Supplementary Figure 9 and related legend of the revised manuscript.

Supplementary Figure 6 and related legend of the revised manuscript.

ABSTRACT The E3 ubiquitin ligase tripartite motif 27 (TRIM27) is a negative regulator of NF‐κB activation and the innate immune response, and TRIM27 deficiency significantly impairs dextran sulfate sodium (DSS)‐induced colitis. The function of TRIM27 in intestinal epithelial cells (IECs), the mechanism by which TRIM27 inhibits the NF‐κB pathway and its dysregulation in ulcerative colitis (UC) remain unclear. Here, it is report that epithelial TRIM27 functions as an anti‐inflammatory factor that inhibited intestinal inflammation in IECs in vitro and in epithelial Trim27 knockout mice in vivo. Mechanistically, TRIM27 destabilized IKKα and TRAF6 via polyubiquitination of IKKα at the K569 site and TRAF6 at the K489 site. In response to TNF‐α, IKKβ phosphorylated TRIM27 at S173 to decrease TRIM27 expression by impairing its binding to ubiquitin‐specific protease 7 (USP7) and USP7‐mediated TRIM27 deubiquitination. Notably, overexpression of TRIM27 enhanced the anti‐inflammatory effect of infliximab (IFX) in IECs. TRIM27 is downregulated in inflamed colons from UC patients and is associated with the therapeutic effect of IFX. Overall, this study identifies epithelial TRIM27 as a bona fide negative modulator of intestinal inflammation and USP7/TRIM27‐IKK as a new double negative feedback mechanism of the NF‐κB pathway, which supports the use of TRIM27 replenishment as a potential therapeutic strategy for UC.

Source data of the revised manuscript.

Supplementary Figure 8 and related legend of the revised manuscript.

BACKGROUND AND AIMS:Drug-induced liver injury (DILI) is a leading cause of acute liver failure. Patients with DILI have disorders of the gut microbiota, yet little is known about the influence of gut microbes on this disease. Herein, we investigated the alterations of gut microbiota in DILI patients, and elucidated the mechanism by which Escherichia coli expressing kpsM gene (kpsM+E. coli) exacerbates DILI, in order to provide targets for intervention of related signaling pathways to improve DILI. METHODS:Full-length 16S sequencing was performed on fecal samples from a prospective cohort of patients with DILI (n = 42). Quantitative PCR was employed for analysis of E. coli and its kpsM gene in human feces. The DILI model was established by intraperitoneal injection of acetaminophen (300 mg/kg) into mice (n = 5-12). Two hours later, kpsM+ or kpsM knockout E. coli strains were gavaged to determine their roles during DILI. Intestinal epithelial Fut2 gene knockout mice (Fut2ΔIEC) and hepatic metabolome were used to assess the pathogenic mechanisms of the kpsM+E. coli. Plasma metabolome of DILI patients was further validated the discoveries in mice. RESULTS:The percentage of subjects carrying kpsM were 14.7 %, 40.0 %, 76.5 % in healthy controls, patients with mild DILI, and patients with moderate-to-severe DILI, respectively. Mice transplanted with kpsM+E. coli exhibited more severe DILI, primarily achieved through impaired gut barrier function and enhanced expression of intestinal Fut2. Fut2ΔIEC mice alleviated the aggravation of DILI caused by E. coli via up-regulating the hepatic levels of taurine and tauroursodeoxycholic acid. In addition, the level of plasma taurine was lower in patients with moderate-to-severe DILI than in those with mild DILI. CONCLUSIONS:The kpsM+ E. coli was associated with the severity of DILI in human. This strain can exacerbate DILI in mice through up-regulating intestinal Fut2 expression and disrupting taurine metabolism.

110 Background: Immune checkpoint blockade (ICB) shows limited efficacy in mismatch repair proficient (pMMR) locally advanced rectal cancer (LARC). Recent clinical studies have shown that neoadjuvant chemoradiotherapy (nCRT) plus ICB improve response. However, radiotherapy is associated with toxicities, including radiation proctitis, anastomotic leakage, and anal dysfunction. Moreover, radiotherapy is not feasible for high position LARC. Radiotherapy-free neoadjuvant strategy warrants further exploration. The FIRM trial is the first phase 2, proof-of-concept study evaluating neoadjuvant immuno-chemotherapy (nICT) with mFOLFOX6 plus PD1 blockade for pMMR LARC. Methods: Patients (pts) with T3/4 or N+, pMMR LARC located ≤15 cm from anal verge were eligible. Pts received 6 cycles of mFOLFOX6 and serplulimab followed by surgery. Primary endpoints were pathological complete response (pCR) rate and major pathological response (MPR) rate. Enrollment of 30 pts was planned, with hypothesis of an increased pCR of 24% compared with reported data of 6.6% after mFOLFOX6 (FOWARC trial, JCO 2016). Multi-omics profiling (single cell RNAseq, bulk RNAseq, whole exome sequencing) was performed to elucidate response mechanisms and develop an immuno-response molecular subtype (IRMS) for immunotherapy stratification. Results: 30 pts were enrolled, 28 completed ≥4 cycles of nICT. 2 discontinued due to adverse events (AE). The pCR and MPR rate were 42.9% and 67.9%, respectively. Tumor regression grade (TRG) 0, 1, 2, and 3 were observed in 44.4%, 11.1%, 40.7%, and 3.7% of pts. 6 pts experienced grade 3 AEs, with no grade 4-5 AE or anastomotic leakage observed. Baseline profiling identified two novel subsets predicting nICT response: inflammatory/migratory tumor cells (IMT) and ITGAX⁺ activated B cells (IAB). Post-nICT samples showed increased enrichment of CD8⁺ T, cDC1, pDC, memory B and Tfh cells, together with reduced exhausted CD8⁺ T, Tregs, extracellular matrix deposition and tumor angiogenesis. IRMS stratified pts into immune-sensitive and immune-resistant types and showed strong predictive performance in internal cohort (AUC =0.87, sensitivity =0.74, specificity =1.0, accuracy =0.81), outperforming conventional biomarkers including CPS (AUC = 0.48) and TMB (AUC =0.47). Notably, IRMS was validated across multiple external immunotherapy cohorts (melanoma, colorectal, lung, breast cancers), showing robust predictive value (AUC =0.70-0.86). Conclusions: These findings provide the first evidence supporting immuno-chemotherapy as a promising radiotherapy-free neoadjuvant strategy for pMMR LARC. nICT converts the immune-cold tumors into immune-activated state by orchestrating a dual-pathway immune remodeling involving IMT-cDC1-CD8 and IAB-CD4 axes. The IRMS provides a high-precision, pan-cancer tool to guide patient selection and personalized immunotherapy. Clinical trial information: NCT06688786 .

Metabolic signals critically shape innate immune responses. Through pharmacological screening of metabolic pathways, we identified aspartate metabolism as a key regulator of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling. Genetically or aminooxyacetic acid-mediated (AOA-mediated) pharmacologically reducing aspartate levels markedly potentiated the cGAS-STING pathway, leading to stronger upregulation of type I interferons and interferon-stimulated genes. Mechanistically, disruption of de novo pyrimidine synthesis, a major downstream pathway of aspartate, induced mtDNA replication stress and increased mtDNA double-strand breaks, promoting mtDNA release into the cytosol. Cytosolic mtDNA synergized with cGAS-STING agonists to upregulate Z-DNA binding protein 1 (ZBP1), which recruits RIPK1/3 to sustain IRF3 phosphorylation, forming a positive feedback loop that amplifies innate immune signaling. In immunocompetent mouse models, AOA enhanced the antitumor efficacy of STING agonists, chemotherapy, or radiotherapy, whereas aspartate supplementation abrogated these effects. Consistently, aspartate levels negatively correlated with antitumor immunity in colorectal cancer patient samples. Together, our study identifies aspartate-pyrimidine metabolism as a critical metabolic checkpoint that licenses STING signaling by enabling mtDNA stress to cooperate with agonist stimulation, driving type I interferon-dependent ZBP1 induction and feed-forward amplification of STING signaling, thus offering a promising strategy to enhance antitumor immunity.

The dynamic assembly and regulation of the IκB kinase (IKK) complex in the NF-κB pathway are central to the pathogenesis and progression of inflammatory bowel disease (IBD). We recently reported that the transcription factor hematopoietically expressed homeobox (HHEX) promotes colitis-associated colorectal cancer, but the potential role of HHEX in intestinal inflammation remains uncharacterized. Here, we found that HHEX is upregulated in inflamed colons in a colitis mouse model and in clinical IBD samples. HHEX overexpression increased inflammatory cytokine expression, and HHEX loss largely abrogated the inflammatory response in vitro and intestinal inflammation in vivo. Mechanistically, IKKα phosphorylates HHEX at S213 to stabilize HHEX in response to TNF-α by inhibiting the interaction of HHEX with the E3 ubiquitin ligase MID2 and subsequent K48-linked ubiquitination and protein degradation. Importantly, HHEX interacted with and stabilized the IKKα/IKKβ complex via its N-terminal domain, thereby activating the NF-κB pathway and establishing a positive feedback loop that exacerbates intestinal inflammation. Our study reveals a transcription-independent function of HHEX in promoting IKK complex assembly and colitis, identifying HHEX as an IBD susceptibility gene and a potential target for IBD treatment.

Supplementary tables of the revised manuscript.

119 Background: PD-1 inhibitor shows poor efficacy in MSS locally advanced rectal cancer (LARC). Radiotherapy activates tumor immunogenicity, and current evidence have shown that combination of neoadjuvant chemoradiotherapy (nCRT) with PD-1 inhibitor enhances tumor regression and improves pathological complete response (pCR) rate in MSS LARC. However, nCRT induces radiation toxicities including radiation proctitis, anastomotic fistula and anal dysfunction. Moreover, radiotherapy is not feasible for high position LARC. Radiotherapy-free neoadjuvant strategy warrants further exploration. Neoadjuvant mFOLFOX6 circumvents radiation toxicity but achieves only 6.6% pCR rate (FOWARC trial, JCO 2016). Preclinical studies suggested oxaliplatin and 5FU have immune-sensitizing effects. Thus, we conducted the first proof of concept trial exploring a radiotherapy-free neoadjuvant immuno-chemotherapy (nICT) regimen, which employed a multicenter, single arm, phase 2 design to evaluate the efficacy and safety of mFOLFOX6 plus serplulimab in MSS/pMMR LARC. Methods: Patients (pts) with cT3/4 or cN+, MSS and pMMR LARC located ≤15 cm from anal verge were eligible. Pts received 6 cycles of neoadjuvant mFOLFOX6 and serplulimab, followed by surgery and adjuvant mFOLFOX6. The primary endpoints were pCR rate and major pathological response (MPR) rate. Enrollment of 30 pts was planned, with hypothesis of an increased pCR of 24% compared with reported data of 6.6% after conventional mFOLFOX6. The per-protocol set (PPS) was defined as pts who completed ≥4 cycles of nICT for primary endpoint assessment. Results: Among 30 enrolled patients, 28 completed ≥4 cycles of nICT and were included in PPS. 2 discontinued due to adverse events. The pCR was achieved in 42.9% (12/28) of pts, and MPR in 67.9% (19/28). For high LARC (>10 cm from anal verge, ineligible for nCRT), the pCR and MPR rates were 71.4% and 85.7%. Radiologic response of tumor regression showed complete response in 57.1% (16/28) and partial response in 39.3% (11/28) of pts. One pt had stable disease and received nCRT. 27 pts proceeded to TME and were included in surgical set. TRG 0, 1, 2, and 3 were observed in 44.4%, 11.1%, 40.7%, and 3.7% of pts. Downstaging was achieved in 22 pts (81.5%). The Grade 3 TRAEs were lymphocyte decrease (4 pts), neutropenia (3 pts) and bilirubin elevation (1 pts). No Grade 4-5 TRAEs or anastomotic fistula were observed. Conclusions: mFOLFOX6 plus serplulimab showed promising efficacy and manageable safety in MSS LARC. pCR rate unexpectedly reached 42.9%, showing an improvement compared with the reported 6.6% pCR rate of chemotherapy, and were comparable to nCRT plus anti-PD-1 regimens. This is the first proof of concept trial to explore a nICT strategy for MSS LARC, which spares pts from radiation toxicities and shows particularly applicability for high LARC. We are launching a randomized controlled trial (FIRM02) for further validation. Clinical trial information: NCT06688786 .

Supplementary Figure 4 and related legend of the revised manuscript.

Liver cirrhosis is an end-stage pathological process caused by various chronic liver diseases, whose incidence is increasing and represents a serious public health burden. The onset of liver cirrhosis is related to various factors such as alcohol, autoimmune, steatosis, and viral infections. Recent research suggests that the gut microbiota also participates in the progression of liver cirrhosis and that supplementation with probiotics improves the progression of liver diseases. We have reviewed the latest studies on the mutual influence between the gut microbiota (including bacteria, fungi and virus) and cirrhosis, and summarized the changes in the gut microbiota, the immune and metabolic mechanisms, and the application of gut microbiota in the clinical management, hoping to provide new strategies for the diagnosis and treatment of liver cirrhosis.

Supplementary Figure 1 and related legend of the revised manuscript.

Supplementary Figure 5 and related legend of the revised manuscript.

Supplementary Figure 10 and related legend of the revised manuscript.

BACKGROUND:Fucosyltransferase 2 (FUT2)-dependent fucosylation of intestinal epithelial cells is vital for preserving gut barrier integrity and microbial balance. Nevertheless, its precise involvement in alcohol-associated liver disease has yet to be fully elucidated. METHODS:We generated mice with intestinal epithelial cell-specific Fut2 knockout (Fut2△IEC) and established a chronic-binge alcohol model. 16S rRNA sequencing and metabolomics analysis were used to reveal differences in the composition and function of faecal bacteria. RESULTS:The loss of intestinal epithelial Fut2 exacerbates alcohol-related hepatic oxidative stress damage, and this effect is dependent on gut bacteria. A marked decrease in the abundance of bacteria carrying nicotinamidase (PncA) in the intestines of Fut2△IEC mice was observed, leading to disrupted nicotinamide metabolism and decreased nicotinic acid production. This reduction in nicotinic acid synthesis results in decreased NAD+ production in the liver via the Preiss-Handler pathway. Administering pncA-overexpressing Escherichia coli promotes hepatic NAD+ synthesis and alleviates alcohol-related oxidative stress damage in Fut2△IEC mice. CONCLUSION:These findings reveal a gut microbiota-Fut2-pncA axis that modulates alcoholic liver injury in mice, which may offer insights into microbial contributions to alcoholic liver disease in people. KEY POINTS:Loss of intestinal epithelial fucosylation exacerbates alcohol-related liver injuries. Intestinal epithelial deletion of Fut2 disrupts nicotinamide (NAM) metabolism due to the decline in bacterial nicotinamidase (PncA). Supplemented with pncA overexpressed E.coli to restore gut PncA levels can improve liver damage in alcohol-induced intestinal epithelial Fut2 deletion mice.

BACKGROUND AND AIMS:Intestinal obstruction caused by fibrosis is a common and serious complication of Crohn's disease (CD). Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motifs (TAZ), the transcriptional effectors of the Hippo signaling pathway, have emerged as key drivers of intestinal fibrosis. Systematic inhibition of YAP/TAZ failed to combat fibrotic progression, probably due to the vital role of epithelial YAP/TAZ in intestinal homeostasis. METHODS:Enzyme-Linked Immunosorbent Assay (ELISA) and immunohistochemical staining were used to detect serum Prostaglandin I2 (PGI2) levels and PGI2 Receptor (PTGIR) in clinical samples derived from CD patients. Dual luciferase reporter and Cut & Run assays were performed to explore the transcriptional regulatory mechanisms of PTGIR and PGI2 synthase (PTGIS) by tumor necrosis factor α (TNF-α) and transforming growth factor-beta (TGF-β), respectively. Primary intestinal fibroblasts and a chronic colitis model were used for assessing the efficacy of a PTGIR agonist in combating fibrosis. RESULTS:The Gαs-coupled PTGIR is expressed in intestinal fibroblasts but is barely expressed in intestinal epithelial cells. PTGIR transcription is directly activated by p65 in fibroblasts upon TNF-α stimulation. Importantly, PTGIS is transcriptionally suppressed by TGF-β, leading to the loss of endogenous antifibrotic PGI2-PTGIR signaling. Serum PGI2 levels are decreased in CD patients with stenosis and are negatively correlated with disease duration. The PTGIR agonist inhibited the profibrotic function of YAP/TAZ in intestinal fibroblasts in vitro and reversed intestinal fibrosis in vivo. CONCLUSIONS:The antifibrotic effects of PGI2-PTGIR signaling are impaired in CD. Restoring PGI2-PTGIR signaling is a pharmacologically tractable and cell-selective approach to targeting YAP/TAZ via PTGIR, which reverses intestinal fibrosis.

Hyperactivation of the YAP/TEAD transcriptional complex in cancers facilitates the development of an immunosuppressive tumor microenvironment. Herein, we observed that the transcription factor SP1 physically interacts with and stabilizes the YAP/TEAD complex at regulatory genomic loci in colorectal cancer (CRC). In response to serum stimulation, PKCζ (protein kinase C ζ) was found to phosphorylate SP1 and enhance its interaction with TEAD4. As a result, SP1 enhanced the transcriptional activity of YAP/TEAD and coregulated the expression of a group of YAP/TEAD target genes. The immune checkpoint V-domain Ig suppressor of T-cell activation (VISTA) was identified as a direct target of the SP1-YAP/TEAD4 complex and found to be widely expressed in CRC cells. Importantly, YAP-induced VISTA upregulation in human CRC cells was found to strongly suppress the antitumor function of CD8+ T cells. Consistently, elevated VISTA expression was found to be correlated with hyperactivation of the SP1-YAP/TEAD axis and associated with poor prognosis of CRC patients. In addition, we found by serendipity that enzymatic deglycosylation significantly improved the anti-VISTA antibody signal intensity, resulting in more accurate detection of VISTA in clinical tumor samples. Overall, our study identified SP1 as a positive modulator of YAP/TEAD for the transcriptional regulation of VISTA and developed a protein deglycosylation strategy to better detect VISTA expression in clinical samples. These findings revealed a new tumor cell-intrinsic mechanism of YAP/TAZ-mediated cancer immune evasion.