Background: Colorectal cancer liver metastasis (CRLM) is characterized by an immunosuppressive microenvironment and a blunted response to immunotherapy. Notably, tumor-associated macrophages (TAMs) play a critical role in modulating immune responses and exhibit significant heterogeneity in CRLM. Sphingosine kinase 1 (SPHK1) serves as a pivotal kinase in maintaining the balance between ceramide and sphingosine-1-phosphate (S1P) levels. However, the effects of SPHK1 within TAMs on tumor immune evasion during CRLM remain elusive. This study aimed at investigating the role of TAM-intrinsic SPHK1 in tumor immunosuppressive microenvironment in CRLM.

Methods: SPHK1 expression levels in TAMs were estimated by immunofluorescence and bioinformatics analysis. Several animal models were established to elucidate the role of SPHK1 in tumor immunity reprogramming in vivo. Flow cytometry, cytokine assay, and transwell assay were conducted to investigate the effects of SPHK1 in TAMs in cell-cell communication in vitro. RNA-sequencing, Western blotting, and quantitative real-time polymerase chain reaction were used to explore the molecular mechanism by which SPHK1 activated NLR family pyrin domain containing 3 (NLRP3) inflammasome in TAMs.

Results: We found that SPHK1 was mainly expressed in TAMs and identified SPHK1⁺ TAMs as associated with CRLM and diminished efficacy of immunotherapy in human patients. These SPHK1⁺ TAMs exhibited strong immunosuppressive activities by inducing CD8⁺ T cell exhaustion with high programmed cell death 1 (PD-1) expression via the interaction between TAMs and CRC cells. Mechanistically, SPHK1-produced S1P exerted an autocrine effect to activate NLRP3 inflammasome and interleukin 1 beta (IL-1β) release via nuclear factor-kappa B (NF-κB) and hypoxia inducible factor 1 subunit alpha (HIF-1α) signaling in TAMs. Paracrine IL-1β then upregulated the expression of monocyte chemoattractants and ADAM metallopeptidase domain 17 (ADAM17) sheddase in CRC cells, resulting in TAM infiltration and CD8⁺ T cell dysfunction in the liver microenvironment. Furthermore, combining SPHK1-targeting treatments with anti-PD-1 therapy or radioimmunotherapy largely stalled liver metastasis and caused a significant extension of lifespan in preclinical mouse models.

Conclusions: Our findings highlighted the role of SPHK1 of TAMs in facilitating CRLM by promoting CD8⁺ T cell dysfunction and immunosuppressive microenvironment. Combining SPHK1 blockade with anti-PD-1 therapy may be a promising treatment regimen for patients with CRLM.

Background: Cadonilimab is a humanized immunoglobulin G1 bispecific antibody targeting programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). This study aimed to evaluate the safety and efficacy of stereotactic body radiotherapy (SBRT) combined with cadonilimab in patients with advanced recurrent or refractory solid tumors.

Methods: Patients with advanced solid tumors who progressed after at least two lines of systemic treatment, including immunotherapy, and were eligible for SBRT were enrolled. SBRT was administered to patients with high-burden/symptomatic lesions in combination with intravenous cadonilimab (6 mg/kg, once every 2 weeks). The primary endpoint was safety.

Results: Sixty-three patients were enrolled from August 28, 2022, to September 14, 2023 (median follow-up: 9.1 months). The median prior treatment line was 3.0 (range 2.0-4.0). Approximately 46.0% (29/63) of patients had received prior PD-1/PD-L1 therapy, 36.5% (23/63) and 12.7% (8/63) of patients had non-small cell lung cancer and soft tissue sarcoma. Treatment-related adverse events (TRAEs) occurred in 38.1% (24/63) of patients, with grade 3 TRAEs reported in 3.2% (2/63). The most common TRAEs included pain (12.7%), elevated transaminases (12.7%), pneumonia (6.4%), fatigue (6.4%), nausea (4.8%), and fever (4.8%). The objective response rate (ORR) was 23.8% (95% confidence interval [CI], 14.0%-36.2%). The median progression-free survival (PFS) was 7.2 months (95% CI, 6.3-8.2 months), and the median overall survival (OS) was 10.0 months (95% CI, 7.7-12.4 months). The 6-month and 12-month local control rates were 98.4% and 93.0%, respectively. In a subgroup analysis of 23 non-small cell lung cancer patients, the ORR was 17.4% (95% CI, 5.0%-38.8%), the median PFS was 6.9 months (95% CI, 4.7-9.1 months), and the median OS was 9.1 months (95% CI, 7.3-10.9 months). Multivariate analysis indicated that receiving ≥6 cycles of cadonilimab and having an Eastern Cooperative Oncology Group performance status score of 0-1 were significantly associated with improved PFS and OS (P < 0.05).

Conclusions: SBRT in combination with cadonilimab demonstrated manageable toxicity and promising efficacy in heavily pretreated patients with refractory solid tumors.

Trial registration: This study was retrospectively registered at ClinicalTrials.gov (NCT05915481) on August 20, 2022.

Post-translational modifications (PTMs) play a pivotal role in epigenetic regulation and are key pathways for modulating protein functionality. PTMs involve the covalent attachment of distinct chemical groups, such as succinyl, crotonyl, and lactyl, at specific protein sites, which alter protein structure, function, stability, and activity, ultimately influencing biological processes. Recently, metabolically derived short-chain acylation modifications (with acyl groups containing fewer than six carbon atoms) have been progressively identified, such as butyrylation, succinylation, crotonylation, and lactylation, differing from traditional acetylation in structure, physicochemical properties, function, and regulation. Aberrant short-chain acyl-PTMs are often associated with tumorigenesis. Research highlights that PTMs like succinylation and lactylation are essential in regulating tumor metabolism, drug resistance, and immune responses. This review elucidates the regulatory mechanisms of eight short-chain acyl-PTMs—butyrylation, succinylation, crotonylation, malonylation, glutarylation, 2-hydroxyisobutyrylation, β-hydroxybutyrylation, and lactylation—that are involved in tumor initiation and progression. Their roles in controlling tumor genomic stability, gene transcription, protein stability, enzyme activity, and nuclear localization are summarized, demonstrating their impact on related biological processes such as tumor metabolism, multi-drug resistance, and immune evasion. Additionally, the review provides an overview of current drug research targeting enzymes that regulate PTMs, offering critical insights to advance therapeutic strategies for cancer treatment.

Background: Eps15 homology domain (EHD) proteins, including EHD1 to EHD4, play vital roles in tumor progression. In this study, we aimed to investigate which specific EHD proteins, if any, are implicated in tumor immune evasion and immunotherapy response.

Methods: The immunotherapy responses of lung adenocarcinoma (LUAD) patients were predicted using tumor immune dysfunction and exclusion (TIDE) analysis. The T cell killing assay was performed by co-culturing activated T cells with LUAD cells. The function of EHD1 as a regulator of programmed death-ligand 1 (PD-L1) endocytic recycling was determined by receptor internalization assays. Methylated RNA immunoprecipitation (MeRIP) was performed to investigate N6-methyladenosine (m⁶A) modification of EHD1 mRNA. The protein-protein interaction was revealed by the molecular docking analysis and validated by immunofluorescence (IF) and immunoprecipitation (IP) assays. RNA immunoprecipitation (RIP) was used to examine the interaction between YTH N6-methyladenosine RNA-binding protein 1 (YTHDF1) and EHD1 mRNA. The regulatory mechanism of YTHDF1 on EHD1 was investigated through the application of m⁶A-binding site mutation analysis. The murine LUAD cells were employed to establish subcutaneous xenograft models within immunocompetent C57BL/6 mice to assess the immunomodulatory impact of EHD1 in vivo.

Results: TIDE algorithms and survival analysis identified that EHD1 promoted LUAD immune escape. EHD1 knockdown enhanced T cell cytotoxicity in killing LUAD cells across all effector-to-target (E/T) ratios. EHD1 overexpression exerted the opposite effect. The molecular docking analysis revealed an interaction between EHD1 and the PD-L1 protein, verified by IF and IP. Furthermore, EHD1 knockdown inhibited PD-L1 recycling, thereby promoting its lysosomal degradation. Disruption of the EHD1/PD-L1 interaction impaired the regulatory function of EHD1 in tumor immune evasion. In an immune-competent mouse model, we found that EHD1 silencing impeded tumor immune evasion and enhanced the efficacy of anti‑PD‑1 therapy. MeRIP-qPCR confirmed obvious m⁶A modification of EHD1. Further, the EHD1 mRNA was found to bind to the YTHDF1 protein, an m⁶A reader. YTHDF1 overexpression up-regulated EHD1 expression by enhancing its mRNA stability in an m⁶A-dependent manner.

Conclusion: Our study illuminates the role of m⁶A-modified EHD1 in tumor immune evasion and immunotherapy responses, thereby offering a novel avenue to potentially enhance immunotherapeutic sensitivity and improve the prognosis for patients with LUAD.

Background: Phosphorylated signal transducer and activator of transcription 3 (p-STAT3) has emerged as a critical modulator of hepatocellular carcinoma (HCC) progression. However, its role in three-dimensional (3D) chromatin conformation and the expression of genes linked to HCC aggressiveness remains largely unexplored. This study aimed to identify HCC 3D chromatin conformations that are regulated by sustained STAT3 activation and validate the molecular mechanisms underlying the aggressiveness of HCC.

Methods: Comparative analyses were performed using HCC cell lines with varying levels of STAT3 activation. Chromatin immunoprecipitation-sequencing (ChIP-seq) for p-STAT3 and H3K27ac was conducted to map p-STAT3-associated genomic regions and assess its influence on chromatin states. Chromatin conformation sequencings (high-throughput chromosome conformation capture and high-throughput chromosome conformation capture followed by immunoprecipitation) were employed to investigate the 3D genome landscape and identify conformational changes linked to sustained p-STAT3 activation. RNA-sequencing was performed to assess transcriptional changes in response to these chromatin rearrangements. Functional assays, including invasion and tube formation assays, were carried out to validate the phenotypic impact of p-STAT3 activation on HCC progressiveness. Pharmacological inhibition of STAT3 was tested to explore potential therapeutic avenues and resistance mechanisms.

Results: We found that sustained activation of p-STAT3 was significantly associated with poor prognostic outcomes in HCC patients. ChIP-seq demonstrated that p-STAT3 regulated chromatin interactions, leading to the formation of frequently interacting regions (FIREs), stable structural units within the 3D genome. Genes within these p-STAT3-associated FIREs exhibited coordinated expression, with many involved in aggressiveness HCC phenotypes like invasion and tube formation. Chromatin conformation data indicated that these FIREs altered topologically associating domains (TADs), potentially influencing broader chromatin organization. Despite STAT3 inhibition, p-STAT3-associated chromatin conformations remained intact, maintaining the expression of genes within FIREs and contributing to drug resistance.

Conclusions: Sustained p-STAT3 activation significantly alters the 3D chromatin conformation in HCC, particularly through the formation of FIREs. These p-STAT3-associated FIREs drive the expression of genes involved in HCC aggressiveness and remain active despite STAT3-targeted treatments, suggesting a mechanism of drug resistance. These findings highlight the potential of targeting 3D chromatin dynamics as a therapeutic strategy in HCC, especially in cases of STAT3 inhibitor resistance.