL induced ICD-associated DAMPs (damage-associated molecular patterns, such as CRT exposure, ATP secretion and HMGB1 release). MCL significantly triggered the regression of established tumors in an immunocompetent mouse vaccine model, and induced ICD (DCs maturation, the stimulation of CD4+, and CD8+ T-cells responses) in vivo. Mechanistically, we found that the magnitude of ICD-associated effects induced upon exposure of HCC cells to MCL was dependent on the generation of reactive oxygen species (ROS)-mediated endoplasmic reticulum stress (ERS). In addition, the suppression of ROS normalized MCL-induced ERS, in contrast, the downregulation of TrxR synergized with the ERS driven by MCL. We also systematically detected the H2O2 generation using Hyper7 sensors in HCC cells exposed to MCL. Notably, MCL inhibited the development of HCC organoids. Collectively, our results reveal a potential association between the TrxR inhibitors and ICD, presenting valuable insights into the MCL-activated ICD in HCC cells.

Recent development of gold(I) and gold(III) complexes as therapeutic agents for cancer diseases.

Metal complexes have demonstrated significant antitumor activities and platinum complexes are well established in the clinical application of cancer chemotherapy. However, the platinum-based treatment of different types of cancers is massively hampered by severe side effects and resistance development. Consequently, the development of novel metal-based drugs with different mechanism of action and pharmaceutical profile attracts modern medicinal chemists to design and synthesize novel metal-based agents. Among non-platinum anticancer drugs, gold complexes have gained considerable attention due to their significant antiproliferative potency and efficacy. In most situations, the gold complexes exhibit anticancer activities by targeting thioredoxin reductase (TrxR) or other thiol-rich proteins and enzymes and trigger cell death via reactive oxygen species (ROS). Interestingly, gold complexes were recently reported to elicit biochemical hallmarks of immunogenic cell death (ICD) as an ICD inducer. In this review, the recent progress of gold(I) and gold(III) complexes is comprehensively summarized, and their activities and mechanism of action are documented.

Effect of a brain-penetrant selective estrogen receptor degrader (SERD) on binge drinking in female mice.

BACKGROUND: Greater circulating levels of the steroid hormone 17ß-estradiol (E2) are associated with higher levels of binge drinking in women. In female mice, estrogen receptors in the ventral tegmental area, a dopaminergic region of the brain involved in the motivation to consume ethanol, regulate binge-like ethanol intake. We recently developed a brain-penetrant selective estrogen receptor degrader (SERD), YL3-122, that could be used to test the behavioral role of brain estrogen receptors. We hypothesized that treating female mice with this compound would reduce binge-like ethanol drinking. METHODS: Female C57BL/6J mice were treated systemically with YL3-122 and a related SERD with low brain penetrance, XR5-27, and tested for binge-like ethanol consumption in the drinking in the dark (DID) test. Mice were also tested for sucrose and water consumption and blood ethanol clearance after treatment with the SERDs. Finally, the effect of ethanol exposure on Esr1 gene expression was measured in the ventral tegmental area (VTA), prefrontal cortex (PFC), and ventral hippocampus (vHPC) of male and female mice by quantitative real-time PCR after 4 DID sessions. RESULTS: YL3-122 reduced ethanol consumption when mice were in diestrus but not estrus. YL3-122 also decreased sucrose consumption but did not alter water intake or blood ethanol clearance. XR5-27 did not affect any of these measures. Binge-like ethanol drinking resulted in increased Esr1 transcript in the VTA of both sexes, male vHPC, and female PFC. CONCLUSIONS: These results indicate that SERD treatment can decrease binge-like ethanol drinking in female mice. Thus, it could be a novel strategy to reduce binge drinking in women, with the caveat that effectiveness may depend on menstrual cycle phase. In addition, Esr1 transcript is increased by binge ethanol exposure in both sexes but in a brain region-specific manner.

Pt(II)-NHC Complex Induces ROS-ERS-Related DAMP Balance to Harness Immunogenic Cell Death in Hepatocellular Carcinoma.

Immunogenic cell death (ICD) can engage a specific immune response and establish a long-term immunity in hepatocellular carcinoma (HCC). Herein, we design and synthesize a series of Pt(II)-N-heterocyclic carbene (Pt(II)-NHC) complexes derived from 4,5-diarylimidazole, which show strong anticancer activities in vitro. Among them, 2c displays much higher anticancer activities than cisplatin and other Pt(II)-NHC complexes, especially in HCC cancer cells. In addition, we find that 2c is a type II ICD inducer, which can successfully induce endoplasmic reticulum stress (ERS) accompanied by reactive oxygen species (ROS) generation and finally lead to the release of damage-associated molecular patterns (DAMPs) in HCC cells. Importantly, 2c shows a great anti-HCC potential in a vaccination mouse model and leads to the in vivo immune cell activation in the CCl4-induced liver injury model.

Synthesis and in vitro anticancer activities of selenium N-heterocyclic carbene compounds.

Fourteen novel selenium N-heterocyclic carbene (Se-NHC) compounds derived from 4,5-diarylimidazole were designed, synthesized, and evaluated as antiproliferative agents. Most of them were more effective toward A2780 ovarian cancer cells than HepG2 hepatocellular carcinoma cells. Among them, the most active compound 2b was about fourfold more active than the positive control ebselen against A2780 cells. In addition, this compound displayed twofold higher cytotoxicity to A2780 cells than to IOSE80 normal ovarian epithelial cells. Further studies revealed that 2b could induce reactive oxygen species production, damage mitochondrial membrane potential, block the cells in the G0/G1 phase, and finally promote A2780 cell apoptosis.

Genetically determined tobacco and alcohol use and risk of atrial fibrillation.

BACKGROUND: The causality between the use of alcohol and cigarettes and atrial fibrillation (AF) remains controversial. We conducted a Mendelian randomization (MR) study to evaluate the association of genetic variants related to tobacco and alcohol use with AF. METHODS: Single nucleotide polymorphisms (SNPs) related to smoking initiation (N = 374), age at initiation of regular smoking (N = 10), cigarettes per day (N = 55), and smoking cessation (N = 24) were derived from a genome-wide association studies (GWAS) of tobacco use (N = 1.2 million individuals). SNPs related to heavy alcohol use (N = 6) were derived from a GWAS of UK biobank (N = 125,249 individuals). The genetically matching instrumented variables were obtained from the GWAS of AF (N = 588,190 individuals). The estimates between tobacco and alcohol use and AF were combined by inverse-variance weighted (IVW), simple median, weighted median, MR-robust adjusted profile score method, MR-PRESSO, and multivariable MR. RESULTS: A total of 65,446 AF patients and 522,744 referents were included. In the IVW analysis, the odds ratio per one-unit increase of smoking initiation was 1.11 (95% CI, 1.06-1.16; P = 3.35 × 10-6) for AF. Genetically predicted age at initiation of regular smoking, cigarettes per day and smoking cessation were not associated with AF. The IVW estimate showed that heavy alcohol consumption increased AF risk (OR, 1.11; 95% CI, 1.04-1.18; P = 0.001). The results were consistent in complementary analyses and multivariable MR. CONCLUSION: Our MR study indicated that regular smoking was associated with increased risk of AF, no matter the age at initiation of regular smoking, or the number of cigarettes smoked per day. Genetically predicted heavy alcohol consumption increased the risk of AF.

Smoking and heart failure: a Mendelian randomization and mediation analysis.

AIMS: We performed a Mendelian randomization (MR) study to elucidate the associations of ever smoking, lifelong smoking duration, and smoking cessation with heart failure (HF) risk. METHODS AND RESULTS: We extracted genetic variants associated with smoking initiation, age at initiation of regular smoking, cigarettes per day, and smoking cessation from the genome-wide association study and Sequencing Consortium of Alcohol and Nicotine use (1.2 million individuals), as well as a composite lifetime smoking index from the UK Biobank (462 690 individuals). The associations between smoking phenotypes and HF were explored in the Heart Failure Molecular Epidemiology for Therapeutic Targets Consortium (47 309 cases; 930 014 controls) employing inverse variance-weighted meta-analysis and multivariable MR. The mediation effects of coronary artery disease and atrial fibrillation on smoking-HF risk were explored using mediation analysis. The odds ratios (ORs) for HF were 1.28 [95% confidence interval (CI), 1.22-1.36; P = 1.5 × 10-18 ] for ever regular smokers compared with never smokers and 1.25 (95% CI, 1.09-1.44; P = 1.6 × 10-3 ) for current smokers vs. former smokers. Genetic liability to smoking more cigarettes per day (OR, 1.37; 95% CI, 1.20-1.58; P = 6.4 × 10-6 ) and a higher composite lifetime smoking index (OR, 1.49; 95% CI, 1.31-1.70; P = 2.5 × 10-9 ) were associated with a higher risk of HF. The results were robust and consistent in all sensitivity analyses and multivariable MR after adjusting for HF risk factors, and their associations were independent of coronary artery disease and atrial fibrillation. CONCLUSIONS: Genetic liability to ever smoking and a higher lifetime smoking burden are associated with a higher risk of HF.