Lactate accelerates cancer progression through the ERK-GCN5 lactylation-phosphorylation feedback cascade

Lactate accelerates cancer progression through the ERK-GCN5 lactylation-phosphorylation feedback cascade | 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 Lactate accelerates cancer progression through the ERK-GCN5 lactylation-phosphorylation feedback cascade Jian Yuan, Bingsong Huang, Yuping Chen, Gaofeng Cui, Georges Mer, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3944681/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Jan, 2026 Read the published version in Nature Chemical Biology → Version 1 posted You are reading this latest preprint version Abstract The Warburg effect released lactate promotes cancer progression, but the mechanisms remain unclear. Here, we found lactate activated MAPK pathway through ERK-lactylation to promote cancer progression. Moreover, we identified the GCN5 as the lactyl-transferase for ERK lactylation. Interestingly, activated ERK phosphorylated GCN5 and promoted GCN5 lactyl-transferase activity for ERK, which formed the positive feedback loop to facilitate lactate-mediated cancer progression. Mechanistically, ERK-K231 lactylation decreased the dissociation energy between ERK and MEK, due to the reduced electrostatic interaction between ERK-K231 and MEK-D217. This facilitated the dissociation of ERK from MEK kinases, which in turn induced ERK dimerization and activation. Hence, we developed a cell-penetrating peptide to specifically inhibit the ERK lactylation, and demonstrated the peptide impaired the tumor growth with KRAS-mutant. Taken together, we define a molecular mechanism that lactate accelerates cancer progression through ERK-GCN5 lactylation-phosphorylation cascade and provide a strategy to target ERK lactylation, especially for RAS-MAPK-driven cancers. Biological sciences/Cancer/Cancer therapy Biological sciences/Cell biology/Post-translational modifications Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Metabolic reprogramming is widely recognized as a hallmark of cancer that enables rapid cell proliferation. One prominent example is the Warburg effect or aerobic glycolysis, which is characterized by elevated glucose uptake and lactate release in cancer cells( 1 ). Despite the inefficiency of this process for energy production, cancer cells preferentially rely on aerobic glycolysis for their energy supply. Lactate was traditionally considered a by-product of aerobic glycolysis, but recent studies have shown that it can serve as a metabolic fuel for cancer cells( 2 ). In fact, lactate contributes to energy production more than glucose does in some cancers. For example, in non-small cell lung carcinoma (NSCLC) models, lactate was found to contribute to the tricarboxylic acid (TCA) cycle more than glucose does( 3 ). In vivo isotopic tracing experiments have also shown that 13 C-labeled lactate more extensively labels TCA intermediates than 13 C-labeled glucose in cancer cells( 3 , 4 ). In addition to its role as an energy source, lactate affects cancer progression in various ways. Lactate inhibited the cytotoxicity T cell proliferation and cytokine production( 5 , 6 ). Furthermore, lactate directly supports tumor-promoting immune cell populations, which impairs immune function and facilitates cancer immune escape( 7 , 8 ). However, the mechanisms by which lactate directly promotes cancer cell proliferation have been poorly investigated. Recently, a newly discovered histone post-translational modification (PTM) called lactylation was identified, which introduces lactate from metabolomics into epigenetics( 9 ). Therefore, we hypothesize that lactate serves as the donor to modify proteins lactylation, which may promote cancer cell proliferation and tumor progression. The RAS-MAPK signaling pathway plays a crucial role in intracellular signal transduction, regulating key cellular processes such as cell growth, differentiation and survival( 10 ). Upon activation by upstream signaling molecules, including receptor tyrosine kinases (RTKs) or G protein-coupled receptors (GPCRs), RAS proteins trigger a phosphorylation cascade that activates the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway ( 11 , 12 ). This pathway comprises a series of kinases, including RAF, MEK, and ERK, which culminates in the activation of transcription factors that govern gene expression and modulate cellular behavior( 11 ). The most common mutations that lead to hyperactivation of the RAS-MAPK signaling in cancer are found in KRAS, NRAS, and BRAF genes, which result in constitutive activation of downstream signaling cascades ( 13 , 14 ). In addition to phosphorylation, other PTM patterns also were also reported to regulate RAS-MAPK signaling. Ubiquitination and acetylation have been reported to occur on RAS-MAPK components( 15 – 19 ). However, their regulatory effects on RAS-MAPK signaling differ considerably. For instance, ubiquitination of RAS inhibits its localization to the plasma membrane( 15 ), while ubiquitination of ERK promotes its degradation( 17 ), leading to the suppression of RAS-MAPK signaling. Conversely, ubiquitination of BRAF results in sustained BRAF activation and subsequent elevation of the RAS-MAPK signaling( 16 ). Moreover, acetylation of RAS inhibits its transforming activity without affecting its plasma membrane localization ( 20 ), thereby suppressing RAS-MAPK signaling. Conversely, acetylation of RAF facilitates its dimerization and promotes RAS-MAPK signaling( 12 ). However, the roles of lactylation on RAS-MAPK signaling is poorly investigated. In this study, we demonstrated that the key component of the RAS-MAPK pathway, ERK is lactylated, which activates the RAS-MAPK signaling. Lysine acetyltransferase 2A (KAT2A, also known as GCN5) functions as a lactyl-transferase for ERK lactylation and activation. Interestingly, ERK directly phosphorylates GCN5, enhancing the lactyl-transferase activity of GCN5 for ERK lactylation. The positive feedback loop between ERK and GCN5 rapidly amplifies the RAS-MAPK signaling and contributes to lactate mediated cancer progression. A cell-penetrating peptide was designed to inhibit ERK lactylation and shut down the ERK-GCN5 feedback loop mediated cancer cell proliferation. Taken together, our findings reveal the mechanism by which protein lactylation modification amplifies RAS-MAPK signaling and imply a potential therapeutic strategy by targeting ERK lactylation to inhibit tumor growth especially for those hyperactive MAPK signaling derived cancer. Results Lactate promotes MAPK signaling through ERK lactylation To explore the physiological function affected by lactate in cancers, we treated U87MG cells with or without lactate and performed RNA-sequencing assays. The differential gene expression analysis revealed 564 up-regulated and 379 down-regulated genes upon lactate stimulation (Extended Data Fig. S1 a). According to GSEA analysis, the top two ranked signaling pathway were cytokine-cytokine receptor interaction and MAPK signaling (Fig. 1 a, b). As described above, the effect of lactate on immune responses has been widely reported( 5 – 8 ). However, the mechanism by which lactate promotes MAPK signaling remains poorly understood. Given the prevalence of RAS-MAPK signaling mutations in cancers, which lead to constitutive pathway activation, we focused on this pathway. To validate the RNA-seq results, we treated U87MG and HeLa cells with lactate or lactate dehydrogenase (LDH) inhibitor sodium oxamate to manipulate lactate level in cells and then examined RAS-MAPK signaling. As shown in Fig. 1 c and d, lactate induced RSK phosphorylation, while sodium oxamate showed the opposite effects. Moreover, lactate increased RAS-MAPK downstream gene expression, and this effect reversed by sodium oxamate(Fig. 1 e). Recent studies have demonstrated that lactate induces protein lactylation, which regulates multiple cellular functions( 9 , 21 , 22 ). We hypothesized lactate may regulate RAS-MAPK pathway through protein lactylation. Interestingly, by screening MAPK components for protein lactylation, we found that only ERK1/2, a critical regulator of RAS-MAPK signaling, could be lactylated(Fig. 1 f), while other MAPK components showed no significant lactylation(Fig. 1 f). Consistently, lactylation of endogenous ERK1/2 was increased after lactate treatment(Fig. 1 g). As earlier study has verified the acetylation of ERK1/2( 23 ).To examine whether acetylation of ERK1/2 was affected by lactate, we also checked the level of ERK1/2 acetylation and found that lactate treatment does not affect ERK1/2 acetylation level(Fig. 1 g). Previous study had presented lactate could directly interact with protein and affect protein function( 24 ). We also synthesized biotin-labeled lactate to perform biotin pull-down assay and found lactate did not bind to ERK1/2 (Fig. 1 h), suggesting that lactate modulation of RAS-MAPK signaling may be mediated through lactylation. Furthermore, we observed that manipulating intracellular lactate levels through lactate or LDH inhibitor treatment dramatically affected ERK1/2 lactylation in dose-dependent manners (Fig. 1 , i and j). Depletion of Lactate Dehydrogenase A (LDHA) decreased ERK2 lactylation, which could be rescued by lactate treatment (Fig. 1 k). Moreover, depletion of MCT1, which transports extracellular lactate into the cell, decreased ERK2 lactylation (Fig. 1 l). Taken together, these results suggest that lactate may activate the RAS-MAPK signaling pathway by inducing ERK lactylation. ERK was Lactylated by GCN5 Acetyltransferase The histone acetyltransferases (HAT) family was also reported to serve as lactyltransferases in vivo ( 9 ). After screening those main acetyltransferases of HAT family, we found GCN5 primarily mediated ERK lactylation(Fig. 2 a; Extended Data Fig. 2 a). Particularly, overexpression of GCN5 increased ERK lactylation in a dose-dependent manner. However, GCN5 did not induce ERK acetylation, which is consistent with previous study that CBP/P300 mediated ERK acetylation( 23 )(Fig. 2 b, Extended Data Fig. 2 b). Furthermore, GCN5 displayed a dose-dependent activation of ERK downstream pathways (Fig. 2 b). On the contrary, depletion of GCN5 sharply decreased ERK lactylation and subsequent downstream signaling activation, witho ut impact on ERK acetylation (Fig. 2 c; Extended Data Fig. 2 c). Given that the interaction between ERK and its upstream activators or substrates is mediated through the common docking (CD) domain on ERK and the D domain-docking site on the activator or substrate ( 26 ) (Fig. 2 d, e), We identified the potential D domain-docking sites on GCN5 (Fig. 2 f). Mutation of these GCN5 D domain-docking sites to alanine (GCN5 4A) and the ERK2 CD domain to asparagine (ERK2 2DN) abrogated the ERK-GCN5 interaction (Fig. 2 g,h; Extended Data Fig. 2 d, e). Additionally, GCN5 4A mutant failed to induce ERK lactylation (Fig. 2 i), and ERK2 2DN mutation led to a decrease in lactylation of ERK2 that could not be induced by lactate treatment (Fig. 2 j). Furthermore, GCN5 directly catalyzed the lactylation, but not the acetylation of ERK2 in vitro (Fig. 2 k, l). Given that GCN5 was well characterized as mainly serving as an acetyltransferase, to further confirm that it also plays a key role in lactylation, we simulated the interaction between lactyl-CoA and GCN5, revealing that GCN5's active site can accommodate lactyl-CoA without causing structural clashes(Extended Data Fig. 2 f). Our modeling suggests that lysine can enter this pocket for lactyl transfer. Particulary, the interaction between lactyl-CoA and GCN5 appears to be stronger compared to that of acetyl-CoA based on the structure of lactyl-CoA and GCN5. The presence of an additional hydroxyl group in lactyl-CoA allows for the formation of two additional hydrogen bonds, potentially enhancing its interaction with GCN5(Extended Data Fig. 2 g). Furthermore, the smaller size and higher electronegativity of the lactyl-CoA potentially facilitate higher efficiency in lysine N(epsilon) proximity, making lactylation more favorable than acetylation(Extended Data Fig. 2 g). Taken together, our findings suggest that GCN5 interacts with ERK and function as a lactyl-transferases for ERK. Lactylation of ERK1/2 at K248/231 facilitate cancer progression ERK1 and ERK2, sharing 84% sequence identity, both contain lysine residues within their kinase domain and C-terminus. To determine the lactylation sites, the lactylation of ERK1/2 kinase domain constructs was analyzed, revealing that both full-length ERK1 and ERK2, as well as their kinase domain constructs, displayed comparable lactylation signals (Extended Data Fig. 3 a,b). These results indicated that the lactylation sites were mainly located within the kinase domain. Within kinase domain, ERK1 and ERK2 possess 15 homologous lysine residues and 3 non-homologous ones. Subsequent mutation of these lysine residues to arginine (R) enabled the assessment of their contribution to lactylation. Mutations of the 15 homologous lysine residues (15KR) resulted in reduced lactylation signals for both kinases, whereas mutations of the non-homologous residues (3KR) did not significantly alter lactylation levels (Extended Data Fig. 3 c-e). Further analysis with subsection mutants revealed that mutations in the ( 10 – 15 ) KR segment led to a marked reduction in lactylation, mirroring the effects observed with the 15KR mutant (Extended Data Fig. 3 f). Single lysine mutant was also evaluated, identifying K231 in ERK2 and K248 in ERK1 as the primary lactylation sites (Fig. 3 a; Extended Data Fig. 3 g). The ERK lactylation sites were further confirmed by mass spectrometry (MS) analysis, which showed that K248 on ERK1 and K231 on ERK2 are able to be lactylated (Fig. 3 b,c), and the sites are highly conserved across multiple species (Extended Data Fig. 3 h). To further explore the ERK1/2 lactylation in vivo, a specific antibody targeting lactylation at K248/231 in ERK1/2 was developed and its specificity was confirmed through dot blot assays (Extended Data Fig. 3 I), terming the antibody as K231lac-ERK. The utilization of the K231lac-ERK antibody revealed that lactate treatment enhances lactylation of ERK wild type, but not the ERK KR mutant (Fig. 3 d; Extended Data Fig. 3 j,k), while inhibition of lactate dehydrogenase (LDH) resulted in the opposite effect (Fig. 3 e; Extended Data Fig. 3 j,k). Notably, K231R mutant did not affect its acetylation level (Fig. 3 d,e), consistent with previous findings that identified K72 in ERK1 and K55 in ERK2 as acetylation sites( 23 ). Additionally, we performed in vitro lactylation assays to verify the lactylation reaction. The results demonstrated that GCN5 selectively enhances ERK2 lactylation in the wild type but not in the K231R mutant(Fig. 3 f; Extended Data Fig. 3 l). Using K231lac-ERK antibody, we also observed the lactylation of endogenous ERK was increased by lactate and decreased by LDH inhibitor or GCN5 depletion in Hela and U87MG cells(Fig. 3 g-i). ERK's involvement in cancer biology is well-established, prompting us to investigate whether lactylation of ERK promotes transcription of downstream genes and cancer cell proliferation. Considering the higher expression of ERK2 over ERK1 in most cell types and their equivalent substrate specificity, our subsequent studies focused on ERK2. Overexpression of ERK2 wild-type (WT) and lactylation-deficient K231R (KR) constructs in Hela cells revealed that lactate treatment increased transcription of ERK downstream targets in ERK2 WT but not in KR mutant cells (Fig. 3 j). Additionally, soft agar assay showed Hela ERK2 KR cells has a reduced growth compared to ERK2 WT cells (Fig. 3 k). Colony formation assay further showed that lactate treatment dramatically increased Hela ERK2 WT cells growth in a dose dependent manner(Fig. 3 l), the effect was not observed in ERK2 KR cells. In contrast, inhibition of LDH resulted in a decreased growth of Hela ERK2 WT cells, with ERK2 KR cells remaining unaffected (Fig. 3 l). In summary, our findings demonstrate ERK1/2 is lactylated at K248/231 in vivo and in vitro, significantly facilitating cancer progression. EGFR-RAS Activation promotes ERK lactylation through ERK-GCN5 feedback loop EGFR and RAS activation are the major inducer of RAS-MAPK cascades, which in turn promote cancer progression. Interestingly, we found EGF stimulation increased ERK lactylation (Fig. 4 a). Furthermore, we overexpressed EGFR VIII and RAS G12D in HEK293T cell, which are constitutive activation mutant forms of EGFR and RAS GTPases, respectively, and found that both EGFR VIII and RAS G12D promote ERK lactylation (Fig. 4 b). In addition, the ERK lactylation was more abundant in RAS-MAPK mutant-driven tumors than in RAS-MAPK wild-type tumors (Extended Data Fig. 4 a). These findings suggest an augmentation of ERK lactylation induced by EGFR and RAS activation. Interestingly, after analyzing the GCN5 sequences, we identified GCN5 as a potential substrate for ERK, with threonine 404 aligning with the PX (S/T)P consensus sequence characteristic of ERK phosphorylating substrates (Fig. 4 C-D)( 27 , 28 ). Thus, we speculate RAS-MAPK activation may induce GCN5 phosphorylation and promote ERK lactylation to form a positive feedback loop. By utilizing the antibody-recognized motif PX(S/T)P, we observed that GCN5 phosphorylation was increased by EGF stimulation (Fig. 4 e). On the contrary, ERK inhibitor reduced the phosphorylation (Fig. 4 f). Consistently, we construct GCN5 T404A mutant and found it totally abolished the phosphorylation signaling, which showed unaltered by EGF stimulation or ERK inhibitor treatment (Fig. 4 f). Subsequently, we speculate ERK phosphorylates GCN5, which enhancing GCN5's lactyl-transferase activity, thereby forming a positive feedback loop. As shown in Fig. 4 g, compared to GCN5 WT, GCN5 TA mutant dramatically inhibited ERK lactylation and ERK downstream signaling. Moreover, ERK inhibitor or MEK inhibitor decreased GCN5 phosphorylation, and impaired ERK lactylation (Fig. 4 h). Meanwhile, lactate treatment could not rescue ERK lactylation in ERK inhibitor or MEK inhibitor treated cells, suggesting that GCN5 phosphorylated by ERK is essential for GCN5 lactyl-transferase activity (Fig. 4 h). Consistently, we also performed the in vitro lactylation assay. As shown in Fig. 4 i, we immunoprecipitated GCN5 from HEK293T cells, and found GCN5 WT but not TA mutant could lactylate ERK2 in vitro. We further confirmed ERK2 phosphorylated GCN5 by in vitro kinase assay. As shown in Fig. 4 j, ERK2 WT but not kinase dead mutant (K54R) phosphorylated GCN5 at T404 in vitro . As previous results showed ERK lactylation facilitating cancer progression. We next examined whether GCN5 phosphorylation is important for lactate mediated cancer cell growth. Depleting endogenous GCN5 and substituting it with GCN5 WT and GCN5 TA revealed that lactate or LDH inhibitor treatment more dramatically impacted cancer cell growth in GCN5 WT cells compared to GCN5 TA mutant cells (Fig. 4 k). Taken together, we found that breaking down ERK-GCN5 lactylation-phosphorylation cascade impaired lactate-induced cancer cell growth. ERK lactylation is essential for EGFR-RAS induced tumor growth and cancer metabolism reprogramming RAS-MAPK components mutant always induce continuous activation of RAS-MAPK signaling, which is common among tumors, such as EGFR VIII and RAS G12D . To further investigate the role of ERK lactylation in RAS-MAPK constitute activation tumor growth. We reconstituted ERK2 WT or the lactylation-deficient KR mutant in the EGFR VIII U87MG and RAS G12D SUM159 cell lines. Soft agar assay showed EGFR VIII U87MG or RAS G12D SUM159 ERK2 KR cells has a reduced growth compared to ERK2 WT cells(Extended Data Fig. 4 b,c). As shown in Fig. 5 a and b, ERK2 KR mutant cells exhibited reduced cell growth compared to ERK2 WT cells. Lactate treatment dramatically promoted cancer cell growth in ERK WT cells compared to ERK KR cells, while the LDH inhibitor showed the opposite effect (Fig. 5 a, b). To further confirm the role of ERK lactylation in cancer cell proliferation in vivo , the reconstructed ERK2 WT or KR mutant cancer cells (EGFR VIII U87MG and RAS G12D SUM159 cells) were injected subcutaneously in nude mice. As shown in Fig. 5 , C to H, the ERK KR mutant cells showed a significant reduction in tumor volume and weight compared to ERK2 WT cells. By intraperitoneal injection with lactate, the tumor growth showed more significant increase in ERK WT cells than ERK KR mutant cells (Fig. c-h). ERK is reported to regulate cancer metabolism reprogramming( 29 , 30 ), we next conducted metabonomic analysis in ERK1/2 WT and KR mutant RAS G12D SUM159 cells. As shown in Fig. 5 i, the metabolites involved in glycolysis (Fig. 5 i; Extended Data Fig. 5 a,b), the pentose phosphate pathway (PPP) (Fig. 5 i; Extended Data Fig. 5 c,d), the TCA cycle (Fig. 5 i; Extended Data Fig. 5 e,f), and amino acid metabolism (Fig. 5 i; Extended Data Fig. 5 g) were downregulated in ERK KR cells compared to ERK WT cells. Furthermore, ERK KR mutant cells exhibited a lower glucose uptake ability (Extended Data Fig. 5 h). Overall, our findings suggest that lactylation of ERK enhances ERK activity and promotes EGFR-RAS induced tumor growth and cancer metabolism reprogramming. Lactylation promotes ERK dissociation from MEK and subsequent dimerization Next, we aim to investigate the mechanism by which ERK lactylation promotes RAS-MAPK signaling. In the resting state, ERK forms a complex with its upstream MEK, which serves as an anchor( 31 , 32 ). Upon receiving an activating stimulus, ERK is phosphorylated and separates from MEK, thereafter ERK forms dimers and exhibits autophosphorylation( 31 – 36 )( 32 , 35 , 37 – 40 ) . Our results confirmed that EGF treatment or serum stimulation causes ERK dissociation from MEK (Fig. 6 a, b). Interestingly, lactate treatment promotes ERK dissociation from MEK, while LDH inhibitor blocked this process (Fig. 6 c). Interestingly, the ERK KR mutant exhibited a tighter association with MEK, which could not be disrupted by EGF or lactate stimulation (Fig. 6 d, e). These results suggested that ERK lactylation affects the ERK-MEK complex dissociation. We next examined whether ERK lactylation regulates its dimerization. As shown in Fig. 6 f, ERK lactylation deficient KR mutant significantly decreased its self-binding compared to ERK WT. Lactate treatment promotes ERK WT self-binding, while LDH inhibitor suppressed this progress (Fig. 6 f). However, ERK KR exhibits a very weak ability for self-binding, which cannot be affected by Lactate or LDH inhibitor treatment (Fig. 6 f). We further proved that lactylation was key for ERK self-binding and dimerization by Native PAGE (Fig. 6 g). After dimerization, ERK generated autophosphorylation at T190( 41 , 42 ). Consistently, EGF stimulation increased T190 autophosphorylation in ERK2 WT, but not in ERK2 KR (Fig. 6 h). Consistently, depletion of GCN5 significantly enhanced ERK-MEK interaction, which could be rescued with reconstituted GCN5 WT but not GCN5 TA mutant(Fig. 6 i, j). To understand the impact of ERK K231 lactylation on its interaction with MEK, we used AlphaFold2 (version 2.3.1) to probe the MEK-ERK complex. All 25 structures generated by AlphaFold2 consistently indicated the same binding interface (Fig. 6 k). Notably, ERK K231 is a part of this interface, in close vicinity to MEK D217, possibly forming hydrogen bonds with D217 (Fig. 6 l, Video 1). We then subjected the MEK-ERK structure to a 100-ns molecular dynamics (MD) simulation, which highlighted the stability of the complex (Fig. S6 , Video 2), giving us confidence in the reliability of AlphaFold2 predictions. During the simulation, ERK K231 consistently maintained proximity to MEK D217, underscoring its potential significance (Video 2). The MD simulation identified 25 hydrogen bonds or salt bridge pairs between MEK and ERK (Extended Data Fig. 6 b). We used this information to guide the generation of mutations, which were subsequently tested in vivo to assess their impact on the MEK-ERK binding interface (Fig. 6 m, n; Extended Data Fig. 6 e, f). The experimental results from mutagenesis experiments aligned with the predicted interface. A lactyl group modeled on K231 Nε did not induce clashes with other residues (Fig. 6 l). ERK K231 lactylation may not disrupt the potential hydrogen bond formation between K231 Nε and MEK D217 Oδ; however, by removing one positive charge from K231, it could reduce the binding affinity. We used the Potential of Mean Force (PMF) method to assess the impact of ERK K231 lactylation on the MEK-ERK complex (Fig. 6 o; Extended Data Fig. 7 ). The dissociation free energy between wild-type MEK and ERK was 29.08 ± 0.57 kcal/mol, which decreased to 27.36 ± 0.52 kcal/mol when ERK was lactylated at K231. This predicted reduction in affinity caused by ERK lactylation aligns with the findings of our in vivo experiments (Fig. 6 d, e). Collectively, our results suggest that ERK lactylation promotes its dissociation from MEK, facilitating ERK dimerization and activation. Based on all these findings, we propose a model that ERK-GCN5 axis facilitate MAPK signaling activation in a lactylation-phosphorylation feedback loop dependent manner. Upon RAS-MAPK activation, MEK activated ERK and subsequently ERK phosphorylates and activate GCN5, which in turn lactylates ERK. Lactylation of ERK promotes its dissociation from MEK and facilitates ERK dimerization and further activation. The lactylation-phosphorylation cascade of ERK-GCN5 accelerates ERK activation, which rapidly amplifies RAS-MAPK signaling in tumor progression. Inhibiting ERK lactylation enhances chemosensitivity We next wanted to clarify the clinical relevance of ERK-GCN5 lactylation axis. We performed an immunohistochemical (IHC) analysis on human NSCLC and colorectal cancer (CRC) samples, as well as matched tumor-adjacent normal tissues (Extended Data Fig. 8a, b). Our findings demonstrated that levels of ERK lactylation were significantly higher in tumors compared to adjacent normal tissues (Fig. 7 a, b). Furthermore, ERK lactylation levels were positively correlated with Ki-67 scores (Fig. 7 c), and significantly elevated in RAS-mutant tumors compared to RAS-WT samples (Fig. 7 d). Moreover, we found a positive correlation between GCN5 expression and the transcriptional levels of ERK target genes at the in various cancer types (Extended Data Fig. 8c). These results provide evidence that ERK lactylation is upregulated in tumors, particularly those with RAS mutations, and suggest that increased GCN5 expression is a potential risk factor positively associated with MAPK signaling. Given ERK lactylation plays an important role in promoting tumor progression, targeting ERK lactylation may be an effective approach for tumor therapy. Previous studies have shown that synthetic peptides can act as analog inhibitors for endogenous protein post-translational modifications( 20 , 43 , 44 ). Recently, competing peptides fused with cell-penetrating peptides have emerged as an efficient strategy for inhibiting specific protein PTMs( 45 – 48 ). Based on the conservative motif surrounding the lactylation site in ERK1 and ERK2, we synthesized five peptides, which all covered the lactylation site, and fused with a cell-penetrating sequence (Fig. 7 e). Our screening experiments revealed that the peptide P-3 effectively inhibited ERK lactylation (Fig. 7 F). The peptides P-3(R), the K231 residue replaced with R, was utilized as a negative control. We found that P-3 but not P-3(R) significantly decreased MAPK signaling in a dose-dependent manner (Fig. 7 g), which led to reduced cancer cell growth (Fig. 7 h, i). In addition, the P-3 peptide inhibited the cell growth in ERK WT cells but not ERK KR mutant cells (Fig. 7 j), suggesting the P-3 peptide inhibits cancer cell growth by blocking ERK lactylation. We next utilized patient-derived xenograft (PDX) models of colorectal cancer harboring the KRAS G12V mutation to examine the P3 peptide therapeutic effect in vivo ( Fig. 7 j). The P3 peptide treatment individually inhibited tumor growth in vivo (Fig. 7 k-n). Moreover, combination treatment with the p3 peptide and 5-Fluorouracil (5-FU) showed synergistic killing effect (Fig. 7 k-n). In addition, IHC staining of tumor sample showed that the P3 peptide dramatically decreased ERK lactylation, phospho-RSK, and Ki-67 in vivo ( Extended Data Fig. 8d). In conclusion, our findings indicate that inhibiting ERK lactylation may be a potential strategy for cancer therapy, especially the tumor with RAS-MAPK overactivation. DISCUSSION Post-translational modifications (PTMs) are essential for regulating protein function and modulating many physiological and pathological processes( 49 ). Eukaryotic proteomes contain hundreds of different types of PTMs, but only a few of them have been extensively studied( 50 ). However, recent developments in high-resolution mass spectrometry have enabled the detection of low-abundance PTM patterns, expanding our understanding of the PTM landscape( 51 ). Newly discovered PTM patterns, such as lactylation( 9 ), succinylation( 52 ), and crotonylation( 53 ), have revealed the non-metabolic functions of metabolites. For example, lactate, in addition to serving as fuel and biosynthesis precursor, can also act as a substrate for lysine lactylation. Despite decades of research on the Warburg effect, the mechanisms by which lactate contributes directly to cancer progression and chemoresistance remain largely unknown ( 1, 2 ). In this study, we have discovered the post-translational modification pattern called lactylation, which directly modifies ERK kinase, a key downstream component of the MAPK pathway. We have shown that lactylation of ERK plays a crucial role in regulating ERK activity, ultimately promoting tumor growth and cancer metabolism reprogramming. In addition, we have identified GCN5 as the lactylation writer for ERK. GCN5 is a member of the GNAT (GCN5-related N-acetyltransferase) family of acetyltransferases that transfer acetyl groups from acetyl-CoA to lysine residues on histones and other proteins( 54 ). Our study identified the lactyl-transferase activity of GCN5, which was previously unknown. Under basal conditions, the ERK interacted with the MEK. Upon activation of the MAPK pathway by a stimulus, MEK was phosphorylated and became kinase-active, leading to phosphorylation of ERK on its TEY motif( 10 ). Phosphorylated ERK dissociated from MEK and formed a dimer with another phosphorylated ERK molecule( 31, 33, 35 ). The ERK dimer was a kinase-active form that could freely phosphorylate downstream targets( 31, 34, 35 ). Negative regulation of the MAPK/ERK pathway was mediated by DUSP4/6, which dephosphorylated the TEY motif of ERK( 55 ). Following cessation of the stimulus, dephosphorylated ERK re-associated with MEK, leading to the return of the system to the basal state( 35 ). Interesting, ERK lactylation facilitated detachment of ERK from MEK, thereby promoting ERK dimer formation. However, in ERK lactylation deficient KR mutant cells, EGF cannot lead ERK to disassociate from MEK, suggesting ERK lactylation is essential for ERK disassembling from MEK following stimulus. Structure study is needed to further clarify the MEK-ERK dynamic interaction regulated by phosphorylation-lactylation cascade. The product of the Warburg effect, accumulated lactate in cancer cells may lead to hyper-lactylation and activation of ERK, which accelerates tumor progression. Inhibiting abnormal ERK lactylation in tumors may have mild effect on normal cells, which may reduce side effects compared to inhibitors directly targeting ERK. We designed a cell-penetrating peptide to specifically block ERK lactylation. The peptide inhibited tumor growth in vivo effectively and showed synergistic killing effect together with 5-FU in KRAS G12V mutation cancers, suggesting targeting ERK lactylation may be a prospective strategy for cancer therapy. In conclusion, we demonstrated that GCN5 function as a lactyl-transferase for ERK. In addition, ERK directly phosphorylates GCN5 and promotes its lactyl-transferase activity for ERK, forming a positive feedback loop that regulates ERK lactylation. Our findings confirm that the Warburg effect induced lactate accumulation in tumors triggers an ERK-GCN5 lactylation-phosphorylation cascade effect, which plays a crucial role in accelerating tumor progression in a positive feedback loop dependent manner. More importantly, we demonstrate that inhibiting ERK lactylation may be a potential strategy for cancer therapy, especially the tumor with RAS-MAPK overactivation. Methods Cell culture, Plasmids, Reagents and Antibodies Human Embryonic Kidney 293T cells (HEK293T), human cervical cancer cell Hela, human glioblastoma cell line U87MG, human triple-negative breast cancer cell line cancer cell line SUM159PT were purchased from ATCC. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% (v/v) CO2. ERK1, ERK2, MEK1 and GCN5 were subcloned into Puro-Lentiviral Expression Vector (PLVX), Purified E.coli Glycogen Express Vector-X-4T-2 (PEGX-4T-2) or pT7-based expression vector. Mutant plasmids were established by a two-step mutation method. EGFR VIII (P26653) and RAS G12D (P39721) were obtained from MiaoLingBio, China. The acetyl-transferases plasmids, CBP, P300, GCN5, PCAF, KAT5, KAT8 were gifts from Dr. Jun Huang. And KAT1 (P37556) and KAT7 (P46543) were obtained from MiaoLingBio, China. L-lactate (L1750), LDH inhibitor oxamate (O2751), puromycin were purchased from Sigma-Aldrich. The antibodies used in this study: anti-Tubulin (Abmart, M20005S,1: 2000), anti-ERK1/2 (Abmart, T40071S,1: 2000), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit mAb (Cell Signaling Technology, Cat# 4370,1:2000), anti-Flag (Sigma: F1804, 1: 2000), anti-HA (Cell Signaling Technology: no.3724, 1: 2000), anti-L-pan-Kla (PTM Biolab: PTM-1401RM, 1:1000), anti-LDHA (Cell Signaling Technology: no. 2012S, 1: 1000), and anti-ERK1/2-K248/231-lac was generated by PTM Biolab (1:1000). Anti-Phospho-RSK1 p90 (T359+S363) (Abmart, T55344, 1:1000), anti-RSK1 (Abclonal, A4695, 1:1000),anti-MCT1 (Abclonal,A3013, 1:1000) anti-MYC tag (Abmart, M20019,1:2000) anti-GCN5 (Santa Cruz Biotechnology, sc-365321,1:1000), anti-MEK1 (Abclonal, A19565, 1:1000), anti-Phospho-MAPK Substrates Motif (Cell Signaling Technology, #14378, 1: 1000),anti-Phospho-ERK1/2-T188 (badrilla, A010-40AP, 1:500), anti-Histone-H3 (proteintech,17168-1-AP,1:2000), Anti-Acetyl-Histone H3 (Lys9) (PTM Biolab, PTM-156, 1:1000). RNA-seq and data analysis Total RNA was extracted from the U87MG using TRIzol® Reagent according to the manufacturer’s instructions (Magen). RNA samples were detected based on the A260/A280 absorbance ratio with a Nanodrop ND-2000 system (Thermo Scientific, USA), and the RIN of RNA was determined by an Agilent Bioanalyzer 4150 system (Agilent Technologies, CA, USA). Only qualified samples will be used for library construction. Paired-end libraries were prepared using a ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) following the manufacturer’s instructions. The mRNA was purified from 1 μg total RNA using oligo (dT) magnetic beads followed by fragmentation carried out using divalent cations at elevated temperatures in ABclonal First Strand Synthesis Reaction Buffer. Subsequently, first-strand cDNAs were synthesized with random hexamer primers and Reverse Transcriptase (RNase H) using mRNA fragments as templates, followed by second-strand cDNA synthesis using DNA polymerase I, RNAseH, buffer, and dNTPs. The synthesized double stranded cDNA fragments were then adapter- ligated for preparation of the paired-end library. Adaptor-ligated cDNA were used for PCR amplification. PCR products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 4150 system. Finally, the library preparations were sequenced on an Illumina Novaseq 6000 (or MGISEQ-T7) and 150 bp paired-end reads were generated. Mass spectrometry analysis Stable HEK293T cells expressing HA-tagged ERK2 were treated with 25mM lactate for 24 hours before harvesting and lysing the cells. The lysates were subjected to purification using anti-HA-agarose beads. The resulting pellet containing the purified HA-ERK2 protein was separated via SDS-PAGE and visualized by Coomassie blue staining. The HA-ERK2 gel band was excised and subjected to destaining using a solution of 30% ACN/100 mM NH4HCO3 until complete removal of stains from the gel. Then the protein was digested using in-gel digestion method. In brief, the gel band was cut into 1 mm3 particles. The cysteine residues on the protein were reduced with dithiothreitol (10 mM DTT/ 100 mM NH4HCO3) for 30 min at 56°C and alkylated with iodoacetamide (200 mM IAA/100 mM NH4HCO3) in the dark at room temperature for 30 minutes. Subsequently, the protein was digested overnight in 12.5 ng/μL sequence grade trypsin in 25 mM NH4HCO. The tryptic-digested peptides were extracted three times with 60% ACN/0.1% TFA. The peptide extracts were pooled and dried completely by a vacuum centrifuge. The peptides were analyzed by Easy-nanoLC 1000 system (Thermo Scientific) tandem with Q Exactive mass spectrometer (Thermo Scientific) in a 60 min gradient with positive ion mode. For the full scan, the ions with a mass range of 300 m/z to 1800 m/z were detected by Orbitrap analyzer with a resolution of 70,000 at m/z 100. The automatic gain control (AGC) target was set to 1e6, maximum inject time to 50 ms. For the MS/MS, the top 20 most abundant precursor ions were selected for HCD fragmentation and detected by Orbitrap analyzer with a resolution of 17,500 at m/z 100. The normalized collision energy was 27%. The automatic gain control (AGC) target was set to 1e5, isolation width was set to 1.5 m/z, maximum inject time to 50 ms, and the dynamic exclusion duration was set to 30.0 s. The mass spectrometry data was converted into MGF format and searched by Mascot search engine (Matrix Science, London, UK; version 2.2) against the nonredundant International Protein Index arabidopsis sequence database v3.85 (released in September 2011; 39679 sequences) from the European Bioinformatics Institute (http://www.ebi.ac.uk/). The search parameters were as following: Precursor ion peaks were searched with an initial mass tolerance of ± 20 ppm, fragment mass tolerance of ± 0.1 Da. Enzyme specificity with trypsin was used. Up to two missed cleavages were allowed. Carbamidomethylation of cysteine was set as a fixed modification. The oxidation of methionine was set as variable modifications. Metabolomics analysis ERK2 WT and ERK2 KR SUM159PT cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% (v/v) CO2 for 24h. Each group were counted and collected for targeted metabolomics analysis on XploreMET platform (Metabo-Profile, Shanghai, China). The raw metabolomic data generated by UPLC-MS/MS were analyzed using iMAP (Metabo-Pro- file, Shanghai, China) platform for peak identification and quantification of each metabolite. The different biomarkers between the ERK2 WT and ERK2 KR were evaluated using orthogonal partial least squares discriminant analysis (OPLS-DA) by setting the thresholds of variable importance in projection (VIP) to > 1, fold change (FC) > 1 and P < 0.05. Measurement of Lactate level Intracellular lactate level was measured by using lactate Colorimetric/Fluorometric assay kit (Abcam ab65331) according to manufacturer’s protocol. Co-immunoprecipitation and Western blotting For transient transfection and co-immunoprecipitation assays, plasmids encoding HA-tagged, MYC-tagged, or Flag-tagged constructs were transiently co-transfected into HEK293T cells. The transfected cells were lysed on ice for 25 minutes with NETN buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) supplemented with 1× protease inhibitors. After centrifugation at 12,000 rpm for 10 minutes, the soluble fractions were collected and incubated with anti-HA, anti-MYC, or anti-Flag beads for 2 hours at 4°C (Sigma: E6779 for anti-HA beads, E6654 for anti-MYC beads, F2426 for anti-Flag beads). The beads were then washed three times with NETN buffer prior to incubation. Following incubation, the samples were boiled in 1× SDS loading buffer for 5 minutes and separated by SDS-PAGE. The membranes were blocked with 5% milk in TBST buffer and subsequently probed with the respective antibodies. Biotin pull down assay In the biotin-lactate pull-down assay, Dynabeads MyOne Streptavidin T1 were first incubated with biotin or biotin-labeled lactate in PBS for an hour at room temperature. Then, they were mixed with cell lysates overnight at 4°C with rotation. Following 3-4 washes, the beads were analyzed by immunoblotting. Molecular modeling and molecular dynamics simulations Human ERK protein sequence spanning amino acid 11 to 360, and human MEK protein sequence spanning amino acid 19 to 393 were used as input for AlphaFold2 (version 2.3.1) ( 56 ). From the 25 models generated, the best ranked model was selected for structural analysis and molecular dynamics (MD) simulation. MD simulation were performed using GROMACS (version 2021.4) with the Amber ff14sb force field ( 57 ) and TIP3P water model ( 58 ). The ERK-MEK complex was charge-neutralized with sodium ions and was positioned in a periodic triclinic box with dimension of 10.497 ´ 10.776 ´ 12.166 nm containing aqueous solution with 150 mM sodium chloride. The system was energy minimized under the steepest descent algorithm and equilibrated under constant volume and temperature (NVT) and constant pressure and temperature (NPT), with heavy atoms restrained in positions. The system temperature was maintained at 300 K with a V-rescale thermostat ( 59 ), while the system pressure was kept at 1 atmosphere using a Berendsen barostat ( 60 ). Bond lengths were constrained using the LINCS algorithm ( 61 ). An unrestrained simulation with leap-frog integration ( 62 ) was performed for 100 ns with a time step of 2 fs. Potential of mean force calculations The restrained electrostatic potential (RESP ) ( 63 ) of lactate-modified lysine (KLA) was calculated using Gaussian 16 at the B3LYP/6-31G* level. A two-step RESP fit was used to assign partial charges to KLA. Subsequently, wild type (WT) MEK/ERK and MEK/ERK with ERK lactylated at K231 were placed in a periodic triclinic box with dimension of 9.444 ´ 20.047 ´ 10.450 nm and processed and equilibrated as described above. The equilibrated ERK and MEK molecules were then pulled away with a force of 2000 kJ/mol/nm 2 at a speed of 0.01 nm/ps along the y-axis. Twenty-eight and 27 snapshots or windows from the pull trajectories were prepared for the WT and KLA system. Each window was subjected to 10 ns of production with a time step of 2 fs using leap-frog integration. This simulation was repeated 5 times for each system. All the data were analyzed using the weighted histogram analysis method (WHAM) ( 64 ). Error estimation was derived from a 100-step Bootstrap analysis ( 65 ). 2-D and soft agar colony formation assay. For the 2D colony formation assay, cells (500–2000) were seeded in triplicate in each well of a 12-well plate. After 24 hours, cells were treated with lactate or oxamate and incubated at 37°C for 10–14 days to allow colony formation. The colonies were stained with Coomassie Brilliant Blue (CBB) and counted. For the soft agar colony formation assay, the bottom layer of agar (1.2%) containing 1×DMEM was plated first and allowed to solidify at room temperature. And then the upper layer of agar (0.7%) containing cells (500–1000 in 1×DMEM). Cells were cultured for approximately 2–3 weeks and observed under a microscope. The results were normalized to account for differences in plating efficiencies. Expression and purification of recombinant proteins Bacterial expression constructs, specifically pGEX-4T-2 and pT7-based expression vectors, carrying the target genes were transformed into Escherichia coli DH5α cells. Protein expression was induced by adding 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and incubating the cells at 18°C with 180 rpm agitation overnight. The cells were then resuspended in PBS containing 0.5% Triton X-100 and 2 mM β-mercaptoethanol, followed by ultrasonication to disrupt the cells. The expressed proteins were purified using the GST-tag/His-tag Protein Purification Kit from Beyotime Biotechnology according to the manufacturer's instructions. In vitro lactylation assay Recombinant GST-ERK2 proteins were incubated with HA-tagged GCN5 proteins, which were purified from HEK293T cells, in a reaction buffer containing 50 mM HEPES (pH 7.8), 30 mM KCl, 0.25 mM EDTA, 5.0 mM MgCl2, 5.0 mM sodium butyrate, and 2.5 mM DTT, supplemented with 20 μM lactyl-CoA. The reactions were incubated at 30°C for 30 minutes. Subsequently, 5× SDS loading buffer was added to the reaction mixture and boiled for 5 minutes at 100°C. The samples were then separated by SDS-PAGE and subjected to immunoblotting using the specified antibodies. In vitro phosphorylation assay The His-GCN5 protein was expressed in BL21 bacteria and purified using a nickel-agarose column as a substrate for ERK2. HA-ERK2 was immunoprecipitated from cells using HA Beads, and the immunoprecipitates were then incubated with the purified His-GCN5 protein for 30 minutes at 30°C in a 20 μl reaction buffer (25 mM Tris-HCl [pH 7.5], 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 0.5 mM ATP). Following the incubation, the proteins were eluted in SDS-sample buffer and analyzed by immunoblotting. Animal All animal experiments and procedures were carried out in strict accordance with the Guidelines for the Care and Use of Laboratory Animals set by the U.S. National Institutes of Health (National Academies Press; 2011) and were performed following the ethical guideline’s protocols approved by Tongji University school of medicine. For in vivo animal experiments, lactate (100ul of 5M lactate) was intraperitoneally injected three times a week. The Cell Line-Derived Xenograft and Patient-derived xenograft models The CDX (Cell Line-Derived Xenograft) model (cell lines, 5×10 ^ 6) and Colon cancer patient-derived xenografts (PDXs) were subcutaneously transplanted into 6-week-old female nu/nu mice. Mice bearing tumors of 100 mm 3 were randomly assigned into each group. Tumor volume was measured and measured as mentioned in tumor xenograft assay. Peptide synthesis All peptides were synthetized by Guoping Pharmaceutic Inc. (Hehui, China). Synthetic peptides were purified to >98% purity by high-pressure liquid chromatography for both in vitro and in vivo as. The amino acid sequences of peptides in vivo use were in D isoform. For in vitro experiments, peptides were dissolved in PBS to generate a 10 mM stock solution. For in vivo use, peptides were dissolved in PBS and kept on ice until injection. Before injection, the solution was brought to room temperature. Immunohistochemical analysis The colorectal Cancer and lung cancer tissue microarray with clinic and pathological data was obtained from Shanghai Zhuoli Biotechnology Co., Ltd. (Shanghai, China). Statistical analysis Error bars represent the SEM or SD, as indicated in the Fig. legends. Statistical significance was determined using Prism version 8.0 software (GraphPad Software, CA, USA). Differences were deemed significant at P < 0.05. Two-way or one-way ANOVA followed by Dunnett’s post-test, Tukey’s multiple-comparisons test, or the Kruskall–Wallis test (for subgroup analyses) was performed for multiple comparisons, and Student’s t test was performed for other experiments to compare mean values. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P 0.05). Declarations Author contributions Jian Yuan and Chunlong Zhong conceived and designed the study. Bingsong Huang performed most of the experiments and wrote the manuscript. Yuping Chen and Georges Mer. reviewed and edited the manuscript. Gaofeng Cui carried out the molecular modeling and MD simulation studies. Acknowledgments For funding, this work is supported by National Natural Science Foundation of China, Grant/Award Number: 81571184, 81771332, 82172820 and 82271406; Fundamental Research for the Central University; Natural Science Foundation of Shanghai, Grant/Award Number: 22ZR1466200 and 22ZR1451200; Pudong Health Committee of Shanghai, Grant/Award Number: PWYgy 2021- 07; Shanghai Pudong New Area Health Commission, Grant/Award Number: PWZxg2022-10; Pudong Health Bureau of Shanghai, Grant/Award Number: PWR12018-07 Declaration of interests The authors declare no competing interests. References W. H. Koppenol, P. L. Bounds, C. V. Dang, Otto Warburg's contributions to current concepts of cancer metabolism. Nat Rev Cancer 11 , 325-337 (2011). M. G. Vander Heiden, L. C. Cantley, C. B. 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Molecular Simulation 42 , 1079-1089 (2016). J. S. Hub, B. L. de Groot, D. van der Spoel, g_wham-A Free Weighted Histogram Analysis Implementation Including Robust Error and Autocorrelation Estimates. Journal of Chemical Theory and Computation 6 , 3713-3720 (2010). Additional Declarations There is NO Competing Interest. Supplementary Files Mov1XXXXXX.mpg Movie 1 Mov2XXXXXX.mpg Movie 2 SF1.pdf Fig. S1. (a) Volcano plot illustrating differential gene expression analysis between the control group and the lactate treatment group (20mM, 24h) using RNA-seq data. Each data point represents a gene, with the x-axis showing the logarithm (base 2) of the fold change, and the y-axis indicating the negative logarithm (base 10) of the adjusted p-value (-log10(p-value)). SF2.pdf Fig. S2. (a) HEK293T cells transfected with the indicated MYC-tagged acetyltransferase plasmids and HA-tagged ERK1 plasmids. (b) HEK293T cells transfected with the indicated MYC-tagged GCN5 plasmids (0, 0.5, 1, 2, 3 μg) and HA-tagged ERK1 plasmids. (c) HEK293T cells depleted of GCN5 were transfected with HA-tagged ERK1. (d) HEK293T cells transfected with the indicated MYC-tagged GCN5 WT/GCN5 4A mutant or (e) HA-tagged ERK1 WT/ERK2 2DN mutant. (f) A model of Lactyl-CoA binding to GCN5 was constructed based on the crystal structure (pdb ID:IQSR) of GCN5 and the Acetyl-CoA complex. Lactate has 2 more heavy atoms than acetate, but this replacement neither introduces any steric clash, nor interferes its transferring to substrate Lysine. On the left, steric clash analysis using coot was conducted. The distance between atoms within 3.5 angstroms was depicted by dashed lines. Red represents oxygen atoms, blue for nitrogen atoms, cyan for carbon atoms, and yellow for sulfur atoms. On the right, the accessibility of Lactyl-CoA and Acetyl-CoA to the substrate lysine was compared. Blue surface denotes positively charged residues, while red surface indicates negatively charged residues. Lactyl-CoA and Acetyl-CoA were represented in stick models. (g) The hydroxyl group of Lactyl-CoA is capable of forming 2 hydrogen bonds with GCN5. Lactyl-CoA and Acetyl-CoA were depicted in stick models. Oxygen atoms were shown in red, nitrogens in blue, carbon atoms in green, and sulfur atoms in yellow. GCN5 was represented in a cartoon representation for the backbone and a line representation for side chains. SF3.pdf Fig. S3. (a) The location of kinase domain on ERK1 and ERK2 and the sequence of kinase domain constructs. (b) HEK293T cells transfected with HA-tagged ERK1/ERK2 WT or kinase domain constract plasmids. (c) HEK293T cells transfected with HA-tagged ERK1/ERK2 WT or 15 homologous lysine to arginine mutant plasmids. (d and e) HEK293T cells transfected with HA-tagged ERK1/ERK2 WT, 3 non-homologous lysine to arginine mutant, 15 homologous lysine arginine mutant plasmids. (f) HEK293T cells transfected with HA-tagged ERK2 WT and indicated homologous lysine to arginine mutant plasmids. (g) HEK293T cells transfected with HA-tagged ERK1 WT and ERK1 K248R mutant plasmids. (h) The comparison of the sequence surrounding the lactylation site of ERK1/2 among species. (i) Characterization of the anti-K231lac-ERK antibody by a dot blot assay. Indicated amounts of unlactylated or lactylated ERK peptides were spotted onto a nitrocellulose membrane and immunoblotted with the anti-K231lac-ERK antibody. (j) HEK293T cells transfected with HA-tagged ERK1 WT, ERK1 K248R mutant, and treated with lactate (20mM, 24h) or (k) oxamate (30mM, 24h). (l) Coomassie blue staining of the bacterially expressed GST protein, GST-ERK WT, and GST-ERK KR fusion protein. SF4.pdf Fig. S4. (a) Different tumor cell lines with RAS gene wild-type or mutant were lysed and blotted with the indicated antibodies. (b) Soft agar assay of EGFR VIII and (c) RAS G12D background cells with ERK2 WT or KR stable expression. SF5.pdf Fig. S5. Metabolomic differences between ERK2 WT and ERK2 KR cells (a) Pathway diagram of glycolysis, red bold fonts represent upregulated genes in ERK2 WT compared to ERK2 KR cells. (b) Relative levels of differential metabolites related to glycolysis between ERK2 WT and ERK2 KR cells. (c) Pathway diagram of the pentose phosphate pathway (PPP), red bold fonts represent upregulated genes in ERK2 WT compared to ERK2 KR cells. (d) Relative levels of differential metabolites related to PPP between ERK2 WT and ERK2 KR cells. (e) Pathway diagram of the TCA cycle, red bold fonts represent upregulated genes in ERK2 WT compared to ERK2 KR cells. (f) Relative levels of differential metabolites related to the TCA cycle between ERK2 WT and ERK2 KR cells. (g) Relative levels of differential metabolites related to amino acid metabolism between ERK2 WT and ERK2 KR cells. (h) Relative level of glucose in the culture medium between ERK2 WT and ERK2 KR cells. SF6.pdf Fig. S6. ERK-MEK form a stable complex. (a) Root of mean square deviation analysis of the MEK-ERK complex calculated from 5,000 snapshots from the MD simulation with fitting on ERK. (b) Intermolecular hydrogen bonds calculated from 5,000 snapshots from the MD simulation. (c) The ERK-MEK binding interface area calculated from 5,000 snapshots from the MD simulation. (d) Center of Mass (COM) distances between MEK and ERK calculated from 5,000 snapshots from the MD simulation. Hydrogen bonds/salt bridge pairs of MEK1(e) and ERK2(f) identified from MD simulation results (b). SF7.pdf Fig. S7. Potential of mean force calculation (a) Umbrella pull force applied to WT MEK-ERK (left) and ERK-MEK harboring K231-lactylated (Klac) ERK (right) to separate MEK from ERK. (b) The Center of Mass (COM) distance between MEK and ERK for WT MEK-ERK (left) and MEK-ERK with KLA-harboring ERK (right). (c) Histograms of WHAM fit for WT MEK-ERK (left, 28 windows) and MEK-ERK with KLA-harboring ERK (right, 27 windows) SF8compressed.pdf Fig. S8. (a) Representative IHC staining images of NSCLC and (b) CRC with their paired adjacent normal tissues, respectively. Scale bars, 100 μm. (c) Correlation analysis of GCN5 and RAS-MAPK downstream gene by GEPIA (http://gepia.cancer-pku.cn/index.html). (d) Representative IHC staining images with Ki-67, K231lac-ERK, p-RSK of PDX model related to Fig. 7(m). Scale bars, 100 μm. Cite Share Download PDF Status: Published Journal Publication published 13 Jan, 2026 Read the published version in Nature Chemical Biology → 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. 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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-3944681","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":273978444,"identity":"b3824df2-b312-4cd9-8469-eebc7ed44e1a","order_by":0,"name":"Jian Yuan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+klEQVRIiWNgGAWjYLACCQMGBn725oOPwTxm5gbitEj2HEs2ZmAAspgZidACAgY3csykwVoYCGgxuJH87IFFgV0ew4Ecs+qCij/R/O1ALT8qtuHRkmZuIGGQXMzYcKzs9owzBrkzDjM2MPacuY1HS4KZhIQBc2IzY/O227xtBrkNQC3MjG34tKR/A2qpT2xjZjArBmmZT1hLDsiWw4k9bCxmzCAtGwhpkTzzpgyo5XjiDB62ZGmeM8a5G4FaDuLzC9/x9G3SEn+qE/fff3zwM0+FXO6884cPPvhRgVuLwgFg3Emgix7AqR4I5BuAUfcBn4pRMApGwSgYBQAtKFkhZ1QeHAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2801-8849","institution":"Tongji University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Jian","middleName":"","lastName":"Yuan","suffix":""},{"id":273978445,"identity":"4714f487-9d6c-484b-9f77-fd0529cded5b","order_by":1,"name":"Bingsong Huang","email":"","orcid":"","institution":"Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Bingsong","middleName":"","lastName":"Huang","suffix":""},{"id":273978446,"identity":"4e14e267-e1dc-45ed-b5a2-0c86fe86c6cc","order_by":2,"name":"Yuping Chen","email":"","orcid":"","institution":"Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yuping","middleName":"","lastName":"Chen","suffix":""},{"id":273978447,"identity":"73b23649-26c8-426c-bf27-a6a4a28bef2a","order_by":3,"name":"Gaofeng Cui","email":"","orcid":"https://orcid.org/0000-0001-5267-9033","institution":"Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine and Science","correspondingAuthor":false,"prefix":"","firstName":"Gaofeng","middleName":"","lastName":"Cui","suffix":""},{"id":273978448,"identity":"87405e42-9376-4a91-8a1c-2780c578b782","order_by":4,"name":"Georges Mer","email":"","orcid":"https://orcid.org/0000-0002-1900-1578","institution":"Mayo Clinic","correspondingAuthor":false,"prefix":"","firstName":"Georges","middleName":"","lastName":"Mer","suffix":""},{"id":273978449,"identity":"e07979f7-e3c2-4712-8e15-29201f6d5c55","order_by":5,"name":"Chunlong Zhong","email":"","orcid":"https://orcid.org/0000-0002-0605-7273","institution":"Tongji University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chunlong","middleName":"","lastName":"Zhong","suffix":""}],"badges":[],"createdAt":"2024-02-10 02:40:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3944681/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3944681/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41589-025-02107-8","type":"published","date":"2026-01-13T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":51472517,"identity":"b9297597-29bd-4745-8bba-0e8eeaf8758b","added_by":"auto","created_at":"2024-02-22 08:25:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":471561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactate promotes MAPK signaling through ERK lactylation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Dot plots of GSEA results between the control group and the lactate treatment group(20mM,24h).\u003c/p\u003e\n\u003cp\u003e(b) GSEA plot indicating enrichment of MAPK signaling pathway in the lactate treatment group(20mM,24h), core enrichment genes of MAPK signaling pathway were plotted.\u003c/p\u003e\n\u003cp\u003e(c) Hela cells or (d) U87MG cells treated with lactate (20mM, 24h) or oxamate (30mM, 24h). The cells were lysed and blotted with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(e) Relative mRNA expression of the indicated genes in U87MG cells, which were treated with lactate (20mM, 24h) as indicated. n = 3 per group.\u003c/p\u003e\n\u003cp\u003e(f) HEK293T cells transfected with the indicated MAPK component plasmids were lysed and purified using anti-HA-agarose beads. The immunoprecipitates were then blotted with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(g) Cell lysates from HEK293T treated with lactate(20mM,24h) or not were subjected to immunoprecipitation using anti-ERK1/2 or IgG as a control followed by immunoblotting with indicated antibody. Inputs (total cell lysates) are included as loading controls.\u003c/p\u003e\n\u003cp\u003e(h) Biotin-labeled lactate or biotin control incubated with cell extracts from Hela and U87MG cells. The incubation complexes were then blotted with the ERK1/2 antibodies.\u003c/p\u003e\n\u003cp\u003e(i) HEK293T cells transfected with HA-tagged ERK1 or ERK2 plasmids and treated with lactate (0, 10, 20mM) or (j) oxamate (0, 15, 30mM) for 24h.\u003c/p\u003e\n\u003cp\u003e(k) HEK293T cells depleted of LDHA or (l) MCT1 were transfected with HA-tagged ERK2 and treated with lactate (24h) as indicated.\u003c/p\u003e","description":"","filename":"Binder11.png","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/2ea9dcf7f57afef4cbd8a91b.png"},{"id":51471920,"identity":"462a5ec2-abf2-4021-9943-a96d8f0fc836","added_by":"auto","created_at":"2024-02-22 08:17:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":379823,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eERK is lactylated by GCN5 Acetyltransferase.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) HEK293T cells transfected with the indicated MYC-tagged acetyltransferase plasmids and HA-tagged ERK2 plasmids. The cells were lysed and purified using anti-HA-agarose beads. The immunoprecipitates were then blotted with the indicated antibodies. Inputs confirm protein expression levels.\u003c/p\u003e\n\u003cp\u003e(b) HEK293T cells transfected with the indicated MYC-tagged GCN5 plasmids (0, 0.5, 1, 2, 3 μg) and HA-tagged ERK2 plasmids. The immunoprecipitates were then blotted with the indicated antibodies. Inputs confirm protein expression levels and pathway alterations.\u003c/p\u003e\n\u003cp\u003e(c) HEK293T cells depleted of GCN5 were transfected with HA-tagged ERK2. The immunoprecipitates were then blotted with the indicated antibodies. Inputs confirm protein expression levels and pathway alterations.\u003c/p\u003e\n\u003cp\u003e(d) CD domains of ERK2 and ERK1.\u003c/p\u003e\n\u003cp\u003e(e) The conserved sequence of the D-domain docking site for MAP kinase phosphatases and MAP kinase substrates, Φ represents a hydrophobic amino acid.\u003c/p\u003e\n\u003cp\u003e(f) The location of the D-domain docking site in GCN5.\u003c/p\u003e\n\u003cp\u003e(g) HEK293T cells transfected with the indicated MYC-tagged GCN5 WT/GCN5 4A mutant or (h) HA-tagged ERK2 WT/ERK2 2DN mutant.\u003c/p\u003e\n\u003cp\u003e(i) Stable GCN5-depleted HEK293T cell lines transfected with the indicated plasmids (control vector, MYC-tagged GCN5 WT, and GCN5 4A mutant).\u003c/p\u003e\n\u003cp\u003e(j) HEK293T cells transfected with the indicated HA-tagged ERK2 WT/ERK2 2DN mutant and treated with lactate (20mM, 24h).\u003c/p\u003e\n\u003cp\u003e(k) Purified GST protein and GST-fusion ERK2 proteins were incubated with MYC-tagged GCN5 proteins extracted from HEK293T to perform in vitro lactylation assays as indicated.\u003c/p\u003e\n\u003cp\u003e(l) Purified GST protein and GST-fusion ERK2 proteins were incubated with MYC-tagged GCN5 proteins extracted from HEK293T to perform in vitro acetylation assays as indicated.\u003c/p\u003e","description":"","filename":"Binder12.png","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/a5429cf49221c23aac0411d8.png"},{"id":51471915,"identity":"4d677e2f-d691-4bd9-9d9f-35cfcd2bbef8","added_by":"auto","created_at":"2024-02-22 08:17:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":336184,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactylation of ERK1/2 at K248/231 facilitate cancer progression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) HEK293T cells transfected with the indicated ERK2 WT or ERK2 mutant plasmids. The cells were lysed and purified using anti-HA-agarose beads. The immunoprecipitates were then blotted with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(b) Lactylation of ERK1/2 at K248/231 was detected by mass spectrometry.\u003c/p\u003e\n\u003cp\u003e(c) The location of the lysine lactylation site of ERK1 (K248) and ERK2 (K231).\u003c/p\u003e\n\u003cp\u003e(d) HEK293T cells transfected with the indicated ERK2 plasmid and treated with lactate (20mM, 24h) or (e) oxamate (30mM, 24h). The samples were lysed and purified using anti-HA-agarose beads. The immunoprecipitates were blotted with the ERK lactylation site-specific antibodies or pan-acetylation antibodies.\u003c/p\u003e\n\u003cp\u003e(f) Purified GST fusion ERK2 WT and ERK2 KR mutant proteins were incubated with MYC-tagged GCN5 proteins extracted from HEK293T to perform in vitro lactylation assays as indicated.\u003c/p\u003e\n\u003cp\u003e(g) Hela cells or (h) U87MG cells treated with lactate (20mM, 24h) or oxamate (30mM, 24h). The cells were lysed and blotted with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(i) HEK293T cells depleted of GCN5. The cells were lysed and blotted with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(j) Relative mRNA expression of the indicated genes in Hela ERK2 WT or KR stable expression cell lines, which were treated with lactate (20mM, 24h) as indicated. n = 3 per group.\u003c/p\u003e\n\u003cp\u003e(k) Soft agar assay of Hela ERK2 WT or KR stable expression cell lines. Right, representative image, Scale bars, 100 mm. Left, quantitative analysis of the soft agar assay for HeLa cells stably expressing ERK2 WT or ERK2 KR. n = 3 per group.\u003c/p\u003e\n\u003cp\u003e(l) Colony formation assay of Hela ERK2 WT or KR stable expression cell lines treated with lactate (0, 7.5, 15, 25mM) or oxamate (0, 7.5, 15, 30mM). n = 3 per group.\u003c/p\u003e","description":"","filename":"Binder13.png","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/c3d675658bb86c592f96544f.png"},{"id":51471916,"identity":"8f5f839b-f267-404d-bd70-0b084d1b56ac","added_by":"auto","created_at":"2024-02-22 08:17:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":311707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEGFR-RAS Activation promotes ERK lactylation through ERK-GCN5 feedback loop\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a)HEK293T cells treated with EGF (100 ng/ml, 15 min). The cells were lysed and blotted with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(b)HEK293T cells transfected with the indicated EGFR\u003csup\u003eVIII\u003c/sup\u003e mutant or RAS\u003csup\u003eG12D\u003c/sup\u003e mutant plasmids. The cells were lysed and blotted with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(c) The conserved sequence of the ERK phosphorylation motif.\u003c/p\u003e\n\u003cp\u003e(d) The location of the ERK phosphorylation site of GCN5.\u003c/p\u003e\n\u003cp\u003e(e) HEK293T cells transfected with the Flag-tagged GCN5 plasmids and treated with or without EGF (100ng/ml, 15min). The cells were lysed, and then purified using anti-Flag-agarose beads. The immunoprecipitates were blotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(f) HEK293T cells transfected with the Flag-tagged GCN5 WT or GCN5 TA mutant plasmids and treated with or without the ERK inhibitor (SCH772984, 1μM, 6h), EGF (100ng/ml, 15min). The cells were lysed, and then purified using anti-Flag-agarose beads. The immunoprecipitates were blotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(g) GCN5 depletion HEK293T cells transfected with the Flag-tagged GCN5 plasmids and treated with or without EGF (100ng/ml, 15min). The cells were lysed, and then purified using anti-Flag-agarose beads. The immunoprecipitates were blotted with indicated antibodies. Inputs confirm protein pathway alterations and expression levels.\u003c/p\u003e\n\u003cp\u003e(h) GCN5 depletion HEK293T cells transfected with the Flag-tagged GCN5 treated with or without lactate (20mM, 24h), ERK inhibitor (SCH772984, 1μM, 6h), MEK inhibitor (Selumetinib, 1μM, 6h). The cells were lysed, and then purified using anti-Flag-agarose beads. The immunoprecipitates were blotted with indicated antibodies. Inputs confirm protein pathway alterations and expression levels.\u003c/p\u003e\n\u003cp\u003e(i) Purified GST fusion ERK2 proteins were incubated with Flag-tagged GCN5 WT or TA mutant proteins extracted from HEK293T to perform in vitro lactylation assays as indicated.\u003c/p\u003e\n\u003cp\u003e(j) Purified His fusion GCN5 WT or GCN5 TA mutant proteins were incubated with HA-tagged ERK2 WT or K54R (kinase dead mutant) proteins extracted from HEK293T to perform in vitro phosphorylation assays as indicated.\u003c/p\u003e\n\u003cp\u003e(k) Colony formation assays were performed using stable cell lines expressing exogenous GCN5 WT or TA mutant in GCN5-depleted SUM159PT cells, treated with lactate (0, 7.5, 15, or 25 mM) or oxamate (0, 7.5, 15, or 30 mM). Each group consisted of three independent replicates (n = 3 per group).\u003c/p\u003e","description":"","filename":"Binder14.png","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/5cd1d380d5a24bb4e7a657ea.png"},{"id":51471926,"identity":"b1334163-14eb-4f33-87ec-8001bbfde22d","added_by":"auto","created_at":"2024-02-22 08:17:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":534094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eERK lactylation is essential for EGFR-RAS induced tumor growth and cancer metabolism reprogramming\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Colony formation assay of U87MG EGFR\u003csup\u003eVIII\u003c/sup\u003e ERK2 WT or U87MG EGFR\u003csup\u003eVIII\u003c/sup\u003e ERK2 KR stable expression cell lines treated with lactate (0, 7.5, 15, 25mM) or (E) oxamate (0, 7.5, 15, 30mM). n = 3 per group.\u003c/p\u003e\n\u003cp\u003e(b) Colony formation assay of SUM159PT RAS\u003csup\u003eG12D\u003c/sup\u003e ERK2 WT or SUM159PT RAS\u003csup\u003eG12D\u003c/sup\u003e ERK2 KR stable expression cell lines treated with lactate (0, 7.5, 15, 25mM) or oxamate (0, 7.5, 15, 30mM). n = 3 per group.\u003c/p\u003e\n\u003cp\u003e(c)-(h)Xenograft tumor formation (n = 5 animals) by ERK2 WT/KR U87MG EGFR\u003csup\u003eVIII\u003c/sup\u003e cells and SUM159PT RAS\u003csup\u003eG12D\u003c/sup\u003e in nude mice that were treated with or without lactate intraperitoneally three times a week (5M of 100 μl). The representative tumor images, (c) and (f); tumor weight, (d) and (g); tumor volume, (e) and (h).\u003c/p\u003e\n\u003cp\u003e(i) Heatmap of metabolomics for Hela ERK2 WT or KR stable expression cell lines. Z-score represents the relative level of each metabolite. n = 3 per group.\u003c/p\u003e","description":"","filename":"Binder15.png","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/3d1345f0bb10515eb85f366c.png"},{"id":51471925,"identity":"23fd4be8-103e-4b4f-8cab-641be3a2e2bd","added_by":"auto","created_at":"2024-02-22 08:17:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":793062,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLactylation promotes ERK dissociation from MEK and subsequent dimerization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) HEK293T cells transfected with the ERK2 WT plasmids and treated with or without EGF (100 ng/ml, 5 min, 15 min, 30 min). The cells were lysed, and then purified using anti-HA-agarose beads.\u003c/p\u003e\n\u003cp\u003e(b) HA-tagged ERK2 stable expression HEK293T cells were cultured with serum-free medium for 12 h, and after serum starvation, cultured with 10 % fetal bovine serum medium for 6 h. The cells were lysed, and then purified using anti-HA-agarose beads.\u003c/p\u003e\n\u003cp\u003e(c) HEK293T cells transfected with the indicated ERK2 WT plasmids and treated with or without lactate (20 mM, 24 h), oxamate (30 mM, 24 h). The cells were lysed, and then purified using anti-HA-agarose beads.\u003c/p\u003e\n\u003cp\u003e(d and e) HEK293T cells transfected with the indicated ERK2 WT or ERK2 mutant plasmids and treated with or without lactate (20 mM, 24 h), oxamate (30 mM, 24 h), or EGF (100 ng/ml, 15 min). The cells were lysed, and then purified using anti-HA-agarose beads.\u003c/p\u003e\n\u003cp\u003e(f) HEK293T cells transfected with the indicated HA-tagged ERK2 WT/KR and MYC-tagged ERK2 WT/KR and treated with or without lactate (20 mM, 24 h), oxamate (30 mM, 24 h).\u003c/p\u003e\n\u003cp\u003e(g) HEK293T cells transfected with the indicated ERK2 WT or ERK2 mutant plasmids and treated with or without lactate (20 mM, 24 h). The cells were lysed and separated into two parts. One part was prepared for Native PAGE, and the other for SDS PAGE. (h) HEK293T cells transfected with the indicated ERK2 WT or ERK2 mutant plasmids and treated with or without EGF (100 ng/ml, 15 min). The cells were lysed, and then purified using anti-HA-agarose beads.\u003c/p\u003e\n\u003cp\u003e(i) HEK293T cells depleted of GCN5 were transfected with HA-tagged ERK2. The immunoprecipitates were then blotted with the indicated antibodies. Inputs confirm protein expression levels and pathway alterations.\u003c/p\u003e\n\u003cp\u003e(j) GCN5-depleted HEK293T cells expressing GCN5 WT or TA mutant were transfected with HA-ERK2, followed by HA-immunoprecipitation and immunoblotting. The cells were lysed, and then purified using anti-HA-agarose beads. The immunoprecipitates were then blotted with the indicated antibodies. Inputs confirm protein expression levels and pathway alterations.\u003c/p\u003e\n\u003cp\u003e(k) Ribbon and surface representation of ERK-MEK complex model build from AlphaFold2. ERK in teal and MEK in orange.\u003c/p\u003e\n\u003cp\u003e(l) ERK K231 is part of the ERK-MEK binding interface, forming polar contacts with MEK D217 (left). ERK K231 lactylation does not causes clashes with other residues. The yellow dash line indicates distances between ERK K231 N\u003csup\u003eε\u003c/sup\u003e and MEK D217 O\u003csup\u003eδ\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e(m) HEK293T cells transfected with the MYC-tagged ERK2 plasmids and indicated HA-tagged MEK1 WT or Mutant plasmids.\u003c/p\u003e\n\u003cp\u003e(n) HEK293T cells transfected with the MYC-tagged MEK1 plasmids and indicated HA-tagged ERK2 WT or Mutant plasmids.\u003c/p\u003e\n\u003cp\u003e(o) Potential of mean force evaluation of the dissociation free energy for WT ERK-MEK and for ERK-MEK harboring K231-lactylated (Klac) ERK. The error bars were calculated from 100 steps of Bootstrap analysis.\u003c/p\u003e","description":"","filename":"Binder16.png","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/cb119277a58f0f0d97ef2051.png"},{"id":51471921,"identity":"64149cae-8116-4c59-999c-6fd45a8dc65c","added_by":"auto","created_at":"2024-02-22 08:17:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":359114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibiting ERK lactylation enhances chemosensitivity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) IHC analyses of 61 human NSCLC and (b) 48 human colorectal cancers (CRC) with their paired adjacent normal tissues were performed, respectively.\u003c/p\u003e\n\u003cp\u003e(c) The correlation between ERK lactylation expression and Ki-67 levels was analyzed.\u003c/p\u003e\n\u003cp\u003e(d) IHC analyses of 21 colorectal cancers with RAS mutant and 21 colorectal cancers with RAS WT.\u003c/p\u003e\n\u003cp\u003e(e) Design scheme of peptides targetig ERK1/2 lactylation sites. The blue-highlighted sequence represents the motif of the cell-penetrating peptide.\u003c/p\u003e\n\u003cp\u003e(f) SUM159PT cells were treated with or without peptides as indicated (100μM, every 12h, three times). After treatment, the cells were lysed and blotted with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(g) SUM159PT cells treated with indicated concentrations of peptides every 12h for three times. After treatment, cells were lysed, and the lysis solution was blotted with indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(h) Colony formation assays were performed using Hela cells or (i) stable cell lines expressing ERK2 WT or KR mutant in SUM159PT cells, treated with or without the peptide as indicated (100μM). Cells were treated with peptides every 12h for seven days. Each group consisted of three independent replicates (n = 3 per group).\u003c/p\u003e\n\u003cp\u003e(j) Schematic diagram of patient-derived xenografts (PDXs) establishment and dosage regimen. (k) Growth curves, (l) tumor weight, and (m) images of the tumors of PDX models treated with saline, P-3, P-3(R), 5-FU, or combined treatment (n = 7 mice for each group).\u003c/p\u003e","description":"","filename":"Binder17.png","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/762bd9aac0fcc23cd4e1fea1.png"},{"id":100213191,"identity":"4bacb805-2d29-41d9-a7a9-bcb79e54fac9","added_by":"auto","created_at":"2026-01-14 08:07:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4674488,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/d5bba0a1-d85d-47f1-89c8-29fe2845753e.pdf"},{"id":51471919,"identity":"d990a840-5df7-4484-965b-71ac04da55a1","added_by":"auto","created_at":"2024-02-22 08:17:41","extension":"mpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4801102,"visible":true,"origin":"","legend":"\u003cp\u003eMovie 1\u003c/p\u003e","description":"","filename":"Mov1XXXXXX.mpg","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/73af883827cd5e03ccfec200.mpg"},{"id":51472516,"identity":"89b9b995-3715-4a44-b196-b07311274c39","added_by":"auto","created_at":"2024-02-22 08:25:41","extension":"mpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3502080,"visible":true,"origin":"","legend":"\u003cp\u003eMovie 2\u003c/p\u003e","description":"","filename":"Mov2XXXXXX.mpg","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/b38c4eb1c179162aabf2148c.mpg"},{"id":51471927,"identity":"86d6d3e7-8b92-40cf-a2ad-97cd315c4776","added_by":"auto","created_at":"2024-02-22 08:17:42","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":15929010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Volcano plot illustrating differential gene expression analysis between the control group and the lactate treatment group (20mM, 24h) using RNA-seq data. Each data point represents a gene, with the x-axis showing the logarithm (base 2) of the fold change, and the y-axis indicating the negative logarithm (base 10) of the adjusted p-value (-log10(p-value)).\u003c/p\u003e","description":"","filename":"SF1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/ea0c5c16b480534067c58948.pdf"},{"id":51471929,"identity":"61be4173-770e-4c4c-82dd-377c419a98fc","added_by":"auto","created_at":"2024-02-22 08:17:42","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12772708,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) HEK293T cells transfected with the indicated MYC-tagged acetyltransferase plasmids and HA-tagged ERK1 plasmids.\u003c/p\u003e\n\u003cp\u003e(b) HEK293T cells transfected with the indicated MYC-tagged GCN5 plasmids (0, 0.5, 1, 2, 3 μg) and HA-tagged ERK1 plasmids.\u003c/p\u003e\n\u003cp\u003e(c) HEK293T cells depleted of GCN5 were transfected with HA-tagged ERK1.\u003c/p\u003e\n\u003cp\u003e(d) HEK293T cells transfected with the indicated MYC-tagged GCN5 WT/GCN5 4A mutant or (e) HA-tagged ERK1 WT/ERK2 2DN mutant.\u003c/p\u003e\n\u003cp\u003e(f) A model of Lactyl-CoA binding to GCN5 was constructed based on the crystal structure (pdb ID:IQSR) of GCN5 and the Acetyl-CoA complex. Lactate has 2 more heavy atoms than acetate, but this replacement neither introduces any steric clash, nor interferes its transferring to substrate Lysine. On the left, steric clash analysis using coot was conducted. The distance between atoms within 3.5 angstroms was depicted by dashed lines. Red represents oxygen atoms, blue for nitrogen atoms, cyan for carbon atoms, and yellow for sulfur atoms. On the right, the accessibility of Lactyl-CoA and Acetyl-CoA to the substrate lysine was compared. Blue surface denotes positively charged residues, while red surface indicates negatively charged residues. Lactyl-CoA and Acetyl-CoA were represented in stick models.\u003c/p\u003e\n\u003cp\u003e(g) The hydroxyl group of Lactyl-CoA is capable of forming 2 hydrogen bonds with GCN5. Lactyl-CoA and Acetyl-CoA were depicted in stick models. Oxygen atoms were shown in red, nitrogens in blue, carbon atoms in green, and sulfur atoms in yellow. GCN5 was represented in a cartoon representation for the backbone and a line representation for side chains.\u003c/p\u003e","description":"","filename":"SF2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/7087512175831d02cbd72f23.pdf"},{"id":51471924,"identity":"9ea487ac-f8d5-48c9-93a4-732965a40b35","added_by":"auto","created_at":"2024-02-22 08:17:42","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":5099142,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) The location of kinase domain on ERK1 and ERK2 and the sequence of kinase domain constructs.\u003c/p\u003e\n\u003cp\u003e(b) HEK293T cells transfected with HA-tagged ERK1/ERK2 WT or kinase domain constract plasmids.\u003c/p\u003e\n\u003cp\u003e(c) HEK293T cells transfected with HA-tagged ERK1/ERK2 WT or 15 homologous lysine to arginine mutant plasmids.\u003c/p\u003e\n\u003cp\u003e(d and e) HEK293T cells transfected with HA-tagged ERK1/ERK2 WT, 3 non-homologous lysine to arginine mutant, 15 homologous lysine arginine mutant plasmids. (f) HEK293T cells transfected with HA-tagged ERK2 WT and indicated homologous lysine to arginine mutant plasmids.\u003c/p\u003e\n\u003cp\u003e(g) HEK293T cells transfected with HA-tagged ERK1 WT and ERK1 K248R mutant plasmids.\u003c/p\u003e\n\u003cp\u003e(h) The comparison of the sequence surrounding the lactylation site of ERK1/2 among species.\u003c/p\u003e\n\u003cp\u003e(i) Characterization of the anti-K231lac-ERK antibody by a dot blot assay. Indicated amounts of unlactylated or lactylated ERK peptides were spotted onto a nitrocellulose membrane and immunoblotted with the anti-K231lac-ERK antibody.\u003c/p\u003e\n\u003cp\u003e(j) HEK293T cells transfected with HA-tagged ERK1 WT, ERK1 K248R mutant, and treated with lactate (20mM, 24h) or (k) oxamate (30mM, 24h).\u003c/p\u003e\n\u003cp\u003e(l) Coomassie blue staining of the bacterially expressed GST protein, GST-ERK WT, and GST-ERK KR fusion protein.\u003c/p\u003e","description":"","filename":"SF3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/4c3cfe2ba161d568825cbedd.pdf"},{"id":51471932,"identity":"66e68c57-290d-4719-b858-1cd3d888b8aa","added_by":"auto","created_at":"2024-02-22 08:17:43","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":14163115,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.\u0026nbsp; S4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Different tumor cell lines with RAS gene wild-type or mutant were lysed and blotted with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e(b) Soft agar assay of EGFR\u003csup\u003eVIII\u003c/sup\u003e and (c) RAS\u003csup\u003eG12D\u003c/sup\u003e background cells with ERK2 WT or KR stable expression.\u003c/p\u003e","description":"","filename":"SF4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/01229946837dade04cda593c.pdf"},{"id":51471923,"identity":"89b089f7-1d5a-4923-bd0f-f58fb7ee8085","added_by":"auto","created_at":"2024-02-22 08:17:42","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1117626,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S5. Metabolomic differences between ERK2 WT and ERK2 KR cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Pathway diagram of glycolysis, red bold fonts represent upregulated genes in ERK2 WT compared to ERK2 KR cells.\u003c/p\u003e\n\u003cp\u003e(b) Relative levels of differential metabolites related to glycolysis between ERK2 WT and ERK2 KR cells.\u003c/p\u003e\n\u003cp\u003e(c) Pathway diagram of the pentose phosphate pathway (PPP), red bold fonts represent upregulated genes in ERK2 WT compared to ERK2 KR cells.\u003c/p\u003e\n\u003cp\u003e(d) Relative levels of differential metabolites related to PPP between ERK2 WT and ERK2 KR cells.\u003c/p\u003e\n\u003cp\u003e(e) Pathway diagram of the TCA cycle, red bold fonts represent upregulated genes in ERK2 WT compared to ERK2 KR cells.\u003c/p\u003e\n\u003cp\u003e(f) Relative levels of differential metabolites related to the TCA cycle between ERK2 WT and ERK2 KR cells.\u003c/p\u003e\n\u003cp\u003e(g) Relative levels of differential metabolites related to amino acid metabolism between ERK2 WT and ERK2 KR cells.\u003c/p\u003e\n\u003cp\u003e(h) Relative level of glucose in the culture medium between ERK2 WT and ERK2 KR cells.\u003c/p\u003e","description":"","filename":"SF5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/5f68b7a64a7ca88ad3cc0c14.pdf"},{"id":51471928,"identity":"f56f34c9-aa5f-4ced-ae14-7f6d760691f5","added_by":"auto","created_at":"2024-02-22 08:17:42","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":723428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S6. ERK-MEK form a stable complex.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Root of mean square deviation analysis of the MEK-ERK complex calculated from 5,000 snapshots from the MD simulation with fitting on ERK.\u003c/p\u003e\n\u003cp\u003e(b) Intermolecular hydrogen bonds calculated from 5,000 snapshots from the MD simulation.\u003c/p\u003e\n\u003cp\u003e(c) The ERK-MEK binding interface area calculated from 5,000 snapshots from the MD simulation.\u003c/p\u003e\n\u003cp\u003e(d) Center of Mass (COM) distances between MEK and ERK calculated from 5,000 snapshots from the MD simulation. Hydrogen bonds/salt bridge pairs of MEK1(e) and ERK2(f) identified from MD simulation results (b).\u003c/p\u003e","description":"","filename":"SF6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/59d4f43c1c6a41a669d909b9.pdf"},{"id":51471931,"identity":"16a3d65d-0fbd-40a0-9998-c7faaafad137","added_by":"auto","created_at":"2024-02-22 08:17:43","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":594977,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S7. Potential of mean force calculation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Umbrella pull force applied to WT MEK-ERK (left) and ERK-MEK harboring K231-lactylated (Klac) ERK (right) to separate MEK from ERK.\u003c/p\u003e\n\u003cp\u003e(b) The Center of Mass (COM) distance between MEK and ERK for WT MEK-ERK (left) and MEK-ERK with KLA-harboring ERK (right).\u003c/p\u003e\n\u003cp\u003e(c) Histograms of WHAM fit for WT MEK-ERK (left, 28 windows) and MEK-ERK with KLA-harboring ERK (right, 27 windows)\u003c/p\u003e","description":"","filename":"SF7.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/b8d4b83b83618f7de6b970aa.pdf"},{"id":51471930,"identity":"2f573704-14fd-4886-ae2e-c50697b12c2e","added_by":"auto","created_at":"2024-02-22 08:17:43","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":467718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S8.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Representative IHC staining images of NSCLC and (b) CRC with their paired adjacent normal tissues, respectively. Scale bars, 100 μm.\u003c/p\u003e\n\u003cp\u003e(c) Correlation analysis of GCN5 and RAS-MAPK downstream gene by GEPIA (http://gepia.cancer-pku.cn/index.html).\u003c/p\u003e\n\u003cp\u003e(d) Representative IHC staining images with Ki-67, K231lac-ERK, p-RSK of PDX model related to Fig. 7(m). Scale bars, 100 μm.\u003c/p\u003e","description":"","filename":"SF8compressed.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3944681/v1/4ac597ebe57f0fb979905b42.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Lactate accelerates cancer progression through the ERK-GCN5 lactylation-phosphorylation feedback cascade","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMetabolic reprogramming is widely recognized as a hallmark of cancer that enables rapid cell proliferation. One prominent example is the Warburg effect or aerobic glycolysis, which is characterized by elevated glucose uptake and lactate release in cancer cells(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Despite the inefficiency of this process for energy production, cancer cells preferentially rely on aerobic glycolysis for their energy supply. Lactate was traditionally considered a by-product of aerobic glycolysis, but recent studies have shown that it can serve as a metabolic fuel for cancer cells(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). In fact, lactate contributes to energy production more than glucose does in some cancers. For example, in non-small cell lung carcinoma (NSCLC) models, lactate was found to contribute to the tricarboxylic acid (TCA) cycle more than glucose does(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In vivo isotopic tracing experiments have also shown that \u003csup\u003e13\u003c/sup\u003eC-labeled lactate more extensively labels TCA intermediates than \u003csup\u003e13\u003c/sup\u003eC-labeled glucose in cancer cells(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to its role as an energy source, lactate affects cancer progression in various ways. Lactate inhibited the cytotoxicity T cell proliferation and cytokine production(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Furthermore, lactate directly supports tumor-promoting immune cell populations, which impairs immune function and facilitates cancer immune escape(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). However, the mechanisms by which lactate directly promotes cancer cell proliferation have been poorly investigated. Recently, a newly discovered histone post-translational modification (PTM) called lactylation was identified, which introduces lactate from metabolomics into epigenetics(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Therefore, we hypothesize that lactate serves as the donor to modify proteins lactylation, which may promote cancer cell proliferation and tumor progression.\u003c/p\u003e \u003cp\u003eThe RAS-MAPK signaling pathway plays a crucial role in intracellular signal transduction, regulating key cellular processes such as cell growth, differentiation and survival(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Upon activation by upstream signaling molecules, including receptor tyrosine kinases (RTKs) or G protein-coupled receptors (GPCRs), RAS proteins trigger a phosphorylation cascade that activates the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). This pathway comprises a series of kinases, including RAF, MEK, and ERK, which culminates in the activation of transcription factors that govern gene expression and modulate cellular behavior(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). The most common mutations that lead to hyperactivation of the RAS-MAPK signaling in cancer are found in KRAS, NRAS, and BRAF genes, which result in constitutive activation of downstream signaling cascades (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). In addition to phosphorylation, other PTM patterns also were also reported to regulate RAS-MAPK signaling. Ubiquitination and acetylation have been reported to occur on RAS-MAPK components(\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). However, their regulatory effects on RAS-MAPK signaling differ considerably. For instance, ubiquitination of RAS inhibits its localization to the plasma membrane(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), while ubiquitination of ERK promotes its degradation(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), leading to the suppression of RAS-MAPK signaling. Conversely, ubiquitination of BRAF results in sustained BRAF activation and subsequent elevation of the RAS-MAPK signaling(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Moreover, acetylation of RAS inhibits its transforming activity without affecting its plasma membrane localization (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), thereby suppressing RAS-MAPK signaling. Conversely, acetylation of RAF facilitates its dimerization and promotes RAS-MAPK signaling(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). However, the roles of lactylation on RAS-MAPK signaling is poorly investigated.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrated that the key component of the RAS-MAPK pathway, ERK is lactylated, which activates the RAS-MAPK signaling. Lysine acetyltransferase 2A (KAT2A, also known as GCN5) functions as a lactyl-transferase for ERK lactylation and activation. Interestingly, ERK directly phosphorylates GCN5, enhancing the lactyl-transferase activity of GCN5 for ERK lactylation. The positive feedback loop between ERK and GCN5 rapidly amplifies the RAS-MAPK signaling and contributes to lactate mediated cancer progression. A cell-penetrating peptide was designed to inhibit ERK lactylation and shut down the ERK-GCN5 feedback loop mediated cancer cell proliferation. Taken together, our findings reveal the mechanism by which protein lactylation modification amplifies RAS-MAPK signaling and imply a potential therapeutic strategy by targeting ERK lactylation to inhibit tumor growth especially for those hyperactive MAPK signaling derived cancer.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLactate promotes MAPK signaling through ERK lactylation\u003c/h2\u003e \u003cp\u003eTo explore the physiological function affected by lactate in cancers, we treated U87MG cells with or without lactate and performed RNA-sequencing assays. The differential gene expression analysis revealed 564 up-regulated and 379 down-regulated genes upon lactate stimulation (Extended Data Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). According to GSEA analysis, the top two ranked signaling pathway were cytokine-cytokine receptor interaction and MAPK signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). As described above, the effect of lactate on immune responses has been widely reported(\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). However, the mechanism by which lactate promotes MAPK signaling remains poorly understood. Given the prevalence of RAS-MAPK signaling mutations in cancers, which lead to constitutive pathway activation, we focused on this pathway. To validate the RNA-seq results, we treated U87MG and HeLa cells with lactate or lactate dehydrogenase (LDH) inhibitor sodium oxamate to manipulate lactate level in cells and then examined RAS-MAPK signaling. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec and d, lactate induced RSK phosphorylation, while sodium oxamate showed the opposite effects. Moreover, lactate increased RAS-MAPK downstream gene expression, and this effect reversed by sodium oxamate(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRecent studies have demonstrated that lactate induces protein lactylation, which regulates multiple cellular functions(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). We hypothesized lactate may regulate RAS-MAPK pathway through protein lactylation. Interestingly, by screening MAPK components for protein lactylation, we found that only ERK1/2, a critical regulator of RAS-MAPK signaling, could be lactylated(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef), while other MAPK components showed no significant lactylation(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef). Consistently, lactylation of endogenous ERK1/2 was increased after lactate treatment(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). As earlier study has verified the acetylation of ERK1/2(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).To examine whether acetylation of ERK1/2 was affected by lactate, we also checked the level of ERK1/2 acetylation and found that lactate treatment does not affect ERK1/2 acetylation level(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg). Previous study had presented lactate could directly interact with protein and affect protein function(\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). We also synthesized biotin-labeled lactate to perform biotin pull-down assay and found lactate did not bind to ERK1/2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eh), suggesting that lactate modulation of RAS-MAPK signaling may be mediated through lactylation.\u003c/p\u003e \u003cp\u003eFurthermore, we observed that manipulating intracellular lactate levels through lactate or LDH inhibitor treatment dramatically affected ERK1/2 lactylation in dose-dependent manners (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, i and j). Depletion of Lactate Dehydrogenase A (LDHA) decreased ERK2 lactylation, which could be rescued by lactate treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ek). Moreover, depletion of MCT1, which transports extracellular lactate into the cell, decreased ERK2 lactylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003el). Taken together, these results suggest that lactate may activate the RAS-MAPK signaling pathway by inducing ERK lactylation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eERK was Lactylated by GCN5 Acetyltransferase\u003c/h2\u003e \u003cp\u003eThe histone acetyltransferases (HAT) family was also reported to serve as lactyltransferases \u003cem\u003ein vivo\u003c/em\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). After screening those main acetyltransferases of HAT family, we found GCN5 primarily mediated ERK lactylation(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Particularly, overexpression of GCN5 increased ERK lactylation in a dose-dependent manner. However, GCN5 did not induce ERK acetylation, which is consistent with previous study that CBP/P300 mediated ERK acetylation(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e)(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Furthermore, GCN5 displayed a dose-dependent activation of ERK downstream pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the contrary, depletion of GCN5 sharply decreased ERK lactylation and subsequent downstream signaling activation, witho\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eut\u003c/span\u003e impact on ERK acetylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Given that the interaction between ERK and its upstream activators or substrates is mediated through the common docking (CD) domain on ERK and the D domain-docking site on the activator or substrate (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e), We identified the potential D domain-docking sites on GCN5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Mutation of these GCN5 D domain-docking sites to alanine (GCN5 4A) and the ERK2 CD domain to asparagine (ERK2 2DN) abrogated the ERK-GCN5 interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eg,h; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Additionally, GCN5\u003csup\u003e4A\u003c/sup\u003e mutant failed to induce ERK lactylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ei), and ERK2\u003csup\u003e2DN\u003c/sup\u003e mutation led to a decrease in lactylation of ERK2 that could not be induced by lactate treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). Furthermore, GCN5 directly catalyzed the lactylation, but not the acetylation of ERK2 in vitro (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ek, l). Given that GCN5 was well characterized as mainly serving as an acetyltransferase, to further confirm that it also plays a key role in lactylation, we simulated the interaction between lactyl-CoA and GCN5, revealing that GCN5's active site can accommodate lactyl-CoA without causing structural clashes(Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Our modeling suggests that lysine can enter this pocket for lactyl transfer. Particulary, the interaction between lactyl-CoA and GCN5 appears to be stronger compared to that of acetyl-CoA based on the structure of lactyl-CoA and GCN5. The presence of an additional hydroxyl group in lactyl-CoA allows for the formation of two additional hydrogen bonds, potentially enhancing its interaction with GCN5(Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Furthermore, the smaller size and higher electronegativity of the lactyl-CoA potentially facilitate higher efficiency in lysine N(epsilon) proximity, making lactylation more favorable than acetylation(Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). Taken together, our findings suggest that GCN5 interacts with ERK and function as a lactyl-transferases for ERK.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLactylation of ERK1/2 at K248/231 facilitate cancer progression\u003c/h2\u003e \u003cp\u003eERK1 and ERK2, sharing 84% sequence identity, both contain lysine residues within their kinase domain and C-terminus. To determine the lactylation sites, the lactylation of ERK1/2 kinase domain constructs was analyzed, revealing that both full-length ERK1 and ERK2, as well as their kinase domain constructs, displayed comparable lactylation signals (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea,b). These results indicated that the lactylation sites were mainly located within the kinase domain. Within kinase domain, ERK1 and ERK2 possess 15 homologous lysine residues and 3 non-homologous ones. Subsequent mutation of these lysine residues to arginine (R) enabled the assessment of their contribution to lactylation. Mutations of the 15 homologous lysine residues (15KR) resulted in reduced lactylation signals for both kinases, whereas mutations of the non-homologous residues (3KR) did not significantly alter lactylation levels (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis with subsection mutants revealed that mutations in the (\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) KR segment led to a marked reduction in lactylation, mirroring the effects observed with the 15KR mutant (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Single lysine mutant was also evaluated, identifying K231 in ERK2 and K248 in ERK1 as the primary lactylation sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). The ERK lactylation sites were further confirmed by mass spectrometry (MS) analysis, which showed that K248 on ERK1 and K231 on ERK2 are able to be lactylated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,c), and the sites are highly conserved across multiple species (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eh). To further explore the ERK1/2 lactylation in vivo, a specific antibody targeting lactylation at K248/231 in ERK1/2 was developed and its specificity was confirmed through dot blot assays (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), terming the antibody as K231lac-ERK.\u003c/p\u003e \u003cp\u003eThe utilization of the K231lac-ERK antibody revealed that lactate treatment enhances lactylation of ERK wild type, but not the ERK KR mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ed; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ej,k), while inhibition of lactate dehydrogenase (LDH) resulted in the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ee; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ej,k). Notably, K231R mutant did not affect its acetylation level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ed,e), consistent with previous findings that identified K72 in ERK1 and K55 in ERK2 as acetylation sites(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Additionally, we performed in vitro lactylation assays to verify the lactylation reaction. The results demonstrated that GCN5 selectively enhances ERK2 lactylation in the wild type but not in the K231R mutant(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ef; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003el). Using K231lac-ERK antibody, we also observed the lactylation of endogenous ERK was increased by lactate and decreased by LDH inhibitor or GCN5 depletion in Hela and U87MG cells(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-i).\u003c/p\u003e \u003cp\u003eERK's involvement in cancer biology is well-established, prompting us to investigate whether lactylation of ERK promotes transcription of downstream genes and cancer cell proliferation. Considering the higher expression of ERK2 over ERK1 in most cell types and their equivalent substrate specificity, our subsequent studies focused on ERK2. Overexpression of ERK2 wild-type (WT) and lactylation-deficient K231R (KR) constructs in Hela cells revealed that lactate treatment increased transcription of ERK downstream targets in ERK2 WT but not in KR mutant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). Additionally, soft agar assay showed Hela ERK2 KR cells has a reduced growth compared to ERK2 WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ek). Colony formation assay further showed that lactate treatment dramatically increased Hela ERK2 WT cells growth in a dose dependent manner(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003el), the effect was not observed in ERK2 KR cells. In contrast, inhibition of LDH resulted in a decreased growth of Hela ERK2 WT cells, with ERK2 KR cells remaining unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003el).\u003c/p\u003e \u003cp\u003eIn summary, our findings demonstrate ERK1/2 is lactylated at K248/231 in vivo and in vitro, significantly facilitating cancer progression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEGFR-RAS Activation promotes ERK lactylation through ERK-GCN5 feedback loop\u003c/h2\u003e \u003cp\u003eEGFR and RAS activation are the major inducer of RAS-MAPK cascades, which in turn promote cancer progression. Interestingly, we found EGF stimulation increased ERK lactylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Furthermore, we overexpressed EGFR\u003csup\u003eVIII\u003c/sup\u003e and RAS\u003csup\u003eG12D\u003c/sup\u003e in HEK293T cell, which are constitutive activation mutant forms of EGFR and RAS GTPases, respectively, and found that both EGFR\u003csup\u003eVIII\u003c/sup\u003e and RAS\u003csup\u003eG12D\u003c/sup\u003e promote ERK lactylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). In addition, the ERK lactylation was more abundant in RAS-MAPK mutant-driven tumors than in RAS-MAPK wild-type tumors (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). These findings suggest an augmentation of ERK lactylation induced by EGFR and RAS activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, after analyzing the GCN5 sequences, we identified GCN5 as a potential substrate for ERK, with threonine 404 aligning with the PX (S/T)P consensus sequence characteristic of ERK phosphorylating substrates (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D)(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Thus, we speculate RAS-MAPK activation may induce GCN5 phosphorylation and promote ERK lactylation to form a positive feedback loop.\u003c/p\u003e \u003cp\u003eBy utilizing the antibody-recognized motif PX(S/T)P, we observed that GCN5 phosphorylation was increased by EGF stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). On the contrary, ERK inhibitor reduced the phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Consistently, we construct GCN5 T404A mutant and found it totally abolished the phosphorylation signaling, which showed unaltered by EGF stimulation or ERK inhibitor treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). Subsequently, we speculate ERK phosphorylates GCN5, which enhancing GCN5's lactyl-transferase activity, thereby forming a positive feedback loop. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eg, compared to GCN5 WT, GCN5 TA mutant dramatically inhibited ERK lactylation and ERK downstream signaling. Moreover, ERK inhibitor or MEK inhibitor decreased GCN5 phosphorylation, and impaired ERK lactylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Meanwhile, lactate treatment could not rescue ERK lactylation in ERK inhibitor or MEK inhibitor treated cells, suggesting that GCN5 phosphorylated by ERK is essential for GCN5 lactyl-transferase activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Consistently, we also performed the \u003cem\u003ein vitro\u003c/em\u003e lactylation assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ei, we immunoprecipitated GCN5 from HEK293T cells, and found GCN5 WT but not TA mutant could lactylate ERK2 in vitro.\u003c/p\u003e \u003cp\u003eWe further confirmed ERK2 phosphorylated GCN5 by \u003cem\u003ein vitro\u003c/em\u003e kinase assay. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ej, ERK2 WT but not kinase dead mutant (K54R) phosphorylated GCN5 at T404 \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAs previous results showed ERK lactylation facilitating cancer progression. We next examined whether GCN5 phosphorylation is important for lactate mediated cancer cell growth. Depleting endogenous GCN5 and substituting it with GCN5 WT and GCN5 TA revealed that lactate or LDH inhibitor treatment more dramatically impacted cancer cell growth in GCN5 WT cells compared to GCN5 TA mutant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ek).\u003c/p\u003e \u003cp\u003eTaken together, we found that breaking down ERK-GCN5 lactylation-phosphorylation cascade impaired lactate-induced cancer cell growth.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eERK lactylation is essential for EGFR-RAS induced tumor growth and cancer metabolism reprogramming\u003c/h2\u003e \u003cp\u003eRAS-MAPK components mutant always induce continuous activation of RAS-MAPK signaling, which is common among tumors, such as EGFR\u003csup\u003eVIII\u003c/sup\u003e and RAS\u003csup\u003eG12D\u003c/sup\u003e. To further investigate the role of ERK lactylation in RAS-MAPK constitute activation tumor growth. We reconstituted ERK2 WT or the lactylation-deficient KR mutant in the EGFR\u003csup\u003eVIII\u003c/sup\u003e U87MG and RAS\u003csup\u003eG12D\u003c/sup\u003e SUM159 cell lines. Soft agar assay showed EGFR\u003csup\u003eVIII\u003c/sup\u003e U87MG or RAS\u003csup\u003eG12D\u003c/sup\u003e SUM159 ERK2 KR cells has a reduced growth compared to ERK2 WT cells(Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,c). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and b, ERK2 KR mutant cells exhibited reduced cell growth compared to ERK2 WT cells. Lactate treatment dramatically promoted cancer cell growth in ERK WT cells compared to ERK KR cells, while the LDH inhibitor showed the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further confirm the role of ERK lactylation in cancer cell proliferation \u003cem\u003ein vivo\u003c/em\u003e, the reconstructed ERK2 WT or KR mutant cancer cells (EGFR\u003csup\u003eVIII\u003c/sup\u003e U87MG and RAS\u003csup\u003eG12D\u003c/sup\u003e SUM159 cells) were injected subcutaneously in nude mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e, C to H, the ERK KR mutant cells showed a significant reduction in tumor volume and weight compared to ERK2 WT cells. By intraperitoneal injection with lactate, the tumor growth showed more significant increase in ERK WT cells than ERK KR mutant cells (Fig. c-h).\u003c/p\u003e \u003cp\u003eERK is reported to regulate cancer metabolism reprogramming(\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), we next conducted metabonomic analysis in ERK1/2 WT and KR mutant RAS\u003csup\u003eG12D\u003c/sup\u003e SUM159 cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei, the metabolites involved in glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea,b), the pentose phosphate pathway (PPP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec,d), the TCA cycle (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ee,f), and amino acid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ei; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eg) were downregulated in ERK KR cells compared to ERK WT cells. Furthermore, ERK KR mutant cells exhibited a lower glucose uptake ability (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eOverall, our findings suggest that lactylation of ERK enhances ERK activity and promotes EGFR-RAS induced tumor growth and cancer metabolism reprogramming.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLactylation promotes ERK dissociation from MEK and subsequent dimerization\u003c/h2\u003e \u003cp\u003eNext, we aim to investigate the mechanism by which ERK lactylation promotes RAS-MAPK signaling. In the resting state, ERK forms a complex with its upstream MEK, which serves as an anchor(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Upon receiving an activating stimulus, ERK is phosphorylated and separates from MEK, thereafter ERK forms dimers and exhibits autophosphorylation(\u003cspan additionalcitationids=\"CR32 CR33 CR34 CR35\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eOur results confirmed that EGF treatment or serum stimulation causes ERK dissociation from MEK (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). Interestingly, lactate treatment promotes ERK dissociation from MEK, while LDH inhibitor blocked this process (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Interestingly, the ERK KR mutant exhibited a tighter association with MEK, which could not be disrupted by EGF or lactate stimulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). These results suggested that ERK lactylation affects the ERK-MEK complex dissociation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next examined whether ERK lactylation regulates its dimerization. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ef, ERK lactylation deficient KR mutant significantly decreased its self-binding compared to ERK WT. Lactate treatment promotes ERK WT self-binding, while LDH inhibitor suppressed this progress (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). However, ERK KR exhibits a very weak ability for self-binding, which cannot be affected by Lactate or LDH inhibitor treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ef). We further proved that lactylation was key for ERK self-binding and dimerization by Native PAGE (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). After dimerization, ERK generated autophosphorylation at T190(\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Consistently, EGF stimulation increased T190 autophosphorylation in ERK2 WT, but not in ERK2 KR (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eConsistently, depletion of GCN5 significantly enhanced ERK-MEK interaction, which could be rescued with reconstituted GCN5 WT but not GCN5 TA mutant(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ei, j).\u003c/p\u003e \u003cp\u003eTo understand the impact of ERK K231 lactylation on its interaction with MEK, we used AlphaFold2 (version 2.3.1) to probe the MEK-ERK complex. All 25 structures generated by AlphaFold2 consistently indicated the same binding interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ek). Notably, ERK K231 is a part of this interface, in close vicinity to MEK D217, possibly forming hydrogen bonds with D217 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003el, Video 1). We then subjected the MEK-ERK structure to a 100-ns molecular dynamics (MD) simulation, which highlighted the stability of the complex (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e, Video 2), giving us confidence in the reliability of AlphaFold2 predictions. During the simulation, ERK K231 consistently maintained proximity to MEK D217, underscoring its potential significance (Video 2). The MD simulation identified 25 hydrogen bonds or salt bridge pairs between MEK and ERK (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). We used this information to guide the generation of mutations, which were subsequently tested in vivo to assess their impact on the MEK-ERK binding interface (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003em, n; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f). The experimental results from mutagenesis experiments aligned with the predicted interface.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA lactyl group modeled on K231 Nε did not induce clashes with other residues (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003el). ERK K231 lactylation may not disrupt the potential hydrogen bond formation between K231 Nε and MEK D217 Oδ; however, by removing one positive charge from K231, it could reduce the binding affinity. We used the Potential of Mean Force (PMF) method to assess the impact of ERK K231 lactylation on the MEK-ERK complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eo; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The dissociation free energy between wild-type MEK and ERK was 29.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 kcal/mol, which decreased to 27.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52 kcal/mol when ERK was lactylated at K231. This predicted reduction in affinity caused by ERK lactylation aligns with the findings of our in vivo experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, e). Collectively, our results suggest that ERK lactylation promotes its dissociation from MEK, facilitating ERK dimerization and activation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on all these findings, we propose a model that ERK-GCN5 axis facilitate MAPK signaling activation in a lactylation-phosphorylation feedback loop dependent manner. Upon RAS-MAPK activation, MEK activated ERK and subsequently ERK phosphorylates and activate GCN5, which in turn lactylates ERK. Lactylation of ERK promotes its dissociation from MEK and facilitates ERK dimerization and further activation. The lactylation-phosphorylation cascade of ERK-GCN5 accelerates ERK activation, which rapidly amplifies RAS-MAPK signaling in tumor progression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eInhibiting ERK lactylation enhances chemosensitivity\u003c/h2\u003e \u003cp\u003eWe next wanted to clarify the clinical relevance of ERK-GCN5 lactylation axis. We performed an immunohistochemical (IHC) analysis on human NSCLC and colorectal cancer (CRC) samples, as well as matched tumor-adjacent normal tissues (Extended Data Fig.\u0026nbsp;8a, b). Our findings demonstrated that levels of ERK lactylation were significantly higher in tumors compared to adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b). Furthermore, ERK lactylation levels were positively correlated with Ki-67 scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ec), and significantly elevated in RAS-mutant tumors compared to RAS-WT samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Moreover, we found a positive correlation between GCN5 expression and the transcriptional levels of ERK target genes at the in various cancer types (Extended Data Fig.\u0026nbsp;8c). These results provide evidence that ERK lactylation is upregulated in tumors, particularly those with RAS mutations, and suggest that increased GCN5 expression is a potential risk factor positively associated with MAPK signaling.\u003c/p\u003e \u003cp\u003eGiven ERK lactylation plays an important role in promoting tumor progression, targeting ERK lactylation may be an effective approach for tumor therapy. Previous studies have shown that synthetic peptides can act as analog inhibitors for endogenous protein post-translational modifications(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Recently, competing peptides fused with cell-penetrating peptides have emerged as an efficient strategy for inhibiting specific protein PTMs(\u003cspan additionalcitationids=\"CR46 CR47\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Based on the conservative motif surrounding the lactylation site in ERK1 and ERK2, we synthesized five peptides, which all covered the lactylation site, and fused with a cell-penetrating sequence (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). Our screening experiments revealed that the peptide P-3 effectively inhibited ERK lactylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). The peptides P-3(R), the K231 residue replaced with R, was utilized as a negative control. We found that P-3 but not P-3(R) significantly decreased MAPK signaling in a dose-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eg), which led to reduced cancer cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003eh, i). In addition, the P-3 peptide inhibited the cell growth in ERK WT cells but not ERK KR mutant cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ej), suggesting the P-3 peptide inhibits cancer cell growth by blocking ERK lactylation.\u003c/p\u003e \u003cp\u003eWe next utilized patient-derived xenograft (PDX) models of colorectal cancer harboring the KRAS G12V mutation to examine the P3 peptide therapeutic effect \u003cem\u003ein vivo (\u003c/em\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ej). The P3 peptide treatment individually inhibited tumor growth in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ek-n). Moreover, combination treatment with the p3 peptide and 5-Fluorouracil (5-FU) showed synergistic killing effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e7\u003c/span\u003ek-n). In addition, IHC staining of tumor sample showed that the P3 peptide dramatically decreased ERK lactylation, phospho-RSK, and Ki-67 \u003cem\u003ein vivo (\u003c/em\u003eExtended Data Fig.\u0026nbsp;8d). In conclusion, our findings indicate that inhibiting ERK lactylation may be a potential strategy for cancer therapy, especially the tumor with RAS-MAPK overactivation.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003ePost-translational modifications (PTMs) are essential for regulating protein function and modulating many physiological and pathological processes(\u003cem\u003e49\u003c/em\u003e). Eukaryotic proteomes contain hundreds of different types of PTMs, but only a few of them have been extensively studied(\u003cem\u003e50\u003c/em\u003e). However, recent developments in high-resolution mass spectrometry have enabled the detection of low-abundance PTM patterns, expanding our understanding of the PTM landscape(\u003cem\u003e51\u003c/em\u003e). Newly discovered PTM patterns, such as lactylation(\u003cem\u003e9\u003c/em\u003e), succinylation(\u003cem\u003e52\u003c/em\u003e), and crotonylation(\u003cem\u003e53\u003c/em\u003e), have revealed the non-metabolic functions of metabolites. For example, lactate, in addition to serving as fuel and biosynthesis precursor, can also act as a substrate for lysine lactylation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite decades of research on the Warburg effect, the mechanisms by which lactate contributes directly to cancer progression and chemoresistance remain largely unknown\u0026nbsp;(\u003cem\u003e1, 2\u003c/em\u003e). In this study, we have discovered the post-translational modification pattern called lactylation, which directly modifies ERK kinase, a key downstream component of the MAPK pathway. We have shown that lactylation of ERK plays a crucial role in regulating ERK activity, ultimately promoting tumor growth and cancer metabolism reprogramming.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, we have identified GCN5 as the lactylation writer for ERK. GCN5 is a member of the GNAT (GCN5-related N-acetyltransferase) family of acetyltransferases that transfer acetyl groups from acetyl-CoA to lysine residues on histones and other proteins(\u003cem\u003e54\u003c/em\u003e). Our study identified the lactyl-transferase activity of GCN5, which was previously unknown. Under basal conditions, the ERK interacted with the MEK. Upon activation of the MAPK pathway by a stimulus, MEK was phosphorylated and became kinase-active, leading to phosphorylation of ERK on its TEY motif(\u003cem\u003e10\u003c/em\u003e). Phosphorylated ERK dissociated from MEK and formed a dimer with another phosphorylated ERK molecule(\u003cem\u003e31, 33, 35\u003c/em\u003e). The ERK dimer was a kinase-active form that could freely phosphorylate downstream targets(\u003cem\u003e31, 34, 35\u003c/em\u003e). Negative regulation of the MAPK/ERK pathway was mediated by DUSP4/6, which dephosphorylated the TEY motif of ERK(\u003cem\u003e55\u003c/em\u003e). Following cessation of the stimulus, dephosphorylated ERK re-associated with MEK, leading to the return of the system to the basal state(\u003cem\u003e35\u003c/em\u003e). Interesting, ERK lactylation facilitated detachment of ERK from MEK, thereby promoting ERK dimer formation. However, in ERK lactylation deficient KR mutant cells, EGF cannot lead ERK to disassociate from MEK, suggesting ERK lactylation is essential for ERK disassembling from MEK following stimulus. Structure study is needed to further clarify the MEK-ERK dynamic interaction regulated by phosphorylation-lactylation cascade.\u003c/p\u003e\n\u003cp\u003eThe product of the Warburg effect, accumulated lactate in cancer cells may lead to hyper-lactylation and activation of ERK, which accelerates tumor progression. Inhibiting abnormal ERK lactylation in tumors may have mild effect on normal cells, which may reduce side effects compared to inhibitors directly targeting ERK. We designed a cell-penetrating peptide to specifically block ERK lactylation. The peptide inhibited tumor growth in vivo effectively and showed synergistic killing effect together with 5-FU in KRAS G12V mutation cancers, suggesting targeting ERK lactylation may be a prospective strategy for cancer therapy.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we demonstrated that GCN5 function as a lactyl-transferase for ERK. In addition, ERK directly phosphorylates GCN5 and promotes its lactyl-transferase activity for ERK, forming a positive feedback loop that regulates ERK lactylation. Our findings confirm that the Warburg effect induced lactate accumulation in tumors triggers an ERK-GCN5 lactylation-phosphorylation cascade effect, which plays a crucial role in accelerating tumor progression in a positive feedback loop dependent manner. More importantly, we demonstrate that inhibiting ERK lactylation may be a potential strategy for cancer therapy, especially the tumor with RAS-MAPK overactivation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture, Plasmids, Reagents and Antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman Embryonic Kidney 293T cells (HEK293T), human cervical cancer cell Hela, human glioblastoma cell line U87MG, human triple-negative breast cancer cell line cancer cell line SUM159PT were purchased from ATCC. Cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% (v/v) CO2. ERK1, ERK2, MEK1 and GCN5 were subcloned into Puro-Lentiviral Expression Vector (PLVX), Purified E.coli Glycogen Express Vector-X-4T-2 (PEGX-4T-2) or pT7-based expression vector. Mutant plasmids were established by a two-step mutation method. EGFR\u003csup\u003eVIII\u003c/sup\u003e (P26653) and RAS\u003csup\u003eG12D\u003c/sup\u003e (P39721) were obtained from MiaoLingBio, China. The acetyl-transferases plasmids, CBP, P300, GCN5, PCAF, KAT5, KAT8 were gifts from Dr. Jun Huang. And KAT1 (P37556)\u0026nbsp;and\u0026nbsp;KAT7 (P46543) were obtained from MiaoLingBio, China. L-lactate (L1750), LDH inhibitor oxamate (O2751), puromycin were purchased from Sigma-Aldrich.\u003c/p\u003e\n\u003cp\u003eThe antibodies used in this study: anti-Tubulin (Abmart, M20005S,1: 2000), anti-ERK1/2 (Abmart, T40071S,1: 2000), anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit mAb (Cell Signaling Technology, Cat# 4370,1:2000), anti-Flag (Sigma: F1804, 1: 2000), anti-HA (Cell Signaling Technology: no.3724, 1: 2000), anti-L-pan-Kla (PTM Biolab: PTM-1401RM, 1:1000), anti-LDHA (Cell Signaling Technology: no. 2012S, 1: 1000), and anti-ERK1/2-K248/231-lac was generated by PTM Biolab (1:1000). Anti-Phospho-RSK1 p90 (T359+S363) (Abmart, T55344, 1:1000), anti-RSK1 (Abclonal, A4695, 1:1000),anti-MCT1 (Abclonal,A3013, 1:1000) anti-MYC tag (Abmart, M20019,1:2000) anti-GCN5 (Santa Cruz Biotechnology, sc-365321,1:1000), anti-MEK1 (Abclonal, A19565, 1:1000), anti-Phospho-MAPK Substrates Motif (Cell Signaling Technology, #14378, 1: 1000),anti-Phospho-ERK1/2-T188 (badrilla, A010-40AP, 1:500), anti-Histone-H3 (proteintech,17168-1-AP,1:2000), Anti-Acetyl-Histone H3 (Lys9) (PTM Biolab, PTM-156, 1:1000).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA-seq and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from the U87MG using TRIzol® Reagent according to the manufacturer’s instructions (Magen). RNA samples were detected based on the A260/A280 absorbance ratio with a Nanodrop ND-2000 system (Thermo Scientific, USA), and the RIN of RNA was determined by an Agilent Bioanalyzer 4150 system (Agilent Technologies, CA, USA). Only qualified samples will be used for library construction. Paired-end libraries were prepared using a ABclonal mRNA-seq Lib Prep Kit (ABclonal, China) following the manufacturer’s instructions. The mRNA was purified from 1 μg total RNA using oligo (dT) magnetic beads followed by fragmentation carried out using divalent cations at elevated temperatures in ABclonal First Strand Synthesis Reaction Buffer. Subsequently, first-strand cDNAs were synthesized with random hexamer primers and Reverse Transcriptase (RNase H) using mRNA fragments as templates, followed by second-strand cDNA synthesis using DNA polymerase I, RNAseH, buffer, and dNTPs. The synthesized double stranded cDNA fragments were then adapter- ligated for preparation of the paired-end library. Adaptor-ligated cDNA were used for PCR amplification. PCR products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 4150 system. Finally, the library preparations were sequenced on an Illumina Novaseq 6000 (or MGISEQ-T7) and 150 bp paired-end reads were generated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMass spectrometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStable HEK293T cells expressing HA-tagged ERK2 were treated with 25mM lactate for 24 hours before harvesting and lysing the cells. The lysates were subjected to purification using anti-HA-agarose beads. The resulting pellet containing the purified HA-ERK2 protein was separated via SDS-PAGE and visualized by Coomassie blue staining. The HA-ERK2 gel band was excised and subjected to destaining using a solution of 30% ACN/100 mM NH4HCO3 until complete removal of stains from the gel. Then the protein was digested using in-gel digestion method. In brief, the gel band was cut into 1 mm3 particles. The cysteine residues on the protein were reduced with dithiothreitol (10 mM DTT/ 100 mM NH4HCO3) for 30 min at 56°C and alkylated with iodoacetamide (200 mM IAA/100 mM NH4HCO3) in the dark at room temperature for 30 minutes. Subsequently, the protein was digested overnight in 12.5 ng/μL sequence grade trypsin in 25 mM NH4HCO. The tryptic-digested peptides were extracted three times with 60% ACN/0.1% TFA. The peptide extracts were pooled and dried completely by a vacuum centrifuge.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe peptides were analyzed by Easy-nanoLC 1000 system (Thermo Scientific) tandem with Q Exactive mass spectrometer (Thermo Scientific) in a 60 min gradient with positive ion mode. For the full scan, the ions with a mass range of 300 m/z to 1800 m/z were detected by Orbitrap analyzer with a resolution of 70,000 at m/z 100. The automatic gain control (AGC) target was set to 1e6, maximum inject time to 50 ms. For the MS/MS, the top 20 most abundant precursor ions were selected for HCD fragmentation and detected by Orbitrap analyzer with a resolution of 17,500 at m/z 100. The normalized collision energy was 27%. The automatic gain control (AGC) target was set to 1e5, isolation width was set to 1.5 m/z, maximum inject time to 50 ms, and the dynamic exclusion duration was set to 30.0 s.\u003c/p\u003e\n\u003cp\u003eThe mass spectrometry data was converted into MGF format and searched by Mascot search engine (Matrix Science, London, UK; version 2.2) against the nonredundant International Protein Index arabidopsis sequence database v3.85 (released in September 2011; 39679 sequences) from the European Bioinformatics Institute (http://www.ebi.ac.uk/). The search parameters were as following: Precursor ion peaks were searched with an initial mass tolerance of ± 20 ppm, fragment mass tolerance of ± 0.1 Da. Enzyme specificity with trypsin was used. Up to two missed cleavages were allowed. Carbamidomethylation of cysteine was set as a fixed modification. The oxidation of methionine was set as variable modifications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolomics analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eERK2 WT and ERK2 KR SUM159PT cell lines were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C in 5% (v/v) CO2 for 24h. Each group were counted and collected for targeted metabolomics analysis on XploreMET platform (Metabo-Profile, Shanghai, China). The raw metabolomic data generated by UPLC-MS/MS were analyzed using iMAP (Metabo-Pro- file, Shanghai, China) platform for peak identification and quantification of each metabolite. The different biomarkers between the ERK2 WT and ERK2 KR were evaluated using orthogonal partial least squares discriminant analysis (OPLS-DA) by setting the thresholds of variable importance in projection (VIP) to \u0026gt; 1, fold change (FC) \u0026gt; 1 and P \u0026lt; 0.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of Lactate level\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntracellular lactate level was measured by using lactate Colorimetric/Fluorometric assay kit (Abcam ab65331) according to manufacturer’s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation and Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor transient transfection and co-immunoprecipitation assays, plasmids encoding HA-tagged, MYC-tagged, or Flag-tagged constructs were transiently co-transfected into HEK293T cells. The transfected cells were lysed on ice for 25 minutes with NETN buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) supplemented with 1× protease inhibitors. After centrifugation at 12,000 rpm for 10 minutes, the soluble fractions were collected and incubated with anti-HA, anti-MYC, or anti-Flag beads for 2 hours at 4°C (Sigma: E6779 for anti-HA beads, E6654 for anti-MYC beads, F2426 for anti-Flag beads). The beads were then washed three times with NETN buffer prior to incubation. Following incubation, the samples were boiled in 1× SDS loading buffer for 5 minutes and separated by SDS-PAGE. The membranes were blocked with 5% milk in TBST buffer and subsequently probed with the respective antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiotin pull down assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the biotin-lactate pull-down assay, Dynabeads MyOne Streptavidin T1 were first incubated with biotin or biotin-labeled lactate in PBS for an hour at room temperature. Then, they were mixed with cell lysates overnight at 4°C with rotation. Following 3-4 washes, the beads were analyzed by immunoblotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular modeling and molecular dynamics simulations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman ERK protein sequence spanning amino acid 11 to 360, and human MEK protein sequence spanning amino acid 19 to 393 were used as input for AlphaFold2 (version 2.3.1)\u0026nbsp;(\u003cem\u003e56\u003c/em\u003e). From the 25 models generated, the best ranked model was selected for structural analysis and molecular dynamics (MD) simulation.\u003c/p\u003e\n\u003cp\u003eMD simulation were performed using GROMACS (version 2021.4) with the Amber ff14sb force field\u0026nbsp;(\u003cem\u003e57\u003c/em\u003e)\u0026nbsp;and TIP3P water model\u0026nbsp;(\u003cem\u003e58\u003c/em\u003e). The ERK-MEK complex was charge-neutralized with sodium ions and was positioned in a periodic triclinic box with dimension of 10.497\u0026nbsp;´\u0026nbsp;10.776\u0026nbsp;´\u0026nbsp;12.166 nm containing aqueous solution with 150 mM sodium chloride. The system was energy minimized under the steepest descent algorithm and equilibrated under constant volume and temperature (NVT) and constant pressure and temperature (NPT), with heavy atoms restrained in positions. The system temperature was maintained at 300 K with a V-rescale thermostat\u0026nbsp;(\u003cem\u003e59\u003c/em\u003e), while the system pressure was kept at 1 atmosphere using a Berendsen barostat\u0026nbsp;(\u003cem\u003e60\u003c/em\u003e). Bond lengths were constrained using the LINCS algorithm\u0026nbsp;(\u003cem\u003e61\u003c/em\u003e). An unrestrained simulation with leap-frog integration\u0026nbsp;(\u003cem\u003e62\u003c/em\u003e)\u0026nbsp;was performed for 100 ns with a time step of 2 fs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePotential of mean force calculations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe restrained electrostatic potential (RESP )\u0026nbsp;(\u003cem\u003e63\u003c/em\u003e)\u0026nbsp;of lactate-modified lysine (KLA) was calculated using Gaussian 16 at the B3LYP/6-31G* level. A two-step RESP fit was used to assign partial charges to KLA. Subsequently, wild type (WT) MEK/ERK and MEK/ERK with ERK lactylated at K231 were placed in a periodic triclinic box with dimension of 9.444\u0026nbsp;´\u0026nbsp;20.047\u0026nbsp;´\u0026nbsp;10.450 nm and processed and equilibrated as described above. The equilibrated ERK and MEK molecules were then pulled away with a force of 2000 kJ/mol/nm\u003csup\u003e2\u003c/sup\u003e at a speed of 0.01 nm/ps along the y-axis. Twenty-eight and 27 snapshots or windows from the pull trajectories were prepared for the WT and KLA system. Each window was subjected to 10 ns of production with a time step of 2 fs using leap-frog integration. This simulation was repeated 5 times for each system. All the data were \u0026nbsp;analyzed using the weighted histogram analysis method (WHAM)\u0026nbsp;(\u003cem\u003e64\u003c/em\u003e). Error estimation was derived from a 100-step Bootstrap analysis\u0026nbsp;(\u003cem\u003e65\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2-D and soft agar colony formation assay.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the 2D colony formation assay, cells (500–2000) were seeded in triplicate in each well of a 12-well plate. After 24 hours, cells were treated with lactate or oxamate and incubated at 37°C for 10–14 days to allow colony formation. The colonies were stained with Coomassie Brilliant Blue (CBB) and counted.\u003c/p\u003e\n\u003cp\u003eFor the soft agar colony formation assay, the bottom layer of agar (1.2%) containing 1×DMEM was plated first and allowed to solidify at room temperature. And then the upper layer of agar (0.7%) containing cells (500–1000 in 1×DMEM). Cells were cultured for approximately 2–3 weeks and observed under a microscope. The results were normalized to account for differences in plating efficiencies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and purification of recombinant proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacterial expression constructs, specifically pGEX-4T-2 and pT7-based expression vectors, carrying the target genes were transformed into Escherichia coli DH5α cells. Protein expression was induced by adding 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) and incubating the cells at 18°C with 180 rpm agitation overnight. The cells were then resuspended in PBS containing 0.5% Triton X-100 and 2 mM β-mercaptoethanol, followed by ultrasonication to disrupt the cells. The expressed proteins were purified using the GST-tag/His-tag Protein Purification Kit from Beyotime Biotechnology according to the manufacturer's instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro lactylation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant GST-ERK2 proteins were incubated with HA-tagged GCN5 proteins, which were purified from HEK293T cells, in a reaction buffer containing 50 mM HEPES (pH 7.8), 30 mM KCl, 0.25 mM EDTA, 5.0 mM MgCl2, 5.0 mM sodium butyrate, and 2.5 mM DTT, supplemented with 20 μM lactyl-CoA. The reactions were incubated at 30°C for 30 minutes. Subsequently, 5× SDS loading buffer was added to the reaction mixture and boiled for 5 minutes at 100°C. The samples were then separated by SDS-PAGE and subjected to immunoblotting using the specified antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro phosphorylation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe His-GCN5 protein was expressed in BL21 bacteria and purified using a nickel-agarose column as a substrate for ERK2. HA-ERK2 was immunoprecipitated from cells using HA Beads, and the immunoprecipitates were then incubated with the purified His-GCN5 protein for 30 minutes at 30°C in a 20 μl reaction buffer (25 mM Tris-HCl [pH 7.5], 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 1 mM EGTA, 0.4 mM EDTA, 0.5 mM ATP). Following the incubation, the proteins were eluted in SDS-sample buffer and analyzed by immunoblotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments and procedures were carried out in strict accordance with the Guidelines for the Care and Use of Laboratory Animals set by the U.S. National Institutes of Health (National Academies Press; 2011) and were performed following the ethical guideline’s protocols approved by Tongji University school of medicine. For in vivo animal experiments, lactate (100ul of 5M lactate) was intraperitoneally injected three times a week.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Cell Line-Derived Xenograft and Patient-derived xenograft models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe CDX (Cell Line-Derived Xenograft) model (cell lines, 5×10\u003csup\u003e^\u003c/sup\u003e6) and Colon cancer patient-derived xenografts (PDXs) were subcutaneously transplanted into 6-week-old female nu/nu mice. Mice bearing tumors of 100 mm\u003csup\u003e3\u003c/sup\u003e were randomly assigned into each group. Tumor volume was measured and measured as mentioned in tumor xenograft assay.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeptide synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll peptides were synthetized by Guoping Pharmaceutic Inc. (Hehui, China). Synthetic peptides were purified to \u0026gt;98% purity by high-pressure liquid chromatography for both in vitro and in vivo as. The amino acid sequences of peptides in vivo use were in D isoform. For in vitro experiments, peptides were dissolved in PBS to generate a 10 mM stock solution. For in vivo use, peptides were dissolved in PBS and kept on ice until injection. Before injection, the solution was brought to room temperature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe colorectal Cancer and lung cancer tissue microarray with clinic and pathological data was obtained from Shanghai Zhuoli Biotechnology Co., Ltd. (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eError bars represent the SEM or SD, as indicated in the Fig. \u0026nbsp;legends. Statistical significance was determined using Prism version 8.0 software (GraphPad Software, CA, USA). Differences were deemed significant at P \u0026lt; 0.05. Two-way or one-way ANOVA followed by Dunnett’s post-test, Tukey’s multiple-comparisons test, or the Kruskall–Wallis test (for subgroup analyses) was performed for multiple comparisons, and Student’s t test was performed for other experiments to compare mean values. ****P \u0026lt; 0.0001; ***P \u0026lt; 0.001; **P \u0026lt; 0.01; *P \u0026lt; 0.05; and ns, not significant (P \u0026gt; 0.05).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJian Yuan and Chunlong Zhong conceived and designed the study. Bingsong Huang performed most of the experiments and wrote the manuscript. Yuping Chen and Georges Mer. reviewed and edited the manuscript. Gaofeng Cui carried out the molecular modeling and MD simulation studies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor funding, this work is supported by National Natural Science Foundation of China, Grant/Award Number: 81571184, 81771332, 82172820 and 82271406; Fundamental Research for the Central University; Natural Science Foundation of Shanghai, Grant/Award Number: 22ZR1466200 and 22ZR1451200; Pudong Health Committee of Shanghai, Grant/Award Number: PWYgy 2021- 07; Shanghai Pudong New Area Health Commission, Grant/Award Number: PWZxg2022-10; Pudong Health Bureau of Shanghai, Grant/Award Number: PWR12018-07\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eW. H. Koppenol, P. L. Bounds, C. V. 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L. de Groot, D. van der Spoel, g_wham-A Free Weighted Histogram Analysis Implementation Including Robust Error and Autocorrelation Estimates. \u003cem\u003eJournal of Chemical Theory and Computation\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 3713-3720 (2010).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3944681/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3944681/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe Warburg effect released lactate promotes cancer progression, but the mechanisms remain unclear. Here, we found lactate activated MAPK pathway through ERK-lactylation to promote cancer progression. Moreover, we identified the GCN5 as the lactyl-transferase for ERK lactylation. Interestingly, activated ERK phosphorylated GCN5 and promoted GCN5 lactyl-transferase activity for ERK, which formed the positive feedback loop to facilitate lactate-mediated cancer progression. Mechanistically, ERK-K231 lactylation decreased the dissociation energy between ERK and MEK, due to the reduced electrostatic interaction between ERK-K231 and MEK-D217. This facilitated the dissociation of ERK from MEK kinases, which in turn induced ERK dimerization and activation. Hence, we developed a cell-penetrating peptide to specifically inhibit the ERK lactylation, and demonstrated the peptide impaired the tumor growth with KRAS-mutant. Taken together, we define a molecular mechanism that lactate accelerates cancer progression through ERK-GCN5 lactylation-phosphorylation cascade and provide a strategy to target ERK lactylation, especially for RAS-MAPK-driven cancers.\u003c/p\u003e","manuscriptTitle":"Lactate accelerates cancer progression through the ERK-GCN5 lactylation-phosphorylation feedback cascade","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-22 08:17:35","doi":"10.21203/rs.3.rs-3944681/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-chemical-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nchembio","sideBox":"Learn more about [Nature Chemical Biology](http://www.nature.com/nchembio/)","snPcode":"","submissionUrl":"","title":"Nature Chemical Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4cfe84ee-0748-43c7-ae22-de83f5ee81ab","owner":[],"postedDate":"February 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28878081,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":28878082,"name":"Biological sciences/Cell biology/Post-translational modifications"}],"tags":[],"updatedAt":"2026-01-14T08:07:13+00:00","versionOfRecord":{"articleIdentity":"rs-3944681","link":"https://doi.org/10.1038/s41589-025-02107-8","journal":{"identity":"nature-chemical-biology","isVorOnly":false,"title":"Nature Chemical Biology"},"publishedOn":"2026-01-13 05:00:00","publishedOnDateReadable":"January 13th, 2026"},"versionCreatedAt":"2024-02-22 08:17:35","video":"","vorDoi":"10.1038/s41589-025-02107-8","vorDoiUrl":"https://doi.org/10.1038/s41589-025-02107-8","workflowStages":[]},"version":"v1","identity":"rs-3944681","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3944681","identity":"rs-3944681","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}