Multi-omics reveals crosstalk between lactylation and m6A methylation promotes angiogenesis in lung adenocarcinoma

Multi-omics reveals crosstalk between lactylation and m6A methylation promotes angiogenesis in lung adenocarcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Multi-omics reveals crosstalk between lactylation and m6A methylation promotes angiogenesis in lung adenocarcinoma Dexin Jia, Zihan Jing, Xingmei Ren, Ruqiong Wang, Bo An, Weitong Gao, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7466899/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bevacizumab (Bev) is pivotal in metastatic lung adenocarcinoma (LUAD) therapy, though lactate's regulatory mechanisms remain incompletely characterized. We reveal significant lactate accumulation in Bev-resistant tumors, driving elevated histone lactylation. EZH2-mediated glycolysis enhances lactylation, repressing TIMP2 transcription to promote mitochondrial transfer between endothelial and malignant cells, thereby accelerating angiogenesis and metastasis. Lactate further induces YTHDF2 K17-lactylation, enhancing nuclear translocation and m 6 A recognition to stabilize EZH2 mRNA. FTO suppresses EZH2 via m 6 A demethylation, negatively regulating glycolysis. Clinical data associate high lactylation with poor prognosis. Dual targeting of lactylation and m 6 A combined with Bev demonstrates potent efficacy. These findings provide novel insights into epigenetic mechanisms of metabolic reprogramming and offer therapeutic strategies for patients with Bev-refractory LUAD. Health sciences/Oncology/Cancer/Lung cancer/Non-small-cell lung cancer Health sciences/Oncology/Cancer/Cancer metabolism Health sciences/Oncology/Cancer/Metastasis Health sciences/Oncology/Cancer/Cancer therapy/Cancer therapeutic resistance Lactylation m6A Bevacizumab resistance Metabolic reprogramming Mitochondrial transfer EZH2 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Lung adenocarcinoma (LUAD) has emerged as a critical global health challenge characterized by high incidence rates and poor clinical outcomes 1 . Notably, LUAD cells persistently utilize glycolytic pathways to take up excessive glucose and generate substantial lactate even under oxygen-sufficient conditions 2 , 3 . Initially dismissed as a metabolic waste product, lactate has now been redefined as a multifunctional signaling mediator that mediates microenvironmental acidification and orchestrating immune evasion mechanisms 4 . Most notably, the recently identified post-translational modification known as lactylation has unveiled lactate's direct involvement in epigenetic regulation 5 . The role of this novel covalent modification of lysine residues 4 , 6 – 8 in bevacizumab (Bev) resistance remains undefined. EZH2 (Enhancer of Zeste Homolog 2) catalyzes the methylation of histone H3 at lysine 27 residue using S-adenosyl-L-methionine (SAM), predominantly producing di- (H3K27me2) and tri-methylated (H3K27me3) epigenetic modifications. H3K27me3-mediated epigenetic silencing dynamically governs cellular differentiation programs and proliferation 9 . Compelling clinical observations demonstrate that EZH2 hyperactivation(manifested through gain-of-function mutations or dysregulation)drives tumor and metastatic progression via genome-wide aberrations of H3K27me3 deposition. Mitochondria, which serve as central hubs of cellular energy metabolism, sustain functional cellular integrity through critical processes encompassing ATP synthesis, regulation of metabolic homeostasis, calcium buffering, and stress response modulation. Through dynamic molecular crosstalk, they not only regulate intracellular compartments but also orchestrate endothelial cell (EC)-mediated intercellular communication networks 10 . Studies have demonstrated that small extracellular vesicles (sEVs) 11 and tunneling nanotubes (TNTs) 12 function as essential conduits for mitochondrial transcellular transfer, playing a pivotal role in maintaining tissue microenvironment homeostasis. Astrocyte-to-neuron mitochondrial transfer is orchestrated by LRP1-dependent intercellular trafficking machinery, which mitigates cerebrovascular ischemia-reperfusion (IR) injury through lactylation-dependent regulation of ARF1 13 . Based on these mechanistic foundations, we propose that lactate serves as a pivotal mediator of mitochondrial transfer between cancer cells and endothelial cells (ECs). In this study, integrative multi-omics profiling delineates a quintuple regulatory axis comprising lactate-FTO/YTHDF2-EZH2/H3K27la-TIMP2-mitochondrial transfer, mechanistically bridging metabolic-epigenetic crosstalk with angiogenesis-mediated LUAD progression. This framework establishes a rationale for developing combined epigenetic-metabolic therapeutic strategies to counteract Bev resistance. 2. Results 2 .1 . Bevacizumab resistance shows increased lactylation levels correlating with poor survival Following Bev administration, tumor cells may significantly activate histone lactylation through compensatory glycolysis, enhancing tumor cell survival and treatment resistance 14,15 . To assess post-antiangiogenic lactate dynamics, we quantified serum and tissue lactate levels in 40 patients (20 therapy-resistant vs 20 sensitive), revealing significantly elevated lactate accumulation in non-responders (Fig. 1A-B). Histone lactylation, a recently identified post-translational modification first reported by Zhang, is metabolically fueled by glycolytic or alternative pathways generating cytoplasmic lactate 5 . The substantial lactate produced by metabolic reprogramming provides a substrate for histone lactylation, manifested by increased pan-lysine lactylation (Pan Kla) (Fig. 1C) and significantly elevated histone H3 lysine 27 lactylation (H3K27la) levels in therapy-resistant tissues (n = 6) (Fig. 1D). Next, we assessed the clinical relevance of H3K27la in 84 LUAD cases (42 sensitive vs. 42 resistant) using immunofluorescence (IF) and immunohistochemistry (IHC). Fig. 1E revealed a significant correlation between H3K27la and pan-lactylation (Pan Kla) levels. Kaplan-Meier analysis stratified by H3K27la levels revealed significantly worse overall survival (OS), indicating their association with poor prognosis (Figs. 1F-G). Furthermore, clinical assessments revealed an inverse correlation between H3K27la levels and therapeutic response rates (Fig. 1H). PET-CT imaging quantifies tumor glucose uptake in LUAD patients 16 . PET-CT imaging analysis of 84 patients utilized maximum standardized uptake value (SUV max) to quantify 18F-FDG uptake. Analysis demonstrated a positive correlation between SUV max and H3K27la levels (IHC) (Fig. 1I). Elevated H3K27la levels significantly correlated with metastatic dissemination ( P < 0.0001) and elevated serum CEA levels ( P < 0.0001) (Table 1). Clinical analyses reveal that aberrant H3K27la is linked to Bev resistance in LUAD, promoting metastasis and poor prognosis. Table 1. Correlation between H3K27la expression and clinicopathological features in patients with LUAD (n = 84) Characteristic n H3K27la High expression(%) Low expression(%) P Gender Male 36 21(58.3) 15(41.6.7) 0.7577 Female 48 25(52) 23(47.9) Age ＜55 29 19(65.5) 10(34.5) 0.1580 ≥55 55 27(49.1) 28(50.9) Tumor Location Left 40 25(62.5) 15(37.5) 0.7587 Right 44 21(47.7) 23(52.3) Smoking Yes 54 29(53.7) 25(46.3) 0.0130* No 30 17(56.7) 13(43.3) Tumor Size ≤3cm 59 24(40.7) 35(59.3) 0.0009* ＞3cm 25 22(88) 3(12) Lymph Node Metastasis Yes 67 30(44.8)) 37(55.2) ＜0.0001* No 17 16(94.1) 1(5.9) CEA High 67 33(49.3) 34(50.7) ＜0.0001* Low 17 13(76.5) 4(23.5) Distance metastasis Yes 9 7(15.2) 2(5.3) ＜0.0001* No 75 39(84.8) 36(94.7) * P < 0.05 The model showed the key enzymes and inhibitors of glycolysis (Fig. S1A). Levels of glucose transporter (GLUT1), hexokinase (HK2), and lactate dehydrogenase LDHA, all of which directly regulate intracellular lactate synthesis 17 , were coordinately upregulated in LUAD samples alongside VEGFA (Fig. S1B-C), indicating that bevacizumab resistance may involve glycolysis-lactylation axis-driven mechanisms. Survival analysis demonstrated significant associations between these factors and adverse prognosis (Fig. S1D). To characterize lactate metabolic remodeling in LUAD, we analyzed public single-cell RNA sequencing datasets (GSE131907) from the GEO database, revealing tumor-specific LDHA overexpression (Fig. S1E). Western blotting analysis confirmed effective LDHA knockdown (Fig. S1F). These findings collectively validate the significant correlation between heightened glycolytic activity and adverse survival outcomes in LUAD patients. 2.2. EZH2 promotes LUAD metastasis by enhancing glycolysis EZH2, as a crucial epigenetic regulator, possesses histone methyltransferase and acetyltransferase activities, catalyzing H3K27, leading to the silencing of target genes 9 . Therefore, we propose that EZH2 may catalyze H3K27 lactylation. EZH2 exhibited marked transcriptional elevation in the UALCAN -LUAD samples and was associated with unfavorable outcomes from the GEPIA database (Fig. S2A). Cell line screening (Fig. S2B) revealed that EZH2-high cells showed elevated bevacizumab IC₅₀ values (Fig. S2C). A549 and H1993 cells showed significantly higher lactylation and H3K27la levels compared to normal lung epithelial cells (HBE) (Fig. S2D). To assess EZH2's lactylation regulatory function, we established knockdown models (Fig. S2E). GO analysis of RNA-seq data revealed significant enrichment of glycolysis pathways upon shEZH2 treatment (Fig. S2F). Seahorse extracellular flux analysis showed that shEZH2 significantly decreased extracellular acidification rate (ECAR), indicating EZH2-driven enhancement of glycolysis (Fig. S2G). EZH2 knockdown cells exhibited reduced GLUT1/HK2/LDHA expression, and lactate production (Fig. S2H-I). EZH2 overexpression induced opposite effects (Fig. S2J-K). Collectively, these findings systematically demonstrate that EZH2 promotes the glycolysis process in lung adenocarcinoma. Clinical data indicated EZH2's association with metastasis in bevacizumab-resistant patients. RNA-seq GO analysis revealed shEZH2-mediated enrichment of metastasis-related pathways (Fig. S3A). Functional validation established EZH2 as a key driver of tumor metastasis (Fig. S3B-E). Western blotting revealed elevated N-cadherin/Vimentin (EMT markers) and reduced E-cadherin in EZH2-overexpressing cells, whereas knockdown cells exhibited the inverse pattern (Fig. S3F). These data demonstrate how EZH2 drives metastasis by promoting EMT. 2.3. EZH2 is a potential writer enzyme of histone lactylation in LUAD cells Inhibiting EZH2 via histone lysine methyltransferase (KMT) targeting represents a pivotal epigenetic therapeutic strategy 18 . We designed a targeted strategy combining Tazemetostat (Taz; first FDA-approved EZH2 inhibitor), 2-deoxy-D-glucose (2-DG; glycolysis inhibitor), and Oxamate (LDH inhibitor) to coordinately block the EZH2-glycolysis-lactylation axis. Taz, 2-DG, and Oxamate showed a dose-dependent reduction of both pan-lactylation and H3K27la in Figure 2A. To validate histone lactylation function, LDHA knockdown reduced pan-lactylation and H3K27 lactylation, with sodium lactate (NaLa) supplementation rescuing these effects (Fig. 2B). Fig. 2C shows reduced Pan Kla and H3K27la levels upon EZH2 inhibition. Under NaLa treatment conditions, shEZH2 still markedly reduced Pan Kla and H3K27la levels. For in vivo validation, 2×10⁶ HUVECs and 5×10⁵ A549 cells were subcutaneously co-implanted in BALB/c mice. Bev (10 mg/kg) was administered via intraperitoneal injection. Taz (100 mg/kg) and DMSO were orally administered to nude mice. 18F-FDG PET-CT imaging demonstrated significantly reduced glucose uptake in Taz-treated xenografts versus controls (Fig. 2D-H). These findings establish EZH2 and lactate as regulators of histone H3K27 lactylation. Angiogenesis as a hallmark of solid tumor progression shows hyperactivation strongly associated with poor prognosis. Recent studies have revealed that glycolysis-derived lactate can remodel angiogenic phenotypes by inducing histone lactylation modification (H3K18la) 19 , while EZH2 may mediate this process through the "metabolic reprogramming-chromatin remodeling" interaction network. A model was constructed by Transwell to identify the influence on ECs (Fig. S4A). Co-culture of EZH2-knockdown tumor cells (shEZH2) with ECs revealed significant impairment of EC proliferation, migration, and tube formation capacity (Fig. 2I-K). These results demonstrate that EZH2, as a potential writer of lactylation, drives LUAD progression through the "metabolic reprogramming-epigenetic remodeling-angiogenesis" cascade pathway. 2.4. Transcriptional repression of TIMP2 by EZH2 regulates angiogenesis in LUAD Next, we elucidated the EZH2-H3K27la epigenetic axis using a multi-omics strategy. CUT&Tag genome-wide profiling with H3K27la-specific antibody showed reduced TSS-proximal occupancy upon EZH2 knockdown (Fig. 3A), while 53% enrichment localized to promoters (Fig. 3B). GO/KEGG analyses of EZH2 knockdown-induced promoter-localized silenced peaks showed angiogenic pathway enrichment (Fig. 3C-D). RNA-seq analysis revealed shEZH2-regulated potential targets (Fig. 3E). KEGG analysis demonstrated that downregulated genes in the shEZH2-treated group were significantly enriched in angiogenesis-related signaling pathways (Fig. 3F). Integration of GEPIA-derived 3130 downregulated genes with CUT&Tag/RNA-seq DEGs identified 275 candidates showing elevated mRNA levels and decreased H3K27la promoter enrichment upon EZH2 knockdown (Fig. 3G). Focus on the angiogenesis pathway revealed significantly reduced H3K27la enrichment at the TIMP2 (Tissue Inhibitor of Metalloproteinases-2) promoter (Fig. 3H). TIMP2 acts as a key angiogenesis inhibitor via an Matrix Metalloproteinase (MMP)-independent regulatory mechanism 20,21 . ChIP-qPCR analysis confirmed reduced H3K27la occupancy at the TIMP2 promoter following treatment with Taz, 2-DG, or Oxamate (Fig. 3I). TIMP2 protein levels were significantly upregulated by EZH2 knockdown, Taz treatment, Oxamate treatment, or 2-DG treatment (Fig. 3J-M). These findings indicate that TIMP2 modulates LUAD progression via H3K27la, providing a rationale for developing epigenetic-metabolic dual-targeting anti-angiogenic therapies. 2.5. TIMP2 upregulation suppresses LUAD angiogenesis Analysis of TCGA datasets revealed significant downregulation of TIMP2 expression in LUAD, correlating with favorable patient prognosis (Fig. S4B). To investigate TIMP2's functional role in LUAD, we conducted TIMP2 knockdown using siRNA, with validation by Western blotting (Fig. S4C). Notably, siTIMP2 significantly enhanced EC proliferation, migration, and tube formation (Fig. S4D-F). These findings suggest that TIMP2 is one of the regulators of angiogenesis in LUAD. To investigate TIMP2's role in H3K27 lactylation-driven LUAD pathogenesis, we transfected A549 and H1993 cells with siTIMP2 in combination with Oxamate and 2-DG. Compared to controls, treatment with glycolysis inhibitors reversed the TIMP2 knockdown-induced enhancement of EC proliferation, migration, and tube formation (Fig. S5A-C). Experimental data indicate TIMP2 regulates H3K27la-mediated angiogenesis in LUAD. TIMP2 regulates tumor metastasis 22,23 . To examine TIMP2's role in metastasis, A549 and H1993 cells were transfected with siTIMP2 and shEZH2. TIMP2 knockdown promoted partial metastatic ability following shEZH2 (Fig. S6A-C). These results indicate that TIMP2 regulates EZH2-driven LUAD metastasis. 2.6. m 6 A lactylation regulates angiogenesis in LUAD Lactate induces histone and non-histone protein lactylation 24 . To characterize the lactylation landscape in LUAD, we performed 4D label-free lactylome analysis to identify differentially lactylated proteins in Bev-resistant A549 cells. We identified 19,265 peptides including 4,607 lactylated species. In A549 cells, 4,841 lactylation sites were mapped to 1,900 proteins (Fig. S7A). Quality control confirmed peptide distributions met predefined analytical criteria (Fig. S7B). Comparative analysis of Klac site flanking sequences versus the human proteome revealed lysine enrichment and cysteine/leucine depletion at most positions (Fig. S7C-D). WikiPathways also revealed significant enrichment of VEGFA-VEGFR2 signaling (Fig. S7E). These findings demonstrate that lactylation regulates angiogenesis in Bev-resistant LUAD. Aberrant m⁶A modification drives key gene reprogramming to promote cancer progression 25,26 . To delineate protein lactylation in tumor angiogenesis, we analyzed the m⁶A pathway and identified lactylated components including METTL3, METTL14, and ALKBH5 (Fig. S7F). Resistant tissues exhibited elevated global m⁶A methylation levels (Fig. S7G). TCGA analysis demonstrated FTO downregulation in LUAD, with low-FTO patients exhibiting significantly shorter overall survival compared to cohorts stratified by other m⁶A writers/erasers (Fig. S8). FTO, but not METTL3, METTL14, or ALKBH5, significantly modulated bevacizumab resistance. IHC validation revealed pronounced FTO decrease in Bev-resistant specimens (Fig. S7H-I). Nuclear-cytoplasmic fractionation with qPCR and IF confirmed predominant nuclear localization of FTO (Fig. S7J-K). These findings uncover lactylation (Kla)-m⁶A crosstalk mediating Bev resistance via an epigenetic regulatory network. 2.7. RNA-seq and MeRIP-seq identify EZH2 as a direct target for FTO To assess FTO-regulated m⁶A on EZH2, we generated shFTO and conducted epitranscriptomic microarray for mRNA m⁶A profiling (Fig. S9A). The Heatmap and scatter plots illustrated the profiles of m 6 A modification and fold changes upon FTO knockdown, revealing widespread hypermethylation/overexpression including EZH2 (Fig. S9B-C). MeRIP-qPCR detected m⁶A enrichment in EZH2 (Fig. S9D). SRAMP analysis revealed high-confidence m⁶A sites in EZH2 (Fig. S9E). To study FTO-regulated m⁶A on EZH2, we performed a luciferase assay showing significant wild-type EZH2 activation post-FTO depletion, unlike unresponsive mutants (Fig. S9F). RNA immunoprecipitation (RIP) assays revealed FTO binding to EZH2 mRNA (Fig. S9G). IF and Western blotting analysis revealed nuclear colocalization of FTO with EZH2 following treatment with the global methylation inhibitor DAA. Intriguingly, DAA treatment significantly suppressed EZH2 protein levels (Fig. S9H-I). Western blotting confirmed FTO knockdown-induced EZH2 upregulation (Fig. S9J). DAA suppressed EZH2 in LUAD cells and attenuated FTO knockdown-induced EZH2 upregulation, validating FTO-dependent m⁶A demethylation of EZH2 (Fig. S9K). FTO knockout increases m⁶A methylation on EZH2, upregulating its expression. 2.8. FTO inhibits glycolysis and metastasis in LUAD FTO maintains metabolic homeostasis by regulating energy metabolism pathways 27 . GO analysis of m 6 A-modified transcripts revealed FTO regulates glycolysis (Fig. S10A). Untargeted metabolomics was used to analyze metabolic changes in shFTO groups. The shFTO group exhibited 12 differentially abundant metabolites (2 downregulated, 10 upregulated) (Fig. S10B). Notably, lactate was among the altered metabolites. KEGG analysis revealed enriched central carbon metabolism pathways (Fig. S10C). GLUT1, HK, LDHA, and lactate levels were significantly elevated upon FTO knockdown (Fig. S10D-G). FTO overexpression significantly suppressed ECAR levels in H1650 cells, indicating its role in inhibiting glycolysis in LUAD (Fig. S10H). These results indicate that FTO influences glycolysis in LUAD. Wound healing and Transwell assays demonstrated that FTO overexpression inhibited metastasis, whereas its knockdown promoted metastasis (Fig. S11A-D). Western blotting analysis revealed significant downregulation of EMT-related proteins (N-cadherin, Vimentin) upon FTO overexpression. Conversely, FTO knockdown exhibited reversed effects (Fig. S11E). These results demonstrate that FTO inhibits metastasis in LUAD through EMT. 2.9. Disrupting FTO-EZH2 interaction increases glycolysis To define EZH2's mechanistic role in FTO-mediated m 6 A modification, we performed rescue experiments. ELISA revealed that EZH2 inhibition attenuated shFTO-induced elevation of GLUT1, HK, LDHA, and LA levels (Fig. S12A). Wound healing and Transwell assays exhibited Taz-mediated suppression of shFTO-induced metastatic potential (Fig. S12B-C). These findings demonstrate that FTO modulates EZH2 to perturb glycolysis, thereby suppressing metastasis in LUAD. To further explore the biological impact of the FTO-EZH2 regulatory axis on tumor angiogenesis, we co-cultured ECs with cancer cells. The results showed that, compared to DMSO, Taz inhibited the proliferation, migration, and tube formation ability of HUVECs (Fig. S13A-C). These findings indicate that EZH2 promotes tumor angiogenesis and metastasis via glycolytic reprogramming under epigenetic control of FTO-mediated m 6 A modification. 2.10. YTHDF2 specifically recognizes EZH2 as an m 6 A reader in LUAD m⁶A modification is coordinated by writers (methyltransferases) and erasers (demethylases), with functional outcomes determined by readers' site-specific recognition of methylated motifs 28,29 . Thus, identifying functional readers is crucial for elucidating m⁶A-mediated biological mechanisms. We investigated the impact of lactylation on m⁶A modification by profiling readers, identifying lactylated regulators YTHDF2/YTHDF3 (Fig. S14A). Western blotting analysis demonstrated YTHDF2 elevates EZH2 protein levels through m⁶A-specific recognition of EZH2 mRNA (Fig. S14B-C). IF showed that YTHDF2 was predominantly localized in the cytoplasm (Fig. S14D). RIP-qPCR validated YTHDF2 specifically binds m⁶A-modified EZH2 transcripts, showing enhanced binding upon FTO knockdown (Fig. S14E). RNA secondary structure predictions confirmed accessibility of the m⁶A consensus motif (Fig. S14F). RNA stability analysis demonstrated that knockdown of YTHDF2 significantly reduced the half-life of EZH2 mRNA (Fig. S14G). These findings define YTHDF2 as a pivotal m⁶A reader regulating EZH2 expression. 2.11. Tumor lactylation elevates YTHDF2 expression in LUAD We examined lactate's regulatory role on YTHDF2 by quantifying its levels in Transwell cocultures, observing significant accumulation (Fig. S15A). YTHDF2 and EZH2 expression exhibited a positive correlation with lactate levels (Fig. S15B). Exogenous lactate dose-dependently increased YTHDF2 expression (Fig. S15C), whereas Oxamate suppressed its expression (Fig. S15D). LDHA knockdown reduced lactate production, consequently attenuating YTHDF2 expression (Fig. S15E-F). Notably, NALA treatment restored YTHDF2 levels (Fig. S15G). We next investigated if YTHDF2 lactylation modulated binding to m⁶A-RNAs. FLAG-YTHDF2 immunoprecipitation (IP) in A549 cells revealed enhanced m⁶A recognition ability upon NALA treatment (Fig. S15H). We assessed YTHDF2 stability with/without NALA treatment under cycloheximide (CHX) exposure. The half-life of YTHDF2 was significantly prolonged (Fig. S15I). Proteasome-dependent degradation of YTHDF2 (evidenced by MG132-induced accumulation) was attenuated by NALA treatment (Fig. S15J). The above experiments demonstrated that NALA increased the stability of YTHDF2. These findings collectively establish that lactate enhances YTHDF2 expression and potentiates its m 6 A recognition capacity. Using 4D label-free quantitative proteomics, we identified a specific lactylation at the K17 site of YTHDF2 (Fig. 4A-B). This site was highly evolutionarily conserved (Fig. 4C). Molecular docking predicted stronger L-lactate binding to YTHDF2-K17, supported by lower binding energy (Fig. 4D). To confirm K17 as the primary lactylation site, we generated K17R mutants and analyzed lactylation levels (Fig. 4E). We next explored YTHDF2 lactylation's functional impact. K17R mutation suppressing lactylation reduced both EZH2 protein and H3K27la levels upon 25 mM NALA treatment (Fig. 4F). IP analysis revealed significant enhancement of lactylation signals that positively correlated with YTHDF2 protein levels (Fig. 4G). The YTHDF2 K17R mutant exhibited near-complete loss of lactylation signals, confirming K17 as the predominant lactylation site (Fig. 4H). Subcellular fractionation combined with IF analysis revealed lactate-induced nuclear translocation of YTHDF2 (Fig. 4I). Notably, NALA increased WT-YTHDF2 nuclear localization but did not alter K17R mutant localization (Fig. 4J). IF imaging showed WT YTHDF2 displayed nucleocytoplasmic distribution, whereas the K17R mutant was predominantly cytoplasmic (Fig. 4K). These findings establish K17 as an evolutionarily conserved lactylation hotspot in YTHDF2, with lactate-induced K17 lactylation enhancing nuclear translocation. We established ECs-LUAD co-cultures with four treatment groups: siNC, siYTHDF2, siYTHDF2+siNC, and siYTHDF2+siTIMP2. Transwell assays showed that siYTHDF2 reduced transmigration, which was partially reversed in the dual-knockdown group (Fig. S16A-B). Endothelial functional assays revealed that siYTHDF2 decreased HUVECs proliferation and tube formation (Fig. S16C-E). These findings establish that YTHDF2 drives metastasis and angiogenesis via lactylation-dependent nuclear translocation in LUAD. 2.12. TIMP2 mediates intercellular mitochondrial transfer post-bevacizumab Mitochondrial transfer mediates EC formation through mitophagy 10 and drives multidrug resistance. IF revealed TIMP2-mitochondrial marker colocalization (Fig. 5A). Transmission electron microscopy (TEM) revealed abnormal mitochondrial morphology with reduced cristae in ECs and cancer cells from the TIMP2-overexpression group (Fig. 5B). Mitochondrial DNA (mtDNA) mutations drive mitochondrial transfer 30 . qPCR revealed TIMP2 overexpression reduced mtDNA copies in ECs and cancer cells (Fig. 5C). TIMP2 overexpression reduced mitochondrial membrane potential (Fig. 5D). CX43-mediated mitochondrial transfer sustains leukemic stemness via metabolic reprogramming 31 . The TIMP2-overexpressing group exhibited a significant reduction in oxygen consumption rate (OCR) (Fig. 5E). To monitor mitochondrial transfer, we transfected the mitochondria-specific fluorescent protein Mito-DsRed into cancer cells. Treatment with TNT inhibitor cytochalasin B and EV inhibitor GW4869 revealed extracellular vesicles predominantly mediate mitochondrial transfer (Fig. 5F). We found GW4869 inhibited angiogenesis (Fig. 5G). These results demonstrate that TIMP2 regulates mitochondrial transfer to promote angiogenesis, thereby driving metastasis in LUAD. 2.13. EZH2-targeted therapy enhances bevacizumab response in LUAD Bev-induced m⁶A-lactylation interplay in LUAD is linked to angiogenesis in metastases. In vivo and vitro models demonstrated EZH2 upregulation drives angiogenesis in Bev-resistant tumors. We developed a targeted therapeutic strategy combining Taz and Bev to synergistically block the EZH2-glycolysis-lactylation regulatory axis. This dual targeting suppressed LUAD metastasis and angiogenesis (Fig. 6A-B). Serum analysis showed increased lactate dehydrogenase in Bev-resistant patients (Fig. 6C). CT scans demonstrated poor treatment response in LDH-high LUAD patients following Bev treatment (Fig. 6D). PET-CT showed elevated SUV max with metastases in lactate dehydrogenase-high cohorts (Fig. 6E). EZH2 and LDHA expression was increased in the LDH-high group (Fig. 6F). Tail vein injection of A549 cells in nude mice established lung metastasis models, showing Bev/Taz combination synergistically reduced metastatic burden (Fig. 6G). These findings demonstrate that Taz disrupts tumor vasculature and overcomes lactylation-mediated bevacizumab resistance. 3. Discussion While substantial progress has been achieved in current LUAD therapy, elucidating the mechanisms underlying treatment resistance remains a pivotal challenge in oncology 32 . Integrated multi-omics analyses, we were the first to reveal that lactate accumulation induced by Bev resistance drives LUAD progression through dual epigenetic axes: coordinated histone/nonhistone lactylation and m 6 A-mediated regulatory networks. Our key findings delineate four mechanistic pillars: (1) The EZH2-glycolysis-H3K27la-TIMP2 signaling axis mediates pathological angiogenesis; (2) An FTO/YTHDF2-m 6 A-EZH2 circuit integrates metabolic-epigenetic crosstalk; (3) K17 lactylation on YTHDF2 potentiates m 6 A recognition through nucleo-cytoplasmic trafficking; (4) Mitochondrial transfer networks remodel tumor-endothelial metabolic crosstalk. These findings establish a novel conceptual framework for overcoming anti-angiogenic therapy resistance. Lactate, a pivotal Warburg effect metabolite, exerts epigenetic regulatory functions via histone lysine lactylation (such as H3K18la), activating pro-tumorigenic transcription 24 , 26 . Mechanistic investigations revealed that Bev resistance enhances glycolysis via EZH2-mediated regulation, driving lactate accumulation in the tumor microenvironment and sustaining substrate supply for histone lactylation. Clinical cohort analysis demonstrated significantly elevated H3K27la levels in resistant tumors, while inhibition of histone lactylation effectively suppressed metastasis and angiogenesis in LUAD (Fig. 1 ). These results were further substantiated in murine models. These findings may provide a novel therapeutic strategy to enhance bevacizumab efficacy in LUAD through targeting histone lactylation. Serving as the enzymatic center of the PRC2 33 , EZH2 mediates H3K27 trimethylation (H3K27me3) to establish epigenetic silencing 34 . Pan-cancer analyses demonstrate that EZH2 orchestrates metabolic reprogramming through glycolysis regulation while suppressing tumor suppressor gene transcription 35 . Mechanistic studies reveal that EZH2 modulates post-bevacizumab metastasis through glycolysis-driven metabolic reprogramming (Fig. 2 ). Notably, tazemetostat, an EZH2 inhibitor initially approved by the FDA, may provide therapeutic benefits for bevacizumab-resistant patients. This discovery provides novel insights for precision oncology and opens new therapeutic avenues for cancer patients. Aberrant histone modifications are prevalent across diverse pathological processes (including malignancies), and their imbalance significantly impacts disease progression 36 . Emerging evidence suggests that histone lactylation is a novel epigenetic driver that induces the expression of genes associated with tumorigenesis and drug resistance 37 , 38 . To our knowledge, this study provides the first evidence that aberrant histone lactylation machinery drives bevacizumab resistance. Integrated multi-omics analysis demonstrated that EZH2-mediated H3K27la transcriptionally suppresses TIMP2, thereby promoting angiogenesis (Fig. 3 ). We have delineated a novel glycolysis-H3K27la-TIMP2 regulatory axis that provides actionable therapeutic targets to overcome resistance to anti-angiogenic therapies. As the pioneer m⁶A demethylase, FTO orchestrates pivotal RNA metabolic pathways while exhibiting dual functionality in energy metabolism dysregulation and tumor evolution 39 . Nevertheless, the regulatory function of FTO in LUAD glycolytic reprogramming remains undefined, underscoring the critical need to delineate its epigenetic mechanisms driving bevacizumab resistance. We demonstrated that FTO-mediated m⁶A modification of EZH2 attenuates glycolytic reprogramming in LUAD, thereby suppressing angiogenesis. As far as we are aware, EZH2 represents the first histone methyltransferase reported to undergo FTO-mediated regulation in LUAD, thereby elucidating novel roles of histone-modifying enzymes in LUAD pathogenesis. Warburg effect-derived lactate not only serves as a signaling molecule in biological processes but also remodels the epigenetic landscape via histone and non-histone lactylation 40 . Intriguingly, lactylation cooperates with m⁶A modifications to orchestrate oncogenic transcriptional programs 24 , 26 , 41 – 43 . Unlike YTHDF3, YTHDF2 predominantly mediates degradation of m⁶A-modified mRNAs through lactylation-dependent regulation 41 . This groundbreaking study demonstrates that hyperactivated glycolysis triggers lactylation at lysine 17 (K17) of YTHDF2, which enhances its m⁶A-binding affinity to stabilize EZH2 mRNA, thereby orchestrating metabolic-epigenetic crosstalk that drives LUAD angiogenesis and metastatic progression (Fig. 4 ). By delineating the lactate-m⁶A-EZH2-glycolysis regulatory axis, we pioneer the identification of YTHDF2's lactylation-driven nuclear translocation pathway, providing novel mechanistic insights into metabolic-epigenetic crosstalk. Metabolic coupling through lactate drives intercellular mitochondrial trafficking between hematopoietic progenitors (HSPCs) and a distinct BM stromal subset (PDGFRα+/Sca-1⁻/CD48low), mediated by Cx43 channels and AMPK activation. This bidirectional crosstalk establishes redox equilibrium through coordinated ROS modulation in these compartments 44 . Experimental evidence from Zhou's group demonstrates that astrocyte-derived LRP1 promotes neuronal mitochondrial transfer by dually suppressing lactate biosynthesis and ARF1 lactylation, thereby attenuating cerebrovascular IR injury in rodent models 13 . Under lactate-rich conditions, cancer cells transfer mitochondria to adjacent endothelial cells through TNTLs and EVs, promoting tumor progression. Mechanistic studies demonstrate that EZH2 promotes EV secretion via TIMP2 to regulate mitochondrial transfer between cancer cells and ECs, whereas GW4869 inhibits this process (Fig. 5 ). This study defines the lactate-mitochondrial transfer-metabolic axis, establishing a framework for mitochondrial transfer-based therapeutics. EZH2 drives malignant transformation by catalyzing H3K27 trimethylation and serves as a master epigenetic regulator of oncogenic programs 45 , 46 . EZH2 elevates H3K27 methylation at the DLC1 promoter to promote proliferation and glycolysis in esophageal cancer cells 47 . Our study revealed a marked accumulation of lactate serving as the substrate for histone H3K27la. And EZH2-driven glycolytic enhancement promotes angiogenesis in LUAD. Integrated multi-omics analyses uncovered the H3K27la-mediated transcriptional repression mechanism, with TIMP2 established as the central regulator driving angiogenesis within this pathway. Lactylome profiling revealed lactylation of m 6 A regulators, with lactylated YTHDF2 stabilizing EZH2 mRNA via m 6 A recognition. This study delineated a five-tiered "lactate-FTO/YTHDF2-EZH2/H3K27la-TIMP2-mitochondrial transfer" axis, showing that pharmacological inhibition of this axis enhances Bev responsiveness, thereby nominating lactylation-m 6 A crosstalk as a novel therapeutic target. 4. Material and methods 4.1. Human sample collection and cell culture Clinical specimens and associated patient data were sourced through collaboration with Harbin Medical University Cancer Center. The study subjects were patients with lung adenocarcinoma and included 20 pairs of fresh-frozen tumors and adjacent non-neoplastic tissues. Pathological sections from 84 patients enrolled between December 2016 and December 2019 were used for IHC, and the clinicopathological characteristics collected included age, gender, smoking history, tumor location, size, lymph node metastasis, survival rate, and CEA level (Supplementary Data 2). 18F-FDG PET/CT scans were analyzed to quantify intratumoral glucose uptake (SUVmax). Blood sera from healthy controls and LUAD cases were included in this study. Ethical approval for this research was obtained from the Institutional Review Board. Human bronchial epithelial (HBE) cells and LUAD cell lines (H1993, A549, H1650) were obtained from the Cancer Experimental Center at Harbin Medical University Cancer Hospital. Cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel) under standard conditions (37°C, 5% CO₂). 4.2. Tumor Xenograft Implantation and 18FFDG PET imaging Female immunodeficient BALB/c mice (aged six weeks; body weight range 18–20 g) were obtained from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). For tumor angiogenesis assay, HUVEC 2×10 6 and 5×10 5 cells were co-implanted in the right axillary of mice, bioluminescence images were taken on day 28, and the mice were euthanized, tumors were excised, and weighed. Mice were anesthetized with 1% sodium pentobarbital the day before euthanasia, following an 8-hour fasting regimen, 200 µl of 18F-FDG tracer was delivered via intraperitoneal bolus injection under controlled metabolic conditions. Then, the mice were positioned on the examination table for PET/CT imaging. The 18F-FDG uptake was achieved by delineating regions of interest (ROI) and measuring the SUVmax using Metis Viewer software. For metastasis analysis, cells were implanted into the body from the tail vein of mice, bioluminescence images were taken after 60 days. All murine studies strictly complied with ethical protocols established by Harbin Medical University's Animal Research Ethics Review Board. 4.3. Statistical analysis Quantification of cellular baseline assays was performed with Image software, with data presented as mean ± SEM from three independent experimental replicates. Statistical analyses and visualization were conducted utilizing R software package alongside GraphPad Prism 9.0. Intergroup comparisons were assessed via two-tailed Student’s t-test, with statistical significance defined at P < 0.05. Additional methodologies are provided in Supplementary Methods 1. Abbreviations 2-DG: 2-deoxy-D-glucose; Bev: bevacizumab; Co-IP: CHX: cycloheximide; ChIP: Chromatin immunoprecipitation; Co-immunoprecipitation; CMs: cardiomyocytes; EED: Embryonic Ectoderm Development; ECs: endocardial cells; EZH2: Enhancer of Zeste Homolog 2; ELISA: enzyme-linked immunosorbent assay; EMT: epithelial-mesenchymal transition; ECAR: extracellular acidification rate; EVs: extracellular vesicle; FTO: fat mass and obesity-associated protein; GLUT1: Solute Carrier Family 2 Member 1; H3K27la: H3K27 lactylation modification; HK2: Hexokinase 2; IF: immunofluorescence; LA: lactate; LDH: lactate dehydrogenase; LUAD: lung adenocarcinoma; m 6 A: N6-methyladenosine; NSCLC: non-small cell lung cancer; OCR: oxygen consumption rate; PRC2: Polycomb Repressive Complex 2; RbAp46/48: Retinoblastoma-Associated Proteins 46/48; SAM: S-adenosyl-L-methionine; SUZ12: Suppressor of Zeste 12 Homolog; SUV max: Standardized Uptake Value maximum; Taz: Tazemetostat; TIMP2: Tissue Inhibitor of Metalloproteinases-2; TEM: Transmission electron microscopy; TNTs: tunneling nanotube; TNTs: tunneling nanotubes; VEGF: vascular endothelial growth factor. Declarations This study was approved by the Institutional Review Board of Harbin Medical University Cancer Center. All participants provided written informed consent, and the study was conducted in accordance with the principles of the Declaration of Helsinki. Authors’ Disclosures No disclosures were reported. Availability of data and materials All materials are available in the main text or supplementary materials. Further information and requests for resources and reagents are available from the corresponding authors on reasonable request. The raw data from the human mRNA and lncRNA epitranscriptomic microarray sequencing have been archived in NCBI's Sequence Read Archive (SRA) under accession number PRJNA1208732, and the data of CUT&Tag assay has also been archived in NCBI's sequence reading archive (SRA) with the registration number of PRJNA1253089. The data of 4D label-free lactylome analysis is archived in NCBI's Identification Protein Groups (IPG) database with the registration number IPX0011458000, while RNA-Seq data can be obtained in NCBI's Gene Expression Comprehensive Database (GEO) with the registration number GSE286409. Ethical approval The study was approved by the Ethics Committee of Harbin Medical University Cancer Hospital (KY2023-68). The ethical protocol stipulates that the maximum diameter of subcutaneous transplanted tumors should not exceed 20 mm, and it is confirmed that all experiments were conducted in accordance with the approved protocol and other relevant guidelines and regulations. Consent for publication All authors agree to publish. Conflict of Interest The authors have declared that no competing interest exists. References Sung H, Ferlay J, Siegel R L, Laversanne M, Soerjomataram I, Jemal A et al. 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Supplementary Files SupplementaryMethods1.docx Supplementary Methods 1 SupplementaryData2.xlsx Supplementary Data 2 SupplementaryData3.xlsx Supplementary Data 3 SupplementaryFigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7466899","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":509802175,"identity":"446f6222-4b9f-4272-b8a5-77272fc84695","order_by":0,"name":"Dexin Jia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYBACxmYGBgkQ4pdgYIMIHSBWi+QMYrWAgASIMLhBrBbmduaHNz62WeQZ3+599uhmG4Mc340Exs8FeB3GZmw544xEsdmd4+bGuW0MxpI3EpilZ+D3i5k0T4VE4rYbaWzSQC2JG24ksDHz4NXC/k36j4FE4uYZEC31RGjhMZNmANqyQQKiJcGACC3Flj1nJBJn3EhjN845J2E488zDZml8Wgz7j2+88bOtLrEf6LDHOWU28nzHkw9+xqulAZUPiiPGBiwKEUAer+woGAWjYBSMAhAAACczRrIwsam8AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0009-2820-6709","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":true,"prefix":"","firstName":"Dexin","middleName":"","lastName":"Jia","suffix":""},{"id":509802176,"identity":"da3faafd-0dea-41e8-9c27-86b9fd31aeb0","order_by":1,"name":"Zihan Jing","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zihan","middleName":"","lastName":"Jing","suffix":""},{"id":509802177,"identity":"0414c89b-9a0f-4c51-bdd7-f16fdfd42145","order_by":2,"name":"Xingmei Ren","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xingmei","middleName":"","lastName":"Ren","suffix":""},{"id":509802178,"identity":"71ff8895-f186-4359-86c0-9acf316fcf31","order_by":3,"name":"Ruqiong Wang","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ruqiong","middleName":"","lastName":"Wang","suffix":""},{"id":509802179,"identity":"1ceb8d2b-8660-4235-808a-3b586a5cb4c6","order_by":4,"name":"Bo An","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"An","suffix":""},{"id":509802180,"identity":"3fcd9b4e-c86d-4f4b-9cb9-ca453a935000","order_by":5,"name":"Weitong Gao","email":"","orcid":"","institution":"Harbin Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weitong","middleName":"","lastName":"Gao","suffix":""},{"id":509802181,"identity":"863eda99-835b-4d42-b9e1-92ad54aad677","order_by":6,"name":"Lihua Shang","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lihua","middleName":"","lastName":"Shang","suffix":""},{"id":509802182,"identity":"1645e807-7252-4c84-9f3f-84bf4e4fdc88","order_by":7,"name":"Yan Yu","email":"","orcid":"","institution":"Harbin Medical University Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2025-08-27 02:00:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7466899/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7466899/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90891809,"identity":"70b8eda3-4d2a-4f55-9ea6-ac98ce728f2c","added_by":"auto","created_at":"2025-09-09 11:13:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":436924,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBevacizumab resistance correlates with enhanced lactylation and poor prognosis in LUAD.\u003c/strong\u003e A.Serum lactate levels in bevacizumab-resistant (n = 20) and sensitive (n = 20) LUAD patients detected by ELISA. B. Lactate levels in lung tissues from resistant versus sensitive LUAD patients (n = 20) measured by ELISA. C. Global lysine lactylation (Pan Kla) levels in tumor tissues from 6 resistant and 6 sensitive patients analyzed by Western blotting. D. H3K27la levels in resistant and sensitive tissues determined by Western blotting (n = 6). E. Representative images of H3K27la and Pan Kla levels in drug-resistant and sensitive LUAD tissues detected by IF. F. H3K27la levels in drug-resistant and sensitive LUAD tissues by IHC (42 resistant vs. 42 sensitive). G-H. Kaplan-Meier survival analysis and rate stratified by H3K27la levels. I. Representative PET-CT images. Data were shown as mean ± SEM. Two-tailed t-tests. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns not significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/e24eca3cd7a36235464eb845.png"},{"id":90894162,"identity":"c22d1a40-89ce-4c78-a5d9-47580eaaf363","added_by":"auto","created_at":"2025-09-09 11:29:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":412873,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEZH2 modulates histone lactylation and angiogenesis in LUAD. \u003c/strong\u003eA. Global lactylation and H3K27la levels treated with Tazemetostat (0-20 μM), 2-DG (0-20 mM), or Oxamate (0-10 mM) in LUAD cells detected by Western blotting. B. Pan Kla and H3K27la levels after LDHA knockdown with or without sodium lactate (NaLa, 25 mM) by Western blotting. C. Pan-lactylation and H3K27la levels in EZH2-inhibited (Tazemetostat, 10 μM) A549 and H1993 cells with or without NaLa treatment (25 mM) analyzed by Western blotting. D-H. Glucose uptake in xenograft tumors of BALB/c nude mice treated with Tazemetostat (100 mg/kg, oral gavage) or vehicle (DMSO) assessed by 18F-FDG PET-CT imaging (n = 6). I. Proliferation of HUVECs co-cultured with shEZH2 or shNC tumor cells measured by CCK-8 assay. J. Tube formation ability of HUVECs co-cultured with shEZH2 or shNC tumor cells determined by Matrigel assay (n = 3). K. Migration capacity of HUVECs co-cultured with shEZH2 or shNC tumor cells assessed by Transwell assay (n = 3). Data were shown as mean ±SEM. Two-tailed t-tests. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/92ba28ca58dabfef0ff07494.png"},{"id":90891813,"identity":"878aca52-6005-4ac3-a208-9608f8ec9157","added_by":"auto","created_at":"2025-09-09 11:13:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":433769,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEZH2-H3K27la axis regulates angiogenesis via TIMP2 in LUAD. \u003c/strong\u003eA. Genome-wide H3K27la chromatin profiling in shEZH2 versus control LUAD cells analyzed by CUT\u0026amp;Tag (n = 3). B. Distribution of H3K27la peaks in promoter regions from CUT\u0026amp;Tag data. C-D GO and KEGG analysis of downregulated genes in shEZH2-specific promoter regions showing angiogenesis pathway enrichment. E. RNA-seq volcano plot of differentially expressed genes in shEZH2 LUAD cells. F. KEGG pathway analysis showed that downregulated genes in the shEZH2 group were enriched in angiogenesis-related pathways. G. Integration of GEPIA LUAD downregulated genes (n = 3,130), CUT\u0026amp;Tag peaks, and RNA-seq differentially expressed genes through Venn diagram analysis identified 275 overlapping candidate genes. H. A significant attenuation of H3K27la signal intensity at the TIMP2 promoter region was observed in the shEZH2-treated group by CUT\u0026amp;Tag analysis. I. H3K27la occupancy at TIMP2 promoter after Tazemetostat (Taz), Oxamate, or 2-DG treatment quantified by ChIP-qPCR (n = 3). J. TIMP2 protein levels in shEZH2 cells were analyzed by Western blotting. K. TIMP2 protein levels in Taz-treated LUAD cells were analyzed by IF. L-M. TIMP2 expression levels were altered in Oxamate-, 2-DG-, and Taz-treated cells as determined by Western blotting. Data were shown as mean ± SEM. Two-tailed t-tests. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; ns not significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/3129ee53287c7f31e13ee464.png"},{"id":90894168,"identity":"12b0f9a8-d169-4a20-84e5-4d1aa09b59a5","added_by":"auto","created_at":"2025-09-09 11:29:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":420145,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactate drives YTHDF2 nuclear translocation in LUAD.\u003c/strong\u003e A-B. 4D label-free quantitative proteomics analysis identified lactylation at the K17 site of YTHDF2. C. Evolutionary conservation analysis of the K17 lactylation site across species. D. Molecular docking analysis predicted preferential binding of L-lactate to YTHDF2-K17 with lower binding energy compared to other residues. E. A549 cells were transfected with Flag- YTHDF2 (K17R) or Flag- YTHDF2 (WT) plasmids and analyzed for Pan Kla expression by Western blotting. F. The protein levels of Pan Kla, EZH2, and H3K27la were analyzed by Western blotting in cells expressing Flag- YTHDF2 (K17R) mutant. G. Coomassie Brilliant Blue staining was performed on total protein lysates following NALA (25 mM) treatment. H. A549 cells were transfected with Flag-YTHDF2 (K17R) or Flag-YTHDF2 (WT) plasmids and analyzed by Co-IP. I. Nuclear and cytoplasmic fractions from LUAD cells (A549 cells and H1993 cells) treated with NALA (25 mM, 24 h) were analyzed for YTHDF2 expression by Western blotting. Lamin B1 and GAPDH served as loading controls. J. Nuclear and cytoplasmic fractions from cells transfected with Flag-tagged YTHDF2 (WT) or Flag-tagged YTHDF2 (K17R) plasmids, with or without NALA treatment, were analyzed for YTHDF2 expression by Western blotting. Lamin B1 and GAPDH served as loading controls. K. IF analysis was performed to detect the protein expression of YTHDF2 and EZH2 in cells transfected with Flag-YTHDF2 (WT) or Flag-YTHDF2 (K17R) plasmids, with or without NALA treatment.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/5d01376d7936fa5300123901.png"},{"id":90894164,"identity":"9052478b-9f19-484b-b265-26002cd264b4","added_by":"auto","created_at":"2025-09-09 11:29:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":453245,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTIMP2-mediated mitochondrial transfer controls endothelial metabolism. \u003c/strong\u003eA. IF analysis demonstrated spatial colocalization of TIMP2 with mitochondrial markers in A549 cells and HUVEC cells. B. The ultrastructure of mitochondria was analyzed by TEM. C. The content of mtDNA in NC group and TIMP2 overexpression group was detected by qPCR. D. The JC-1 mitochondrial membrane potential assay revealed a significant decrease in the TIMP2-overexpressing group compared to controls. E. Seahorse XF analysis demonstrated a significant reduction in basal OCR, maximal OCR, and spare respiratory capacity in HUVECs co-cultured with the TIMP2-overexpressing group compared to controls. F. Inhibition of mitochondrial transfer pathways by cytochalasin B (TNT inhibitor) and GW4869 (EV inhibitor), assessed via MitoTracker staining. G. Angiogenesis assays demonstrated that GW4869 inhibited angiogenic capacity of TIMP2-overexpressing cells by compared to vehicle controls. Data are presented as means ± SEMs. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, Two-tailed t-tests.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/14c26793ae55f554e36afb73.png"},{"id":90892464,"identity":"d48643a2-cd6d-4488-9bcc-f03f15e19373","added_by":"auto","created_at":"2025-09-09 11:21:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":528901,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEZH2-glycolysis-lactylation axis targeting overcomes bevacizumab resistance in LUAD.\u003c/strong\u003e A. Transwell invasion assays demonstrated that Taz and Bev treatment inhibited the invasive and migratory abilities of A549 and H1993 cells (n = 3). B. Endothelial functional analysis demonstrated that Taz and Bev treatment impaired the tube-forming capacity of HUVECs (n = 3). C. Serum LDH activity in Bev-resistant versus Bev-sensitive patients (n = 10). D. CT imaging showed tumors in high LDH and low LDH groups before and after treatment. E. PET/CT imaging showed SUV max values in high-LDH and low-LDH groups. F. IF analysis revealed the protein expression of EZH2 and LDHA between the high-LDH and low-LDH groups. G. Schematic diagram of synergistic inhibition by Bev combined with Taz in A549 xenograft mice. Fluorescence intensity and pulmonary metastatic nodule count of metastatic tumors in xenograft mice (n = 5). Fluorescence imaging and gross lung images of xenograft mice (n = 5). Representative IHC images of pulmonary metastatic tumors. Data are presented as means ± SEMs. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, Two-tailed t-tests.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/a59cd12c004ba592b44c4051.png"},{"id":90894163,"identity":"49a00f1d-a297-4f89-aa4c-f2674e69fd35","added_by":"auto","created_at":"2025-09-09 11:29:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":239695,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA-lactylation modification crosstalk.\u003c/strong\u003e Lactate suppresses TIMP2 via EZH2-mediated lactylation, synergizing with FTO/YTHDF2-driven m\u003csup\u003e6\u003c/sup\u003eA dysregulation to induce bevacizumab resistance. Targeting this epigenetic axis blocks mitochondrial transfer, reverses angiogenic metastasis, and inhibits metastatic LUAD progression.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/59949074df0c97041a9af3a7.png"},{"id":92963298,"identity":"2ef15967-074e-4fe5-a754-48e6ab6ce9b8","added_by":"auto","created_at":"2025-10-07 15:19:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4158861,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/303bd0b8-8f21-438c-a4ea-dd90789fb90b.pdf"},{"id":90891807,"identity":"d7a9c286-edc3-47a4-b5a7-2e9888313216","added_by":"auto","created_at":"2025-09-09 11:13:19","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23942,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Methods 1\u003c/p\u003e","description":"","filename":"SupplementaryMethods1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/c4ee316a598db1cb0787b522.docx"},{"id":90891808,"identity":"8c8d6e6c-ac04-4ce1-8f3e-79f604eb1792","added_by":"auto","created_at":"2025-09-09 11:13:19","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":16415,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 2\u003c/p\u003e","description":"","filename":"SupplementaryData2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/d072d0dcb4adbe2c71ed7383.xlsx"},{"id":90892460,"identity":"d10f9e93-e1fb-4324-9ff4-2ee3cb0da21b","added_by":"auto","created_at":"2025-09-09 11:21:19","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11770,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 3\u003c/p\u003e","description":"","filename":"SupplementaryData3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/9303d0142c71bf93f3c3282a.xlsx"},{"id":90891848,"identity":"faf33f0b-4e1c-4c87-9217-b273dde51a79","added_by":"auto","created_at":"2025-09-09 11:13:20","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":25834368,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7466899/v1/5f70cce49b85d15b720a8475.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Multi-omics reveals crosstalk between lactylation and m6A methylation promotes angiogenesis in lung adenocarcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eLung adenocarcinoma (LUAD) has emerged as a critical global health challenge characterized by high incidence rates and poor clinical outcomes\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Notably, LUAD cells persistently utilize glycolytic pathways to take up excessive glucose and generate substantial lactate even under oxygen-sufficient conditions\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Initially dismissed as a metabolic waste product, lactate has now been redefined as a multifunctional signaling mediator that mediates microenvironmental acidification and orchestrating immune evasion mechanisms\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Most notably, the recently identified post-translational modification known as lactylation has unveiled lactate's direct involvement in epigenetic regulation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. The role of this novel covalent modification of lysine residues\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e in bevacizumab (Bev) resistance remains undefined.\u003c/p\u003e\u003cp\u003eEZH2 (Enhancer of Zeste Homolog 2) catalyzes the methylation of histone H3 at lysine 27 residue using S-adenosyl-L-methionine (SAM), predominantly producing di- (H3K27me2) and tri-methylated (H3K27me3) epigenetic modifications. H3K27me3-mediated epigenetic silencing dynamically governs cellular differentiation programs and proliferation\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Compelling clinical observations demonstrate that EZH2 hyperactivation(manifested through gain-of-function mutations or dysregulation)drives tumor and metastatic progression via genome-wide aberrations of H3K27me3 deposition.\u003c/p\u003e\u003cp\u003eMitochondria, which serve as central hubs of cellular energy metabolism, sustain functional cellular integrity through critical processes encompassing ATP synthesis, regulation of metabolic homeostasis, calcium buffering, and stress response modulation. Through dynamic molecular crosstalk, they not only regulate intracellular compartments but also orchestrate endothelial cell (EC)-mediated intercellular communication networks\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Studies have demonstrated that small extracellular vesicles (sEVs)\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e and tunneling nanotubes (TNTs)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e function as essential conduits for mitochondrial transcellular transfer, playing a pivotal role in maintaining tissue microenvironment homeostasis. Astrocyte-to-neuron mitochondrial transfer is orchestrated by LRP1-dependent intercellular trafficking machinery, which mitigates cerebrovascular ischemia-reperfusion (IR) injury through lactylation-dependent regulation of ARF1\u003csup\u003e13\u003c/sup\u003e. Based on these mechanistic foundations, we propose that lactate serves as a pivotal mediator of mitochondrial transfer between cancer cells and endothelial cells (ECs).\u003c/p\u003e\u003cp\u003eIn this study, integrative multi-omics profiling delineates a quintuple regulatory axis comprising lactate-FTO/YTHDF2-EZH2/H3K27la-TIMP2-mitochondrial transfer, mechanistically bridging metabolic-epigenetic crosstalk with angiogenesis-mediated LUAD progression. This framework establishes a rationale for developing combined epigenetic-metabolic therapeutic strategies to counteract Bev resistance.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003cstrong\u003e.1\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eBevacizumab resistance shows increased lactylation levels correlating with poor survival\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing Bev administration, tumor cells may significantly activate histone lactylation through compensatory glycolysis, enhancing tumor cell survival and treatment resistance\u003csup\u003e14,15\u003c/sup\u003e. To assess post-antiangiogenic lactate dynamics, we quantified serum and tissue lactate levels in 40 patients (20 therapy-resistant vs 20 sensitive), revealing significantly elevated lactate accumulation in non-responders (Fig. 1A-B). Histone lactylation, a recently identified post-translational modification first reported by Zhang, is metabolically fueled by glycolytic or alternative pathways generating cytoplasmic lactate\u003csup\u003e5\u003c/sup\u003e. The substantial lactate produced by metabolic reprogramming provides a substrate for histone lactylation, manifested by increased pan-lysine lactylation (Pan Kla) (Fig. 1C) and significantly elevated histone H3 lysine 27 lactylation (H3K27la) levels in therapy-resistant tissues (n = 6) (Fig. 1D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNext, we assessed the clinical relevance of H3K27la in 84 LUAD cases (42 sensitive vs. 42 resistant) using immunofluorescence (IF) and immunohistochemistry (IHC). Fig. 1E revealed a significant correlation between H3K27la and pan-lactylation (Pan Kla) levels. Kaplan-Meier analysis stratified by H3K27la levels revealed significantly worse overall survival (OS), indicating their association with poor prognosis (Figs. 1F-G). Furthermore, clinical assessments revealed an inverse correlation between H3K27la levels and therapeutic response rates (Fig. 1H). PET-CT imaging quantifies tumor glucose uptake in LUAD patients\u003csup\u003e16\u003c/sup\u003e. PET-CT imaging analysis of 84 patients utilized maximum standardized uptake value (SUV max) to quantify 18F-FDG uptake. Analysis demonstrated a positive correlation between SUV max and H3K27la levels (IHC) (Fig. 1I). Elevated H3K27la levels significantly correlated with metastatic dissemination (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001) and elevated serum CEA levels (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001) (Table 1). Clinical analyses reveal that aberrant H3K27la is linked to Bev resistance in LUAD, promoting metastasis and poor prognosis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e \u003cstrong\u003eCorrelation between H3K27la expression and clinicopathological features in patients with LUAD (n = 84)\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"605\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\u0026nbsp;\u003cp\u003e\u003cstrong\u003eCharacteristic\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eH3K27la\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eHigh expression(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 134px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eLow expression(%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eP\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 188px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" rowspan=\"2\" valign=\"top\" style=\"width: 125px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eGender\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e21(58.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e15(41.6.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" rowspan=\"2\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e0.7577\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e25(52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e23(47.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;＜55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e19(65.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e10(34.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e0.1580\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026ge;55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e27(49.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e28(50.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor Location\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eLeft\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e25(62.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e15(37.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e0.7587\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eRight\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e21(47.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e23(52.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSmoking\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;Yes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e29(53.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e25(46.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e0.0130*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;No\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e17(56.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e13(43.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTumor Size\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026le;3cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e24(40.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e35(59.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e0.0009*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;＞3cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e22(88)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e3(12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLymph Node Metastasis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;Yes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e30(44.8))\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e37(55.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e＜0.0001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;No\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e16(94.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e1(5.9)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCEA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;High\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e33(49.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e34(50.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e＜0.0001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u0026nbsp;Low\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e13(76.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e4(23.5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDistance metastasis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e7(15.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e2(5.3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e＜0.0001*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 138px;\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 54px;\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 196px;\"\u003e\n \u003cp\u003e39(84.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 101px;\"\u003e\n \u003cp\u003e36(94.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05\u003c/p\u003e\n\u003cp\u003eThe model showed the key enzymes and inhibitors of glycolysis (Fig. S1A). Levels of glucose transporter (GLUT1), hexokinase (HK2), and lactate dehydrogenase LDHA, all of which directly regulate intracellular lactate synthesis\u003csup\u003e17\u003c/sup\u003e, were coordinately upregulated in LUAD samples alongside VEGFA (Fig. S1B-C), indicating that bevacizumab resistance may involve glycolysis-lactylation axis-driven mechanisms. Survival analysis demonstrated significant associations between these factors and adverse prognosis (Fig. S1D). To characterize lactate metabolic remodeling in LUAD, we analyzed public single-cell RNA sequencing datasets (GSE131907) from the GEO database, revealing tumor-specific LDHA overexpression (Fig. S1E). Western blotting analysis confirmed effective LDHA knockdown (Fig. S1F). These findings collectively validate the significant correlation between heightened glycolytic activity and adverse survival outcomes in LUAD patients.\u003c/p\u003e\n\u003ch2\u003e2.2. EZH2 promotes LUAD metastasis by enhancing glycolysis\u003c/h2\u003e\n\u003cp\u003eEZH2, as a crucial epigenetic regulator, possesses histone methyltransferase and acetyltransferase activities, catalyzing H3K27, leading to the silencing of target genes\u003csup\u003e9\u003c/sup\u003e. Therefore, we propose that EZH2 may catalyze H3K27 lactylation. EZH2 exhibited marked transcriptional elevation in the UALCAN -LUAD samples and was associated with unfavorable outcomes from the GEPIA database (Fig. S2A). Cell line screening (Fig. S2B) revealed that EZH2-high cells showed elevated bevacizumab IC₅₀ values (Fig. S2C). A549 and H1993 cells showed significantly higher lactylation and H3K27la levels compared to normal lung epithelial cells (HBE) (Fig. S2D). To assess EZH2\u0026apos;s lactylation regulatory function, we established knockdown models (Fig. S2E). GO analysis of RNA-seq data revealed significant enrichment of glycolysis pathways upon shEZH2 treatment (Fig. S2F). Seahorse extracellular flux analysis showed that shEZH2 significantly decreased extracellular acidification rate (ECAR), indicating EZH2-driven enhancement of glycolysis (Fig. S2G). EZH2 knockdown cells exhibited reduced GLUT1/HK2/LDHA expression, and lactate production (Fig. S2H-I). EZH2 overexpression induced opposite effects (Fig. S2J-K). Collectively, these findings systematically demonstrate that EZH2 promotes the glycolysis process in lung adenocarcinoma.\u003c/p\u003e\n\u003cp\u003eClinical data indicated EZH2\u0026apos;s association with metastasis in bevacizumab-resistant patients. RNA-seq GO analysis revealed shEZH2-mediated enrichment of metastasis-related pathways (Fig. S3A). Functional validation established EZH2 as a key driver of tumor metastasis (Fig. S3B-E). Western blotting revealed elevated N-cadherin/Vimentin (EMT markers) and reduced E-cadherin in EZH2-overexpressing cells, whereas knockdown cells exhibited the inverse pattern (Fig. S3F). These data demonstrate how EZH2 drives metastasis by promoting EMT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. EZH2 is a potential writer enzyme of histone lactylation in LUAD cells\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInhibiting EZH2 via histone lysine methyltransferase (KMT) targeting represents a pivotal epigenetic therapeutic strategy\u003csup\u003e18\u003c/sup\u003e. We designed a targeted strategy combining Tazemetostat (Taz; first FDA-approved EZH2 inhibitor), 2-deoxy-D-glucose (2-DG; glycolysis inhibitor), and Oxamate (LDH inhibitor) to coordinately block the EZH2-glycolysis-lactylation axis. Taz, 2-DG, and Oxamate showed a dose-dependent reduction of both pan-lactylation and H3K27la\u0026nbsp;in Figure 2A. To validate histone lactylation function, LDHA knockdown reduced pan-lactylation and H3K27 lactylation, with sodium lactate (NaLa) supplementation rescuing these effects\u0026nbsp;(Fig.\u0026nbsp;2B). Fig. 2C shows reduced Pan Kla and H3K27la levels upon EZH2 inhibition.\u0026nbsp;Under NaLa treatment conditions,\u0026nbsp;shEZH2 still markedly reduced\u0026nbsp;Pan\u0026nbsp;Kla and H3K27la levels. For \u003cem\u003ein vivo\u003c/em\u003e validation, 2\u0026times;10⁶ HUVECs and 5\u0026times;10⁵ A549 cells were\u0026nbsp;subcutaneously\u0026nbsp;co-implanted in BALB/c mice. Bev (10\u0026nbsp;mg/kg) was administered via intraperitoneal injection. Taz\u0026nbsp;(100 mg/kg)\u0026nbsp;and DMSO were orally administered to nude mice.\u0026nbsp;18F-FDG PET-CT imaging demonstrated significantly reduced glucose uptake in Taz-treated xenografts versus controls (Fig.\u0026nbsp;2D-H). These findings establish EZH2 and lactate as regulators of histone H3K27 lactylation.\u003c/p\u003e\n\u003cp\u003eAngiogenesis as a hallmark of solid tumor progression shows hyperactivation strongly associated with poor prognosis. Recent studies have revealed that glycolysis-derived lactate can remodel angiogenic phenotypes by inducing histone lactylation modification (H3K18la)\u003csup\u003e19\u003c/sup\u003e, while EZH2 may mediate this process through the \u0026quot;metabolic reprogramming-chromatin remodeling\u0026quot; interaction network. A model was constructed by Transwell to identify the influence on ECs (Fig. S4A). Co-culture of EZH2-knockdown tumor cells (shEZH2) with ECs revealed significant impairment of EC proliferation, migration, and tube formation capacity (Fig. 2I-K). These results demonstrate that EZH2, as a potential writer of lactylation, drives LUAD progression through the \u0026quot;metabolic reprogramming-epigenetic remodeling-angiogenesis\u0026quot; cascade pathway. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Transcriptional repression of TIMP2 by EZH2 regulates angiogenesis in LUAD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we elucidated the EZH2-H3K27la epigenetic axis using a multi-omics strategy. CUT\u0026amp;Tag genome-wide profiling with H3K27la-specific antibody showed reduced TSS-proximal occupancy upon EZH2 knockdown (Fig. 3A), while 53% enrichment localized to promoters (Fig. 3B). GO/KEGG analyses of EZH2 knockdown-induced promoter-localized silenced peaks showed angiogenic pathway enrichment (Fig. 3C-D). RNA-seq analysis revealed shEZH2-regulated potential targets (Fig. 3E). KEGG analysis demonstrated that downregulated genes in the shEZH2-treated group were significantly enriched in angiogenesis-related signaling pathways (Fig. 3F). Integration of GEPIA-derived 3130 downregulated genes with CUT\u0026amp;Tag/RNA-seq DEGs identified 275 candidates showing elevated mRNA levels and decreased H3K27la promoter enrichment upon EZH2 knockdown (Fig. 3G). Focus on the angiogenesis pathway revealed significantly reduced H3K27la enrichment at the TIMP2 (Tissue Inhibitor of Metalloproteinases-2) promoter (Fig. 3H). TIMP2 acts as a key angiogenesis inhibitor via an Matrix Metalloproteinase (MMP)-independent regulatory mechanism\u003csup\u003e20,21\u003c/sup\u003e. ChIP-qPCR analysis confirmed reduced H3K27la occupancy at the TIMP2 promoter following treatment with Taz, 2-DG, or Oxamate (Fig. 3I). TIMP2 protein levels were significantly upregulated by EZH2 knockdown, Taz treatment, Oxamate treatment, or 2-DG treatment (Fig. 3J-M). These findings indicate that TIMP2 modulates LUAD progression via H3K27la, providing a rationale for developing epigenetic-metabolic dual-targeting anti-angiogenic therapies. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. TIMP2 upregulation suppresses LUAD angiogenesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of TCGA datasets revealed significant downregulation of TIMP2 expression in LUAD, correlating with favorable patient prognosis (Fig. S4B). To investigate TIMP2\u0026apos;s functional role in LUAD, we conducted TIMP2 knockdown using siRNA, with validation by Western blotting (Fig. S4C). Notably, siTIMP2 significantly enhanced EC proliferation, migration, and tube formation (Fig. S4D-F). These findings suggest that TIMP2 is one of the regulators of angiogenesis in LUAD. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate TIMP2\u0026apos;s role in H3K27 lactylation-driven LUAD pathogenesis, we transfected A549 and H1993 cells with siTIMP2 in combination with Oxamate and 2-DG. Compared to controls, treatment with glycolysis inhibitors reversed the TIMP2 knockdown-induced enhancement of EC proliferation, migration, and tube formation (Fig. S5A-C). Experimental data indicate TIMP2 regulates H3K27la-mediated angiogenesis in LUAD.\u003c/p\u003e\n\u003cp\u003eTIMP2 regulates tumor metastasis\u003csup\u003e22,23\u003c/sup\u003e. To examine TIMP2\u0026apos;s role in metastasis, A549 and H1993 cells were transfected with siTIMP2 and shEZH2. TIMP2 knockdown promoted partial metastatic ability following shEZH2 (Fig. S6A-C). These results indicate that TIMP2 regulates EZH2-driven LUAD metastasis.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. m\u003csup\u003e6\u003c/sup\u003eA lactylation regulates angiogenesis in LUAD\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLactate induces histone and non-histone protein lactylation\u003csup\u003e24\u003c/sup\u003e. To characterize the lactylation landscape in LUAD, we performed 4D label-free lactylome analysis to identify differentially lactylated proteins in Bev-resistant A549 cells. We identified 19,265 peptides including 4,607 lactylated species. In A549 cells, 4,841 lactylation sites were mapped to 1,900 proteins (Fig. S7A). Quality control confirmed peptide distributions met predefined analytical criteria (Fig. S7B). Comparative analysis of Klac site flanking sequences versus the human proteome revealed lysine enrichment and cysteine/leucine depletion at most positions (Fig. S7C-D). WikiPathways also revealed significant enrichment of VEGFA-VEGFR2 signaling (Fig. S7E). These findings demonstrate that lactylation regulates angiogenesis in Bev-resistant LUAD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAberrant m⁶A modification drives key gene reprogramming to promote cancer progression\u003csup\u003e25,26\u003c/sup\u003e. To delineate protein lactylation in tumor angiogenesis, we analyzed the m⁶A pathway and identified lactylated components including METTL3, METTL14, and ALKBH5 (Fig. S7F). Resistant tissues exhibited elevated global m⁶A methylation levels (Fig. S7G). TCGA analysis demonstrated FTO downregulation in LUAD, with low-FTO patients exhibiting significantly shorter overall survival compared to cohorts stratified by other m⁶A writers/erasers (Fig. S8). FTO, but not METTL3, METTL14, or ALKBH5, significantly modulated bevacizumab resistance. IHC validation revealed pronounced FTO decrease in Bev-resistant specimens (Fig. S7H-I). Nuclear-cytoplasmic fractionation with qPCR and IF confirmed predominant nuclear localization of FTO (Fig. S7J-K). These findings uncover lactylation (Kla)-m⁶A crosstalk mediating Bev resistance via an epigenetic regulatory network.\u003c/p\u003e\n\u003ch2\u003e2.7. RNA-seq and MeRIP-seq identify EZH2 as a direct target for FTO\u003c/h2\u003e\n\u003cp\u003eTo assess FTO-regulated m⁶A on EZH2, we generated shFTO and conducted epitranscriptomic microarray for mRNA m⁶A profiling (Fig. S9A). The Heatmap and scatter plots illustrated the profiles of m\u003csup\u003e6\u003c/sup\u003eA modification and fold changes upon FTO knockdown, revealing widespread hypermethylation/overexpression including EZH2 (Fig. S9B-C). MeRIP-qPCR detected m⁶A enrichment in EZH2 (Fig. S9D). SRAMP analysis revealed high-confidence m⁶A sites in EZH2 (Fig. S9E). To study FTO-regulated m⁶A on EZH2, we performed a luciferase assay showing significant wild-type EZH2 activation post-FTO depletion, unlike unresponsive mutants (Fig. S9F). RNA immunoprecipitation (RIP) assays revealed FTO binding to EZH2 mRNA (Fig. S9G). IF and Western blotting analysis revealed nuclear colocalization of FTO with EZH2 following treatment with the global methylation inhibitor DAA. Intriguingly, DAA treatment significantly suppressed EZH2 protein levels (Fig. S9H-I). Western blotting confirmed FTO knockdown-induced EZH2 upregulation (Fig. S9J). DAA suppressed EZH2 in LUAD cells and attenuated FTO knockdown-induced EZH2 upregulation, validating FTO-dependent m⁶A demethylation of EZH2 (Fig. S9K). FTO knockout increases m⁶A methylation on EZH2, upregulating its expression.\u003c/p\u003e\n\u003ch2\u003e2.8. FTO inhibits glycolysis and metastasis in LUAD\u003c/h2\u003e\n\u003cp\u003eFTO maintains metabolic homeostasis by regulating energy metabolism pathways\u003csup\u003e27\u003c/sup\u003e. GO analysis of m\u003csup\u003e6\u003c/sup\u003eA-modified transcripts revealed FTO regulates glycolysis (Fig. S10A). Untargeted metabolomics was used to analyze metabolic changes in shFTO groups. The shFTO group exhibited 12 differentially abundant metabolites (2 downregulated, 10 upregulated) (Fig. S10B). Notably, lactate was among the altered metabolites. KEGG analysis revealed enriched central carbon metabolism pathways (Fig. S10C). GLUT1, HK, LDHA, and lactate levels were significantly elevated upon FTO knockdown (Fig. S10D-G). FTO overexpression significantly suppressed ECAR levels in H1650 cells, indicating its role in inhibiting glycolysis in LUAD (Fig. S10H). These results indicate that FTO influences glycolysis in LUAD.\u003c/p\u003e\n\u003cp\u003eWound healing and Transwell assays demonstrated that FTO overexpression inhibited metastasis, whereas its knockdown promoted metastasis (Fig. S11A-D). Western blotting analysis revealed significant downregulation of EMT-related proteins (N-cadherin, Vimentin) upon FTO overexpression. Conversely, FTO knockdown exhibited reversed effects (Fig. S11E). These results demonstrate that FTO inhibits metastasis in LUAD through EMT.\u003c/p\u003e\n\u003ch2\u003e2.9. Disrupting FTO-EZH2 interaction increases glycolysis\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eTo define EZH2\u0026apos;s mechanistic role in FTO-mediated m\u003csup\u003e6\u003c/sup\u003eA modification, we performed rescue experiments. ELISA revealed that EZH2 inhibition attenuated shFTO-induced elevation of GLUT1, HK, LDHA, and LA levels (Fig. S12A). Wound healing and Transwell assays exhibited Taz-mediated suppression of shFTO-induced metastatic potential (Fig. S12B-C). These findings demonstrate that FTO modulates EZH2 to perturb glycolysis, thereby suppressing metastasis in LUAD.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To further explore the biological impact of the FTO-EZH2 regulatory axis on tumor angiogenesis, we co-cultured ECs with cancer cells. The results showed that, compared to DMSO, Taz inhibited the proliferation, migration, and tube formation ability of HUVECs (Fig. S13A-C). These findings indicate that EZH2 promotes tumor angiogenesis and metastasis via glycolytic reprogramming under epigenetic control of FTO-mediated m\u003csup\u003e6\u003c/sup\u003eA modification.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.10. YTHDF2 specifically recognizes EZH2 as an m\u003csup\u003e6\u003c/sup\u003eA reader in LUAD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003em⁶A modification is coordinated by writers (methyltransferases) and erasers (demethylases), with functional outcomes determined by readers\u0026apos; site-specific recognition of methylated motifs\u003csup\u003e28,29\u003c/sup\u003e. Thus, identifying functional readers is crucial for elucidating m⁶A-mediated biological mechanisms. We investigated the impact of lactylation on m⁶A modification by profiling readers, identifying lactylated regulators YTHDF2/YTHDF3 (Fig. S14A). Western blotting analysis demonstrated YTHDF2 elevates EZH2 protein levels through m⁶A-specific recognition of EZH2 mRNA (Fig. S14B-C). IF showed that YTHDF2 was predominantly localized in the cytoplasm (Fig. S14D). RIP-qPCR validated YTHDF2 specifically binds m⁶A-modified EZH2 transcripts, showing enhanced binding upon FTO knockdown (Fig. S14E). RNA secondary structure predictions confirmed accessibility of the m⁶A consensus motif (Fig. S14F). RNA stability analysis demonstrated that knockdown of YTHDF2 significantly reduced the half-life of EZH2 mRNA (Fig. S14G). These findings define YTHDF2 as a pivotal m⁶A reader regulating EZH2 expression.\u003c/p\u003e\n\u003ch2\u003e2.11. Tumor lactylation elevates YTHDF2 expression in LUAD\u003c/h2\u003e\n\u003cp\u003eWe examined lactate\u0026apos;s regulatory role on YTHDF2 by quantifying its levels in Transwell cocultures, observing significant accumulation (Fig. S15A). YTHDF2 and EZH2 expression exhibited a positive correlation with lactate levels (Fig. S15B). Exogenous lactate dose-dependently increased YTHDF2 expression (Fig. S15C), whereas Oxamate suppressed its expression (Fig. S15D). LDHA\u0026nbsp;knockdown reduced lactate production, consequently attenuating YTHDF2 expression (Fig. S15E-F). Notably, NALA treatment restored YTHDF2 levels (Fig. S15G). We next investigated if YTHDF2 lactylation modulated binding to m⁶A-RNAs. FLAG-YTHDF2 immunoprecipitation (IP) in A549 cells revealed enhanced m⁶A recognition ability upon NALA treatment (Fig. S15H). We assessed YTHDF2 stability with/without NALA treatment under cycloheximide (CHX) exposure. The half-life of YTHDF2 was significantly prolonged (Fig. S15I). Proteasome-dependent degradation of YTHDF2 (evidenced by MG132-induced accumulation) was attenuated by NALA treatment (Fig. S15J). The above experiments demonstrated that NALA increased the stability of YTHDF2. These findings collectively establish that lactate enhances YTHDF2 expression and potentiates its m\u003csup\u003e6\u003c/sup\u003eA recognition capacity.\u003c/p\u003e\n\u003cp\u003eUsing 4D label-free quantitative proteomics, we identified a specific lactylation at the K17 site of YTHDF2 (Fig. 4A-B). This site was highly evolutionarily conserved (Fig. 4C). Molecular docking predicted stronger L-lactate binding to YTHDF2-K17, supported by lower binding energy (Fig. 4D). To confirm K17 as the primary lactylation site, we generated K17R mutants and analyzed lactylation levels (Fig. 4E). We next explored YTHDF2 lactylation\u0026apos;s functional impact. K17R mutation suppressing lactylation reduced both EZH2 protein and H3K27la levels upon 25 mM NALA treatment (Fig. 4F). IP analysis revealed significant enhancement of lactylation signals that positively correlated with YTHDF2 protein levels (Fig. 4G). The YTHDF2 K17R mutant exhibited near-complete loss of lactylation signals, confirming K17 as the predominant lactylation site (Fig. 4H). Subcellular fractionation combined with IF analysis revealed lactate-induced nuclear translocation of YTHDF2 (Fig. 4I). Notably, NALA increased WT-YTHDF2 nuclear localization but did not alter K17R mutant localization (Fig. 4J). IF imaging showed WT YTHDF2 displayed nucleocytoplasmic distribution, whereas the K17R mutant was predominantly cytoplasmic (Fig. 4K). These findings establish K17 as an evolutionarily conserved lactylation hotspot in YTHDF2, with lactate-induced K17 lactylation enhancing nuclear translocation.\u003c/p\u003e\n\u003cp\u003eWe established ECs-LUAD co-cultures with four treatment groups: siNC, siYTHDF2, siYTHDF2+siNC, and siYTHDF2+siTIMP2. Transwell assays showed that siYTHDF2 reduced transmigration, which was partially reversed in the dual-knockdown group (Fig. S16A-B). Endothelial functional assays revealed that siYTHDF2 decreased HUVECs proliferation and tube formation (Fig. S16C-E). These findings establish that YTHDF2 drives metastasis and angiogenesis via lactylation-dependent nuclear translocation in LUAD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.12. TIMP2 mediates intercellular mitochondrial transfer post-bevacizumab\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial transfer mediates EC formation through mitophagy\u003csup\u003e10\u003c/sup\u003e and drives multidrug resistance. IF revealed TIMP2-mitochondrial marker colocalization (Fig. 5A). Transmission electron microscopy (TEM) revealed abnormal mitochondrial morphology with reduced cristae in ECs and cancer cells from the TIMP2-overexpression group (Fig. 5B). Mitochondrial DNA (mtDNA) mutations drive mitochondrial transfer\u003csup\u003e30\u003c/sup\u003e. qPCR revealed TIMP2 overexpression reduced mtDNA copies in ECs and cancer cells (Fig. 5C). TIMP2 overexpression reduced mitochondrial membrane potential (Fig. 5D). CX43-mediated mitochondrial transfer sustains leukemic stemness via metabolic reprogramming\u003csup\u003e31\u003c/sup\u003e. The TIMP2-overexpressing group exhibited a significant reduction in oxygen consumption rate (OCR) (Fig. 5E). To monitor mitochondrial transfer, we transfected the mitochondria-specific fluorescent protein Mito-DsRed into cancer cells. Treatment with TNT inhibitor cytochalasin B and EV inhibitor GW4869 revealed extracellular vesicles predominantly mediate mitochondrial transfer (Fig. 5F). We found GW4869 inhibited angiogenesis (Fig. 5G). These results demonstrate that TIMP2 regulates mitochondrial transfer to promote angiogenesis, thereby driving metastasis in LUAD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.13.\u003c/strong\u003e \u003cstrong\u003eEZH2-targeted therapy enhances bevacizumab response in LUAD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBev-induced m⁶A-lactylation interplay in LUAD is linked to angiogenesis in metastases.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eIn vivo and vitro\u0026nbsp;\u003c/em\u003emodels demonstrated EZH2 upregulation drives angiogenesis in Bev-resistant tumors. We developed a targeted therapeutic strategy combining Taz and Bev to synergistically block the EZH2-glycolysis-lactylation regulatory axis. This dual targeting suppressed LUAD metastasis and angiogenesis (Fig. 6A-B). Serum analysis showed increased lactate dehydrogenase in Bev-resistant patients (Fig. 6C). CT scans demonstrated poor treatment response in LDH-high LUAD patients following Bev treatment (Fig. 6D). PET-CT showed elevated SUV max with metastases in lactate dehydrogenase-high cohorts (Fig. 6E). EZH2 and LDHA expression was increased in the LDH-high group (Fig. 6F). Tail vein injection of A549 cells in nude mice established lung metastasis models, showing Bev/Taz combination synergistically reduced metastatic burden (Fig. 6G). These findings demonstrate that Taz disrupts tumor vasculature and overcomes lactylation-mediated bevacizumab resistance.\u003c/p\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eWhile substantial progress has been achieved in current LUAD therapy, elucidating the mechanisms underlying treatment resistance remains a pivotal challenge in oncology\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Integrated multi-omics analyses, we were the first to reveal that lactate accumulation induced by Bev resistance drives LUAD progression through dual epigenetic axes: coordinated histone/nonhistone lactylation and m\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003eA-mediated regulatory networks. Our key findings delineate four mechanistic pillars: (1) The EZH2-glycolysis-H3K27la-TIMP2 signaling axis mediates pathological angiogenesis; (2) An FTO/YTHDF2-m\u003csup\u003e6\u003c/sup\u003eA-EZH2 circuit integrates metabolic-epigenetic crosstalk; (3) K17 lactylation on YTHDF2 potentiates m\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003eA recognition through nucleo-cytoplasmic trafficking; (4) Mitochondrial transfer networks remodel tumor-endothelial metabolic crosstalk. These findings establish a novel conceptual framework for overcoming anti-angiogenic therapy resistance.\u003c/p\u003e\u003cp\u003eLactate, a pivotal Warburg effect metabolite, exerts epigenetic regulatory functions via histone lysine lactylation (such as H3K18la), activating pro-tumorigenic transcription\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Mechanistic investigations revealed that Bev resistance enhances glycolysis via EZH2-mediated regulation, driving lactate accumulation in the tumor microenvironment and sustaining substrate supply for histone lactylation. Clinical cohort analysis demonstrated significantly elevated H3K27la levels in resistant tumors, while inhibition of histone lactylation effectively suppressed metastasis and angiogenesis in LUAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results were further substantiated in murine models. These findings may provide a novel therapeutic strategy to enhance bevacizumab efficacy in LUAD through targeting histone lactylation.\u003c/p\u003e\u003cp\u003eServing as the enzymatic center of the PRC2\u003csup\u003e33\u003c/sup\u003e, EZH2 mediates H3K27 trimethylation (H3K27me3) to establish epigenetic silencing\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Pan-cancer analyses demonstrate that EZH2 orchestrates metabolic reprogramming through glycolysis regulation while suppressing tumor suppressor gene transcription\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Mechanistic studies reveal that EZH2 modulates post-bevacizumab metastasis through glycolysis-driven metabolic reprogramming (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, tazemetostat, an EZH2 inhibitor initially approved by the FDA, may provide therapeutic benefits for bevacizumab-resistant patients. This discovery provides novel insights for precision oncology and opens new therapeutic avenues for cancer patients.\u003c/p\u003e\u003cp\u003eAberrant histone modifications are prevalent across diverse pathological processes (including malignancies), and their imbalance significantly impacts disease progression\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Emerging evidence suggests that histone lactylation is a novel epigenetic driver that induces the expression of genes associated with tumorigenesis and drug resistance\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. To our knowledge, this study provides the first evidence that aberrant histone lactylation machinery drives bevacizumab resistance. Integrated multi-omics analysis demonstrated that EZH2-mediated H3K27la transcriptionally suppresses TIMP2, thereby promoting angiogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e). We have delineated a novel glycolysis-H3K27la-TIMP2 regulatory axis that provides actionable therapeutic targets to overcome resistance to anti-angiogenic therapies.\u003c/p\u003e\u003cp\u003eAs the pioneer m⁶A demethylase, FTO orchestrates pivotal RNA metabolic pathways while exhibiting dual functionality in energy metabolism dysregulation and tumor evolution\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the regulatory function of FTO in LUAD glycolytic reprogramming remains undefined, underscoring the critical need to delineate its epigenetic mechanisms driving bevacizumab resistance. We demonstrated that FTO-mediated m⁶A modification of EZH2 attenuates glycolytic reprogramming in LUAD, thereby suppressing angiogenesis. As far as we are aware, EZH2 represents the first histone methyltransferase reported to undergo FTO-mediated regulation in LUAD, thereby elucidating novel roles of histone-modifying enzymes in LUAD pathogenesis.\u003c/p\u003e\u003cp\u003eWarburg effect-derived lactate not only serves as a signaling molecule in biological processes but also remodels the epigenetic landscape via histone and non-histone lactylation\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Intriguingly, lactylation cooperates with m⁶A modifications to orchestrate oncogenic transcriptional programs\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Unlike YTHDF3, YTHDF2 predominantly mediates degradation of m⁶A-modified mRNAs through lactylation-dependent regulation\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. This groundbreaking study demonstrates that hyperactivated glycolysis triggers lactylation at lysine 17 (K17) of YTHDF2, which enhances its m⁶A-binding affinity to stabilize EZH2 mRNA, thereby orchestrating metabolic-epigenetic crosstalk that drives LUAD angiogenesis and metastatic progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig19\" class=\"InternalRef\"\u003e4\u003c/span\u003e). By delineating the lactate-m⁶A-EZH2-glycolysis regulatory axis, we pioneer the identification of YTHDF2's lactylation-driven nuclear translocation pathway, providing novel mechanistic insights into metabolic-epigenetic crosstalk.\u003c/p\u003e\u003cp\u003eMetabolic coupling through lactate drives intercellular mitochondrial trafficking between hematopoietic progenitors (HSPCs) and a distinct BM stromal subset (PDGFRα+/Sca-1⁻/CD48low), mediated by Cx43 channels and AMPK activation. This bidirectional crosstalk establishes redox equilibrium through coordinated ROS modulation in these compartments\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Experimental evidence from Zhou's group demonstrates that astrocyte-derived LRP1 promotes neuronal mitochondrial transfer by dually suppressing lactate biosynthesis and ARF1 lactylation, thereby attenuating cerebrovascular IR injury in rodent models\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Under lactate-rich conditions, cancer cells transfer mitochondria to adjacent endothelial cells through TNTLs and EVs, promoting tumor progression. Mechanistic studies demonstrate that EZH2 promotes EV secretion via TIMP2 to regulate mitochondrial transfer between cancer cells and ECs, whereas GW4869 inhibits this process (Fig.\u0026nbsp;\u003cspan refid=\"Fig21\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This study defines the lactate-mitochondrial transfer-metabolic axis, establishing a framework for mitochondrial transfer-based therapeutics.\u003c/p\u003e\u003cp\u003eEZH2 drives malignant transformation by catalyzing H3K27 trimethylation and serves as a master epigenetic regulator of oncogenic programs\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. EZH2 elevates H3K27 methylation at the DLC1 promoter to promote proliferation and glycolysis in esophageal cancer cells\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Our study revealed a marked accumulation of lactate serving as the substrate for histone H3K27la. And EZH2-driven glycolytic enhancement promotes angiogenesis in LUAD. Integrated multi-omics analyses uncovered the H3K27la-mediated transcriptional repression mechanism, with TIMP2 established as the central regulator driving angiogenesis within this pathway. Lactylome profiling revealed lactylation of m\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003eA regulators, with lactylated YTHDF2 stabilizing EZH2 mRNA via m\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003eA recognition. This study delineated a five-tiered \"lactate-FTO/YTHDF2-EZH2/H3K27la-TIMP2-mitochondrial transfer\" axis, showing that pharmacological inhibition of this axis enhances Bev responsiveness, thereby nominating lactylation-m\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003eA crosstalk as a novel therapeutic target.\u003c/p\u003e"},{"header":"4. Material and methods","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e4.1. Human sample collection and cell culture\u003c/h2\u003e\u003cp\u003eClinical specimens and associated patient data were sourced through collaboration with Harbin Medical University Cancer Center. The study subjects were patients with lung adenocarcinoma and included 20 pairs of fresh-frozen tumors and adjacent non-neoplastic tissues. Pathological sections from 84 patients enrolled between December 2016 and December 2019 were used for IHC, and the clinicopathological characteristics collected included age, gender, smoking history, tumor location, size, lymph node metastasis, survival rate, and CEA level (Supplementary Data 2). 18F-FDG PET/CT scans were analyzed to quantify intratumoral glucose uptake (SUVmax). Blood sera from healthy controls and LUAD cases were included in this study. Ethical approval for this research was obtained from the Institutional Review Board. Human bronchial epithelial (HBE) cells and LUAD cell lines (H1993, A549, H1650) were obtained from the Cancer Experimental Center at Harbin Medical University Cancer Hospital. Cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel) under standard conditions (37\u0026deg;C, 5% CO₂).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.2. Tumor Xenograft Implantation and 18FFDG PET imaging\u003c/h2\u003e\u003cp\u003eFemale immunodeficient BALB/c mice (aged six weeks; body weight range 18\u0026ndash;20 g) were obtained from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). For tumor angiogenesis assay, HUVEC 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e and 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were co-implanted in the right axillary of mice, bioluminescence images were taken on day 28, and the mice were euthanized, tumors were excised, and weighed. Mice were anesthetized with 1% sodium pentobarbital the day before euthanasia, following an 8-hour fasting regimen, 200 \u0026micro;l of 18F-FDG tracer was delivered via intraperitoneal bolus injection under controlled metabolic conditions. Then, the mice were positioned on the examination table for PET/CT imaging. The 18F-FDG uptake was achieved by delineating regions of interest (ROI) and measuring the SUVmax using Metis Viewer software. For metastasis analysis, cells were implanted into the body from the tail vein of mice, bioluminescence images were taken after 60 days. All murine studies strictly complied with ethical protocols established by Harbin Medical University's Animal Research Ethics Review Board.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.3. Statistical analysis\u003c/h2\u003e\u003cp\u003eQuantification of cellular baseline assays was performed with Image software, with data presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM from three independent experimental replicates. Statistical analyses and visualization were conducted utilizing R software package alongside GraphPad Prism 9.0. Intergroup comparisons were assessed via two-tailed Student\u0026rsquo;s t-test, with statistical significance defined at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Additional methodologies are provided in Supplementary Methods 1.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e2-DG: 2-deoxy-D-glucose; Bev: bevacizumab; Co-IP: CHX: cycloheximide; ChIP: Chromatin immunoprecipitation; Co-immunoprecipitation; CMs: cardiomyocytes; EED: Embryonic Ectoderm Development; ECs: endocardial cells; EZH2: Enhancer of Zeste Homolog 2; ELISA: enzyme-linked immunosorbent assay; EMT: epithelial-mesenchymal transition; ECAR: extracellular acidification rate; EVs: extracellular vesicle; FTO: fat mass and obesity-associated protein; GLUT1: Solute Carrier Family 2 Member 1; H3K27la: H3K27 lactylation modification; HK2: Hexokinase 2; IF: immunofluorescence; LA: lactate; LDH: lactate dehydrogenase; LUAD: lung adenocarcinoma;\u0026nbsp;m\u003csup\u003e6\u003c/sup\u003eA: N6-methyladenosine; NSCLC: non-small cell lung cancer; OCR: oxygen consumption rate; PRC2: Polycomb Repressive Complex 2; RbAp46/48: Retinoblastoma-Associated Proteins 46/48; SAM: S-adenosyl-L-methionine; SUZ12: Suppressor of Zeste 12 Homolog; SUV max: Standardized Uptake Value maximum; Taz: Tazemetostat; TIMP2: Tissue Inhibitor of Metalloproteinases-2; TEM: Transmission electron microscopy; TNTs: tunneling nanotube; TNTs: tunneling nanotubes; VEGF: vascular endothelial growth factor.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis study was approved by the Institutional Review Board of Harbin Medical University Cancer Center. All participants provided written informed consent, and the study was conducted in accordance with the principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ Disclosures\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo disclosures were reported.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll materials are available in the main text or supplementary materials. Further information and requests for resources and reagents are available from the corresponding authors on reasonable request. The raw data from the human mRNA and lncRNA epitranscriptomic microarray sequencing have been archived in NCBI's Sequence Read Archive (SRA) under accession number PRJNA1208732, and the data of CUT\u0026amp;Tag assay has also been archived in NCBI's sequence reading archive (SRA) with the registration number of PRJNA1253089. The data of 4D label-free lactylome analysis is archived in NCBI's Identification Protein Groups (IPG) database with the registration number IPX0011458000, while RNA-Seq data can be obtained in NCBI's Gene Expression Comprehensive Database (GEO) with the registration number GSE286409.\u003c/p\u003e\n\u003ch6\u003eEthical approval\u0026nbsp;\u003c/h6\u003e\n\u003ch6\u003eThe study was approved by the Ethics Committee of Harbin Medical University Cancer Hospital (KY2023-68).\u0026nbsp;The ethical protocol stipulates that the maximum diameter of subcutaneous transplanted tumors should not exceed 20 mm, and it is confirmed that all experiments were conducted in accordance with the approved protocol and other relevant guidelines and regulations.\u003c/h6\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors agree to publish.\u003c/p\u003e\n\u003ch6\u003eConflict of Interest\u003c/h6\u003e\n\u003ch6\u003eThe authors have declared that no competing interest exists.\u003c/h6\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eSung H, Ferlay J, Siegel R L, Laversanne M, Soerjomataram I, Jemal A et al. 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LINC00114 stimulates growth and glycolysis of esophageal cancer cells by recruiting EZH2 to enhance H3K27me3 of DLC1. Clinical epigenetics 14, 51, doi:10.1186/s13148-022-01258-y (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Lactylation, m6A, Bevacizumab resistance, Metabolic reprogramming, Mitochondrial transfer, EZH2","lastPublishedDoi":"10.21203/rs.3.rs-7466899/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7466899/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBevacizumab (Bev) is pivotal in metastatic lung adenocarcinoma (LUAD) therapy, though lactate's regulatory mechanisms remain incompletely characterized. We reveal significant lactate accumulation in Bev-resistant tumors, driving elevated histone lactylation. EZH2-mediated glycolysis enhances lactylation, repressing TIMP2 transcription to promote mitochondrial transfer between endothelial and malignant cells, thereby accelerating angiogenesis and metastasis. Lactate further induces YTHDF2 K17-lactylation, enhancing nuclear translocation and m\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003eA recognition to stabilize EZH2 mRNA. FTO suppresses EZH2 via m\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003eA demethylation, negatively regulating glycolysis. Clinical data associate high lactylation with poor prognosis. Dual targeting of lactylation and m\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003eA combined with Bev demonstrates potent efficacy. These findings provide novel insights into epigenetic mechanisms of metabolic reprogramming and offer therapeutic strategies for patients with Bev-refractory LUAD.\u003c/p\u003e","manuscriptTitle":"Multi-omics reveals crosstalk between lactylation and m6A methylation promotes angiogenesis in lung adenocarcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-09 11:13:14","doi":"10.21203/rs.3.rs-7466899/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"89c54334-f6ab-4710-b72f-a1c7d18f37d2","owner":[],"postedDate":"September 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54149390,"name":"Health sciences/Oncology/Cancer/Lung cancer/Non-small-cell lung cancer"},{"id":54149391,"name":"Health sciences/Oncology/Cancer/Cancer metabolism"},{"id":54149392,"name":"Health sciences/Oncology/Cancer/Metastasis"},{"id":54149393,"name":"Health sciences/Oncology/Cancer/Cancer therapy/Cancer therapeutic resistance"}],"tags":[],"updatedAt":"2025-10-07T15:11:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-09 11:13:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7466899","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7466899","identity":"rs-7466899","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}