BNIP3-mediated mitophagy boosts the competitive growth of Lenvatinib-resistant cells via energy metabolism reprogramming in HCC.

An increasing evidence supports that cell competition, a vital selection and quality control mechanism in multicellular organisms, is involved in tumorigenesis and development; however, the mechanistic contributions to the association between cell competition and tumor drug resistance remain ill-defined. In our study, based on a contructed lenvitinib-resistant hepatocellular carcinoma (HCC) cells display obvious competitive growth dominance over sensitive cells through reprogramming energy metabolism. Mechanistically, the hyperactivation of BCL2 interacting protein3 (BNIP3) -mediated mitophagy in lenvatinib-resistant HCC cells promotes glycolytic flux via shifting energy production from mitochondrial oxidative phosphorylation to glycolysis, by regulating AMP-activated protein kinase (AMPK) -enolase 2 (ENO2) signaling, which perpetually maintaining lenvatinib-resistant HCC cells' competitive advantage over sensitive HCC cells. Of note, BNIP3 inhibition significantly sensitized the anti-tumor efficacy of lenvatinib in HCC. Our findings emphasize a vital role for BNIP3-AMPK-ENO2 signaling in maintaining the competitive outcome of lenvitinib-resistant HCC cells via regulating energy metabolism reprogramming; meanwhile, this work recognizes BNIP3 as a promising target to overcome HCC drug resistance.

Evaluating soluble Axl as a biomarker for glioblastoma: A pilot study.

With current imaging, discriminating tumor progression from treatment effect following immunotherapy or oncolytic virotherapy of glioblastoma (GBM) is challenging. A blood based diagnostic biomarker would therefore be helpful. Axl is a receptor tyrosine kinase that is highly expressed by many cancers including GBM. Axl expression is regulated through enzymatic cleavage of its extracellular domain. The resulting fragment can be detected in serum as soluble Axl (sAxl). sAxl levels can distinguish patients with melanoma, hepatocellular carcinoma, and pancreatic ductal adenocarcinoma from healthy controls. This is a pilot study to determine if sAxl is a candidate biomarker for GBM. The sAxl levels in the serum of 40 healthy volunteers and 20 GBM patients were determined using an enzyme-linked immunosorbent assay (ELISA). Pre- and post- operative sAxl levels were obtained. Volumetric MRI evaluation provided GBM tumor volume metrics. There was no significant difference in the sAxl levels of the volunteers (30.16±1.88 ng/ml) and GBM patients (30.74±1.96 ng/ml) p = 0.27. The postoperative sAxl levels were significantly higher than preoperative levels (32.32±2.26 ng/ml vs 30.74±1.96 ng/ml, p = 0.03). We found no correlation between tumor volume and sAxl levels. Axl expression was low or absent in 6 of 11 (55%) patient derived GBM cell lines. Given the wide range of Axl expression by GBM tumors, sAxl may not be a reliable indicator of GBM. However, given the small sample size in this study, a larger study may be considered.

Repression of SMAD3 by STAT3 and c-Ski induces conventional dendritic cell differentiation.

A pleiotropic immunoregulatory cytokine, TGF-ß, signals via the receptor-regulated SMADs: SMAD2 and SMAD3, which are constitutively expressed in normal cells. Here, we show that selective repression of SMAD3 induces cDC differentiation from the CD115+ common DC progenitor (CDP). SMAD3 was expressed in haematopoietic cells including the macrophage DC progenitor. However, SMAD3 was specifically down-regulated in CD115+ CDPs, SiglecH- pre-DCs, and cDCs, whereas SMAD2 remained constitutive. SMAD3-deficient mice showed a significant increase in cDCs, SiglecH- pre-DCs, and CD115+ CDPs compared with the littermate control. SMAD3 repressed the mRNA expression of FLT3 and the cDC-related genes: IRF4 and ID2. We found that one of the SMAD transcriptional corepressors, c-SKI, cooperated with phosphorylated STAT3 at Y705 and S727 to repress the transcription of SMAD3 to induce cDC differentiation. These data indicate that STAT3 and c-Ski induce cDC differentiation by repressing SMAD3: the repressor of the cDC-related genes during the developmental stage between the macrophage DC progenitor and CD115+ CDP.

Application of a Fluorescence Recovery-Based Polo-Like Kinase 1 Binding Assay to Polo-Like Kinase 2 and Polo-Like Kinase 3.

Assay systems for evaluating compound protein-binding affinities are essential for developing agonists and/or antagonists. Targeting individual members of a protein family can be extremely important and for this reason it is critical to have methods for evaluating selectivity. We have previously reported a fluorescence recovery assay that employs a fluorescein-labelled probe to determine IC50 values of ATP-competitive type 1 inhibitors of polo-like kinase 1 (Plk1). This probe is based on the potent Plk1 inhibitor BI2536 [fluorescein isothiocyanate (FITC)-polyethylene glycol (PEG)-lysine (Lys) (BI2536) 1]. Herein, we extend this approach to the highly homologous Plk2 and Plk3 members of this kinase family. Our results suggest that this assay system is suitable for evaluating binding affinities against Plk2 and Plk3 as well as Plk1. The new methodology represents the first example of evaluating N-terminal catalytic kinase domain (KD) affinities of Plk2 and Plk3. It represents a simple and cost-effective alternative to traditional kinase assays to explore the KD-binding compounds against Plk2 and Plk3 as well as Plk1.

TurboID-mediated proximity labeling technologies to identify virus co-receptors.

Virus receptors determine the tissue tropism of viruses and have a certain relationship with the clinical outcomes caused by viral infection, which is of great importance for the identification of virus receptors to understand the infection mechanism of viruses and to develop entry inhibitor. Proximity labeling (PL) is a new technique for studying protein-protein interactions, but it has not yet been applied to the identification of virus receptors or co-receptors. Here, we attempt to identify co-receptor of SARS-CoV-2 by employing TurboID-catalyzed PL. The membrane protein angiotensin-converting enzyme 2 (ACE2) was employed as a bait and conjugated to TurboID, and a A549 cell line with stable expression of ACE2-TurboID was constructed. SARS-CoV-2 pseudovirus were incubated with ACE2-TurboID stably expressed cell lines in the presence of biotin and ATP, which could initiate the catalytic activity of TurboID and tag adjacent endogenous proteins with biotin. Subsequently, the biotinylated proteins were harvested and identified by mass spectrometry. We identified a membrane protein, AXL, that has been functionally shown to mediate SARS-CoV-2 entry into host cells. Our data suggest that PL could be used to identify co-receptors for virus entry.

Human DDX6 regulates translation and decay of inefficiently translated mRNAs.

Recent findings indicate that the translation elongation rate influences mRNA stability. One of the factors that has been implicated in this link between mRNA decay and translation speed is the yeast DEAD-box helicase Dhh1p. Here, we demonstrated that the human ortholog of Dhh1p, DDX6, triggers the deadenylation-dependent decay of inefficiently translated mRNAs in human cells. DDX6 interacts with the ribosome through the Phe-Asp-Phe (FDF) motif in its RecA2 domain. Furthermore, RecA2-mediated interactions and ATPase activity are both required for DDX6 to destabilize inefficiently translated mRNAs. Using ribosome profiling and RNA sequencing, we identified two classes of endogenous mRNAs that are regulated in a DDX6-dependent manner. The identified targets are either translationally regulated or regulated at the steady-state-level and either exhibit signatures of poor overall translation or of locally reduced ribosome translocation rates. Transferring the identified sequence stretches into a reporter mRNA caused translation- and DDX6-dependent degradation of the reporter mRNA. In summary, these results identify DDX6 as a crucial regulator of mRNA translation and decay triggered by slow ribosome movement and provide insights into the mechanism by which DDX6 destabilizes inefficiently translated mRNAs.

Metformin synergizes with gilteritinib in treating FLT3-mutated leukemia via targeting PLK1 signaling.

Fms-like tyrosine kinase 3 (FLT3) mutations, present in over 30% of acute myeloid leukemia (AML) cases and dominated by FLT3-internal tandem duplication (FLT3-ITD), are associated with poor outcomes in patients with AML. While tyrosine kinase inhibitors (TKIs; e.g., gilteritinib) are effective, they face challenges such as drug resistance, relapse, and high costs. Here, we report that metformin, a cheap, safe, and widely used anti-diabetic agent, exhibits a striking synergistic effect with gilteritinib in treating FLT3-ITD AML. Metformin significantly sensitizes FLT3-ITD AML cells (including TKI-resistant ones) to gilteritinib. Metformin plus gilteritinib (low dose) dramatically suppresses leukemia progression and prolongs survival in FLT3-ITD AML mouse models. Mechanistically, the combinational treatment cooperatively suppresses polo-like kinase 1 (PLK1) expression and phosphorylation of FLT3/STAT5/ERK/mTOR. Clinical analysis also shows improved survival rates in patients with FLT3-ITD AML taking metformin. Thus, the metformin/gilteritinib combination represents a promising and cost-effective treatment for patients with FLT3-mutated AML, particularly for those with low income/affordability.

Discovery of a first-in-class protein degrader for the c-ros oncogene 1 (ROS1).

The c-ros oncogene 1 (ROS1), an oncogenic driver, is known to induce non-small cell lung cancer (NSCLC) when overactivated, particularly through the formation of fusion proteins. Traditional targeted therapies focus on inhibiting ROS1 activity with ROS 1 inhibitors to manage cancer progression. However, a new strategy involving the design of protein degraders offers a more potent approach by completely degrading ROS1 fusion oncoproteins, thereby effectively blocking their kinase activity and enhancing anti-tumour potential. Utilizing PROteolysis-TArgeting Chimera (PROTAC) technology and informed by molecular docking and rational design, we report the first ROS1-specific PROTAC, SIAIS039. This degrader effectively targets multiple ROS1 fusion oncoproteins (CD74-ROS1, SDC4-ROS1 and SLC34A2-ROS1) in engineered Ba/F3 cells and HCC78 cells, demonstrating anti-tumour effects against ROS1 fusion-driven cancer cells. It suppresses cell proliferation, induces cell cycle arrest, and apoptosis, and inhibits clonogenicity. The anti-tumour efficacy of SIAIS039 surpasses two approved drugs, crizotinib and entrectinib, and matches that of the top inhibitors, including lorlatinib and taletrectinib. Mechanistic studies confirm that the degradation induced by 039 requires the participation of ROS1 ligands and E3 ubiquitin ligases, and involves the proteasome and ubiquitination. In addition, 039 exhibited excellent oral bioavailability in a mouse xenograft model, highlighting its potential for clinical application. In conclusion, our study presents a promising and novel therapeutic strategy for ROS1 fusion-positive NSCLC by targeting ROS1 fusion oncoproteins for degradation, laying the foundation for the development of further PROTAC and offering hope for patients with ROS1 fusion-positive NSCLC.

Dual-localized PPTC7 limits mitophagy through proximal and dynamic interactions with BNIP3 and NIX.

PPTC7 is a mitochondrial-localized phosphatase that suppresses BNIP3- and NIX-mediated mitophagy, but the mechanisms underlying this regulation remain ill-defined. Here, we demonstrate that loss of PPTC7 upregulates BNIP3 and NIX post-transcriptionally and independent of HIF-1α stabilization. Loss of PPTC7 prolongs the half-life of BNIP3 and NIX while blunting their accumulation in response to proteasomal inhibition, suggesting that PPTC7 promotes the ubiquitin-mediated turnover of BNIP3 and NIX. Consistently, overexpression of PPTC7 limits the accumulation of BNIP3 and NIX protein levels, which requires an intact catalytic motif but is surprisingly independent of its targeting to mitochondria. Consistently, we find that PPTC7 is dual-localized to the outer mitochondrial membrane and the matrix. Importantly, anchoring PPTC7 to the outer mitochondrial membrane is sufficient to blunt BNIP3 and NIX accumulation, and proximity labeling and fluorescence co-localization experiments demonstrate that PPTC7 dynamically associates with BNIP3 and NIX within the native cellular environment. Collectively, these data reveal that a fraction of PPTC7 localizes to the outer mitochondrial membrane to promote the proteasomal turnover of BNIP3 and NIX, limiting basal mitophagy.

Optimized Protocol for the Regulation of DNA Methylation and Gene Expression Using Modified dCas9-SunTag Platforms.

DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are considered worthwhile and many epigenome editing technologies have been developed, which were based on ZF, TALE, and CRISPR-dCas9. Here, we describe a protocol for the application of a modified dCas9-SunTag system, which increased the efficiency of targeted demethylation and gene activation at specific DNA loci. The original SunTag system consists of 10 copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve more efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids. Moreover, we describe the co-recruitment of TET1 and VP64 for efficient gene activation. Since we showed the manipulation of DNA methylation at specific loci and gene activation, its application could lead to its future use in the clinic.

Proteomic Changes Induced by the Immunosuppressant Everolimus in Human Podocytes.

mTOR inhibitors (mTOR-Is) may induce proteinuria in kidney transplant recipients through podocyte damage. However, the mechanism has only been partially defined. Total cell lysates and supernatants of immortalized human podocytes treated with different doses of everolimus (EVE) (10, 100, 200, and 500 nM) for 24 h were subjected to mass spectrometry-based proteomics. Support vector machine and partial least squares discriminant analysis were used for data analysis. The results were validated in urine samples from 28 kidney transplant recipients receiving EVE as part of their immunosuppressive therapy. We identified more than 7000 differentially expressed proteins involved in several pathways, including kinases, cell cycle regulation, epithelial-mesenchymal transition, and protein synthesis, according to gene ontology. Among these, after statistical analysis, 65 showed an expression level significantly and directly correlated with EVE dosage. Polo-Like Kinase 1 (PLK1) content was increased, whereas osteopontin (SPP1) content was reduced in podocytes and supernatants in a dose-dependent manner and significantly correlated with EVE dose (p < 0.0001, FDR < 5%). Similar results were obtained in the urine of kidney transplant patients. This study analyzed the impact of different doses of mTOR-Is on podocytes, helping to understand not only the biological basis of their therapeutic effects but also the possible mechanisms underlying proteinuria.

In vivo turnover and biodistribution of soluble AXL: implications for biomarker development.

Soluble biomarkers are paramount to personalized medicine. However, the in vivo turnover and biodistribution of soluble proteins is seldom characterized. The cleaved extracellular domain of the AXL receptor (sAXL) is a prognostic biomarker in several diseases and a predictive marker of AXL targeting agents. Plasma sAXL reflects a balance between production in tissues with lymphatic transport into the circulation and removal from blood by degradation or excretion. It is unclear how this transport cycle affects plasma sAXL levels that are the metric for biomarker development. Radiolabeled mouse sAxl was monitored after intravenous injection to measure degradation and urinary excretion of sAxl, and after intradermal injection to mimic tissue or tumor production. sAxl was rapidly taken-up and degraded by the liver and kidney cortex. Surprisingly, intact sAxl was detectable in urine, indicating passage through the glomerular filter and a unique sampling opportunity. The structure of sAxl showed an elongated, flexible molecule with a length of 18 nm and a thickness of only 3 nm, allowing passage through the glomerulus and excretion into the urine. Intradermally injected sAxl passed through local and distant lymph nodes, followed by uptake in liver and kidney cortex. Low levels of sAxl were seen in the plasma, consistent with an extended transit time from local tissue to circulation. The rapid plasma clearance of sAxl suggests that steady-state levels in blood will sensitively and dynamically reflect the rate of production of sAxl in the tissues but will be influenced by perturbations of liver and kidney function.

HIV-1 Vpr combats the PU.1-driven antiviral response in primary human macrophages.

HIV-1 Vpr promotes efficient spread of HIV-1 from macrophages to T cells by transcriptionally downmodulating restriction factors that target HIV-1 Envelope protein (Env). Here we find that Vpr induces broad transcriptomic changes by targeting PU.1, a transcription factor necessary for expression of host innate immune response genes, including those that target Env. Consistent with this, we find silencing PU.1 in infected macrophages lacking Vpr rescues Env. Vpr downmodulates PU.1 through a proteasomal degradation pathway that depends on physical interactions with PU.1 and DCAF1, a component of the Cul4A E3 ubiquitin ligase. The capacity for Vpr to target PU.1 is highly conserved across primate lentiviruses. In addition to impacting infected cells, we find that Vpr suppresses expression of innate immune response genes in uninfected bystander cells, and that virion-associated Vpr can degrade PU.1. Together, we demonstrate Vpr counteracts PU.1 in macrophages to blunt antiviral immune responses and promote viral spread.

Transcription Factor TAL1 in Erythropoiesis.

Lineage-specific transcription factors (TFs) regulate differentiation of hematopoietic stem cells (HSCs). They are decisive for the establishment and maintenance of lineage-specific gene expression programs during hematopoiesis. For this they create a regulatory network between TFs, epigenetic cofactors, and microRNAs. They activate cell-type specific genes and repress competing gene expression programs. Disturbance of this process leads to impaired lineage fidelity and diseases of the blood system. The TF T-cell acute leukemia 1 (TAL1) is central for erythroid differentiation and contributes to the formation of distinct gene regulatory complexes in progenitor cells and erythroid cells. A TAL1/E47 heterodimer binds to DNA with the TFs GATA-binding factor 1 and 2 (GATA1/2), the cofactors LIM domain only 1 and 2 (LMO1/2), and LIM domain-binding protein 1 (LDB1) to form a core TAL1 complex. Furthermore, cell-type-dependent interactions of TAL1 with other TFs such as with runt-related transcription factor 1 (RUNX1) and Kruppel-like factor 1 (KLF1) are established. Moreover, TAL1 activity is regulated by the formation of TAL1 isoforms, posttranslational modifications (PTMs), and microRNAs. Here, we describe the function of TAL1 in normal hematopoiesis with a focus on erythropoiesis.

TET2 regulates extranodal NK/T cell lymphoma progression through regulation of DNA methylation.

The biological role of Ten-11 translocation 2 (TET2) and the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in the development of extra-nodal natural killer/T-cell lymphoma (ENKTL) remains unclear. The level of 5mC and 5hmC was detected in 112 cases of ENKTL tissue specimens by immunohistochemical (IHC) staining. Subsequently, TET2 knockdown and the overexpression cell models were constructed in ENKTL cell lines. Biochemical analyses were used to assess proliferation, apoptosis, cell cycle and monoclonal formation in cells treated or untreated with L-Ascorbic acid sodium salt (LAASS). Dot-Blots were used to detect levels of genome 5mC and 5hmC. Additionally, the ILLUMINA 850k methylation chip was used to analyze the changes of TET2 regulatory genes. RNA-Seq was used to profile differentially expressed genes regulated by TET2. The global level of 5hmC was significantly decreased, while 5mC was highly expressed in ENKTL tissue. TET2 protein expression was negatively correlated with the ratio of 5mC/5hmC (p < 0.0001). The 5mC/5hmC status were related to the site of disease, clinical stage, PINK score and Ki-67 index, as well as the 5-year OS. TET2 knockdown prolonged the DNA synthesis period, increased the cloning ability of tumor cells, increased the level of 5mC and decreased the level of 5hmC in ENKTL cells. While overexpression of TET2 presented the opposite effect. Furthermore, treatment of ENKTL cells with LAASS significantly induced ENKTL cell apoptosis. These results suggest that TET2 plays an important role in ENKTL development via regulation of 5mC and 5hmC and may serve as a novel therapeutic target for ENKTL.

TET1 displays catalytic and non-catalytic functions in the adult mouse cortex.

TET1/2/3 dioxygenases iteratively demethylate 5-methylcytosine, beginning with the formation of 5-hydroxymethylcytosine (5hmC). The post-mitotic brain maintains higher levels of 5hmC than most peripheral tissues, and TET1 ablation studies have underscored the critical role of TET1 in brain physiology. However, deletion of Tet1 precludes the disentangling of the catalytic and non-catalytic functions of TET1. Here, we dissect these functions of TET1 by comparing adult cortex of Tet1 wildtype (Tet1 WT), a novel Tet1 catalytically dead mutant (Tet1 HxD), and Tet1 knockout (Tet1 KO) mice. Using DNA methylation array, we uncover that Tet1 HxD and KO mutations perturb the methylation status of distinct subsets of CpG sites. Gene ontology (GO) analysis on specific differential 5hmC regions indicates that TET1's catalytic activity is linked to neuronal-specific functions. RNA-Seq further shows that Tet1 mutations predominantly impact the genes that are associated with alternative splicing. Lastly, we performed High-performance Liquid Chromatography Mass-Spectrometry lipidomics on WT and mutant cortices and uncover accumulation of lysophospholipids lysophosphatidylethanolamine and lysophosphatidylcholine in Tet1 HxD cortex. In summary, we show that Tet1 HxD does not completely phenocopy Tet1 KO, providing evidence that TET1 modulates distinct cortical functions through its catalytic and non-catalytic roles.

SPI1-mediated macrophage polarization aggravates age-related macular degeneration.

Objective: This study revealed a core regulator and common upstream mechanisms for the multifaceted pathological processes of age-related macular degeneration (AMD) and provided proof-of-concept for this new therapeutic target. Methods: Comprehensive gene expression analysis was performed using RNA sequencing of eye cup from old mice as well as laser-induced choroidal neovascularization (CNV) mouse model. Through integrative analysis and protein-protein interaction (PPI) analysis, common pathways and key transcription factor was identified simultaneously engaged in age-related retinal degeneration and CNV, the two typical pathological process of AMD. Subsequently, the expression changes of Spi1, the key regulator, as well as the alternation of the downstream mechanisms were validated in both models through qRT-PCR, Elisa, flow cytometry and immunofluorescence. Further, we assessed the impact of Spi1 knockdown in vitro and in vivo using gene intervention vectors carried by adeno-associated virus or lentivirus to test its potential as a therapeutic target. Results: Compared to corresponding controls, we found 1,939 and 1,319 genes differentially expressed in eye cups of old and CNV mice respectively. The integrative analysis identified a total of 275 overlapping DEGs, of which 150 genes were co-upregulated. PPI analysis verified a central transcription factor, SPI1. The significant upregulation of Spi1 expression was then validated in both models, accompanied by macrophage polarization towards the M1 phenotype. Finally, SPI1 suppression significantly inhibited M1 polarization of BMDMs and attenuated neovascularization in CNV mice. Conclusion: This study demonstrates that SPI1 exerts a pivotal role in AMD by regulation of macrophage polarization and innate immune response, offering promise as an innovative target for treating AMD.

Targeting polo-like kinase 1 to treat kidney diseases.

Globally, ∼850 million individuals suffer from some form of kidney disease. This staggering figure underscores the importance of continued research and innovation in the field of nephrology to develop effective treatments and improve overall global kidney health. In current research, the polo-like kinase (Plk) family has emerged as a group of highly conserved enzyme kinases vital for proper cell cycle regulation. Plks are defined by their N-terminal kinase domain and C-terminal polo-box domain, which regulate their catalytic activity, subcellular localization, and substrate recognition. Among the Plk family members, Plk1 has garnered significant attention due to its pivotal role in regulating multiple mitotic processes, particularly in the kidneys. It is a crucial serine-threonine (Ser-Thr) kinase involved in cell division and genomic stability. In this review, we delve into the types and functions of Plks, focusing on Plk1's significance in processes such as cell proliferation, spindle assembly, and DNA damage repair. The review also underscores Plk1's vital contributions to maintaining kidney homeostasis, elucidating its involvement in nuclear envelope breakdown, anaphase-promoting complex/cyclosome activation, and the regulation of mRNA translation machinery. Furthermore, the review discusses how Plk1 contributes to the development and progression of kidney diseases, emphasizing its overexpression in conditions such as acute kidney injury, chronic kidney disease, and so forth. It also highlights the importance of exploring Plk1 modulators as targeted therapies for kidney diseases in future. This review will help in understanding the role of Plk1 in kidney disease development, paving the way for the discovery and development of novel therapeutic approaches to manage kidney diseases effectively.

Epigenetic regulation of DNA repair gene program by Hippo/YAP1-TET1 axis mediates sorafenib resistance in HCC.

Hepatocellular carcinoma (HCC) is a malignancy that occurs worldwide and is generally associated with poor prognosis. The development of resistance to targeted therapies such as sorafenib is a major challenge in clinical cancer treatment. In the present study, Ten-eleven translocation protein 1 (TET1) was found to be highly expressed in sorafenib-resistant HCC cells and knockdown of TET1 can substantially improve the therapeutic effect of sorafenib on HCC, indicating the potential important roles of TET1 in sorafenib resistance in HCC. Mechanistic studies determined that TET1 and Yes-associated protein 1 (YAP1) synergistically regulate the promoter methylation and gene expression of DNA repair-related genes in sorafenib-resistant HCC cells. RNA sequencing indicated the activation of DNA damage repair signaling was extensively suppressed by the TET1 inhibitor Bobcat339. We also identified TET1 as a direct transcriptional target of YAP1 by promoter analysis and chromatin-immunoprecipitation assays in sorafenib-resistant HCC cells. Furthermore, we showed that Bobcat339 can overcome sorafenib resistance and synergized with sorafenib to induce tumor eradication in HCC cells and mouse models. Finally, immunostaining showed a positive correlation between TET1 and YAP1 in clinical samples. Our findings have identified a previously unrecognized molecular pathway underlying HCC sorafenib resistance, thus revealing a promising strategy for cancer therapy.