NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated PTX3 mRNA in rheumatoid arthritis.

OBJECTIVE: Recent studies indicate that N-acetyltransferase 10 (NAT10)-mediated ac4C modification plays unique roles in tumour metastasis and immune infiltration. This study aimed to uncover the role of NAT10-mediated ac4C in fibroblast-like synoviocytes (FLSs) functions and synovial immune cell infiltration in rheumatoid arthritis (RA). METHODS: FLSs were obtained from active established patients with RA. Protein expression was determined by western blotting or immunohistochemistry or multiplexed immunohistochemistry. Cell migration was measured using a Boyden chamber. ac4C-RIP-seq combined with RNA-seq was performed to identify potential targets of NAT10. RNA immunoprecipitation was used to validate the interaction between protein and mRNA. NAT10 haploinsufficiency, inhibitor remodelin or intra-articular Adv-NAT10 was used to suppress arthritis in mice with delayed-type hypersensitivity arthritis (DYHA) and collagen II-induced arthritis (CIA) and rats with CIA. RESULTS: We found elevated levels of NAT10 and ac4C in FLSs and synovium from patients with RA. NAT10 knockdown or specific inhibitor treatment reduced the migration and invasion of RA FLSs. Increased NAT10 level in the synovium was positively correlated with synovial infiltration of multiple types of immune cells. NAT10 inhibition in vivo attenuated the severity of arthritis in mice with CIA and DTHA, and rats with CIA. Mechanistically, we explored that NAT10 regulated RA FLS functions by promoting stability and translation efficiency of N4-acetylated PTX3 mRNA. PTX3 also regulated RA FLS aggression and is associated with synovial immune cell infiltration. CONCLUSION: Our findings uncover the important roles of NAT10-mediated ac4C modification in promoting rheumatoid synovial aggression and inflammation, indicating that NAT10 may be a potential target for the treatment of RA, even other dysregulated FLSs-associated disorders.

CD276-dependent efferocytosis by tumor-associated macrophages promotes immune evasion in bladder cancer.

Interplay between innate and adaptive immune cells is important for the antitumor immune response. However, the tumor microenvironment may turn immune suppressive, and tumor associated macrophages are playing a role in this transition. Here, we show that CD276, expressed on tumor-associated macrophages (TAM), play a role in diminishing the immune response against tumors. Using a model of tumors induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in BLCA male mice we show that genetic ablation of CD276 in TAMs blocks efferocytosis and enhances the expression of the major histocompatibility complex class II (MHCII) of TAMs. This in turn increases CD4 + and cytotoxic CD8 + T cell infiltration of the tumor. Combined single cell RNA sequencing and functional experiments reveal that CD276 activates the lysosomal signaling pathway and the transcription factor JUN to regulate the expression of AXL and MerTK, resulting in enhanced efferocytosis in TAMs. Proving the principle, we show that simultaneous blockade of CD276 and PD-1 restrain tumor growth better than any of the components as a single intervention. Taken together, our study supports a role for CD276 in efferocytosis by TAMs, which is potentially targetable for combination immune therapy.

RNA modification-related genes illuminate prognostic signature and mechanism in esophageal squamous cell carcinoma.

Emerging studies have demonstrated the link between RNA modifications and various cancers, while the predictive value and functional mechanisms of RNA modification-related genes (RMGs) in esophageal squamous cell carcinoma (ESCC) remain unclear. Here we established a prognostic signature for ESCC based on five RMGs. The analysis of ESCC clinical samples further verified the prognostic power of the prognostic signature. Moreover, we found that the knockdown of NSUN6 promotes ESCC progression in vitro and in vivo, whereas the overexpression of NSUN6 inhibits the malignant phenotype of ESCC cells. Mechanically, NSUN6 mediated tRNA m5C modifications selectively enhance the translation efficiency of CDH1 mRNA in a codon dependent manner. Rescue assays revealed that E-cadherin is an essential downstream target that mediates NSUN6's function in the regulation of ESCC progression. These findings offer additional insights into the link between ESCC and RMGs, as well as provide potential strategies for ESCC management and therapy.

RNA modification-mediated mRNA translation regulation in liver cancer: mechanisms and clinical perspectives.

Malignant liver cancer is characterized by rapid tumour progression and a high mortality rate, whereas the molecular mechanisms underlying liver cancer initiation and progression are still poorly understood. The dynamic and reversible RNA modifications have crucial functions in gene expression regulation by modulating RNA processing and mRNA translation. Emerging evidence has revealed that alterations in RNA modifications facilitate the selective translation of oncogenic transcripts and promote the diverse tumorigenic processes of liver cancer. In this Review, we first highlight the current progress on the functions and mechanisms underlying RNA modifications in the regulation of mRNA translation and then summarize the exciting discoveries on aberrant RNA modification-mediated mRNA translation in the regulation of tumour initiation, metastasis, metabolism, tumour microenvironment, and drug and radiotherapy resistance in liver cancer. Finally, we discuss the diagnostic and therapeutic potentials of targeting RNA modifications and mRNA translation for the clinical management of liver cancer.

ALKBH5-Mediated RNA m6 A Methylation Regulates the Migration, Invasion, and Proliferation of Rheumatoid Fibroblast-Like Synoviocytes.

OBJECTIVE: Fibroblast-like synoviocytes (FLSs) are critical for promoting joint damage in rheumatoid arthritis (RA). N6 -methyladenosine (m6 A) modification plays key roles in various diseases, but its role in the pathogenesis of RA is largely unknown. Here, we investigate increased demethylase ALKBH5 promotion of proliferation, migration, and invasion of RA FLSs via regulating JARID2 expression. METHODS: ALKBH5 expression in FLSs was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. 5-ethynyl-2'-deoxyuridine, scratch wound healing, and transwell assays were implemented to determine the role of ALKBH5 on RA FLS proliferation, mobility, and migration. Then, m6 A sequencing combined with RNA sequencing was performed to identify the potential targets of ALKBH5. RNA immunoprecipitation and RNA pulldown were then used to validate the interaction between the protein and messenger RNA (mRNA). Collagen-induced arthritis (CIA) and delayed-type hypersensitivity arthritis (DTHA) models were further established to assess the therapeutic potency of ALKBH5 in vivo. RESULTS: We demonstrated that ALKBH5 expression was increased in FLSs and synovium from RA. Functionally, ALKBH5 knockdown inhibited the proliferation, migration, and invasion of RA FLSs, whereas overexpression of ALKBH5 displayed the opposite effect. Mechanistically, ALKBH5 mediated m6 A modification in the JARID2 mRNA and enhanced its mRNA stability in cooperation with IGF2BP3. Intriguingly, the severity of arthritis was attenuated in mice with DTHA and ALKBH5 knockout or rats with CIA and intra-articular injection of ALKBH5 short hairpin RNA. CONCLUSION: Our findings suggest that ALKBH5-mediated m6 A modification is crucial for synovial hyperplasia and invasion in RA. ALKBH5 might be a potential therapeutic target for RA and even for dysregulated fibroblasts in a wide range of diseases.

METTL5-mediated 18S rRNA m6A modification promotes oncogenic mRNA translation and intrahepatic cholangiocarcinoma progression.

Intrahepatic cholangiocarcinoma (ICC) is a deadly cancer with rapid tumor progression. While hyperactive mRNA translation caused by mis-regulated mRNA or tRNA modifications promotes ICC development, the role of rRNA modifications remains elusive. Here, we found that 18S rRNA m6A modification and its methyltransferase METTL5 were aberrantly upregulated in ICC and associated with poorer survival (log rank test, p < 0.05). We further revealed the critical role of METTL5-mediated 18S rRNA m6A modification in regulation of ICC cell growth and metastasis using loss- and gain-of function assays in vitro and in vivo. The oncogenic function of METTL5 is corroborated using liver-specific knockout and overexpression ICC mouse models. Mechanistically, METTL5 depletion impairs 18S rRNA m6A modification that hampers ribosome synthesis and inhibits translation of G-quadruplex-containing mRNAs that are enriched in the transforming growth factor (TGF)-ß pathway. Our study uncovers the important role of METTL5-mediated 18S rRNA m6A modification in ICC and unravels the mechanism of rRNA m6A modification-mediated oncogenic mRNA translation control.

Single-dose injectable nanovaccine-in-hydrogel for robust immunotherapy of large tumors with abscopal effect.

Current cancer immunotherapy [e.g., immune checkpoint blockade (ICB)] only benefits small subsets of patients, largely due to immunosuppressive tumor microenvironment (TME). In situ tumor vaccination can reduce TME immunosuppression and thereby improve cancer immunotherapy. Here, we present single-dose injectable (nanovaccines + ICBs)-in-hydrogel (NvIH) for robust immunotherapy of large tumors with abscopal effect. NvIH is thermo-responsive hydrogel co-encapsulated with ICB antibodies and novel polymeric nanoparticles loaded with three immunostimulatory agonists for Toll-like receptors 7/8/9 (TLR7/8/9) and stimulator of interferon genes (STING). Upon in situ tumor vaccination, NvIH undergoes rapid sol-to-gel transformation, prolongs tumor retention, sustains the release of immunotherapeutics, and reduces acute systemic inflammation. In multiple poorly immunogenic tumor models, single-dose NvIH reduces multitier TME immunosuppression, elicits potent TME and systemic innate and adaptive antitumor immunity with memory, and regresses both local (vaccinated) and distant large tumors with abscopal effect, including distant orthotopic glioblastoma. Overall, NvIH holds great potential for tumor immunotherapy.

NAT10-mediated ac4C tRNA modification promotes EGFR mRNA translation and gefitinib resistance in cancer.

Aberrant RNA modifications are frequently associated with cancers, while the underlying mechanisms and clinical significance remain poorly understood. Here, we find that the ac4C RNA acetyltransferase NAT10 is significantly upregulated in esophageal cancers (ESCAs) and associated with poor ESCA prognosis. In addition, using ESCA cell lines and mouse models, we confirm the critical functions of NAT10 in promoting ESCA tumorigenesis and progression in vitro and in vivo. Mechanistically, NAT10 depletion reduces the abundance of ac4C-modified tRNAs and decreases the translation efficiencies of mRNAs enriched for ac4C-modified tRNA-decoded codons. We further identify EGFR as a key downstream target that facilitates NAT10's oncogenic functions. In terms of clinical significance, we demonstrate that NAT10 depletion and gefitinib treatment synergistically inhibit ESCA progression in vitro and in vivo. Our data indicate the mechanisms underlying ESCA progression at the layer of mRNA translation control and provide molecular insights for the development of effective cancer therapeutic strategies.

METTL1/WDR4-mediated tRNA m7G modification and mRNA translation control promote oncogenesis and doxorubicin resistance.

Osteosarcoma is the most common bone tumor that leads to high mortality in adolescents and children. The tRNA N7-methylguanosine methyltransferase METTL1 is located in chromosome 12q14.1, a region that is frequently amplified in osteosarcoma patients, while its functions and underlying mechanisms in regulation of osteosarcoma remain unknown. Herein we show that METTL1 and WDR4 are overexpressed in osteosarcoma and associated with poor patient prognosis. Knockdown of METTL1 or WDR4 causes decreased tRNA m7G modification level and impairs osteosarcoma progression in vitro and in vivo. Conversely, METTL1/WDR4 overexpression promotes osteosarcoma proliferation, migration and invasion capacities. tRNA methylation and mRNA translation profiling indicate that METTL1/WDR4 modified tRNAs enhance translation of mRNAs with more m7G tRNA-decoded codons, including extracellular matrix (ECM) remodeling effectors, which facilitates osteosarcoma progression and chemoresistance to doxorubicin. Our study demonstrates METTL1/WDR4 mediated tRNA m7G modification plays crucial oncogenic functions to enhance osteosarcoma progression and chemoresistance to doxorubicin via alteration of oncogenic mRNA translation, suggesting METTL1 inhibition combined with chemotherapy is a promising strategy for treatment of osteosarcoma patients.

mRNA alternative polyadenylation (APA) in regulation of gene expression and diseases.

The mRNA polyadenylation plays essential function in regulation of mRNA metabolism. Mis-regulations of mRNA polyadenylation are frequently linked with aberrant gene expression and disease progression. Under the action of polyadenylate polymerase, poly(A) tail is synthesized after the polyadenylation signal (PAS) sites on the mRNAs. Alternative polyadenylation (APA) often occurs in mRNAs with multiple poly(A) sites, producing different 3' ends for transcript variants, and therefore plays important functions in gene expression regulation. In this review, we first summarize the classical process of mRNA 3'-terminal formation and discuss the length control mechanisms of poly(A) in nucleus and cytoplasm. Then we review the research progress on alternative polyadenylation regulation and the APA site selection mechanism. Finally, we summarize the functional roles of APA in the regulation of gene expression and diseases including cancers.

N6-methyladenosine (m6A) modification of ribosomal RNAs (rRNAs): Critical roles in mRNA translation and diseases.

As key components of the ribosome and the most abundant RNA species, the rRNAs are modified during ribosome formation. N6-methyladenosine (m6A) is a conserved RNA modification occurring on different RNA species including rRNAs. Recently, it has been reported that ZCCHC4 and METTL5 are methyltransferases that mediate m6A modification of human 28S and 18S rRNA, respectively. The newly discovered biological functions of the two methyltransferases include regulation of mRNA translation, cell proliferation, cell differentiation, stress response, and other biological processes. Both of them, especially METTL5, have been proved to be associated with a variety of diseases such as intellectual disability, cancer, congenital dysplasia and have potential clinical application as biomarkers and therapeutic targets.

METTL3-m6A-EGFR-axis drives lenvatinib resistance in hepatocellular carcinoma.

Lenvatinib is emerging as the first-line therapeutic option for advanced hepatocellular carcinoma (HCC), but drug resistance remains a major hurdle for its long-term therapy efficiency in clinic. N6-methyladenosine (m6A) is the most abundant mRNA modification. Here, we aimed to investigate the modulatory effects and underlying mechanisms of m6A in lenvatinib resistance in HCC. Our data revealed that m6A mRNA modification was significantly upregulated in the HCC lenvatinib resistance (HCC-LR) cells compared to parental cells. Methyltransferase-like 3 (METTL3) was the most significantly upregulated protein among the m6A regulators. Either genetic or pharmacological inhibition of m6A methylation through METTL3 deactivation in primary resistant cell line MHCC97H and acquired resistant Huh7-LR cells decreased cell proliferation and increased cell apoptosis upon lenvatinib treatment in vitro and in vivo. In addition, the specific METTL3 inhibitor STM2457 improved tumor response to lenvatinib in multiple mouse HCC models, including subcutaneous, orthotopic and hydrodynamic models. The MeRIP-seq results showed that epidermal growth factor receptor (EGFR) was a downstream target of METTL3. EGFR overexpression abrogated the METTL3 knocked down-induced cell growth arrest upon lenvatinib treatment in HCC-LR cells. Thus, we concluded that targeting METTL3 using specific inhibitor STM2457 improved the sensitivity to lenvatinib in vitro and in vivo, indicating that METTL3 may be a potential therapeutic target to overcome lenvatinib resistance in HCC.

Ionizable polymeric nanocarriers for the codelivery of bi-adjuvant and neoantigens in combination tumor immunotherapy.

Ionizable lipid nanocarriers have made historical contribution to COVID-19 mRNA vaccines. Here, we report ionizable polymeric nanoparticles that co-deliver bi-adjuvant and neoantigen peptides for cancer immunotherapy in combination with immune checkpoint blockade (ICB). Current cancer ICB benefits only a small subset of patients, largely due to a lack of pre-existing target cells and checkpoint targets for ICB, tumor antigenic heterogeneity, and tumor immunosuppression. Therapeutic vaccines hold the potential to enhance ICB therapeutic efficacy by expanding antitumor cell repertoires, upregulating immune checkpoint levels and hence sensitizing ICB, and reducing tumor immunosuppression. Chemically defined peptide vaccines are attractive, but their current therapeutic efficacy has been limited due to 1) poor vaccine delivery to immunomodulatory lymph nodes (LNs) and antigen (Ag)-presenting cells (APCs), 2) poor immunostimulant adjuvant efficacy with restricted target cell subsets in humans, 3) limited adjuvant/Ag codelivery to enhance Ag immunogenicity, and 4) limited ability to overcome tumor antigenic heterogeneity. Here, we developed nanovaccines (NVs) using pH-responsive polymeric micellular nanoparticles (NPs) for the codelivery of bi-adjuvant [Toll-like receptor (TLR) 7/8 agonist R848 and TLR9 agonist CpG] and peptide neoantigens (neoAgs) to draining LNs for efficient Ag presentation in a broad range of APC subsets. These NVs potentiated the immunogenicity of peptide Ags and elicits robust antitumor T cell responses with memory, and remodeled the tumor immune milium with reduced tumor immunosuppression. As a result, NVs significantly enhanced ICB therapeutic efficacy for murine colorectal tumors and orthotopic glioblastoma multiforme (GBM). These results suggest marked potential of bi-adjuvant/neoAg-codelivering NVs for combination cancer immunotherapy.

Targeting tumour-intrinsic N7-methylguanosine tRNA modification inhibits MDSC recruitment and improves anti-PD-1 efficacy.

OBJECTIVE: Intrahepatic cholangiocarcinoma (ICC) exhibits very low response rate to immune checkpoint inhibitors (ICIs) and the underlying mechanism is largely unknown. We investigate the tumour immune microenvironment (TIME) of ICCs and the underlying regulatory mechanisms with the aim of developing new target to inhibit tumour growth and improve anti-programmed cell death protein-1 (PD-1) efficacy. DESIGN: Tumour tissues from patients with ICC together with hydrodynamic ICC mouse models were employed to identify the key cell population in TIME of ICCs. Functional analysis and mechanism studies were performed using cell culture, conditional knockout mouse model and hydrodynamic transfection ICC model. The efficacy of single or combined therapy with anti-PD-1 antibody, gene knockout and chemical inhibitor were evaluated in vivo. RESULTS: Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are enriched in advanced ICCs and significantly correlated with N7-methylguanosine tRNA methyltransferase METTL1. Using diverse in vivo cancer models, we demonstrate the crucial immunomodulator function of METTL1 in regulation of PMN-MDSC accumulation in TIME and ICC progression. Mechanistically, CXCL8 in human and Cxcl5 in mouse are key translational targets of METTL1 that facilitate its function in promoting PMN-MDSC accumulation in TIME and ICC progression in vivo. Co-blockade of METTL1 and its downstream chemokine pathway enhances the anti-PD-1 efficacy in ICC preclinical mouse models. CONCLUSIONS: Our data uncover novel mechanisms underlying chemokine regulation and TIME shaping at the layer of messenger RNA translation level and provide new insights for development of efficient cancer immunotherapeutic strategies.

Emerging functions of tRNA modifications in mRNA translation and diseases.

tRNAs are essential modulators that recognize mRNA codons and bridge amino acids for mRNA translation. The tRNAs are heavily modified, which are essential for forming a complex secondary structure that facilitates codon recognition and mRNA translation. In recent years, studies have identified the regulatory roles of tRNA modifications in mRNA translation networks. Misregulation of tRNA modifications is closely related to the progression of developmental diseases and cancers. In this review, we summarize the tRNA biogenesis process and then discuss the effects and mechanisms of tRNA modifications on tRNA processing and mRNA translation. Finally, we provide a comprehensive overview of the physiological and pathological functions of tRNA modifications, focusing on diseases including cancers.

Methyltransferase 1 is required for nonhomologous end-joining repair and renders hepatocellular carcinoma resistant to radiotherapy.

BACKGROUND AND AIMS: Radiotherapy is an increasingly essential therapeutic strategy in the management of hepatocellular carcinoma (HCC). Nevertheless, resistance to radiotherapy is one of the primary obstacles to successful treatment outcomes. Hence, we aim to elucidate the mechanisms underlying radioresistance and identify reliable biotargets that would be inhibited to enhance the efficacy of radiotherapy in HCC. APPROACH AND RESULTS: From a label-free quantitative proteome screening, we identified transfer RNA (tRNA; guanine- N [7]-) methyltransferase 1 (METTL1), a key enzyme for N7-methylguanosine (m 7 G) tRNA modification, as an essential driver for HCC cells radioresistance. We reveal that METTL1 promotes DNA double-strand break (DSB) repair and renders HCC cells resistant to ionizing radiation (IR) using loss-of-function and gain-of-function assays in vitro and in vivo. Mechanistically, METTL1-mediated m 7 G tRNA modification selectively regulates the translation of DNA-dependent protein kinase catalytic subunit or DNA ligase IV with higher frequencies of m 7 G-related codons after IR treatment, thereby resulting in the enhancement of nonhomologous end-joining (NHEJ)-mediated DNA DSB repair efficiency. Clinically, high METTL1 expression in tumor tissue is significantly correlated with poor prognosis in radiotherapy-treated patients with HCC. CONCLUSIONS: Our findings show that METTL1 is a critical enhancer for HCC cell NHEJ-based DNA repair following IR therapy. These findings give insight into the role of tRNA modification in messenger RNA translation control in HCC radioresistance.

Eliminating METTL1-mediated accumulation of PMN-MDSCs prevents hepatocellular carcinoma recurrence after radiofrequency ablation.

BACKGROUND AND AIMS: Radiofrequency ablation (RFA) is an important curative therapy in hepatocellular carcinoma (HCC), but recurrence rate remains as high as all the other HCC therapeutic modalities. Methyltransferase 1 (METTL1), an enzyme for m 7 G tRNA modification, was reported to promote HCC development. Here, we assessed the role of METTL1 in shaping the immunosuppressive tumor microenvironment after insufficient RFA (iRFA). APPROACH AND RESULTS: By immunohistochemistry and multiplex immunofluorescence (mIF) staining, we showed that METTL1 expression was enhanced in post-RFA recurrent HCC, accompanied by increased CD11b + CD15 + polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and decreased CD8 + T cells. Mechanistically, heat-mediated METTL1 upregulation enhanced TGF-ß2 translation to form the immunosuppressive environment by induction of myeloid-derived suppressor cell. Liver-specific overexpression or knockdown of Mettl1 significantly affected the accumulation of PMN-MDSCs and subsequently affected CD8 + T cell infiltration. Complete RFA successfully eliminated the tumor, whereas iRFA-treated mice exhibited enhanced tumor growth and metastasis with increased PMN-MDSC accumulation and decreased CD8 + T cells compared to sham surgery. Interrupting METTL1-TGF-ß2-PMN-MDSC axis by anti-Ly6G antibody, or knockdown of hepatoma-intrinsic Mettl1 or Tgfb2 , or TGF-ß signaling blockade significantly mitigated tumor progression induced by iRFA and restored CD8 + T cell population. CONCLUSIONS: Our study sheds light on the pivotal role of METTL1 in modulating an immunosuppressive microenvironment and demonstrated that interrupting METTL1-TGF-ß2-PMN-MDSC axis could be a therapeutic strategy to restore antitumor immunity and prevent HCC recurrence after RFA treatment, meriting further clinical studies.

N7-methylguanosine (m7G) tRNA modification: a novel autophagy modulator in cancer.

Macroautophagy/autophagy, a physiological process that is involved in tumorigenesis, is regulated at genetic and epigenetic levels. Emerging reports suggest that aberrant RNA modifications cause dysregulated autophagy and affect tumorigenesis, while the role of RNA modifications in the regulation of autophagy in cancers remains unclear. In a recent study, we describe a new role for the tRNA m7G methyltransferase complex components METTL1 and WDR4 as negative regulators of MTORC1-mediated autophagy in esophageal squamous cell carcinoma (ESCC). METTL1 and WDR4 show abnormally high expression in ESCC tissues, and are associated with poor ESCC prognosis. Targeting METTL1 or WDR4 leads to decreased expression of m7G-modified tRNAs and reduces the translation of a subset of oncogenic transcripts, including the genes related to the MTOR signaling pathway and negative regulators of autophagy in an m7G-related codon-dependent manner, thereby resulting in hyperactivated MTORC1-mediated autophagy via dephosphorylation of ULK1 and finally causes cell death in ESCC. Our findings provide a new layer of translation regulation mechanism mediated by tRNA m7G modification, link translational machinery with autophagic machinery, and suggest that METTL1 and its downstream signaling axis could be potential therapeutic targets for ESCC treatment.

METTL1-Mediated m7G tRNA Modification Promotes Lenvatinib Resistance in Hepatocellular Carcinoma.

The tyrosine kinase inhibitor lenvatinib is a first-line drug for treating patients with advanced hepatocellular carcinoma (HCC). However, its efficacy is severely hampered by drug resistance. Insights into the molecular mechanisms underlying lenvatinib resistance could provide new strategies to improve and prolong responses. Here, we performed unbiased proteomic screening of parental and lenvatinib-resistant HCC cells and discovered that methyltransferase-like protein-1 (METTL1) and WD repeat domain 4 protein (WDR4), the two key components of the tRNA N7-methylguanosine (m7G) methyltransferase complex, were dramatically upregulated in lenvatinib-resistant cells. METTL1 knockdown overrode resistance by impairing the proliferation capacity of HCC cells and promoting apoptosis under lenvatinib treatment. In addition, overexpression of wild-type METTL1 but not its catalytic dead mutant induced lenvatinib resistance. Animal experiments including hydrodynamic injection, subcutaneous implantation, and orthotopic xenograft mouse models further demonstrated the critical function of METTL1/WDR4-mediated m7G tRNA modification in promoting lenvatinib resistance in vivo. Mechanistically, METTL1 promoted translation of EGFR pathway genes to trigger drug resistance. This work reveals the important role of METTL1-mediated m7G tRNA modification in promoting lenvatinib resistance and provides a promising prediction marker and intervention target for resistance. SIGNIFICANCE: Upregulation of tRNA m7G methyltransferase complex components METTL1 and WDR4 promotes lenvatinib resistance in HCC and confers a sensitivity to METTL1 targeting, providing a promising strategy to override resistance.