Antagonistic SUMOylation and Ubiquitination of ACSL4 Control Ferroptosis in Colorectal Cancer

Antagonistic SUMOylation and Ubiquitination of ACSL4 Control Ferroptosis in Colorectal Cancer | 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 Antagonistic SUMOylation and Ubiquitination of ACSL4 Control Ferroptosis in Colorectal Cancer Xuelai Luo, Jiakun Zhang, Shengjie Feng1, Guang Shi, Haokun Zhang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8870994/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract Ferroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation and represents a therapeutic vulnerability in cancer. ACSL4 is a critical regulator of ferroptosis, yet how its activity is post-translationally regulated remains unclear. Here, we identify a SUMO–ubiquitin switch that controls ACSL4 dimerization, enzymatic activity, and ferroptosis. ACSL4 undergoes both SUMOylation and ubiquitination at K661. SUMOylation suppresses ACSL4 dimer formation and activity, whereas ubiquitination promotes dimerization and ferroptotic function. This antagonistic regulation is driven by competitive binding of the SUMO E3 ligase PIAS1 and the ubiquitin E3 ligase CBL to ACSL4. Inhibition of ACSL4 SUMOylation enhances lipid peroxidation, promotes ferroptosis, and suppresses tumor growth. These findings uncover a post-translational mechanism that precisely regulates ACSL4 activity and ferroptosis, and suggest that targeting ACSL4 SUMOylation may enhance the efficacy of ferroptosis-based antitumor therapy. Health sciences/Diseases/Cancer Biological sciences/Cell biology ACSL4 SUMOylation PIAS1 Ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation and is widely implicated in various diseases, particularly cancer [ 1 – 3 ] . Its execution involves inhibition of solute carrier family 7 member 11 (SLC7A11; also known as xCT), decreased glutathione peroxidase 4 (GPX4) activity, disruption of iron homeostasis, and accumulation of phospholipid peroxides mediated by Acyl-CoA synthetase long-chain family member 4 (ACSL4) [ 3 , 4 ] . Ferroptosis not only directly kills tumor cells but also enhances the efficacy of chemotherapy, radiotherapy, and immunotherapy [ 5 – 8 ] . Although immunogenic cell death (ICD) has been widely incorporated into combination treatment strategies in clinical settings and has achieved substantial therapeutic benefits, a considerable proportion of patients still fail to derive clinical benefit [ 9 – 11 ] . Therefore, combining ferroptosis-inducing therapy with immunotherapy represents a promising strategy, and elucidating the regulatory mechanisms underlying tumor cell ferroptosis is of considerable clinical significance. ACSL4 plays a pivotal role in ferroptosis [ 4 ] . As a key enzyme in lipid metabolism, ACSL4 functions as a dimer to activate polyunsaturated fatty acids (PUFAs), converting them into oxidizable phospholipid substrates [ 4 , 12 ] . Under iron overload, reactive oxygen species (ROS) generated via the Fenton reaction amplify lipid peroxidation, ultimately triggering ferroptosis when cellular antioxidant defenses are overwhelmed [ 13 ] . High ACSL4 expression has been associated with improved responses to chemotherapy and immune checkpoint blockade in colorectal cancer [ 8 , 14 – 16 ] . These findings indicate that ACSL4 may serve not only as a prognostic biomarker for combination therapies but also as a potential therapeutic target. Post-translational modifications (PTMs) dynamically modulate protein properties and constitute a central mechanism for signal integration and cell fate determination. SUMOylation, a reversible PTM mediated by small ubiquitin-like modifiers (SUMO), regulates protein localization, interactions, stability, and enzymatic activity [ 17 ] . Certain lysine residues can undergo both SUMOylation and ubiquitination, and competitive SUMOylation can regulate protein activity and interactions. This competitive interplay may be facilitated by the structural homology between SUMO and ubiquitin [ 18 ] . For instance, SUMOylation of IκBα at Lys21 antagonizes ubiquitination at the same residue, thereby modulating NF-κB signaling [ 19 ] . Increasing evidence indicates that dysregulated SUMOylation contributes to tumor progression and immune evasion [ 20 , 21 ] . Previous studies have shown that SUMOylation of PD-L1 in gastric cancer stabilizes the protein by antagonizing ubiquitination, leading to enhanced immune checkpoint signaling and impaired antitumor immunity [ 22 ] . Accordingly, inhibition of SUMOylation has been shown to enhance tumor sensitivity to immunotherapy. However, whether ACSL4 is subject to a similar SUMO–ubiquitin antagonism, and how this modification crosstalk regulates ACSL4 activity and ferroptosis in tumors, remains unknown. In this study, we found that ACSL4 undergoes both SUMOylation and ubiquitination at K661. SUMOylation at this site restrains ACSL4 dimerization and activity, whereas ubiquitination reverses this inhibition. This antagonistic regulation is driven by the differential binding affinity of the SUMO E3 ligase PIAS1 and the ubiquitin E3 ligase CBL for ACSL4. Inhibition of ACSL4 SUMOylation enhances ferroptosis in tumor cells by promoting ACSL4 dimer formation, indicating that modulation of ACSL4 SUMOylation may enhance tumor sensitivity to therapy. Results 1.ACSL4 can undergo SUMOylation at K661. In order to identify the key lysine residues mediating ACSL4 SUMOylation in tumor cells, we used three independent SUMOylation prediction tools (JASSA, SUMOPLOT, and GPS-SUMO). The overlapping results from these algorithms identified two potential SUMOylation sites, K532 and K661(Fig. 1 a). Mammals encode four SUMO isoforms (SUMO1–4), of which SUMO1–3 are well characterized, whereas SUMO4 is less studied and shows a more restricted expression pattern [ 23 ] . Then we determine the SUMOylation status of ACSL4. We enriched ACSL4 from human colorectal cancer HCT116 cells and performed immunoprecipitation (IP). The results showed that ACSL4 could be modified by SUMO1, SUMO2, and SUMO3 (Fig. 1 b, c). Immunofluorescence (IF) analysis further demonstrated that ACSL4 colocalized with SUMO1 and SUMO2/3 in cells (Fig. 1 d). 2-D08, a SUMOylation inhibitor [ 24 ] , suppressed ACSL4 SUMOylation (Fig. 1 e). We then generated lysine-to-arginine mutants at these positions and examined their SUMOylation levels. Both K532R and K661R mutants exhibited reduced SUMOylation, with a more pronounced decrease observed in the K661R mutant (Fig. 1 f-h and Figure S1 a). Interestingly, interrogation of protein modification databases (Figure S1 b), together with our mass spectrometry analysis of ACSL4 modifications (Figure S1 c), revealed that K661 is also a ubiquitination site of ACSL4. Notably, this lysine residue is highly conserved across species (Fig. 1 i–j). These observations prompted us to specifically examine how post-translational modifications at K661 regulate ACSL4 protein function and abundance. 2. ACSL4 K661 SUMOylation regulates ACSL4 dimerization and ferroptosis. We established that K661 of ACSL4 is a SUMOylation site and may serve as a hotspot for additional PTMs. These findings prompted us to further dissect how this residue governs the ACSL4-driven ferroptosis. We generated LoVo and HCT116 cell lines stably expressing either wild-type (WT) ACSL4 or the K661R mutant (Figure S2 a). Cycloheximide (CHX) chase assays revealed no appreciable difference in protein half-life between WT and K661R ACSL4 (Figure S2 b). Previous studies have shown that dimerization of ACSL4 is essential for its enzyme activation. Our results showed that the ACSL4-K661R mutant displayed reduced ACSL4 dimerization compared with ACSL4-WT, indicating that K661 is essential for ACSL4 enzymatic activity (Fig. 2 a, b). To define the role of K661 in ferroptosis, LoVo and HCT116 cells stably expressing ACSL4-WT or K661R were treated with the ferroptosis inducer RSL3 [ 25 ] . CCK-8 assays showed that, compared with WT, expression of K661R significantly increased cell viability (Fig. 2 c). Given that ferroptosis is characterized by the accumulation of lipid peroxides and malondialdehyde (MDA), we next assessed these biochemical hallmarks. Lipid peroxides and MDA levels were markedly reduced in ACSL4-K661R cells relative to WT controls. (Fig. 2 d, e). Because ferroptosis is also associated with collapse of the mitochondrial membrane potential, we assessed the mitochondrial membrane potential using the JC-1 probe [ 26 ] . ACSL4-K661R cells exhibited a higher mitochondrial membrane potential than WT cells, indicating reduced ferroptosis (Fig. 2 f). Given that changes in mitochondrial morphology represent a key ultrastructural feature of ferroptosis, transmission electron microscopy (TEM) was used to assess the mitochondrial status in cells [ 27 ] . Consistently, TEM revealed that mitochondria in ACSL4-K661R cells displayed less shrinkage and cristae disruption than those in WT cells (Fig. 2 g). We further evaluated the effect of the ACSL4 K661R mutation on tumor growth in vivo . ACSL4 knockout (KO), WT and K661R mutant CT26 murine colorectal cancer cell lines were established (Figure S2 c). Subcutaneous tumor models were then generated in mice, and all tumors were treated with intratumoral injections of the ferroptosis inducer RSL3 (100 mg/kg). Tumor growth curves showed that ACSL4-K661R significantly promoted tumor growth compared with ACSL4-WT (Fig. 2 h, i). Consistently, tumors derived from ACSL4-K661R cells exhibited higher weights and reduced MDA levels (Fig. 2 j, k). IHC analysis of 4-hydroxynonenal (4-HNE), a ferroptosis marker, showed markedly decreased 4-HNE staining in ACSL4-K661R tumors (Fig. 2 l). These findings collectively indicate that ACSL4 K661 regulates ferroptosis in tumors by modulating ACSL4 dimer formation. 3. PIAS1 mediates ACSL4 SUMOylation in colorectal cancer To further elucidate the functional role of SUMOylation at the ACSL4-K661 site in regulating ACSL4-mediated ferroptosis, we first sought to identify the SUMO E3 ligase responsible for catalyzing SUMO conjugation at this residue. Using mass spectrometry, we analyzed ACSL4-interacting proteins and cross-referenced these results with candidate SUMO E3 ligases predicted by the STRING protein–protein interaction database. The intersection yielded three potential ligases: PIAS1, TRIM28, and ZBED1 (Fig. 3a). IP assays demonstrated endogenous interactions between ACSL4 and each of these ligases (Figure S3a). Subsequent assessment of the SUMOylation of ACSL4 WT and the K661R mutant in the presence of each candidate ligase demonstrated that PIAS1 specifically catalyzes SUMOylation of ACSL4 at K661 (Fig. 3b). Subsequently, we conducted a more in-depth characterization of the interaction between PIAS1 and ACSL4. As PIAS1 was detected by ACSL4 mass spectrometry (Fig. 3c), co-immunoprecipitation (co-IP) analysis using anti-ACSL4 or anti-PIAS1 antibodies was performed to validate the endogenous interaction between ACSL4 and PIAS1 (Fig. 3d). In vitro pull-down assays showed that ACSL4 interacted with GST–PIAS1 (but not to GST alone) (Fig. 3e). IF analysis revealed clear cytoplasmic colocalization of PIAS1 and ACSL4 (Fig. 3f). The PIAS1 C351S mutant represents a SUMO E3 ligase–deficient variant of PIAS1 [ 28 ] . Whereas PIAS1 WT promotes ACSL4 SUMOylation, the E3 ligase–inactive C351S mutant fails to induce this modification (Fig. 3g). We established the stable PIAS1 knockdown (KD) cell lines, and found PIAS1 KD markedly decreased ACSL4 SUMOylation (Fig. 3h and Figure S3b). Additionally, 2-D08 effectively abrogated the PIAS1-driven enhancement of ACSL4 SUMOylation in a dose-dependent manner, with higher concentrations progressively diminishing SUMOylation (Fig. 3i). We next examined whether PIAS1 influences ACSL4 expression or enzymatic activity. In both HCT116 and LoVo cells, neither ectopic overexpression (OE) nor KD of PIAS1 altered total ACSL4 protein expression (Figure S3c, d). Given that SUMOylation at K661 regulates ACSL4 dimerization, we further assessed the impact of PIAS1 on this process. Notably, PIAS1 KD markedly increased the level of ACSL4 dimers (Fig. 3j). Additionally, 2-D08 restored ACSL4 dimerization in PIAS1 OE cells, further confirming that PIAS1 restrains ACSL4 dimer formation through SUMOylation (Fig. 3k). We further examined the functional consequences of PIAS1 in ferroptosis. We found PIAS1 KD markedly enhanced lipid peroxidation and MDA accumulation while significantly reducing cell viability (Figure S3e-g). TEM and JC-1 staining showed that PIAS1 KD cells exhibited increased mitochondrial shrinkage, disrupted cristae, and elevated membrane density, indicative of ferroptosis (Figure S3h-i). We also generated subcutaneous tumors using PIAS1 KD CT26 cells and administered intratumoral injections of RSL3 at a therapeutic dose (Figure S3j). PIAS1 KD significantly delayed tumor growth, reduced tumor weight, and increased intratumoral MDA and 4-HNE levels (Figure S3k–o). Collectively, these findings demonstrate that PIAS1 acts as a SUMO E3 ligase for ACSL4, suppressing ACSL4 dimer formation and ferroptosis. 4. SUMOylation of ACSL4 at K661 is required for PIAS1-mediated regulation of ferroptosis To determine whether PIAS1 regulates ferroptosis via ACSL4 K661, we generated stable PIAS1 KD CRC cell lines expressing ACSL4-WT or ACSL4-K661R (Figure S4a). PIAS1 OE did not alter ACSL4-WT or ACSL4-K661R expression levels (Figure S4b). However, non-reducing electrophoresis showed that PIAS1 KD markedly increased ACSL4 dimer formation in ACSL4-WT cells but not in ACSL4-K661R cells (Fig. 4a). Consistently, the K661R mutation largely abolished the ferroptosis-promoting effects of PIAS1 KD. The PIAS1 KD–induced increase in MDA levels observed in ACSL4-WT cells was significantly attenuated in ACSL4-K661R cells (Fig. 4b). Similarly, lipid peroxidation, JC-1 staining, and cell viability assays further confirmed that PIAS1 KD enhanced ferroptosis in ACSL4-WT cells but not in ACSL4-K661R cells (Fig. 4c and Figure S4c, d). TEM further revealed increased mitochondrial shrinkage and cristae loss following PIAS1 KD in ACSL4-WT cells, whereas these changes were minimal in ACSL4-K661R cells (Figure S4e). To assess in vivo relevance, subcutaneous tumor models were established using ACSL4-WT or ACSL4-K661R CT26 cells with or without PIAS1 KD (Fig. 4d), followed by intratumoral RSL3 treatment. PIAS1 KD significantly suppressed tumor growth in ACSL4-WT tumors, whereas this effect was largely reversed in ACSL4-K661R tumors (Fig. 4e–g). IHC analysis showed increased 4-HNE staining and elevated MDA levels in PIAS1-KD ACSL4-WT tumors, but not in ACSL4-K661R tumors (Fig. 4h, i). 5. Inhibition of ACSL4 SUMOylation enhances ACSL4 ubiquitination Because SUMOylation typically antagonizes ubiquitination and ACSL4 K661 may undergo ubiquitin modification, we sought to evaluate the relationship between ACSL4 SUMOylation and ubiquitination. We first collected CRC specimens from patients who were treated with neoadjuvant immunotherapy. IP of ACSL4 revealed that ACSL4 SUMOylation levels were markedly lower in the treatment-sensitive group than in the resistant group. Conversely, ACSL4 ubiquitination was significantly higher in the sensitive group. These findings suggest that the antagonistic interplay between ACSL4 SUMOylation and ubiquitination may shape CRC responsiveness to immunotherapy (Fig. 5a). Based on our mass spectrometry analysis, K661 was identified as a potential ubiquitination site (Figure S5a, b), and the K661R mutation markedly reduced ACSL4 ubiquitination (Figure S5c). Moreover, we also found that K661 was specifically modified by K63-linked ubiquitin chains (Figure S5d). Collectively, these results indicate that ACSL4 can undergo both SUMOylation and ubiquitination at the conserved K661 site. Importantly, the ACSL4 K661R mutant exhibited diminished SUMOylation and ubiquitination, accompanied by a marked suppression of enzymatic activity. Conversely, inhibition of ACSL4 SUMOylation through 2-D08 treatment or PIAS1 KD restored its enzymatic activity. These findings support a model in which K661 functions as a regulatory switch: Ubiquitination of ACSL4 at K661 promotes its dimer formation, whereas SUMOylation at the same residue maintains ACSL4 in an enzymatically repressed state. The K661R mutation disrupts this regulatory axis and impairs dimerization. Subsequently, we performed a series of ubiquitination assays to test this hypothesis. 2-D08 markedly enhanced ACSL4 ubiquitination (Fig. 5b), whereas PIAS1 OE reduced ACSL4 ubiquitination (Figure S5e). In addition, the SUMO E3 ligase–deficient PIAS1 C351S mutant failed to suppress ACSL4 ubiquitination, indicating that PIAS1-mediated SUMOylation antagonizes ACSL4 ubiquitination (Figure S5g). Notably, PIAS1 OE had minimal impact on the ubiquitination of the ACSL4 K661R mutant (Figure S5f), suggesting that PIAS1 primarily inhibits ubiquitination at K661. Furthermore, PIAS1 mainly suppressed K63-linked, but not K48-linked, ubiquitination of ACSL4 (Fig. 5c). We next sought to identify the E3 ubiquitin ligase responsible for promoting ACSL4 K661 ubiquitination. Based on mass spectrometry data, several potential ACSL4-interacting E3 ligases were identified, including CBL, PJA1, TRIM9, TRIM25, TRIM33, CBLL1, ARIH1, RNF25, and RBBP6. Plasmids encoding these ligases were constructed and individually overexpressed in HCT116 cells. Co-IP confirmed that these candidate ligases interacted with endogenous ACSL4 (Fig. 5d). We individually knocked down these E3 ubiquitin ligases using siRNAs and observed that depletion of CBL, TRIM25, CBLL1, or PJA1 did not significantly alter ACSL4 protein abundance (Figure S5h, i). Next, we examined which E3 ligase specifically mediates ubiquitination at K661. Co-transfection assays revealed that CBL, TRIM25, CBLL1, and PJA1 each enhanced ACSL4-WT ubiquitination to varying extents; however, only CBL overexpression failed to increase ubiquitination of the K661R mutant (Fig. 5e). In vitro pull-down assays further validated a direct interaction between ACSL4 and CBL (Figure S5j). Additionally, co-IP revealed that the K661R mutation significantly impaired ACSL4–CBL binding (Figure S5k). Furthermore, IF analysis revealed reduced ACSL4–CBL colocalization in K661R mutant cells (Fig. 5f, g). These findings support a model in which SUMOylation and ubiquitination at ACSL4 K661 are mutually antagonistic, and identify CBL as the E3 ubiquitin ligase that catalyzes ACSL4 K661 ubiquitination. 6. CBL promotes ACSL4 dimerization to facilitate ferroptosis After identifying CBL as the E3 ligase that mediates ACSL4 K661 ubiquitination, we further investigated its regulatory role in CRC ferroptosis. Ubiquitination assays confirmed that CBL selectively promotes K63-linked ubiquitination of ACSL4, with minimal impact on K48-linked chains (Figure S6a, b). Importantly, the CBL-mediated increase in K63-linked ubiquitination was abolished in the ACSL4-K661R mutant (Fig. 6a). In addition, 2-D08 further enhanced CBL-mediated ubiquitination of ACSL4 (Fig. 6b). Given that K63-linked ubiquitination correlates with ACSL4 dimerization, we next examined whether CBL regulates ACSL4 dimer formation. In HCT116 and LoVo cells stably expressing shNC or shPIAS1, CBL was silenced using siRNA (Figure S6c). Non-reducing immunoblotting demonstrated that CBL KD significantly reversed the PIAS1 KD–induced increase in ACSL4 dimer formation (Fig. 6c). We therefore propose that CBL promotes ACSL4 dimerization via K63-linked ubiquitination. Subsequently, we assessed whether CBL is involved in PIAS1-mediated regulation of ferroptosis. The results showed that CBL KD significantly attenuated PIAS1 KD–induced ferroptosis (Fig. 6d, e). JC-1 staining, MDA quantification and cell viability assays corroborated these findings (Fig. 6f, g). Moreover, TEM revealed that CBL KD attenuated mitochondrial damage (Fig. 6h). Collectively, these data indicate that CBL is required for PIAS1 KD–induced ferroptosis. 7. PIAS1 competes with CBL for ACSL4 binding ACSL4 SUMOylation and ubiquitination at K661 function antagonistically, with SUMOylation restraining and ubiquitination promoting ACSL4 activity; accordingly, PIAS1 suppresses, whereas CBL enhances, its enzymatic function. These findings led us to hypothesize that the antagonism between SUMOylation and ubiquitination, as well as the preferential occurrence of SUMOylation, arises from competitive binding of the two E3 ligases to ACSL4. To test this hypothesis, we first performed co-IP assays. The results showed that increased PIAS1 expression weakened the interaction between ACSL4 and CBL (Fig. 7a), whereas reduced PIAS1 levels enhanced their association (Fig. 7b). We then carried out ubiquitination and SUMOylation assays. PIAS1 OE impaired CBL-mediated ACSL4 ubiquitination (Fig. 7c), whereas CBL OE had minimal impact on PIAS1-driven ACSL4 SUMOylation (Figure S7a). Next, we examined ACSL4–CBL co-localization in PIAS1-KD cells and found that PIAS1 KD markedly increased their colocalization (Fig. 7d, e and Figure S7b). We subsequently performed molecular-docking simulations to model PIAS1–ACSL4 and CBL–ACSL4 interactions (Fig. 7f, g), which predicted a greater number of hydrogen bonds at the PIAS1–ACSL4 interface (Figure S7c). Sequential BLI analyses were performed to examine the binding hierarchy between ACSL4, PIAS1, and CBL. When CBL was first allowed to associate with immobilized ACSL4, subsequent addition of PIAS1 led to a further increase in the binding signal, indicating that PIAS1 can efficiently bind ACSL4 even in the presence of pre-bound CBL. In contrast, when PIAS1 was pre-bound to ACSL4, subsequent addition of CBL resulted in only a minimal increase in the binding response (Fig. 7h, i). These findings indicate that PIAS1 preferentially associates with ACSL4 and thereby restricts the access of CBL, providing a mechanistic explanation for why SUMOylation predominates over ubiquitination at this residue, given the stronger binding affinity of PIAS1. From a therapeutic perspective, this regulatory hierarchy further implies that targeting ACSL4 SUMOylation may represent a potential strategy to enhance the sensitivity of colorectal tumors to neoadjuvant therapy (Fig. 7j). 8. Targeting SUMOylation sensitizes CRC to chemoimmunotherapy We previously demonstrated that CRC sensitivity to neoadjuvant immunotherapy is associated with the SUMOylation and ubiquitination status of ACSL4 (Fig. 5a). We therefore assessed PIAS1 and CBL expression, as well as ferroptosis levels and immune infiltration, in clinical specimens obtained after neoadjuvant immunotherapy. IHC analysis revealed that patients who responded better to neoadjuvant immunotherapy exhibited higher levels of lipid peroxidation and stronger CD8⁺ T-cell infiltration and function. Notably, compared with treatment-insensitive patients—those with stable disease (SD) or progressive disease (PD)—treatment-sensitive patients who achieved a complete response (CR) or partial response (PR) showed lower PIAS1 and higher CBL expression (Fig. 8a–c and Figure S8a, b). Among the 11 immunotherapy-sensitive cases, PIAS1 expression negatively correlated with ferroptosis and CD8⁺ T-cell infiltration and function, whereas CBL displayed the opposite trend (Fig. 8d, e). Furthermore, Kaplan–Meier analysis of the TCGA cohort revealed that high PIAS1 expression predicts poorer overall survival in CRC (Figure S8c). Clinically, the XELOX regimen combined with immune-checkpoint inhibitors has become a standard neoadjuvant approach for resectable CRC [ 29 , 30 ] . Additionally, the SUMOylation inhibitor 2-D08 has been reported to enhance antitumor T-cell responses within the tumor microenvironment [ 24 ] . Given the limited response rates of current combination regimens, we next tested whether 2‑D08 could increase sensitivity to regimens that include immune‑checkpoint blockade. Using CT26 subcutaneous tumor model, we evaluated the efficacy of XELOX plus anti-PD-1 with or without 2-D08. In vivo , 2-D08 monotherapy produced only modest reductions in tumor volume and weight, with all CT26 tumors exhibiting PD. XELOX combined with anti-PD-1 improved tumor control, resulting in 80% PD and 20% SD. Strikingly, the triple combination markedly suppressed tumor growth and induced tumor regression, achieving PR in all treated mice (Fig. 8g, h and Figure S8d, e). Analysis of tumor tissues showed that 2-D08 enhanced intratumoral ferroptosis and enhanced CD8⁺ T-cell infiltration, accompanied by higher proportions of IFN-γ⁺ and granzyme B⁺ CD8⁺ T cells. Importantly, the triple regimen further enhanced intratumoral ferroptosis and robustly increased CD8⁺ T-cell infiltration, expanding IFN-γ⁺ and granzyme B⁺ CD8⁺ T-cell subsets, thereby synergistically enhancing antitumor immunity (Fig. 8i, j and Figure S8f, g). Together, these findings suggest that targeting ACSL4 SUMOylation may represent a promising strategy to improve the efficacy of neoadjuvant therapy in colorectal cancer. Discussion Inducing ferroptosis has emerged as a promising strategy to overcome the survival advantages of drug-resistant tumor cells and enhance therapeutic efficacy [ 31 , 32 ] . However, some tumor cells can evade ferroptosis and acquire resistant phenotypes by modulating key ferroptosis-related molecules such as GPX4, ferroptosis suppressor protein 1 (FSP1), and ACSL4 [ 6 , 8 , 33 ] . PUFAs, owing to their abundance of double bonds, are highly susceptible to oxidative damage triggered by reactive oxygen species (ROS) generated through the Fenton reaction [ 1 , 25 , 34 ] . The lethal accumulation of lipid peroxides represents a hallmark and critical execution step of ferroptosis [ 35 ] . As a major driver of ferroptosis, ACSL4 plays a central role in lipid metabolic remodeling [ 4 ] . Accumulation of PUFAs may enhance membrane fluidity, thereby weakening PD-L1–PD-1 interactions and increasing the sensitivity of non-small cell lung cancer to immune checkpoint inhibitors (ICIs) [ 36 ] . Therefore, targeting ACSL4 to modulate ferroptosis sensitivity may represent an effective approach to improving immunotherapy responses in cancer. PTMs profoundly impact protein activity, stability, subcellular localization, and protein–protein interactions [ 37 , 38 ] . Previous studies have shown that PKCβII activates ACSL4 through phosphorylation to promote PUFA-phospholipid synthesis and enhance ferroptosis sensitivity [ 39 ] , while CARM1 modulates ACSL4 expression through arginine methylation to augment responses to immunotherapy [ 40 ] . In recent years, SUMOylation has been recognized as a key regulatory mechanism in cancer therapy and cell death, influencing protein stability, transcriptional activity, subcellular localization, and DNA damage repair [ 17 , 20 , 23 ] . SUMOylation of ACSL4 at K532 has been implicated in regulating neuronal ferroptosis after spinal cord injury [ 41 ] , whereas our study identifies K661 as the major SUMOylation site in colorectal cancer that controls ACSL4 enzymatic activity, underscoring the disease-specific regulation of ACSL4 SUMOylation. We further showed that PIAS1-mediated SUMOylation at K661 significantly suppresses ACSL4 activity and inhibits CBL-mediated ACSL4 dimerization. These findings expand the known PTM landscape of ACSL4 and reveal a previously unrecognized mechanism whereby SUMOylation regulates ferroptosis. SUMOylation and ubiquitination often exhibit antagonistic crosstalk [ 22 , 42 ] . We observed that PIAS1 KD led to decreased ACSL4 SUMOylation, accompanied by markedly increased K661 ubiquitination and enhanced dimer formation. Conversely, the K661R ACSL4 mutant showed reduced dimerization capacity and profoundly impaired enzymatic activity. Based on these findings, we propose that SUMOylation may suppress ubiquitination at K661 through a competitive mechanism. Further investigation identified CBL as the E3 ligase responsible for catalyzing ACSL4 K661 ubiquitination and promoting dimerization. Our data indicate that PIAS1 and CBL competitively bind to ACSL4, thereby determining the prioritization of SUMO versus ubiquitin modification. Given the higher binding affinity of PIAS1 for ACSL4, PIAS1 more efficiently recruits the SUMO conjugation machinery, allowing SUMO to occupy K661 and block CBL-mediated ubiquitination through steric hindrance or conformational alteration. Similar competitive mechanisms have been described in other systems, such as SUMO/ubiquitin competition at IκBα K21, where SUMOylation prevents ubiquitin-dependent degradation and suppresses NF-κB activation [ 19 ] . Although the full-length structure of ACSL4 has not yet been resolved, our molecular docking analysis suggests that the stronger PIAS1–ACSL4 interaction may result from the formation of more hydrogen bonds. Collectively, these findings deepen our understanding of how competitive PTMs cooperate or antagonize each other to fine-tune protein function. Immunotherapy has become one of the most transformative advances in cancer treatment [ 9 , 10 ] ; however, a substantial proportion of patients fail to respond, underscoring the urgent need to enhance immunotherapy sensitivity [ 11 ] . Recent studies have suggested that ferroptosis may synergize with immunotherapy to potentiate antitumor immunity. Interferon-γ (IFN-γ) has been shown to synergize with arachidonic acid (AA) to induce ACSL4-dependent ferroptosis in melanoma cells [ 8 ] . Meanwhile, SUMOylation has been shown to reshape the tumor immune microenvironment and impair immune surveillance, thereby promoting immune evasion [ 24 , 43 ] . In addition to suppressing immune cell function, tumor-intrinsic SUMOylation directly contributes to immune escape [ 22 , 44 ] . Thus, targeting SUMOylation may represent a promising approach to improving immunotherapy responses. Indeed, previous studies have shown that inhibition of the SUMO E2 enzyme UBC9 by 2-D08 enhances antitumor immunity [ 24 ] . Given the differential ACSL4 SUMOylation we observed in colorectal cancer samples receiving neoadjuvant XELOX plus ICIs, we further explored whether 2-D08 could enhance responses to combined therapies including anti-PD-1. Our findings demonstrate that inhibition of ACSL4 SUMOylation promotes ferroptosis both in vitro and in vivo . Importantly, 2-D08 synergized with XELOX and anti-PD-1 therapy to amplify ferroptosis and markedly enhance antitumor immune responses. In summary, we identified PIAS1-mediated SUMOylation of ACSL4 at K661 as a key suppressive mechanism of ferroptosis. Combination therapy involving SUMOylation inhibitors, chemotherapy, and immune checkpoint blockade effectively suppressed tumor progression. These findings not only uncover a novel mechanism of ferroptosis regulation but also provide a potential therapeutic strategy for targeting ferroptosis- and SUMOylation-related pathways in colorectal cancer. Materials and Methods Collection of clinical specimens This study was approved by the Ethics Committee of Tongji Hospital (TJ-IRB20220723). Clinical specimens were obtained from the Department of Gastrointestinal Surgery, Tongji Hospital. Written informed consent was obtained from all patients prior to surgery. Routine imaging examinations, such as computed tomography, were performed to evaluate the efficacy of preoperative chemotherapy. According to RECIST version 1.1 [ 45 ] , patients who achieved CR or PR were classified as immunotherapy-sensitive, whereas those with SD or PD were classified as immunotherapy-insensitive. Demographic information, including age and sex, is provided in Table S1 (Supplementary Information). Cell lines, antibodies, and reagents The colorectal cancer cell lines LoVo, HCT116, and CT26 were obtained from the American Type Culture Collection (ATCC). All cell lines were authenticated by short tandem repeat (STR) profiling conducted by Procell. Mycoplasma contamination was routinely monitored every three months using a PCR-based Mycoplasma Detection Kit (Cat. No. C0301S, InvivoGen, Bayogene). Cells were maintained at 37°C in a humidified incubator with 5% CO₂ and cultured in Gibco DMEM, McCoy’s 5A, or RPMI-1640 medium, each supplemented with 10% fetal bovine serum (Cat. No. SV30160.03, HyClone) and 1% penicillin–streptomycin solution. The following antibodies were used in this study: Anti-ACSL4 (ab155282), anti-PIAS1 (ab109388), anti-Ubiquitin (ab134953), anti-TRIM9 (ab300515), anti-TRIM25 (ab167154), anti-TRIM33 (ab300146), and anti-RNF25 (ab140514) were purchased from Abcam. CBL (SAB4503444) and PJA1 (HPA000595) were obtained from Sigma‒Aldrich. CBLL1 (sc-517157), ARIH1 (sc-390763), and RNF25 (sc-398749) were purchased from Santa Cruz Biotechnology. SUMO1 (#4930), SUMO2/3 (#4971), TRIM28 (#4124), GAPDH (#2118), HA-tag (#3724), and Myc-tag (#2276) antibodies were obtained from Cell Signaling Technology. ZBED1 (A6792) and RBBP6 (A14776) were purchased from Abclonal. Secondary antibodies, including DyLight 488 goat anti-mouse IgG (#A23210), DyLight 488 goat anti-rabbit IgG (#A23220), DyLight 549 goat anti-mouse IgG (#A23310), and DyLight 549 goat anti-rabbit IgG (#A23320), were purchased from Abbkine. For flow cytometry, the Zombie NIR Fixable Viability Kit (#423105), BV510-CD45 (#103137), BV650-CD8 (#810742), BV785-CD3 (#100232), PC5.5-GZMB (#372212), and PE-IFN-γ (#505808) were obtained from BioLegend. Reagents including 2-D08 (HY-114166), oxaliplatin (HY-17371), MG132 (HY-13259), and cycloheximide (CHX, HY-12320) were purchased from MedChem Express. The sources of all remaining reagents are provided in the respective sections. Transient transfection of siRNA and plasmids SiRNAs targeting PIAS1, CBL, PJA1, TRIM9, TRIM25, TRIM33, CBLL1, RNF25, ARIH1, and RBBP6 were designed and synthesized by RiboBio (Guangzhou, China), and their sequences are provided in Supplementary Table S2 . Expression plasmids encoding pcDNA3.1-Flag-PIAS1, pcDNA3.1-Flag-TRIM28, pcDNA3.1-Flag-ZBED1, pcDNA3.1-HA-ACSL4, pcDNA3.1-Myc-Ubiquitin, pcDNA3.1-His-SUMO1, pcDNA3.1-His-SUMO2, pcDNA3.1-His-SUMO3, pcDNA3.1-Flag-PJA1, pcDNA3.1-Flag-TRIM9, pcDNA3.1-Flag-TRIM25, pcDNA3.1-Flag-TRIM33, pcDNA3.1-Flag-CBLL1, pcDNA3.1-Flag-RNF25, pcDNA3.1-Flag-CBL, pcDNA3.1-Flag-RBBP6, and pcDNA3.1-Flag-ARIH1 were purchased from AUGCT ( http://www.augct.com ). In addition, the pLVX-ACSL4 construct was generated by inserting the corresponding DNA fragment into the indicated vector. All site-directed mutants were generated using the Mut Express II Fast Mutagenesis Kit V2 (C214-01, Vazyme). Furthermore, ACSL4 deletion mutants were constructed based on the pcDNA3.1-HA-ACSL4 plasmid. The primer sequences used for ACSL4 point mutations and deletion mutants are listed in Supplementary Table S3. All plasmids were sequence-verified prior to use. Syngeneic tumor model All animal experiments were approved by the Institutional Animal Care and Use Committee of Tongji Hospital. BALB/c mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. CT26 cells (2 × 10⁵) were subcutaneously injected into 6-week-old female BALB/c mice. Tumor volume was measured at designated time points and calculated using the formula: 0.5 × L × D² (L: long diameter; D: short diameter). After euthanasia, subcutaneous tumors were weighed, and the tissues were collected for further analysis. In some experiments, mice were treated with RSL3 (100 mg/kg, intratumoral injection, twice per week for 2 weeks). Mice were euthanized 20 days after treatment initiation, and tumors were harvested for subsequent analyses. In combination therapy experiments, mice were treated according to a modified XELOX-based regimen combined with immunotherapy. Oxaliplatin (10 mg/kg) was administered via intraperitoneal injection on day 1. Capecitabine (350 mg/kg) was given by oral gavage once daily from day 1 to day 14. The anti–PD-1 antibody (100 µg/mouse) was administered intraperitoneally every 3 days throughout the treatment period. Where indicated, 2-D08 (10 mg/kg) was delivered via intratumoral injection every 3 days. After 14 days of treatment, mice were euthanized, and tumors were collected for subsequent analyses. For individual mice, PD was defined as 25% increase in the initial volume at the end of treatment. SD was defined as < 50% regression from the initial volume during the treatment and ≤ 25% increase in the initial volume at the end of the treatment. PR was defined as a tumor volume regression ≥ 50% for at least one time point but with measurable tumor (≥ 0.10 cm3). Preparation of single-cell suspension from subcutaneous tumors Subcutaneous tumors were minced into small pieces using scissors and transferred into 3 mL serum-free RPMI-1640 medium containing type IV collagenase (50 µL, 25 mg/mL; Cat. No. V900893, Sigma-Aldrich), hyaluronidase (50 µL, 32 mg/mL; Cat. No. H3506, Sigma-Aldrich), and DNase I (25 µL, 10 mg/mL; Cat. No. 10104159001, Roche). Tumor tissue was digested by shaking at 37°C for 1 hour at 150 r.p.m. After complete digestion, 7 mL of serum-free RPMI-1640 medium was added to dilute the enzymes. The cell suspension was then filtered and centrifuged, followed by treatment with 1 mL of ACK lysis buffer for 1 minute to lyse red blood cells, and subsequently neutralized. Finally, the cells were resuspended and kept on ice until further staining experiments. Western Blotting, IP, and IF Analysis For immunoblot assay, CRC tissues and cells were lysed with NP40 buffer containing protease inhibitors and phosphatase inhibitors. Protein quantification was performed by the BCA Protein Assay Kit (23225, Thermo Fisher Scientific). After incubation at 95°C for 10 min, equal amounts of protein were separated by a 10% SDS-PAGE gel and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). After being blocked by 5% skim milk for 1 h, the membranes were incubated with specific antibodies overnight at 4°C. Subsequently, the membranes were incubated with secondary antibodies for 1 h, followed by enhanced chemiluminescence detection. To examine ACSL4 dimerization, Western blot analysis was performed under non-reducing conditions using sample buffer without β-mercaptoethanol or dithiothreitol. For IP, whole-cell lysates were incubated and rotated overnight with anti-Flag, anti-HA beads, or Protein A/G beads conjugated with specific antibodies. Beads were washed four times with lysis buffer and followed by immunoblot assays. For IF staining, the indicated cells were cultured on round cell slides and fixed with 4% paraformaldehyde for 20 min. After permeabilization with 0.3% Triton X-100, the samples were blocked with 2% bovine serum albumin, and then stained with specific antibodies overnight at 4°C. Subsequently, the samples were incubated with corresponding fluorescently labeled secondary antibodies (Dylight 488, Goat Anti-Rabbit IgG, and Dylight 549, Goat Anti-Mouse IgG) for 2 h at room temperature, and followed by staining with 4,6-diamidino-2-phenylindole (DAPI). The images were obtained by a confocal fluorescence microscope (Zeiss) to assess colocalization. Images were processed using ImageJ software. In Vitro Binding Assays Recombinant human ACSL4 (ABIN7232834, antibodies-online), CBL (TP314069, OriGene), and PIAS1 (H00008554-P01, Abnova) proteins were used for in vitro binding assays. Purified His-tagged ACSL4 was incubated with GST or GST-PIAS1, followed by rotation with anti-GST magnetic beads at 4°C overnight. Likewise, His-ACSL4 was incubated with Flag-tagged CBL and rotated with anti-Flag magnetic beads at 4°C overnight. The beads were washed four times with lysis buffer and subsequently subjected to immunoblot analysis. Cell Viability Assay Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Cat. No. HY-K0301, MedChemExpress). LoVo and HCT116 cells were seeded into 96-well plates at a density of 5 × 10⁴ cells per well and treated with RSL3. At the indicated time points, 10 µL of CCK-8 solution was added to each well and incubated for 5 h. Absorbance was then measured at 450 nm. Cell viability was calculated by normalizing to the viability of the negative control group and presented as a percentage. Ferroptosis detection: Lipid peroxidation was assessed using the fluorescent probe C11 BODIPY 581/591 (Abclonal, Cat. No. RM02821). The probe was dissolved in DMSO to prepare a 10 mM stock solution and stored at − 20°C protected from light. After treatment, cells were incubated with 10 µM C11 BODIPY 581/591 at 37°C for 30 min in the dark, followed by two washes with PBS to remove excess dye. Fluorescence was analyzed by flow cytometry with excitation at 488 nm and emission collected at 505–550 nm. Lipid peroxidation levels were quantified as mean fluorescence intensity. MDA measurement Cells were collected after PBS washing, and tissue samples were homogenized. Supernatants were obtained after centrifugation at 4°C. MDA levels were then measured using a commercial assay kit (Beyotime, Cat. No. S0131S) according to the manufacturer’s instructions. Absorbance was recorded at the specified wavelength, and concentrations were calculated from a standard curve and normalized to protein concentration or tissue weight. TEM Cells were treated with 2.5 µM RSL3 for 8 h, collected, and fixed in 2.5% glutaraldehyde at room temperature before storage at 4°C. Samples were subsequently post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, embedded in epoxy resin, and sectioned into ultrathin slices. Sections were stained with uranyl acetate and lead citrate and examined by transmission electron microscopy to assess mitochondrial ultrastructure. Mitochondrial membrane potential assay Mitochondrial membrane potential was measured using the JC-1 assay kit (Beyotime, Cat. No. C2006). Cells were stained according to the manufacturer’s protocol, and fluorescence was analyzed by flow cytometry. IHC IHC analysis was performed on mouse subcutaneous tumors and CRC tissues from patients. Paraffin-embedded CRC specimens were processed for IHC according to the manufacturer’s instructions. The staining results were independently evaluated by two experienced gastrointestinal pathologists. The IHC score was determined based on both the staining extent and intensity. The percentage of positively stained tumor cells was graded as 0 ( 75%). The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The final IHC score was calculated by multiplying the percentage score by the intensity score, yielding a total score ranging from 0 to 12. Samples with an IHC score ≥ 6 were defined as high expression, whereas those with a score < 6 were classified as low expression. Biolayer Interferometry (BLI) Assay BLI experiments were performed using an Octet system (ForteBio). Recombinant ACSL4 protein was immobilized on biosensors. Prior to the assay, sensors were equilibrated in PBST buffer (PBS containing 0.01% Tween-20) for 15 min. After baseline acquisition (60–120 s), purified CBL and/or PIAS1 proteins were sequentially added at the indicated concentrations to monitor association for 300–600 s, followed by dissociation in PBST buffer for 30 s. Binding data were analyzed using Octet Data Analysis software according to the manufacturer’s instructions. Mass spectrometry analysis The detailed procedures for mass spectrometry analysis have been reported previously. ACSL4 protein samples were enriched by IP and separated by SDS–PAGE. Gel bands of interest were excised and submitted to the HIT Center for Life Sciences at Harbin Institute of Technology for mass spectrometric analysis. Protein identification was performed with technical support from the HIT Center for Life Sciences. Peptide mapping and identification of potential ubiquitination (K) sites were carried out using the ptmRS node in Proteome Discoverer software. Statistical analysis: All statistical analyses were performed using GraphPad Prism 8.0 and Origin 2022. Statistical significance between groups was determined using two-tailed Student’s t-tests, one-way ANOVA followed by Tukey’s post hoc test, or two-way ANOVA followed by Tukey’s post hoc test, as appropriate. Categorical variables were analyzed using the chi-square test. Correlation analyses were conducted using Spearman’s rank correlation test. Overall survival was evaluated using the Kaplan–Meier method, and differences were assessed by the log-rank test. Differences were considered statistically significant at P values less than 0.05. * p < 0.05, ** p < 0.01, and *** p 0.05. Declarations Conflict-of-interest disclosure : The authors declare that there are no conflicts of interest. Competing interests The authors declare no competing interests. Author Contributions XL conceived and designed the experiments. JZ performed most of the experiments. SF performed animal experiments. GS, HZ and WZ collected biological samples and analyzed the data. Other authors provided suggestions for several experiments. JZ and XL organized and analyzed the data and wrote the manuscript. Acknowledgments We appreciate the support from members of Guihua Wang’s laboratory and Junbo Hu’s laboratory. We also acknowledge equipment support from the Experimental Medicine Center of Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology. This work is supported by the National Natural Science Foundation of China (No. 82472829); Huanggang Innovation and Development Joint Fund Project of Hubei Provincial Natural Science Foundation (No. 2025AFD335); The Joint supported by Hubei Provincial Natural Science Foundation and United Imaging of China (No. 2025AFD848) Authors’ Disclosures No disclosures were reported. References DIXON S J, LEMBERG K M, LAMPRECHT M R, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death [J]. Cell, 2012, 149(5): 1060-72. 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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-8870994","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":599986929,"identity":"e18f7d1e-461b-4a5d-95f8-02c4dd2a1062","order_by":0,"name":"Xuelai 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Technology","correspondingAuthor":false,"prefix":"","firstName":"Junbo","middleName":"","lastName":"Hu","suffix":""}],"badges":[],"createdAt":"2026-02-13 11:00:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8870994/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8870994/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104063704,"identity":"efc46564-9e9e-4300-bd72-6a23ba1f56df","added_by":"auto","created_at":"2026-03-06 10:11:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":485801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACSL4 undergoes SUMOylation at lysine 661. \u003c/strong\u003e(a) Schematic illustration of the screening strategy used to identify candidate E3 SUMO ligases interacting with ACSL4. (b) Endogenous ACSL4 was IP from HCT116 cells using an anti-ACSL4 antibody, followed by western blot analysis to detect ACSL4 SUMOylation. (c) HCT116 cells were transfected with the indicated plasmids. 48 h after transfection, cell lysates were subjected to IP using an anti-HA antibody, and ACSL4 SUMOylation was analyzed by western blotting. (d) IF staining of ACSL4 (red) and SUMO1 or SUMO2/3 (green) in HCT116 cells. Nuclei were counterstained with DAPI (blue). Plot profile qualitative analysis is shown on the right. Scale bar, 20 μm. (e) HCT116 cells were transfected with the indicated plasmids and treated with the SUMOylation inhibitor 2-D08 (0, 100, or 200 μM) for 24 h. 48 h after transfection, ACSL4 SUMOylation was examined by anti-HA IP followed by western blot analysis. (f–h) HCT116 cells were transfected with ACSL4 WT, K532R, or K661R mutants, followed by co-transfection with His-SUMO1 (f), His-SUMO2 (g), or His-SUMO3 (h). SUMOylation of ACSL4 was assessed by IP assays. (i, j) Multiple sequence alignment of ACSL4 amino acid sequences from different species was performed using Jalview (i) and visualized using WebLogo (j).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/ad690105a59e0f90ae95d909.jpg"},{"id":104063767,"identity":"3395f975-7df5-4ddf-947d-f9e4bd4f17eb","added_by":"auto","created_at":"2026-03-06 10:12:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":607845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eACSL4 undergoes SUMOylation at lysine 661. \u003c/strong\u003e(a) Flag- or HA-tagged ACSL4 WT or K661R mutant constructs were co-transfected into HCT116 cells. Cell lysates were subjected to IP using an anti-HA antibody, followed by immunoblotting with an anti-Flag antibody. (b) Cell lysates from LoVo and HCT116 cells stably expressing ACSL4 WT or the K661R mutant were analyzed by immunoblotting under non-reducing conditions. (c) The indicated cells were treated with RSL3 (2.5 μM) for 12 h, and cell viability was measured using the CCK-8 assay (n = 5 independent experiments). (d, e) After treatment with RSL3 (2.5 μM) for 12 h, intracellular relative lipid ROS levels and MDA content were quantified in the indicated cells (n = 3 independent experiments). (f) Mitochondrial membrane potential was assessed by JC-1 fluorescent staining (n = 3 independent experiments). (g) Representative TEM images showing mitochondrial ultrastructure in cells treated with RSL3 (2.5 μM) for 12 h. Scale bars, 2 μm (left) and 500 nm (right). (h-j) The indicated stable CT26 cells were subcutaneously injected into mice, and RSL3 was administered by intratumoral injection (100 mg/kg, twice per week). Tumor growth curve (h); tumor image (i); tumor weight (j). (n=5 biologically independent samples). (k) MDA levels in tumor cells isolated from (i) were quantified (n = 5 independent experiments). (l) Representative IHC images of ACSL4 and 4-HNE in tumor sections are shown. Scale bars, 20 μm. Data are presented as mean ± SEM; Comparisons were made by using one-way ANOVA with Tukey’s test. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/fd03e02b36252121eeb78c8d.jpg"},{"id":104063706,"identity":"bd635cd6-c1fa-444b-947e-e8db303e4403","added_by":"auto","created_at":"2026-03-06 10:11:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":490281,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePIAS1 mediates ACSL4 SUMOylation in colorectal cancer. \u003c/strong\u003e(a) Screening strategy for predicting potential SUMO E3 ligases mediating ACSL4 SUMOylation. (b) \u0026nbsp;HCT116 cells were transfected with the indicated plasmids, followed by IP with an anti-HA antibody and western blot analysis to detect ACSL4 SUMOylation. (c) Mass spectrometry analysis identified PIAS1 in the ACSL4-associated protein pool. (d) IP assays were performed in HCT116 cells using antibodies against PIAS1 and ACSL4 to examine their endogenous interaction.\u003cstrong\u003e \u003c/strong\u003e(e) In vitro pull-down assay verifying the interaction between ACSL4 and PIAS1.\u003cstrong\u003e \u003c/strong\u003e(f) IF staining of PIAS1 (green) and ACSL4 (red) in HCT116 cells. Nuclei were counterstained with DAPI (blue). Qualitative analysis of the fluorescence intensity profile is shown below. Scale bar: 20 μm. (g) HCT116 cells were transfected with the indicated plasmids, followed by IP with an anti-HA antibody and western blot analysis to assess ACSL4 SUMOylation. (h) HA-ACSL4 plasmids were transfected into shControl, shPIAS1 #1, and shPIAS1 #2 HCT116 cells. IP with an anti-HA antibody followed by western blotting was performed to detect ACSL4 SUMOylation. (i) HCT116 cells were transfected with the indicated plasmids and treated with 2-D08 (0, 100, 200, or 300 μM) for 24 h, followed by IP to analyze ACSL4 SUMOylation. (j) The indicated cell lysates were analyzed by immunoblotting under non-reducing conditions. (k) HCT116 cells were transfected with the indicated plasmids and treated with 2-D08 (200 μM) for 24 h. Cell lysates were subjected to immunoblotting under non-reducing conditions to assess ACSL4 dimer formation.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/3e910284236937905682ada6.jpg"},{"id":104063701,"identity":"5fdf3684-c874-42e9-91ed-e369bdf0637d","added_by":"auto","created_at":"2026-03-06 10:11:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":554613,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSUMOylation of ACSL4 at K661 is required for PIAS1-mediated regulation of ferroptosis. \u003c/strong\u003e(a) Cell lysates from the indicated cells were subjected to immunoblotting under non-reducing conditions to assess ACSL4 dimer formation. (b, c) After treatment with RSL3 (2.5 μM) for 8 h, intracellular relative lipid ROS levels and MDA content were quantified in the indicated cells (n = 3 independent experiments). (d) PIAS1-KD CT26 cell lines stably expressing ACSL4 WT or ACSL4 K661R were successfully established. (e-g) Indicated stable CT26 cells were subcutaneously injected into BALB/c mice, and the mice were treated with intratumoral injection of RSL3 (100 mg/kg, twice per week). Tumor growth curve (e); tumor image (f); tumor weight (g). (n=5 biologically independent samples). (h) MDA levels in tumor cells isolated from (f) were quantified (n = 5 independent experiments). (i) Representative IHC images of ACSL4, PIAS1 and 4-HNE in tumor sections are shown. Scale bars, 20 μm. Data are presented as mean ± SEM.\u003cem\u003e P \u003c/em\u003evalues were calculated via one-way ANOVA with Tukey’s test; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, not significant difference.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/2f2cc67f1869aa2738dd6459.jpg"},{"id":104063768,"identity":"7aa0da96-17ef-4598-9e14-4d9a61a414b3","added_by":"auto","created_at":"2026-03-06 10:12:08","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":479642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of ACSL4 SUMOylation enhances ACSL4 ubiquitination. \u003c/strong\u003e(a) Proteins were extracted from CRC tissues obtained after neoadjuvant immunotherapy, and co-IP assays were performed to examine the ubiquitination and SUMOylation levels of ACSL4. (b) HCT116 cells were transfected with the indicated plasmids and treated with 2-D08 (0, 100, or 200 μM) for 24 h, followed by treatment with MG132 (10 μM) for 8 h. IP was performed using an anti-HA antibody, and western blotting with an anti-Myc antibody was used to detect the ubiquitination level of ACSL4. (c) HCT116 cells were transfected with the indicated plasmids and treated with MG132 (10 μM) for 8 h. IP with an anti-HA antibody and western blotting with an anti-Myc antibody were performed to detect the ubiquitination level of ACSL4. (d) Co-IP analyses were performed to examine the interaction between the indicated E3 ubiquitin ligases and endogenous ACSL4 using anti-Flag antibodies in HCT116 cells. (e) The indicated plasmids were transfected into ACSL4-WT–HCT116 and ACSL4-K661R–HCT116 cells. IP with an anti-HA antibody and western blotting with an anti-Myc antibody were performed to assess the ubiquitination level of ACSL4. (f) IF staining was performed to assess changes in the colocalization of ACSL4 (red) and CBL (green) in ACSL4-WT and ACSL4-K661R HCT116 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 20 μm. (g) Quantification of ACSL4 and CBL colocalization based on Pearson’s correlation coefficient (30 cells per sample). Data are presented as mean ± SEM. \u003cem\u003eP\u003c/em\u003e values were calculated using a two-tailed Student’s t-test; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/c7f1fecc351f3986e90fac7f.jpg"},{"id":104063776,"identity":"f590c269-8a2b-48d5-845e-b7adff0f578d","added_by":"auto","created_at":"2026-03-06 10:12:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":530563,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCBL promotes ACSL4 dimerization to facilitate ferroptosis. \u003c/strong\u003e(a) HCT116 cells stably expressing ACSL4-WT or ACSL4-K661R were transfected with HA-K63 ubiquitin and Myc-CBL and treated with MG132 (10 μM) for 8 h. ACSL4 ubiquitination was analyzed by anti-Flag IP followed by anti-HA immunoblotting. (b) HCT116 cells were transfected with the indicated plasmids, treated with 2-D08 (0 or 200 μM) for 24 h, and subsequently treated with MG132 (10 μM) for 8 h. ACSL4 ubiquitination was analyzed by anti-HA IP followed by anti-Myc immunoblotting. (c) ACSL4 dimerization in the indicated CRC cells was assessed by immunoblotting under non-reducing conditions. (d-g) The indicated cells were treated with RSL3 (2.5 μM) for 12 h. (d) Lipid ROS levels were measured using C11 BODIPY 581/591 (n = 3 independent experiments); (e) Cell viability was assessed by the CCK-8 assay (n = 5 independent experiments); (f) Mitochondrial membrane potential was evaluated by JC-1 staining (n = 3 independent experiments); (g) Malondialdehyde (MDA) levels were quantified (n = 3 independent experiments). (h) Representative transmission electron microscopy images showing mitochondrial ultrastructure in the indicated cells treated with RSL3 (2.5 μM) for 12 h. Scale bars, 2 μm (upper) and 500 nm (lower). Data presented as mean±SEM. p-value was calculated via one-way ANOVA with Tukey’s test; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, not significant difference.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/1aeed599e58e27c243a0c0c1.jpg"},{"id":104063760,"identity":"81a2eab4-7a49-4c6a-958e-912468b24219","added_by":"auto","created_at":"2026-03-06 10:12:08","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":548181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePIAS1 competes with CBL for ACSL4 binding.\u003c/strong\u003e (a) HCT116 cells were transfected with HA-ACSL4 and increasing amounts of Flag-PIAS1 plasmids. Co-IP with anti-HA antibody and Western blotting with CBL antibody were performed to examine the interaction between ACSL4 and CBL. (b)\u003cstrong\u003e \u003c/strong\u003eHCT116 cells were transfected with increasing doses of siPIAS1. Co-IP with anti-HA antibody and Western blotting with CBL antibody were performed to examine the interaction between ACSL4 and CBL. (c) HCT116 cells were transfected with indicated plasmids and treated with MG132 (10 μM) for 8 h. IP with anti-HA antibody and Western blotting with anti-Myc antibody were performed to detect the ubiquitination level of ACSL4. (d) IF assay was performed to observe the colocalization changes of ACSL4 (red) and CBL (green) in shNC and shPIAS1 HCT116 cells. Nuclei were counterstained with DAPI (blue). Scale bar: 20 μm. (e) Statistics of the colocalization of ACSL4 and CBL, as indicated by Pearson’s correlation (30 cells per sample). (f, g) The protein-protein interactions were predicted and the protein-protein interaction figure was visualized using PyMOL. ACSL4 is shown as a blue cartoon model, whereas PIAS1 (f) and CBL (g) are shown as green cartoon models, with their binding sites depicted as stick structures in the corresponding colors. (h, i) Sequential bio-layer interferometry (BLI) analyses were performed to examine the binding hierarchy between ACSL4, PIAS1, and CBL. ACSL4 was immobilized on the sensor, followed by sequential addition of CBL and PIAS1 or PIAS1 and CBL. Binding responses were monitored to assess competitive interactions. (j) Schematic diagram of our hypothesis about this project. The data shown represent the mean ± SEM. Comparisons were made by using two-tailed, unpaired Student’s t-test; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/1bb9c926d54d454a58e1c34b.jpg"},{"id":104063792,"identity":"e8214fe0-7618-46f4-b812-0036b1d33e36","added_by":"auto","created_at":"2026-03-06 10:12:20","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":730649,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting SUMOylation sensitizes CRC to chemoimmunotherapy. (a) \u003c/strong\u003eRepresentative IHC images of PIAS1, CBL, and 4-HNE, and IF images of GZMB⁺ CD8⁺ T cells in clinical CRC specimens. Scale bars, 50 μm. (b, c) PIAS1 (b) and 4-HNE (c) IHC staining scores were evaluated in clinical CRC tissues (n = 22). Student’s two-tailed t-test; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. (d, e) Three-dimensional scatter plots depicting the coordinated relationship between PIAS1 (d) or CBL (e) expression and two functional parameters—ferroptosis (4-HNE) and CD8⁺ T-cell infiltration—in immunotherapy-insensitive CRC tissues (n = 11). All \u003cem\u003ep\u003c/em\u003e and \u003cem\u003er\u003c/em\u003evalues were calculated using Spearman’s correlation test. (f) Treatment protocol for subcutaneous CT26 tumors in BALB/c mice following treatment with the XELOX regimen and anti–PD-1, with or without 2-D08. (g) Waterfall plot showing the therapeutic responses of CT26 tumor–bearing mice. The black dashed line indicates complete response. (h) CT26 cells were subcutaneously implanted into BALB/c mice, which then received the indicated treatments. After completion of therapy, mice were sacrificed and tumors were excised. Representative tumor images are shown in (h). (i, j) Flow cytometric analysis of interferon-γ and granzyme B levels in tumors derived from (h). Data presented as mean ± SEM. \u003cem\u003eP \u003c/em\u003evalues were calculated via one-way ANOVA with Tukey’s test; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/c96c37cc260411a929845856.jpg"},{"id":104063836,"identity":"a98dbeca-cbef-4ff3-9bc1-14660f28b921","added_by":"auto","created_at":"2026-03-06 10:12:26","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4978007,"visible":true,"origin":"","legend":"Supplementary","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/9156f0e3285b60a1a0135472.docx"},{"id":104063829,"identity":"090d1172-3fa7-4a38-9d09-189a787085a6","added_by":"auto","created_at":"2026-03-06 10:12:24","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2040109,"visible":true,"origin":"","legend":"WB raw data","description":"","filename":"WBrawdata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8870994/v1/e514f6d1783ae6523e998bfd.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Antagonistic SUMOylation and Ubiquitination of ACSL4 Control Ferroptosis in Colorectal Cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFerroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation and is widely implicated in various diseases, particularly cancer\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Its execution involves inhibition of solute carrier family 7 member 11 (SLC7A11; also known as xCT), decreased glutathione peroxidase 4 (GPX4) activity, disruption of iron homeostasis, and accumulation of phospholipid peroxides mediated by Acyl-CoA synthetase long-chain family member 4 (ACSL4)\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Ferroptosis not only directly kills tumor cells but also enhances the efficacy of chemotherapy, radiotherapy, and immunotherapy\u003csup\u003e[\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Although immunogenic cell death (ICD) has been widely incorporated into combination treatment strategies in clinical settings and has achieved substantial therapeutic benefits, a considerable proportion of patients still fail to derive clinical benefit\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Therefore, combining ferroptosis-inducing therapy with immunotherapy represents a promising strategy, and elucidating the regulatory mechanisms underlying tumor cell ferroptosis is of considerable clinical significance.\u003c/p\u003e \u003cp\u003eACSL4 plays a pivotal role in ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. As a key enzyme in lipid metabolism, ACSL4 functions as a dimer to activate polyunsaturated fatty acids (PUFAs), converting them into oxidizable phospholipid substrates\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Under iron overload, reactive oxygen species (ROS) generated via the Fenton reaction amplify lipid peroxidation, ultimately triggering ferroptosis when cellular antioxidant defenses are overwhelmed\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. High ACSL4 expression has been associated with improved responses to chemotherapy and immune checkpoint blockade in colorectal cancer\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. These findings indicate that ACSL4 may serve not only as a prognostic biomarker for combination therapies but also as a potential therapeutic target.\u003c/p\u003e \u003cp\u003ePost-translational modifications (PTMs) dynamically modulate protein properties and constitute a central mechanism for signal integration and cell fate determination. SUMOylation, a reversible PTM mediated by small ubiquitin-like modifiers (SUMO), regulates protein localization, interactions, stability, and enzymatic activity\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Certain lysine residues can undergo both SUMOylation and ubiquitination, and competitive SUMOylation can regulate protein activity and interactions. This competitive interplay may be facilitated by the structural homology between SUMO and ubiquitin\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. For instance, SUMOylation of IκBα at Lys21 antagonizes ubiquitination at the same residue, thereby modulating NF-κB signaling\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Increasing evidence indicates that dysregulated SUMOylation contributes to tumor progression and immune evasion\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that SUMOylation of PD-L1 in gastric cancer stabilizes the protein by antagonizing ubiquitination, leading to enhanced immune checkpoint signaling and impaired antitumor immunity\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Accordingly, inhibition of SUMOylation has been shown to enhance tumor sensitivity to immunotherapy. However, whether ACSL4 is subject to a similar SUMO\u0026ndash;ubiquitin antagonism, and how this modification crosstalk regulates ACSL4 activity and ferroptosis in tumors, remains unknown.\u003c/p\u003e \u003cp\u003eIn this study, we found that ACSL4 undergoes both SUMOylation and ubiquitination at K661. SUMOylation at this site restrains ACSL4 dimerization and activity, whereas ubiquitination reverses this inhibition. This antagonistic regulation is driven by the differential binding affinity of the SUMO E3 ligase PIAS1 and the ubiquitin E3 ligase CBL for ACSL4. Inhibition of ACSL4 SUMOylation enhances ferroptosis in tumor cells by promoting ACSL4 dimer formation, indicating that modulation of ACSL4 SUMOylation may enhance tumor sensitivity to therapy.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003e1.ACSL4 can undergo SUMOylation at K661.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn order to identify the key lysine residues mediating ACSL4 SUMOylation in tumor cells, we used three independent SUMOylation prediction tools (JASSA, SUMOPLOT, and GPS-SUMO). The overlapping results from these algorithms identified two potential SUMOylation sites, K532 and K661(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Mammals encode four SUMO isoforms (SUMO1\u0026ndash;4), of which SUMO1\u0026ndash;3 are well characterized, whereas SUMO4 is less studied and shows a more restricted expression pattern\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Then we determine the SUMOylation status of ACSL4. We enriched ACSL4 from human colorectal cancer HCT116 cells and performed immunoprecipitation (IP). The results showed that ACSL4 could be modified by SUMO1, SUMO2, and SUMO3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). Immunofluorescence (IF) analysis further demonstrated that ACSL4 colocalized with SUMO1 and SUMO2/3 in cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). 2-D08, a SUMOylation inhibitor\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, suppressed ACSL4 SUMOylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). We then generated lysine-to-arginine mutants at these positions and examined their SUMOylation levels. Both K532R and K661R mutants exhibited reduced SUMOylation, with a more pronounced decrease observed in the K661R mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-h and Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). Interestingly, interrogation of protein modification databases (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb), together with our mass spectrometry analysis of ACSL4 modifications (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec), revealed that K661 is also a ubiquitination site of ACSL4. Notably, this lysine residue is highly conserved across species (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei\u0026ndash;j). These observations prompted us to specifically examine how post-translational modifications at K661 regulate ACSL4 protein function and abundance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2. ACSL4 K661 SUMOylation regulates ACSL4 dimerization and ferroptosis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe established that K661 of ACSL4 is a SUMOylation site and may serve as a hotspot for additional PTMs. These findings prompted us to further dissect how this residue governs the ACSL4-driven ferroptosis. We generated LoVo and HCT116 cell lines stably expressing either wild-type (WT) ACSL4 or the K661R mutant (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ea). Cycloheximide (CHX) chase assays revealed no appreciable difference in protein half-life between WT and K661R ACSL4 (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eb). Previous studies have shown that dimerization of ACSL4 is essential for its enzyme activation. Our results showed that the ACSL4-K661R mutant displayed reduced ACSL4 dimerization compared with ACSL4-WT, indicating that K661 is essential for ACSL4 enzymatic activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo define the role of K661 in ferroptosis, LoVo and HCT116 cells stably expressing ACSL4-WT or K661R were treated with the ferroptosis inducer RSL3\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. CCK-8 assays showed that, compared with WT, expression of K661R significantly increased cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Given that ferroptosis is characterized by the accumulation of lipid peroxides and malondialdehyde (MDA), we next assessed these biochemical hallmarks. Lipid peroxides and MDA levels were markedly reduced in ACSL4-K661R cells relative to WT controls. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Because ferroptosis is also associated with collapse of the mitochondrial membrane potential, we assessed the mitochondrial membrane potential using the JC-1 probe\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. ACSL4-K661R cells exhibited a higher mitochondrial membrane potential than WT cells, indicating reduced ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Given that changes in mitochondrial morphology represent a key ultrastructural feature of ferroptosis, transmission electron microscopy (TEM) was used to assess the mitochondrial status in cells\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Consistently, TEM revealed that mitochondria in ACSL4-K661R cells displayed less shrinkage and cristae disruption than those in WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eWe further evaluated the effect of the ACSL4 K661R mutation on tumor growth \u003cem\u003ein vivo\u003c/em\u003e. ACSL4 knockout (KO), WT and K661R mutant CT26 murine colorectal cancer cell lines were established (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003ec). Subcutaneous tumor models were then generated in mice, and all tumors were treated with intratumoral injections of the ferroptosis inducer RSL3 (100 mg/kg). Tumor growth curves showed that ACSL4-K661R significantly promoted tumor growth compared with ACSL4-WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, i). Consistently, tumors derived from ACSL4-K661R cells exhibited higher weights and reduced MDA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej, k). IHC analysis of 4-hydroxynonenal (4-HNE), a ferroptosis marker, showed markedly decreased 4-HNE staining in ACSL4-K661R tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el). These findings collectively indicate that ACSL4 K661 regulates ferroptosis in tumors by modulating ACSL4 dimer formation.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3. PIAS1 mediates ACSL4 SUMOylation in colorectal cancer\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the functional role of SUMOylation at the ACSL4-K661 site in regulating ACSL4-mediated ferroptosis, we first sought to identify the SUMO E3 ligase responsible for catalyzing SUMO conjugation at this residue. Using mass spectrometry, we analyzed ACSL4-interacting proteins and cross-referenced these results with candidate SUMO E3 ligases predicted by the STRING protein\u0026ndash;protein interaction database. The intersection yielded three potential ligases: PIAS1, TRIM28, and ZBED1 (Fig.\u0026nbsp;3a). IP assays demonstrated endogenous interactions between ACSL4 and each of these ligases (Figure S3a). Subsequent assessment of the SUMOylation of ACSL4 WT and the K661R mutant in the presence of each candidate ligase demonstrated that PIAS1 specifically catalyzes SUMOylation of ACSL4 at K661 (Fig.\u0026nbsp;3b). Subsequently, we conducted a more in-depth characterization of the interaction between PIAS1 and ACSL4. As PIAS1 was detected by ACSL4 mass spectrometry (Fig.\u0026nbsp;3c), co-immunoprecipitation (co-IP) analysis using anti-ACSL4 or anti-PIAS1 antibodies was performed to validate the endogenous interaction between ACSL4 and PIAS1 (Fig.\u0026nbsp;3d). In vitro pull-down assays showed that ACSL4 interacted with GST\u0026ndash;PIAS1 (but not to GST alone) (Fig.\u0026nbsp;3e). IF analysis revealed clear cytoplasmic colocalization of PIAS1 and ACSL4 (Fig.\u0026nbsp;3f). The PIAS1 C351S mutant represents a SUMO E3 ligase\u0026ndash;deficient variant of PIAS1\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Whereas PIAS1 WT promotes ACSL4 SUMOylation, the E3 ligase\u0026ndash;inactive C351S mutant fails to induce this modification (Fig.\u0026nbsp;3g). We established the stable PIAS1 knockdown (KD) cell lines, and found PIAS1 KD markedly decreased ACSL4 SUMOylation (Fig.\u0026nbsp;3h and Figure S3b). Additionally, 2-D08 effectively abrogated the PIAS1-driven enhancement of ACSL4 SUMOylation in a dose-dependent manner, with higher concentrations progressively diminishing SUMOylation (Fig.\u0026nbsp;3i).\u003c/p\u003e \u003cp\u003eWe next examined whether PIAS1 influences ACSL4 expression or enzymatic activity. In both HCT116 and LoVo cells, neither ectopic overexpression (OE) nor KD of PIAS1 altered total ACSL4 protein expression (Figure S3c, d). Given that SUMOylation at K661 regulates ACSL4 dimerization, we further assessed the impact of PIAS1 on this process. Notably, PIAS1 KD markedly increased the level of ACSL4 dimers (Fig.\u0026nbsp;3j). Additionally, 2-D08 restored ACSL4 dimerization in PIAS1 OE cells, further confirming that PIAS1 restrains ACSL4 dimer formation through SUMOylation (Fig.\u0026nbsp;3k). We further examined the functional consequences of PIAS1 in ferroptosis. We found PIAS1 KD markedly enhanced lipid peroxidation and MDA accumulation while significantly reducing cell viability (Figure S3e-g). TEM and JC-1 staining showed that PIAS1 KD cells exhibited increased mitochondrial shrinkage, disrupted cristae, and elevated membrane density, indicative of ferroptosis (Figure S3h-i). We also generated subcutaneous tumors using PIAS1 KD CT26 cells and administered intratumoral injections of RSL3 at a therapeutic dose (Figure S3j). PIAS1 KD significantly delayed tumor growth, reduced tumor weight, and increased intratumoral MDA and 4-HNE levels (Figure S3k\u0026ndash;o). Collectively, these findings demonstrate that PIAS1 acts as a SUMO E3 ligase for ACSL4, suppressing ACSL4 dimer formation and ferroptosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4. SUMOylation of ACSL4 at K661 is required for PIAS1-mediated regulation of ferroptosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine whether PIAS1 regulates ferroptosis via ACSL4 K661, we generated stable PIAS1 KD CRC cell lines expressing ACSL4-WT or ACSL4-K661R (Figure S4a). PIAS1 OE did not alter ACSL4-WT or ACSL4-K661R expression levels (Figure S4b). However, non-reducing electrophoresis showed that PIAS1 KD markedly increased ACSL4 dimer formation in ACSL4-WT cells but not in ACSL4-K661R cells (Fig.\u0026nbsp;4a). Consistently, the K661R mutation largely abolished the ferroptosis-promoting effects of PIAS1 KD. The PIAS1 KD\u0026ndash;induced increase in MDA levels observed in ACSL4-WT cells was significantly attenuated in ACSL4-K661R cells (Fig.\u0026nbsp;4b). Similarly, lipid peroxidation, JC-1 staining, and cell viability assays further confirmed that PIAS1 KD enhanced ferroptosis in ACSL4-WT cells but not in ACSL4-K661R cells (Fig.\u0026nbsp;4c and Figure S4c, d). TEM further revealed increased mitochondrial shrinkage and cristae loss following PIAS1 KD in ACSL4-WT cells, whereas these changes were minimal in ACSL4-K661R cells (Figure S4e). To assess in vivo relevance, subcutaneous tumor models were established using ACSL4-WT or ACSL4-K661R CT26 cells with or without PIAS1 KD (Fig.\u0026nbsp;4d), followed by intratumoral RSL3 treatment. PIAS1 KD significantly suppressed tumor growth in ACSL4-WT tumors, whereas this effect was largely reversed in ACSL4-K661R tumors (Fig.\u0026nbsp;4e\u0026ndash;g). IHC analysis showed increased 4-HNE staining and elevated MDA levels in PIAS1-KD ACSL4-WT tumors, but not in ACSL4-K661R tumors (Fig.\u0026nbsp;4h, i).\u003c/p\u003e \u003cp\u003e \u003cb\u003e5. Inhibition of ACSL4 SUMOylation enhances ACSL4 ubiquitination\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBecause SUMOylation typically antagonizes ubiquitination and ACSL4 K661 may undergo ubiquitin modification, we sought to evaluate the relationship between ACSL4 SUMOylation and ubiquitination. We first collected CRC specimens from patients who were treated with neoadjuvant immunotherapy. IP of ACSL4 revealed that ACSL4 SUMOylation levels were markedly lower in the treatment-sensitive group than in the resistant group. Conversely, ACSL4 ubiquitination was significantly higher in the sensitive group. These findings suggest that the antagonistic interplay between ACSL4 SUMOylation and ubiquitination may shape CRC responsiveness to immunotherapy (Fig.\u0026nbsp;5a). Based on our mass spectrometry analysis, K661 was identified as a potential ubiquitination site (Figure S5a, b), and the K661R mutation markedly reduced ACSL4 ubiquitination (Figure S5c). Moreover, we also found that K661 was specifically modified by K63-linked ubiquitin chains (Figure S5d). Collectively, these results indicate that ACSL4 can undergo both SUMOylation and ubiquitination at the conserved K661 site.\u003c/p\u003e \u003cp\u003eImportantly, the ACSL4 K661R mutant exhibited diminished SUMOylation and ubiquitination, accompanied by a marked suppression of enzymatic activity. Conversely, inhibition of ACSL4 SUMOylation through 2-D08 treatment or PIAS1 KD restored its enzymatic activity. These findings support a model in which K661 functions as a regulatory switch: Ubiquitination of ACSL4 at K661 promotes its dimer formation, whereas SUMOylation at the same residue maintains ACSL4 in an enzymatically repressed state. The K661R mutation disrupts this regulatory axis and impairs dimerization. Subsequently, we performed a series of ubiquitination assays to test this hypothesis. 2-D08 markedly enhanced ACSL4 ubiquitination (Fig.\u0026nbsp;5b), whereas PIAS1 OE reduced ACSL4 ubiquitination (Figure S5e). In addition, the SUMO E3 ligase\u0026ndash;deficient PIAS1 C351S mutant failed to suppress ACSL4 ubiquitination, indicating that PIAS1-mediated SUMOylation antagonizes ACSL4 ubiquitination (Figure S5g). Notably, PIAS1 OE had minimal impact on the ubiquitination of the ACSL4 K661R mutant (Figure S5f), suggesting that PIAS1 primarily inhibits ubiquitination at K661. Furthermore, PIAS1 mainly suppressed K63-linked, but not K48-linked, ubiquitination of ACSL4 (Fig.\u0026nbsp;5c).\u003c/p\u003e \u003cp\u003eWe next sought to identify the E3 ubiquitin ligase responsible for promoting ACSL4 K661 ubiquitination. Based on mass spectrometry data, several potential ACSL4-interacting E3 ligases were identified, including CBL, PJA1, TRIM9, TRIM25, TRIM33, CBLL1, ARIH1, RNF25, and RBBP6. Plasmids encoding these ligases were constructed and individually overexpressed in HCT116 cells. Co-IP confirmed that these candidate ligases interacted with endogenous ACSL4 (Fig.\u0026nbsp;5d). We individually knocked down these E3 ubiquitin ligases using siRNAs and observed that depletion of CBL, TRIM25, CBLL1, or PJA1 did not significantly alter ACSL4 protein abundance (Figure S5h, i). Next, we examined which E3 ligase specifically mediates ubiquitination at K661. Co-transfection assays revealed that CBL, TRIM25, CBLL1, and PJA1 each enhanced ACSL4-WT ubiquitination to varying extents; however, only CBL overexpression failed to increase ubiquitination of the K661R mutant (Fig.\u0026nbsp;5e). \u003cem\u003eIn vitro\u003c/em\u003e pull-down assays further validated a direct interaction between ACSL4 and CBL (Figure S5j). Additionally, co-IP revealed that the K661R mutation significantly impaired ACSL4\u0026ndash;CBL binding (Figure S5k). Furthermore, IF analysis revealed reduced ACSL4\u0026ndash;CBL colocalization in K661R mutant cells (Fig.\u0026nbsp;5f, g). These findings support a model in which SUMOylation and ubiquitination at ACSL4 K661 are mutually antagonistic, and identify CBL as the E3 ubiquitin ligase that catalyzes ACSL4 K661 ubiquitination.\u003c/p\u003e \u003cp\u003e \u003cb\u003e6. CBL promotes ACSL4 dimerization to facilitate ferroptosis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter identifying CBL as the E3 ligase that mediates ACSL4 K661 ubiquitination, we further investigated its regulatory role in CRC ferroptosis. Ubiquitination assays confirmed that CBL selectively promotes K63-linked ubiquitination of ACSL4, with minimal impact on K48-linked chains (Figure S6a, b). Importantly, the CBL-mediated increase in K63-linked ubiquitination was abolished in the ACSL4-K661R mutant (Fig.\u0026nbsp;6a). In addition, 2-D08 further enhanced CBL-mediated ubiquitination of ACSL4 (Fig.\u0026nbsp;6b). Given that K63-linked ubiquitination correlates with ACSL4 dimerization, we next examined whether CBL regulates ACSL4 dimer formation. In HCT116 and LoVo cells stably expressing shNC or shPIAS1, CBL was silenced using siRNA (Figure S6c). Non-reducing immunoblotting demonstrated that CBL KD significantly reversed the PIAS1 KD\u0026ndash;induced increase in ACSL4 dimer formation (Fig.\u0026nbsp;6c). We therefore propose that CBL promotes ACSL4 dimerization via K63-linked ubiquitination. Subsequently, we assessed whether CBL is involved in PIAS1-mediated regulation of ferroptosis. The results showed that CBL KD significantly attenuated PIAS1 KD\u0026ndash;induced ferroptosis (Fig.\u0026nbsp;6d, e). JC-1 staining, MDA quantification and cell viability assays corroborated these findings (Fig.\u0026nbsp;6f, g). Moreover, TEM revealed that CBL KD attenuated mitochondrial damage (Fig.\u0026nbsp;6h). Collectively, these data indicate that CBL is required for PIAS1 KD\u0026ndash;induced ferroptosis.\u003c/p\u003e \u003cp\u003e \u003cb\u003e7. PIAS1 competes with CBL for ACSL4 binding\u003c/b\u003e \u003c/p\u003e \u003cp\u003eACSL4 SUMOylation and ubiquitination at K661 function antagonistically, with SUMOylation restraining and ubiquitination promoting ACSL4 activity; accordingly, PIAS1 suppresses, whereas CBL enhances, its enzymatic function. These findings led us to hypothesize that the antagonism between SUMOylation and ubiquitination, as well as the preferential occurrence of SUMOylation, arises from competitive binding of the two E3 ligases to ACSL4. To test this hypothesis, we first performed co-IP assays. The results showed that increased PIAS1 expression weakened the interaction between ACSL4 and CBL (Fig.\u0026nbsp;7a), whereas reduced PIAS1 levels enhanced their association (Fig.\u0026nbsp;7b). We then carried out ubiquitination and SUMOylation assays. PIAS1 OE impaired CBL-mediated ACSL4 ubiquitination (Fig.\u0026nbsp;7c), whereas CBL OE had minimal impact on PIAS1-driven ACSL4 SUMOylation (Figure S7a). Next, we examined ACSL4\u0026ndash;CBL co-localization in PIAS1-KD cells and found that PIAS1 KD markedly increased their colocalization (Fig.\u0026nbsp;7d, e and Figure S7b). We subsequently performed molecular-docking simulations to model PIAS1\u0026ndash;ACSL4 and CBL\u0026ndash;ACSL4 interactions (Fig.\u0026nbsp;7f, g), which predicted a greater number of hydrogen bonds at the PIAS1\u0026ndash;ACSL4 interface (Figure S7c). Sequential BLI analyses were performed to examine the binding hierarchy between ACSL4, PIAS1, and CBL. When CBL was first allowed to associate with immobilized ACSL4, subsequent addition of PIAS1 led to a further increase in the binding signal, indicating that PIAS1 can efficiently bind ACSL4 even in the presence of pre-bound CBL. In contrast, when PIAS1 was pre-bound to ACSL4, subsequent addition of CBL resulted in only a minimal increase in the binding response (Fig.\u0026nbsp;7h, i). These findings indicate that PIAS1 preferentially associates with ACSL4 and thereby restricts the access of CBL, providing a mechanistic explanation for why SUMOylation predominates over ubiquitination at this residue, given the stronger binding affinity of PIAS1. From a therapeutic perspective, this regulatory hierarchy further implies that targeting ACSL4 SUMOylation may represent a potential strategy to enhance the sensitivity of colorectal tumors to neoadjuvant therapy (Fig.\u0026nbsp;7j).\u003c/p\u003e \u003cp\u003e \u003cb\u003e8. Targeting SUMOylation sensitizes CRC to chemoimmunotherapy\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe previously demonstrated that CRC sensitivity to neoadjuvant immunotherapy is associated with the SUMOylation and ubiquitination status of ACSL4 (Fig.\u0026nbsp;5a). We therefore assessed PIAS1 and CBL expression, as well as ferroptosis levels and immune infiltration, in clinical specimens obtained after neoadjuvant immunotherapy. IHC analysis revealed that patients who responded better to neoadjuvant immunotherapy exhibited higher levels of lipid peroxidation and stronger CD8⁺ T-cell infiltration and function. Notably, compared with treatment-insensitive patients\u0026mdash;those with stable disease (SD) or progressive disease (PD)\u0026mdash;treatment-sensitive patients who achieved a complete response (CR) or partial response (PR) showed lower PIAS1 and higher CBL expression (Fig.\u0026nbsp;8a\u0026ndash;c and Figure S8a, b). Among the 11 immunotherapy-sensitive cases, PIAS1 expression negatively correlated with ferroptosis and CD8⁺ T-cell infiltration and function, whereas CBL displayed the opposite trend (Fig.\u0026nbsp;8d, e). Furthermore, Kaplan\u0026ndash;Meier analysis of the TCGA cohort revealed that high PIAS1 expression predicts poorer overall survival in CRC (Figure S8c). Clinically, the XELOX regimen combined with immune-checkpoint inhibitors has become a standard neoadjuvant approach for resectable CRC\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Additionally, the SUMOylation inhibitor 2-D08 has been reported to enhance antitumor T-cell responses within the tumor microenvironment\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Given the limited response rates of current combination regimens, we next tested whether 2‑D08 could increase sensitivity to regimens that include immune‑checkpoint blockade. Using CT26 subcutaneous tumor model, we evaluated the efficacy of XELOX plus anti-PD-1 with or without 2-D08. \u003cem\u003eIn vivo\u003c/em\u003e, 2-D08 monotherapy produced only modest reductions in tumor volume and weight, with all CT26 tumors exhibiting PD. XELOX combined with anti-PD-1 improved tumor control, resulting in 80% PD and 20% SD. Strikingly, the triple combination markedly suppressed tumor growth and induced tumor regression, achieving PR in all treated mice (Fig.\u0026nbsp;8g, h and Figure S8d, e). Analysis of tumor tissues showed that 2-D08 enhanced intratumoral ferroptosis and enhanced CD8⁺ T-cell infiltration, accompanied by higher proportions of IFN-γ⁺ and granzyme B⁺ CD8⁺ T cells. Importantly, the triple regimen further enhanced intratumoral ferroptosis and robustly increased CD8⁺ T-cell infiltration, expanding IFN-γ⁺ and granzyme B⁺ CD8⁺ T-cell subsets, thereby synergistically enhancing antitumor immunity (Fig.\u0026nbsp;8i, j and Figure S8f, g). Together, these findings suggest that targeting ACSL4 SUMOylation may represent a promising strategy to improve the efficacy of neoadjuvant therapy in colorectal cancer.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eInducing ferroptosis has emerged as a promising strategy to overcome the survival advantages of drug-resistant tumor cells and enhance therapeutic efficacy\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. However, some tumor cells can evade ferroptosis and acquire resistant phenotypes by modulating key ferroptosis-related molecules such as GPX4, ferroptosis suppressor protein 1 (FSP1), and ACSL4\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. PUFAs, owing to their abundance of double bonds, are highly susceptible to oxidative damage triggered by reactive oxygen species (ROS) generated through the Fenton reaction\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. The lethal accumulation of lipid peroxides represents a hallmark and critical execution step of ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. As a major driver of ferroptosis, ACSL4 plays a central role in lipid metabolic remodeling\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Accumulation of PUFAs may enhance membrane fluidity, thereby weakening PD-L1\u0026ndash;PD-1 interactions and increasing the sensitivity of non-small cell lung cancer to immune checkpoint inhibitors (ICIs)\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Therefore, targeting ACSL4 to modulate ferroptosis sensitivity may represent an effective approach to improving immunotherapy responses in cancer.\u003c/p\u003e \u003cp\u003ePTMs profoundly impact protein activity, stability, subcellular localization, and protein\u0026ndash;protein interactions\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that PKCβII activates ACSL4 through phosphorylation to promote PUFA-phospholipid synthesis and enhance ferroptosis sensitivity\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e, while CARM1 modulates ACSL4 expression through arginine methylation to augment responses to immunotherapy\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. In recent years, SUMOylation has been recognized as a key regulatory mechanism in cancer therapy and cell death, influencing protein stability, transcriptional activity, subcellular localization, and DNA damage repair\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. SUMOylation of ACSL4 at K532 has been implicated in regulating neuronal ferroptosis after spinal cord injury\u003csup\u003e[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]\u003c/sup\u003e, whereas our study identifies K661 as the major SUMOylation site in colorectal cancer that controls ACSL4 enzymatic activity, underscoring the disease-specific regulation of ACSL4 SUMOylation. We further showed that PIAS1-mediated SUMOylation at K661 significantly suppresses ACSL4 activity and inhibits CBL-mediated ACSL4 dimerization. These findings expand the known PTM landscape of ACSL4 and reveal a previously unrecognized mechanism whereby SUMOylation regulates ferroptosis.\u003c/p\u003e \u003cp\u003eSUMOylation and ubiquitination often exhibit antagonistic crosstalk\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. We observed that PIAS1 KD led to decreased ACSL4 SUMOylation, accompanied by markedly increased K661 ubiquitination and enhanced dimer formation. Conversely, the K661R ACSL4 mutant showed reduced dimerization capacity and profoundly impaired enzymatic activity. Based on these findings, we propose that SUMOylation may suppress ubiquitination at K661 through a competitive mechanism. Further investigation identified CBL as the E3 ligase responsible for catalyzing ACSL4 K661 ubiquitination and promoting dimerization. Our data indicate that PIAS1 and CBL competitively bind to ACSL4, thereby determining the prioritization of SUMO versus ubiquitin modification. Given the higher binding affinity of PIAS1 for ACSL4, PIAS1 more efficiently recruits the SUMO conjugation machinery, allowing SUMO to occupy K661 and block CBL-mediated ubiquitination through steric hindrance or conformational alteration. Similar competitive mechanisms have been described in other systems, such as SUMO/ubiquitin competition at IκBα K21, where SUMOylation prevents ubiquitin-dependent degradation and suppresses NF-κB activation\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Although the full-length structure of ACSL4 has not yet been resolved, our molecular docking analysis suggests that the stronger PIAS1\u0026ndash;ACSL4 interaction may result from the formation of more hydrogen bonds. Collectively, these findings deepen our understanding of how competitive PTMs cooperate or antagonize each other to fine-tune protein function.\u003c/p\u003e \u003cp\u003eImmunotherapy has become one of the most transformative advances in cancer treatment\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e; however, a substantial proportion of patients fail to respond, underscoring the urgent need to enhance immunotherapy sensitivity\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Recent studies have suggested that ferroptosis may synergize with immunotherapy to potentiate antitumor immunity. Interferon-γ (IFN-γ) has been shown to synergize with arachidonic acid (AA) to induce ACSL4-dependent ferroptosis in melanoma cells\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. Meanwhile, SUMOylation has been shown to reshape the tumor immune microenvironment and impair immune surveillance, thereby promoting immune evasion\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. In addition to suppressing immune cell function, tumor-intrinsic SUMOylation directly contributes to immune escape\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Thus, targeting SUMOylation may represent a promising approach to improving immunotherapy responses. Indeed, previous studies have shown that inhibition of the SUMO E2 enzyme UBC9 by 2-D08 enhances antitumor immunity\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Given the differential ACSL4 SUMOylation we observed in colorectal cancer samples receiving neoadjuvant XELOX plus ICIs, we further explored whether 2-D08 could enhance responses to combined therapies including anti-PD-1. Our findings demonstrate that inhibition of ACSL4 SUMOylation promotes ferroptosis both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Importantly, 2-D08 synergized with XELOX and anti-PD-1 therapy to amplify ferroptosis and markedly enhance antitumor immune responses.\u003c/p\u003e \u003cp\u003eIn summary, we identified PIAS1-mediated SUMOylation of ACSL4 at K661 as a key suppressive mechanism of ferroptosis. Combination therapy involving SUMOylation inhibitors, chemotherapy, and immune checkpoint blockade effectively suppressed tumor progression. These findings not only uncover a novel mechanism of ferroptosis regulation but also provide a potential therapeutic strategy for targeting ferroptosis- and SUMOylation-related pathways in colorectal cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cstrong\u003eCollection of clinical specimens\u003c/strong\u003e \u003cp\u003e This study was approved by the Ethics Committee of Tongji Hospital (TJ-IRB20220723). Clinical specimens were obtained from the Department of Gastrointestinal Surgery, Tongji Hospital. Written informed consent was obtained from all patients prior to surgery. Routine imaging examinations, such as computed tomography, were performed to evaluate the efficacy of preoperative chemotherapy. According to RECIST version 1.1\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e, patients who achieved CR or PR were classified as immunotherapy-sensitive, whereas those with SD or PD were classified as immunotherapy-insensitive. Demographic information, including age and sex, is provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (Supplementary Information).\u003c/p\u003e \u003c/p\u003e\n\u003ch3\u003eCell lines, antibodies, and reagents\u003c/h3\u003e\n\u003cp\u003eThe colorectal cancer cell lines LoVo, HCT116, and CT26 were obtained from the American Type Culture Collection (ATCC). All cell lines were authenticated by short tandem repeat (STR) profiling conducted by Procell. Mycoplasma contamination was routinely monitored every three months using a PCR-based Mycoplasma Detection Kit (Cat. No. C0301S, InvivoGen, Bayogene). Cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂ and cultured in Gibco DMEM, McCoy\u0026rsquo;s 5A, or RPMI-1640 medium, each supplemented with 10% fetal bovine serum (Cat. No. SV30160.03, HyClone) and 1% penicillin\u0026ndash;streptomycin solution.\u003c/p\u003e \u003cp\u003eThe following antibodies were used in this study: Anti-ACSL4 (ab155282), anti-PIAS1 (ab109388), anti-Ubiquitin (ab134953), anti-TRIM9 (ab300515), anti-TRIM25 (ab167154), anti-TRIM33 (ab300146), and anti-RNF25 (ab140514) were purchased from Abcam. CBL (SAB4503444) and PJA1 (HPA000595) were obtained from Sigma‒Aldrich. CBLL1 (sc-517157), ARIH1 (sc-390763), and RNF25 (sc-398749) were purchased from Santa Cruz Biotechnology. SUMO1 (#4930), SUMO2/3 (#4971), TRIM28 (#4124), GAPDH (#2118), HA-tag (#3724), and Myc-tag (#2276) antibodies were obtained from Cell Signaling Technology. ZBED1 (A6792) and RBBP6 (A14776) were purchased from Abclonal. Secondary antibodies, including DyLight 488 goat anti-mouse IgG (#A23210), DyLight 488 goat anti-rabbit IgG (#A23220), DyLight 549 goat anti-mouse IgG (#A23310), and DyLight 549 goat anti-rabbit IgG (#A23320), were purchased from Abbkine. For flow cytometry, the Zombie NIR Fixable Viability Kit (#423105), BV510-CD45 (#103137), BV650-CD8 (#810742), BV785-CD3 (#100232), PC5.5-GZMB (#372212), and PE-IFN-γ (#505808) were obtained from BioLegend. Reagents including 2-D08 (HY-114166), oxaliplatin (HY-17371), MG132 (HY-13259), and cycloheximide (CHX, HY-12320) were purchased from MedChem Express. The sources of all remaining reagents are provided in the respective sections.\u003c/p\u003e\n\u003ch3\u003eTransient transfection of siRNA and plasmids\u003c/h3\u003e\n\u003cp\u003eSiRNAs targeting PIAS1, CBL, PJA1, TRIM9, TRIM25, TRIM33, CBLL1, RNF25, ARIH1, and RBBP6 were designed and synthesized by RiboBio (Guangzhou, China), and their sequences are provided in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Expression plasmids encoding pcDNA3.1-Flag-PIAS1, pcDNA3.1-Flag-TRIM28, pcDNA3.1-Flag-ZBED1, pcDNA3.1-HA-ACSL4, pcDNA3.1-Myc-Ubiquitin, pcDNA3.1-His-SUMO1, pcDNA3.1-His-SUMO2, pcDNA3.1-His-SUMO3, pcDNA3.1-Flag-PJA1, pcDNA3.1-Flag-TRIM9, pcDNA3.1-Flag-TRIM25, pcDNA3.1-Flag-TRIM33, pcDNA3.1-Flag-CBLL1, pcDNA3.1-Flag-RNF25, pcDNA3.1-Flag-CBL, pcDNA3.1-Flag-RBBP6, and pcDNA3.1-Flag-ARIH1 were purchased from AUGCT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.augct.com\u003c/span\u003e\u003cspan address=\"http://www.augct.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). In addition, the pLVX-ACSL4 construct was generated by inserting the corresponding DNA fragment into the indicated vector. All site-directed mutants were generated using the Mut Express II Fast Mutagenesis Kit V2 (C214-01, Vazyme). Furthermore, ACSL4 deletion mutants were constructed based on the pcDNA3.1-HA-ACSL4 plasmid. The primer sequences used for ACSL4 point mutations and deletion mutants are listed in Supplementary Table S3. All plasmids were sequence-verified prior to use.\u003c/p\u003e\n\u003ch3\u003eSyngeneic tumor model\u003c/h3\u003e\n\u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee of Tongji Hospital. BALB/c mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. CT26 cells (2 \u0026times; 10⁵) were subcutaneously injected into 6-week-old female BALB/c mice. Tumor volume was measured at designated time points and calculated using the formula: 0.5 \u0026times; L \u0026times; D\u0026sup2; (L: long diameter; D: short diameter). After euthanasia, subcutaneous tumors were weighed, and the tissues were collected for further analysis.\u003c/p\u003e \u003cp\u003eIn some experiments, mice were treated with RSL3 (100 mg/kg, intratumoral injection, twice per week for 2 weeks). Mice were euthanized 20 days after treatment initiation, and tumors were harvested for subsequent analyses. In combination therapy experiments, mice were treated according to a modified XELOX-based regimen combined with immunotherapy. Oxaliplatin (10 mg/kg) was administered via intraperitoneal injection on day 1. Capecitabine (350 mg/kg) was given by oral gavage once daily from day 1 to day 14. The anti\u0026ndash;PD-1 antibody (100 \u0026micro;g/mouse) was administered intraperitoneally every 3 days throughout the treatment period. Where indicated, 2-D08 (10 mg/kg) was delivered via intratumoral injection every 3 days. After 14 days of treatment, mice were euthanized, and tumors were collected for subsequent analyses.\u003c/p\u003e \u003cp\u003eFor individual mice, PD was defined as \u0026lt;\u0026thinsp;50% regression from the initial volume during the treatment and \u0026gt;\u0026thinsp;25% increase in the initial volume at the end of treatment. SD was defined as \u0026lt;\u0026thinsp;50% regression from the initial volume during the treatment and \u0026le;\u0026thinsp;25% increase in the initial volume at the end of the treatment. PR was defined as a tumor volume regression\u0026thinsp;\u0026ge;\u0026thinsp;50% for at least one time point but with measurable tumor (\u0026ge;\u0026thinsp;0.10 cm3).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of single-cell suspension from subcutaneous tumors\u003c/h2\u003e \u003cp\u003eSubcutaneous tumors were minced into small pieces using scissors and transferred into 3 mL serum-free RPMI-1640 medium containing type IV collagenase (50 \u0026micro;L, 25 mg/mL; Cat. No. V900893, Sigma-Aldrich), hyaluronidase (50 \u0026micro;L, 32 mg/mL; Cat. No. H3506, Sigma-Aldrich), and DNase I (25 \u0026micro;L, 10 mg/mL; Cat. No. 10104159001, Roche). Tumor tissue was digested by shaking at 37\u0026deg;C for 1 hour at 150 r.p.m. After complete digestion, 7 mL of serum-free RPMI-1640 medium was added to dilute the enzymes. The cell suspension was then filtered and centrifuged, followed by treatment with 1 mL of ACK lysis buffer for 1 minute to lyse red blood cells, and subsequently neutralized. Finally, the cells were resuspended and kept on ice until further staining experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eWestern Blotting, IP, and IF Analysis\u003c/h3\u003e\n\u003cp\u003eFor immunoblot assay, CRC tissues and cells were lysed with NP40 buffer containing protease inhibitors and phosphatase inhibitors. Protein quantification was performed by the BCA Protein Assay Kit (23225, Thermo Fisher Scientific). After incubation at 95\u0026deg;C for 10 min, equal amounts of protein were separated by a 10% SDS-PAGE gel and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). After being blocked by 5% skim milk for 1 h, the membranes were incubated with specific antibodies overnight at 4\u0026deg;C. Subsequently, the membranes were incubated with secondary antibodies for 1 h, followed by enhanced chemiluminescence detection. To examine ACSL4 dimerization, Western blot analysis was performed under non-reducing conditions using sample buffer without β-mercaptoethanol or dithiothreitol. For IP, whole-cell lysates were incubated and rotated overnight with anti-Flag, anti-HA beads, or Protein A/G beads conjugated with specific antibodies. Beads were washed four times with lysis buffer and followed by immunoblot assays. For IF staining, the indicated cells were cultured on round cell slides and fixed with 4% paraformaldehyde for 20 min. After permeabilization with 0.3% Triton X-100, the samples were blocked with 2% bovine serum albumin, and then stained with specific antibodies overnight at 4\u0026deg;C. Subsequently, the samples were incubated with corresponding fluorescently labeled secondary antibodies (Dylight 488, Goat Anti-Rabbit IgG, and Dylight 549, Goat Anti-Mouse IgG) for 2 h at room temperature, and followed by staining with 4,6-diamidino-2-phenylindole (DAPI). The images were obtained by a confocal fluorescence microscope (Zeiss) to assess colocalization. Images were processed using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eIn Vitro Binding Assays\u003c/h3\u003e\n\u003cp\u003eRecombinant human ACSL4 (ABIN7232834, antibodies-online), CBL (TP314069, OriGene), and PIAS1 (H00008554-P01, Abnova) proteins were used for in vitro binding assays. Purified His-tagged ACSL4 was incubated with GST or GST-PIAS1, followed by rotation with anti-GST magnetic beads at 4\u0026deg;C overnight. Likewise, His-ACSL4 was incubated with Flag-tagged CBL and rotated with anti-Flag magnetic beads at 4\u0026deg;C overnight. The beads were washed four times with lysis buffer and subsequently subjected to immunoblot analysis.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell Viability Assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the Cell Counting Kit-8 (CCK-8; Cat. No. HY-K0301, MedChemExpress). LoVo and HCT116 cells were seeded into 96-well plates at a density of 5 \u0026times; 10⁴ cells per well and treated with RSL3. At the indicated time points, 10 \u0026micro;L of CCK-8 solution was added to each well and incubated for 5 h. Absorbance was then measured at 450 nm. Cell viability was calculated by normalizing to the viability of the negative control group and presented as a percentage.\u003c/p\u003e \u003cp\u003eFerroptosis detection: Lipid peroxidation was assessed using the fluorescent probe C11 BODIPY 581/591 (Abclonal, Cat. No. RM02821). The probe was dissolved in DMSO to prepare a 10 mM stock solution and stored at \u0026minus;\u0026thinsp;20\u0026deg;C protected from light. After treatment, cells were incubated with 10 \u0026micro;M C11 BODIPY 581/591 at 37\u0026deg;C for 30 min in the dark, followed by two washes with PBS to remove excess dye. Fluorescence was analyzed by flow cytometry with excitation at 488 nm and emission collected at 505\u0026ndash;550 nm. Lipid peroxidation levels were quantified as mean fluorescence intensity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMDA measurement\u003c/h2\u003e \u003cp\u003eCells were collected after PBS washing, and tissue samples were homogenized. Supernatants were obtained after centrifugation at 4\u0026deg;C. MDA levels were then measured using a commercial assay kit (Beyotime, Cat. No. S0131S) according to the manufacturer\u0026rsquo;s instructions. Absorbance was recorded at the specified wavelength, and concentrations were calculated from a standard curve and normalized to protein concentration or tissue weight.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTEM\u003c/h2\u003e \u003cp\u003eCells were treated with 2.5 \u0026micro;M RSL3 for 8 h, collected, and fixed in 2.5% glutaraldehyde at room temperature before storage at 4\u0026deg;C. Samples were subsequently post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, embedded in epoxy resin, and sectioned into ultrathin slices. Sections were stained with uranyl acetate and lead citrate and examined by transmission electron microscopy to assess mitochondrial ultrastructure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial membrane potential assay\u003c/h2\u003e \u003cp\u003eMitochondrial membrane potential was measured using the JC-1 assay kit (Beyotime, Cat. No. C2006). Cells were stained according to the manufacturer\u0026rsquo;s protocol, and fluorescence was analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIHC\u003c/h2\u003e \u003cp\u003eIHC analysis was performed on mouse subcutaneous tumors and CRC tissues from patients. Paraffin-embedded CRC specimens were processed for IHC according to the manufacturer\u0026rsquo;s instructions. The staining results were independently evaluated by two experienced gastrointestinal pathologists. The IHC score was determined based on both the staining extent and intensity. The percentage of positively stained tumor cells was graded as 0 (\u0026lt;\u0026thinsp;10%), 1 (10\u0026ndash;25%), 2 (26\u0026ndash;50%), 3 (51\u0026ndash;75%), or 4 (\u0026gt;\u0026thinsp;75%). The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The final IHC score was calculated by multiplying the percentage score by the intensity score, yielding a total score ranging from 0 to 12. Samples with an IHC score\u0026thinsp;\u0026ge;\u0026thinsp;6 were defined as high expression, whereas those with a score\u0026thinsp;\u0026lt;\u0026thinsp;6 were classified as low expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eBiolayer Interferometry (BLI) Assay\u003c/h2\u003e \u003cp\u003eBLI experiments were performed using an Octet system (ForteBio). Recombinant ACSL4 protein was immobilized on biosensors. Prior to the assay, sensors were equilibrated in PBST buffer (PBS containing 0.01% Tween-20) for 15 min. After baseline acquisition (60\u0026ndash;120 s), purified CBL and/or PIAS1 proteins were sequentially added at the indicated concentrations to monitor association for 300\u0026ndash;600 s, followed by dissociation in PBST buffer for 30 s. Binding data were analyzed using Octet Data Analysis software according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry analysis\u003c/h2\u003e \u003cp\u003eThe detailed procedures for mass spectrometry analysis have been reported previously. ACSL4 protein samples were enriched by IP and separated by SDS\u0026ndash;PAGE. Gel bands of interest were excised and submitted to the HIT Center for Life Sciences at Harbin Institute of Technology for mass spectrometric analysis. Protein identification was performed with technical support from the HIT Center for Life Sciences. Peptide mapping and identification of potential ubiquitination (K) sites were carried out using the ptmRS node in Proteome Discoverer software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis:\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using GraphPad Prism 8.0 and Origin 2022. Statistical significance between groups was determined using two-tailed Student\u0026rsquo;s t-tests, one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test, or two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test, as appropriate. Categorical variables were analyzed using the chi-square test. Correlation analyses were conducted using Spearman\u0026rsquo;s rank correlation test. Overall survival was evaluated using the Kaplan\u0026ndash;Meier method, and differences were assessed by the log-rank test. Differences were considered statistically significant at P values less than 0.05. *\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and ***\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; not significant (ns), \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cb\u003eConflict-of-interest disclosure\u003c/b\u003e: The authors declare that there are no conflicts of interest.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contributions\u003c/h2\u003e \u003cp\u003eXL conceived and designed the experiments. JZ performed most of the experiments. SF performed animal experiments. GS, HZ and WZ collected biological samples and analyzed the data. Other authors provided suggestions for several experiments. JZ and XL organized and analyzed the data and wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eWe appreciate the support from members of Guihua Wang\u0026rsquo;s laboratory and Junbo Hu\u0026rsquo;s laboratory. We also acknowledge equipment support from the Experimental Medicine Center of Tongji Hospital, Tongji Medical School, Huazhong University of Science and Technology. This work is supported by the National Natural Science Foundation of China (No. 82472829); Huanggang Innovation and Development Joint Fund Project of Hubei Provincial Natural Science Foundation (No. 2025AFD335); The Joint supported by Hubei Provincial Natural Science Foundation and United Imaging of China (No. 2025AFD848)\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Disclosures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo disclosures were reported.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDIXON S J, LEMBERG K M, LAMPRECHT M R, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death [J]. Cell, 2012, 149(5): 1060-72.\u003c/li\u003e\n\u003cli\u003eJIANG X, STOCKWELL B R, CONRAD M. Ferroptosis: mechanisms, biology and role in disease [J]. Nat Rev Mol Cell Biol, 2021, 22(4): 266-82.\u003c/li\u003e\n\u003cli\u003eYAN H F, ZOU T, TUO Q Z, et al. Ferroptosis: mechanisms and links with diseases [J]. 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BJU Int, 2022, 130(1): 68-75.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ACSL4 SUMOylation, PIAS1, Ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-8870994/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8870994/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFerroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation and represents a therapeutic vulnerability in cancer. ACSL4 is a critical regulator of ferroptosis, yet how its activity is post-translationally regulated remains unclear. Here, we identify a SUMO\u0026ndash;ubiquitin switch that controls ACSL4 dimerization, enzymatic activity, and ferroptosis. ACSL4 undergoes both SUMOylation and ubiquitination at K661. SUMOylation suppresses ACSL4 dimer formation and activity, whereas ubiquitination promotes dimerization and ferroptotic function. This antagonistic regulation is driven by competitive binding of the SUMO E3 ligase PIAS1 and the ubiquitin E3 ligase CBL to ACSL4. Inhibition of ACSL4 SUMOylation enhances lipid peroxidation, promotes ferroptosis, and suppresses tumor growth. These findings uncover a post-translational mechanism that precisely regulates ACSL4 activity and ferroptosis, and suggest that targeting ACSL4 SUMOylation may enhance the efficacy of ferroptosis-based antitumor therapy.\u003c/p\u003e","manuscriptTitle":"Antagonistic SUMOylation and Ubiquitination of ACSL4 Control Ferroptosis in Colorectal Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 10:09:25","doi":"10.21203/rs.3.rs-8870994/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-04-02T14:19:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-29T12:03:09+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-23T14:11:55+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-06T23:07:13+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-03-06T14:14:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-03-03T12:38:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-16T10:29:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2026-02-13T10:56:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-13T10:56:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c195b92d-9f6c-4a35-90c3-a877911bc7cc","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63846140,"name":"Health sciences/Diseases/Cancer"},{"id":63846141,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2026-04-02T14:29:20+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 10:09:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8870994","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8870994","identity":"rs-8870994","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}