Aldehydes alter TGF-ß signaling and induce obesity and cancer.

Obesity and fatty liver diseases-metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH)-affect over one-third of the global population and are exacerbated in individuals with reduced functional aldehyde dehydrogenase 2 (ALDH2), observed in approximately 560 million people. Current treatment to prevent disease progression to cancer remains inadequate, requiring innovative approaches. We observe that Aldh2-/- and Aldh2-/-Sptbn1+/- mice develop phenotypes of human metabolic syndrome (MetS) and MASH with accumulation of endogenous aldehydes such as 4-hydroxynonenal (4-HNE). Mechanistic studies demonstrate aberrant transforming growth factor ß (TGF-ß) signaling through 4-HNE modification of the SMAD3 adaptor SPTBN1 (ß2-spectrin) to pro-fibrotic and pro-oncogenic phenotypes, which is restored to normal SMAD3 signaling by targeting SPTBN1 with small interfering RNA (siRNA). Significantly, therapeutic inhibition of SPTBN1 blocks MASH and fibrosis in a human model and, additionally, improves glucose handling in Aldh2-/- and Aldh2-/-Sptbn1+/- mice. This study identifies SPTBN1 as a critical regulator of the functional phenotype of toxic aldehyde-induced MASH and a potential therapeutic target.

Identification of autoantibodies as potential non-invasive biomarkers for intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) presents a challenging diagnosis due to its nonspecific early clinical manifestations, often resulting in late-stage detection and high mortality. Diagnosing iCCA is further complicated by its limited accuracy, often necessitating multiple invasive procedures for precise identification. Despite carbohydrate antigen 19-9 (CA19-9) having been investigated and employed for iCCA diagnosis, it demonstrates modest diagnostic performance. Consequently, the identification of novel biomarkers with improved sensitivity and specificity remains an imperative yet formidable task. Autoantibodies, as early indicators of the immune response against cancer, offer a promising avenue for enhancing diagnostic accuracy. Our study aimed to identify non-invasive blood-based autoantibody biomarkers capable of distinguishing iCCA patients from healthy individuals (CTRs). We profiled autoantibodies in 26 serum samples (16 iCCAs and 10 CTRs) using protein microarrays containing 1622 functional proteins. Leveraging machine learning techniques, we identified a signature composed of three autoantibody biomarkers (NDE1, PYCR1, and VIM) in conjunction with CA19-9 for iCCA detection. This combined signature demonstrated superior diagnostic performance with an AUC of 96.9%, outperforming CA19-9 alone (AUC: 83.8%). These results suggest the potential of autoantibody biomarkers to develop a complementary non-invasive diagnostic utility for routine iCCA screening.

Identification of intracellular bacteria from multiple single-cell RNA-seq platforms using CSI-Microbes.

The study of the tumor microbiome has been garnering increased attention. We developed a computational pipeline (CSI-Microbes) for identifying microbial reads from single-cell RNA sequencing (scRNA-seq) data and for analyzing differential abundance of taxa. Using a series of controlled experiments and analyses, we performed the first systematic evaluation of the efficacy of recovering microbial unique molecular identifiers by multiple scRNA-seq technologies, which identified the newer 10x chemistries (3' v3 and 5') as the best suited approach. We analyzed patient esophageal and colorectal carcinomas and found that reads from distinct genera tend to co-occur in the same host cells, testifying to possible intracellular polymicrobial interactions. Microbial reads are disproportionately abundant within myeloid cells that up-regulate proinflammatory cytokines like IL1Β and CXCL8, while infected tumor cells up-regulate antigen processing and presentation pathways. These results show that myeloid cells with bacteria engulfed are a major source of bacterial RNA within the tumor microenvironment (TME) and may inflame the TME and influence immunotherapy response.

A global transcriptional activator involved in the iron homeostasis in cyanobacteria.

Cyanobacteria use a series of adaptation strategies and a complicated regulatory network to maintain intracellular iron (Fe) homeostasis. Here, a global activator named IutR has been identified through three-dimensional chromosome organization and transcriptome analysis in a model cyanobacterium Synechocystis sp. PCC 6803. Inactivation of all three homologous IutR-encoding genes resulted in an impaired tolerance of Synechocystis to Fe deficiency and loss of the responses of Fe uptake-related genes to Fe-deplete conditions. Protein-promoter interaction assays confirmed the direct binding of IutR with the promoters of genes related to Fe uptake, and chromatin immunoprecipitation sequencing analysis further revealed that in addition to Fe uptake, IutR could regulate many other physiological processes involved in intracellular Fe homeostasis. These results proved that IutR is an important transcriptional activator, which is essential for cyanobacteria to induce Fe-deficiency response genes. This study provides in-depth insights into the complicated Fe-deficient signaling network and the molecular mechanism of cyanobacteria adaptation to Fe-deficient environments.

PARP-1 selectively impairs KRAS-driven phenotypic and molecular features in intrahepatic cholangiocarcinoma.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options. KRAS mutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated in KRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined. DESIGN: PARP-1 inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition in KRAS-mutant, non-mutant cells. In addition, Parp-1 knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features of Kras-driven and Kras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA. RESULTS: PARP-1 was significantly enhanced in KRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in human KRAS-mutant cell lines. Consistently, loss of Parp-1 provoked distinct phenotype in Kras/Tp53-induced versus Akt/Nicd-induced iCCA and abolished Kras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally, Parp-1 depletion induced molecular switch of KRAS-mutant iCCA recapitulating good prognostic human iCCA patients. CONCLUSION: Our findings identify the novel prognostic and therapeutic role of PARP-1 in iCCA patients with activation of oncogenic KRAS signalling.

[The Preventive Effect of Levofloxacin Combined with G-CSF or Only G-CSF Supportive Therapy on Infection in Autologous Hematopoietic Stem Cell Transplantation].

OBJECTIVE: To investigate the role of levofloxacin combined with recombinant human granulocyte colony-stimulating factor (G-CSF) or only G-CSF supportive therapy in preventing infection in autologous hematopoietic stem cell transplantation(ASCT), and to analyze the length of hospital stay, hospitalization cost and post-transplant survival of the patients. METHODS: A retrospective analysis was performed in the patients with hematological malignancies who accepted ASCT at our hospital from January 2012 to July 2022, the febrile neutropenia, the incidence of bacterial infection and the use rate of intravenous antibiotics in the levofloxacin+G-CSF group and only G-CSF support group during ASCT were observed. The length of hospital stay, total cost during hospitalization and survival after 90 days of transplantation between the two groups were compared. RESULTS: A total of 102 cases were included in this study, including 57 cases of multiple myeloma, 36 cases of acute leukaemia, 7 cases of lymphoma, 3 cases of myelodysplastic syndrome, 1 case of light chain amyloidosis, and 1 case of POEMS syndrome. 47 patients received levofloxacin+ G-CSF antibacterial prophylaxis, and 55 patients received G-CSF supportive therapy. In the levofloxacin+ G-CSF group, 40 cases (85.11%) developed febrile neutropenia, and 13 cases (27.66%) were confirmed as bacterial infection. In the G-CSF group, 44 cases (80.00%) developed febrile neutropenia, and 16 cases (29.09%) were bacterial infection. There was no statistically significant difference in the incidence of febrile neutropenia and bacterial infection between the two groups (χ2=0.46，P =0.50; χ2=0.03，P =0.87). The use rate of intravenous antibiotics in the levofloxacin+ G-CSF group was 85.11% (40/47), which was not statistically different from 85.45% (47/55) in the G-CSF group (χ2=0.04，P =0.84). The detection rates of levofloxacin-resistant bacteria in the levofloxacin+ G-CSF group and G-CSF group were 8.57% (3/35) and 21.43% (6/28), respectively, with no statistical difference (χ2=0.65, P >0.05). The median length and median cost of hospitalization in the levofloxacin+ G-CSF group and G-CSF group were 25 d vs 22 d and 78 216.24 yuan vs 80 724.38 yuan, with no statistically significant differences ( t =3.00，P =0.09; t =0.94，P =0.09). Within 90 days after transplantation, two cases (4.26%) died in the levofloxacin+ G-CSF group and one case (1.82%) died in the G-CSF group, with no statistically significant difference between the two groups (χ2=0.53，P =0.47). CONCLUSION: Application of levofloxacin+ G-CSF showed no significant benefit compared to G-CSF support for the prevention of bacterial infections during ASCT.

HIF-1α facilitates glioma proliferation and invasion by activating pyroptosis signaling axis.

BACKGROUND: HIF-1α is thought to be a novel regulator which contributes to carcinogenesis. However, the mechanism underlying the effect of HIF-1α in gliomas remains largely unknown. METHODS: In the research, we demonstrate that HIF-lα mRNA and protein levels are elevated in glioma cells. The colony formation assays, transwell assays, and wound-healing assays showed that overexpression of HIF-1α promoted proliferation and invasion of glioma cells. RESULTS: Overexpression of HIF-lα also increased the expression of inflammatory factors related to pyrolysis (TNF-α, IL-10, and IL-1ß) and protein related to pyrolysis signal pathway (NLRP3, ASC, caspase-1, GSDMD, and GSDME). CONCLUSIONS: Therefore, we speculate that HIF-1α promotes the proliferation and invasion of glial cells by regulating pyrolysis pathway. These results might provide a novel strategy and target for treatment of glioma.

The landscape of etiological patterns of hepatocellular carcinoma and intrahepatic cholangiocarcinoma in Thailand.

Thailand is among countries with the highest global incidence and mortality rates of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA). While viral hepatitis and liver fluke infections have been associated with HCC and iCCA, respectively, other environmental risk factors, overall risk factor commonality and combinatorial roles, and effects on survival have not been systematically examined. We conducted a TIGER-LC consortium-based population study covering all high-incidence areas of both malignancies across Thailand: 837 HCC, 1474 iCCA, and 1112 controls (2011-2019) were comprehensively queried on lifelong environmental exposures, lifestyle, and medical history. Multivariate logistic regression and Cox proportional hazards analyses were used to evaluate risk factors and associated survival patterns. Our models identified shared risk factors between HCC and iCCA, such as viral hepatitis infection, liver fluke infection, and diabetes, including novel and shared associations of agricultural pesticide exposure (OR range of 1.50; 95% CI: 1.06-2.11 to 2.91; 95% CI: 1.82-4.63) along with vulnerable sources of drinking water. Most patients had multiple risk factors, magnifying their risk considerably. Patients with lower risk levels had better survival in both HCC (HR 0.78; 95% CI: 0.64-0.96) and iCCA (HR 0.84; 95% CI: 0.70-0.99). Risk factor co-exposures and their common associations with HCC and iCCA in Thailand emphasize the importance for future prevention and control measures, especially in its large agricultural sector. The observed mortality patterns suggest ways to stratify patients for anticipated survivorship and develop plans to support medical care of longer-term survivors, including behavioral changes to reduce exposures.

Alcohol-associated liver cancer.

Heavy alcohol intake induces a wide spectrum of liver diseases ranging from steatosis, steatohepatitis, cirrhosis, and HCC. Although alcohol consumption is a well-known risk factor for the development, morbidity, and mortality of HCC globally, alcohol-associated hepatocellular carcinoma (A-HCC) is poorly characterized compared to viral hepatitis-associated HCC. Most A-HCCs develop after alcohol-associated cirrhosis (AC), but the direct carcinogenesis from ethanol and its metabolites to A-HCC remains obscure. The differences between A-HCC and HCCs caused by other etiologies have not been well investigated in terms of clinical prognosis, genetic or epigenetic landscape, molecular mechanisms, and heterogeneity. Moreover, there is a huge gap between basic research and clinical practice due to the lack of preclinical models of A-HCC. In the current review, we discuss the pathogenesis, heterogeneity, preclinical approaches, epigenetic, and genetic profiles of A-HCC, and discuss the current insights into and the prospects for future research on A-HCC. The potential effect of alcohol on cholangiocarcinoma and liver metastasis is also discussed.

Characterization of tumor evolution by functional clonality and phylogenetics in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a molecularly heterogeneous solid malignancy, and its fitness may be shaped by how its tumor cells evolve. However, ability to monitor tumor cell evolution is hampered by the presence of numerous passenger mutations that do not provide any biological consequences. Here we develop a strategy to determine the tumor clonality of three independent HCC cohorts of 524 patients with diverse etiologies and race/ethnicity by utilizing somatic mutations in cancer driver genes. We identify two main types of tumor evolution, i.e., linear, and non-linear models where non-linear type could be further divided into classes, which we call shallow branching and deep branching. We find that linear evolving HCC is less aggressive than other types. GTF2IRD2B mutations are enriched in HCC with linear evolution, while TP53 mutations are the most frequent genetic alterations in HCC with non-linear models. Furthermore, we observe significant B cell enrichment in linear trees compared to non-linear trees suggesting the need for further research to uncover potential variations in immune cell types within genomically determined phylogeny types. These results hint at the possibility that tumor cells and their microenvironment may collectively influence the tumor evolution process.

Deep whole-genome analysis of 494 hepatocellular carcinomas.

Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China1-3. However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited4-8, with current analyses of HCC mainly from non-HBV-enriched populations9,10. Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribed mutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain (FGA), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications.

Single-Cell Characterization of the Tumor Ecosystem in Liver Cancer.

Tumor heterogeneity along with the complex landscape of the tumor microenvironment create critical challenges for effective liver cancer interventions. Characterizing the tumor ecosystem at the single-cell level may provide insight into the collective behaviors of tumor cells and their interplays with stromal and immune cells. Here we introduce the experimental protocol and computational methods for the single-cell study of liver cancer, which may be essential for a mechanistic understanding of the tumor ecosystem in liver cancer and further pave the way for developing novel therapeutics.

Lineage and ecology define liver tumor evolution in response to treatment.

A tumor ecosystem constantly evolves over time in the face of immune predation or therapeutic intervention, resulting in treatment failure and tumor progression. Here, we present a single-cell transcriptome-based strategy to determine the evolution of longitudinal tumor biopsies from liver cancer patients by measuring cellular lineage and ecology. We construct a lineage and ecological score as joint dynamics of tumor cells and their microenvironments. Tumors may be classified into four main states in the lineage-ecological space, which are associated with clinical outcomes. Analysis of longitudinal samples reveals the evolutionary trajectory of tumors in response to treatment. We validate the lineage-ecology-based scoring system in predicting clinical outcomes using bulk transcriptomic data of additional cohorts of 716 liver cancer patients. Our study provides a framework for monitoring tumor evolution in response to therapeutic intervention.

Combined immune checkpoint inhibition with durvalumab and tremelimumab with and without radiofrequency ablation in patients with advanced biliary tract carcinoma.

BACKGROUND: Current standard of care for advanced biliary tract cancer (BTC) is gemcitabine, cisplatin plus anti-PD1/PD-L1, but response rates are modest. The purpose of this study was to explore the efficacy and safety of durvalumab (anti-PD-L1) and tremelimumab (anti-CTLA-4), with and without an interventional radiology (IR) procedure in advanced BTC. METHODS: Eligible patients with advanced BTC who had received or refused at least one prior line of systemic therapy were treated with tremelimumab and durvalumab for four combined doses followed by monthly durvalumab alone with and without an IR procedure until the progression of disease or unacceptable toxicity. Objective response was assessed through CT or MRI by Response Evaluation Criteria in Solid Tumors (RECIST, version 1.1) every 8 weeks. Adverse events (AEs) were recorded and managed. The primary endpoint was 6-month progression-free survival (PFS). RESULTS: Twenty-three patients with advanced BTC were enrolled; 17 patients were assigned to treatment with durvalumab and tremelimumab (Durva/Treme); and 6 patients were treated with the combination of durvalumab, tremelimumab plus IR procedure (Durva/Treme + IR). The best clinical responses in the Durva/Treme arm were partial response (n = 1), stable disease (n = 5), progressive disease (n = 5), and in the Durva/Treme + IR arm: partial response (n = 0), stable disease (n = 3), progressive disease (n = 3). The median PFS was 2.2 months (95% CI: 1.3-3.1 months) in the Durva/Treme arm and 2.9 months (95% CI: 1.9-4.7 months) in the Durva/Treme + IR arm (p = 0.27). The median OS was 5.1 months (95% CI: 2.5-6.9 months) in the Durva/Treme arm and 5.8 months (95% CI: 2.9-40.1 months) in the Durva/Treme + IR arm (p = 0.31). The majority of AEs were grades 1-2. CONCLUSION: Durva/Treme and Durva/Treme + IR showed similar efficacy. With a manageable safety profile. Larger studies are needed to fully characterize the efficacy of Durva/Treme ± IR in advanced BTC.

Immunosuppressive CD29+ Treg accumulation in the liver in mice on checkpoint inhibitor therapy.

OBJECTIVE: Liver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown. DESIGN: Flow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy. RESULTS: We found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29+ (Itgb1, integrin ß1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29+ Tregs as the prominent niche-filling subpopulation in the liver, and CD29+ Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29+ and CD29- hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29+ Tregs was in part IL2-independent. CONCLUSION: We propose that CD29+ Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance.

Spatial proximity of tumor-immune interactions predicts patient outcome in hepatocellular carcinoma.

BACKGROUND AND AIMS: The fitness and viability of a tumor ecosystem are influenced by the spatial organization of its cells. We aimed to study the structure, architecture, and cell-cell dynamics of the heterogeneous liver cancer tumor microenvironment using spatially resolved multiplexed imaging. APPROACH AND RESULTS: We performed co-detection by indexing multiplexed immunofluorescence imaging on 68 HCC biopsies from Thai patients [(Thailand Initiative in Genomics and Expression Research for Liver Cancer (TIGER-LC)] as a discovery cohort, and then validated the results in an additional 190 HCC biopsies from Chinese patients [Liver Cancer Institute (LCI)]. We segmented and annotated 117,270 and 465,632 cells from the TIGER-LC and LCI cohorts, respectively. We observed 4 patient groups of TIGER-LC (IC1, IC2, IC3, and IC4) with distinct tumor-immune cellular interaction patterns. In addition, patients from IC2 and IC4 had much better overall survival than those from IC1 and IC3. Noticeably, tumor and CD8 + T-cell interactions were strongly enriched in IC2, the group with the best patient outcomes. The close proximity between the tumor and CD8 + T cells was a strong predictor of patient outcome in both the TIGER-LC and the LCI cohorts. Bulk transcriptomic data from 51 of the 68 HCC cases were used to determine tumor-specific gene expression features of our classified subtypes. Moreover, we observed that the presence of immune spatial neighborhoods in HCC as a measure of overall immune infiltration is linked to better patient prognosis. CONCLUSIONS: Highly multiplexed imaging analysis of liver cancer reveals tumor-immune cellular heterogeneity within spatial contexts, such as tumor and CD8 + T-cell interactions, which may predict patient survival.

Chromosome 8p engineering reveals increased metastatic potential targetable by patient-specific synthetic lethality in liver cancer.

Large-scale chromosomal aberrations are prevalent in human cancer, but their function remains poorly understood. We established chromosome-engineered hepatocellular carcinoma cell lines using CRISPR-Cas9 genome editing. A 33-mega-base pair region on chromosome 8p (chr8p) was heterozygously deleted, mimicking a frequently observed chromosomal deletion. Using this isogenic model system, we delineated the functional consequences of chr8p loss and its impact on metastatic behavior and patient survival. We found that metastasis-associated genes on chr8p act in concert to induce an aggressive and invasive phenotype characteristic for chr8p-deleted tumors. Genome-wide CRISPR-Cas9 viability screening in isogenic chr8p-deleted cells served as a powerful tool to find previously unidentified synthetic lethal targets and vulnerabilities accompanying patient-specific chromosomal alterations. Using this target identification strategy, we showed that chr8p deletion sensitizes tumor cells to targeting of the reactive oxygen sanitizing enzyme Nudix hydrolase 17. Thus, chromosomal engineering allowed for the identification of novel synthetic lethalities specific to chr8p loss of heterozygosity.

Pan-viral serology uncovers distinct virome patterns as risk predictors of hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

This study evaluates the pan-serological profiles of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA) compared to several diseased and non-diseased control populations to identify risk factors and biomarkers of liver cancer. We used phage immunoprecipitation sequencing, an anti-viral antibody screening method using a synthetic-phage-displayed human virome epitope library, to screen patient serum samples for exposure to over 1,280 strains of pathogenic and non-pathogenic viruses. Using machine learning methods to develop an HCC or iCCA viral score, we discovered that both viral scores were positively associated with several liver function markers in two separate at-risk populations independent of viral hepatitis status. The HCC score predicted all-cause mortality over 8 years in patients with chronic liver disease at risk of HCC, while the viral hepatitis status was not predictive of survival. These results suggest that non-hepatitis viral infections may contribute to HCC and iCCA development and could be biomarkers in at-risk populations.

A genetic basis of mitochondrial DNAJA3 in nonalcoholic steatohepatitis-related hepatocellular carcinoma.

BACKGROUND AND AIMS: NAFLD is the most common form of liver disease worldwide, but only a subset of individuals with NAFLD may progress to NASH. While NASH is an important etiology of HCC, the underlying mechanisms responsible for the conversion of NAFLD to NASH and then to HCC are poorly understood. We aimed to identify genetic risk genes that drive NASH and NASH-related HCC. APPROACH AND RESULTS: We searched genetic alleles among the 24 most significant alleles associated with body fat distribution from a genome-wide association study of 344,369 individuals and validated the top allele in 3 independent cohorts of American and European patients (N=1380) with NAFLD/NASH/HCC. We identified an rs3747579-TT variant significantly associated with NASH-related HCC and demonstrated that rs3747579 is expression quantitative trait loci of a mitochondrial DnaJ Heat Shock Protein Family (Hsp40) Member A3 ( DNAJA3 ). We also found that rs3747579-TT and a previously identified PNPLA3 as a functional variant of NAFLD to have significant additional interactions with NASH/HCC risk. Patients with HCC with rs3747579-TT had a reduced expression of DNAJA3 and had an unfavorable prognosis. Furthermore, mice with hepatocyte-specific Dnaja3 depletion developed NASH-dependent HCC either spontaneously under a normal diet or enhanced by diethylnitrosamine. Dnaja3 -deficient mice developed NASH/HCC characterized by significant mitochondrial dysfunction, which was accompanied by excessive lipid accumulation and inflammatory responses. The molecular features of NASH/HCC in the Dnaja3 -deficient mice were closely associated with human NASH/HCC. CONCLUSIONS: We uncovered a genetic basis of DNAJA3 as a key player of NASH-related HCC.