A novel NFKB1 agonist remodels tumor microenvironment and activates dendritic cells to promote anti-tumor immunity in colorectal cancer

Abstract

Background

The immunosuppressive nature of the tumor microenvironment (TME) and the existence of cancer stem cells (CSCs) present significant hurdles in tumor therapy. The identification of therapeutic agents that can target both CSCs and the TME could be a potential approach to overcome treatment resistance.

Methods

We conducted an in vivo chemical screen to identify F1929-1458, which is capable of eliciting an organism-wide response to destroy stem cell tumors in Drosophila. We then performed functional validation using a mouse colorectal cancer graft tumor model established with the CT26 cell line characterized by its high content of CSCs. Single-cell sequencing was employed to analyze alterations in the TME. Small molecule pull-down mass spectrometry, cellular thermal shift assay, drug affinity experiment, and molecular docking were utilized to identify the target of F1929-1458. An in vitro co-culture system was applied to establish that the damage-associated molecular patterns (DAMPs) released by the tumor cells are accountable for the activation of dendritic cells (DCs).

Results

We demonstrated that F1929-1458 treatment enhanced T cell infiltration and T cell mediated tumor regression, its anti-tumor effect was nullified in nude mice and was abolished after anti-CD3 neutralizing antibody treatment. We found that F1929-1458 binds NFKB1 to activate the NF-κB signaling pathway in tumor cells. The activation further elicits cellular stress, causing tumor cells to release DAMPs (HMGB1-gDNA complex, ATP, and OxLDL). These DAMPs, in turn, stimulate the cGAS-STING and NLRP3 inflammasome pathways in DCs, resulting in the generation of type I IFNs and IL-1β. These cytokines facilitate the maturation of DCs and antigen presentation, ultimately enhancing T cell-mediated anti-tumor immunity. Additionally, we showed that the combination of F1929-1458 and the anti-PD-1 antibody exhibited a synergistic anti-tumor effect.

Conclusion

Our study identified a novel NFKB1 agonist that promotes anti-tumor immunity by remodeling the TME and activating DCs and that may provide a new way to overcome resistance to current anti-tumor immunotherapy in colorectal cancer.

Introduction

Cancer stem cells (CSCs) in colorectal cancer were identified over a decade ago [1, 2]. CD44 and CD133 were identified as surface markers of CSCs in colorectal cancer [3]. Anti-cancer immunotherapies with immune-checkpoint blockades (ICBs) and adoptive T cell therapy (ACT) have exhibited remarkably durable efficacy in some patients due to their inherently adaptive nature [4,5,6]. However, most patients do not respond or respond only incompletely to the current immune therapies [7, 8]. One of the main reasons is that the presence of CSCs poses a significant obstacle to the immunotherapy of colorectal cancer [9]. Thus, the identification of new strategies for improving the effectiveness and durability of current immunotherapies remains an important goal and an unmet clinical need.

Many candidates identified in cell culture systems are ineffective or toxic in patients due to in vivo physiological conditions or metabolic changes. Given that the immune response is an organism-level event, an alternative approach is to conduct in vivo chemical library screening. Genetic screens in model organisms have been extensively used to study genes’ biological functions [10]. However, a chemical library screening platform with model organisms is still absent. Our previous studies and those of others established neoplastic stem cell tumor models in Drosophila by expressing a constitutively activated form of the Ras gene in the kidney stem cells or expressing a dominant negative form of the Notch gene in the intestinal stem cells [11,12,13]. Feeding Drosophila with Arf1 inhibitors selectively killed the tumorigenic stem cells (CSC-like cells) but not normal stem cells [14, 15]. Arf1 knockdown or Arf1 inhibitor treatment promoted death of transformed stem cells via a non-cell autonomous immune-like coordinated cell death (CCD) pathway in Drosophila [14] and a trans-cellular anti-tumor immune response to destroy tumors in mice [16,17,18].

The Drosophila system offers a unique opportunity for high-throughput chemical screening to mobilize the whole-body system (similar to the immune response in mammals) for destroying stem cell tumors. Using the Drosophila stem cell tumor system, we conducted a large-scale chemical screening in vivo with whole animals. We used the 96-well tissue culture plates and identified F1929-1458 for anti-cancer immunotherapy. The present study revealed that F1929-1458 binds to NFKB1, which causes the NF-κB pathway to become active in tumor cells. This activation further induces cellular stress, resulting in the release of the HMGB1-gDNA complex, ATP, and oxidized low-density lipoprotein (OxLDL). These factors subsequently stimulate the cGAS-STING and NLRP3 inflammasome signaling pathways in dendritic cells (DCs), leading to the generation of type I IFNs and IL-1β. Collectively, these cytokines facilitate maturation, activation, and antigen presentation of DCs, ultimately augmenting T cell-mediated anti-tumor immunity. Consequently, F1929-1458, a newly identified agonist of the NF-κB pathway, might offer a novel therapeutic option for surmounting the resistance encountered in current anti-tumor immunotherapies.

Materials and methods

Reagents and antibodies

F1929-1458 (over 98% by HPLC) was purchased from WuXi AppTec, China. Detailed information regarding other reagents and antibodies utilized in the present study is listed in Supplemental Table 1.

Screening of small molecule compounds in Drosophila tumor models

We evaluated the inhibitory effect of novel small molecule compounds on Ras^V12 mutated fruit fly intestinal stem cell tumors using fluorescence microscopy imaging. The specific method is as follows: first, we used a GFP positive labelling system to induce clones in adult fruit flies. Secondly, we added 0.5 μL of test drug to 100 μL of Drosophila unsolidified food, mixed well so that the final drug concentration is 10 μM. After the food was solidified, 3 fruit flies carrying Ras^V12 mutant tumors were placed in each well. A total of 15 fruit flies were allocated for each drug for the experiment, and they were further cultured at 25 °C for 3 days. Finally, we dissected the fruit flies and observed and took photos under a fluorescence microscope.

Animal studies

All animals were maintained under specific pathogen free conditions at the Laboratory Animal Center of Institute of Developmental Biology and School of Life Science, Fudan University. All animal experiments were performed in accordance with the animal study protocols approved by the Animal Care and Use Committee of Fudan University. The registration number of the approved animal study protocols is IDM2021035.

Cell culture

CT26 tumor cell line and DC2.4 dendritic cell line were obtained from the American Type Culture Collection (ATCC), and both were cultured in RPMI 1640 media supplemented with 1% penicillin–streptomycin and 10% fetal bovine serum (FBS). All cell lines were maintained at 37 °C under a humidified atmosphere containing 5% CO₂.

Engrafted tumor models

The mouse graft tumor model was conducted following previously described methods [17]. In the experimental procedure, 5 × 10⁵ CT26 cells were carefully resuspended in sterile phosphate-buffered saline (PBS) and subcutaneously injected into BALB/c mice. Mice were divided into groups and given the indicated treatments. The administration of the substances was performed via intragastric gavage on a daily basis. The tumor volume was monitored at intervals of 2 days using a caliper. To calculate the tumor volume, the following formula was employed: $\text{V}=\frac{1}{2}\times \text{l}\times {\upomega }^{2}$, where $\text{l}$ represents the longitudinal diameter (length) and $\upomega$ represents the greatest transverse diameter (width). Once the tumor volume reached the range of 1000–2000 mm³, the mice were euthanized through cervical dislocation. Subsequently, further analysis was performed on the excised tumors to assess the effects of the treatment.

BMDCs isolation and culture

Mouse bone marrow-derived dendritic cells were extracted following a previous protocol [19]. Briefly, the bone marrow cells were extracted from the femurs and tibias with PBS. After that, the cells were seeded into a 24-well plate at a density of 1 mL per well. Concurrently, the recombinant mouse GM-CSF (20 ng/mL) and IL-4 (10 ng/mL) were added, and the cells were incubated at 37 °C in an incubator under a 5% CO₂ atmosphere. This is the 0 day of cultivation. Gently shake the culture plate at 2-day intervals, then substitute 3/4 of the volume with fresh culture medium and replenish with cytokines. Between the 5 th and 8 th days, collect suspended cells and loosely adherent cells, which are BMDCs.

Immunofluorescent staining

The cells were rinsed with PBS, and subsequently fixed in 4% paraformaldehyde for 10 min. Following that, the cells were permeabilized using 0.1% Triton X-100. Non-specific binding was prevented by incubation with 1% donkey serum for 30 min. The cells were stained with the primary antibodies overnight at 4 °C and afterwards incubated with secondary antibodies conjugated with fluorochrome for 1 h at room temperature. The images were captured using the confocal fluorescence microscopes Zeiss LSM 880. Image analysis was performed using ImageJ software.

Flow cytometry

First, tumor tissues were harvested. Then, they were ground and passed through a 40 μm strainer to prepare single-cell homogenates. After that, the red blood cells were lysed. The conjugated fluorescent primary antibody was diluted with cell staining buffer at a ratio of 1:500. Antibodies were then used to stain the surface markers for 30 min at 4 °C. Flow cytometry was carried out using Beckman Coulter equipment.

Small molecule pull-down and MS

Tumor cells were lysed with NP40 Buffer. The lysate was collected in a centrifuge tube, centrifuged at 12,000g for 5 min, and the supernatant was taken. The supernatant was divided into three groups: the first group served as a negative control, the second group had biotin-labelled F1929-1458 added to it, and the third group had un-labelled small molecules added first, followed by the addition of biotin-labelled F1929-1458. Subsequently, all the groups were incubated at 4 °C for 1 h. Magnetic beads were added to the three groups, then the groups were incubated at 4 °C for 1 h. The magnetic beads were cleaned on the magnetic rack and washed three times with NP40 Lysis Buffer. The magnetic beads were collected, 100 μL of 1 × SDS loading buffer was added, and they were heated at 95 °C for 10 min. SDS-PAGE electrophoresis was performed on the protein sample, Coomassie Brilliant Blue staining was conducted on the protein gel, and the gel was cut for mass spectrometry.

Isothermal titration calorimetry

The protocol was conducted in line with the method previously reported [20]. To prepare the CDC73 and NFKB1 proteins for the corresponding assay, the gene fragments of Cdc73 and Nfkb1 were inserted into the sites of the pET-28a (+) vector. Once the correct sequence was verified, the plasmids were transformed into BL21 (DE3) to enable heterologous expression. When the optical density at 600 nm (OD600) of LB medium reached 0.6, 1 mM of isopropyl-β-d-thiogalactoside was added, and the mixture was then shaken overnight at 16 °C. A protein purification kit (Beyotime, China) was utilized to extract the His-tagged proteins, and their identification was achieved through western blotting. A titration experiment was performed using the MicroCal PEAQ-iTC instrument. 200 μL of the purified CDC73 or NFKB1 protein was added to the sample pool. 40 μL of ligand F1929-1458 was extracted, and the titration curves of ligands and receptors at different concentrations were tested.

Western blot analysis

First, equivalent quantities of proteins underwent size-fractionation on a 12% SDS-PAGE gel. Following this, the proteins on the gel were transferred to polyvinylidene fluoride membranes. Subsequently, these membranes were blocked at room temperature for 1 h. Next, the membranes were treated with primary antibody overnight at 4 °C with shaking. They were then rinsed three times with TBST, with each rinse lasting 5 min. After that, the membranes were treated with the secondary antibody for an hour at room temperature. Following a thorough three-cycle wash with TBST, the proteins were analyzed using a Tanon imaging system.

Cellular thermal shift assay

Tumor cells were treated with control (PBS) or F1929-1458 (100 μM) for 24 h. The treated tumor cells were centrifuged (1000g, 5 min, 4 °C) to collect cells, resuspended in 1 mL of PBS (with added protease inhibitors), and divided equally into different PCR tubes. The test tubes were heated using a PCR thermal cycler at temperatures ranging from 37 to 62 °C at 5 °C intervals for 3 min each. After heating, the test tubes were immediately incubated at room temperature for another 3 min. Three freeze–thaw cycles were performed on the sample in liquid nitrogen. Each cell lysate was centrifuged (15,000g, 20 min, 4 °C) to collect the supernatant. The supernatant was analyzed through protein blotting. Protein levels were quantified using ImageJ.

Drug affinity responsive target stability

These operations were carried out using previously described methods [21]. In brief, the tumor cells were washed twice with pre-cold PBS, and then 1 mL of NP40 Lysis Buffer (containing protease inhibitor) was added, and the cells were lysed on ice for 15 min. The cell lysate was centrifuged (15,000g, 5 min, 4 °C) to obtain the supernatant. The obtained supernatant was then equally divided and incubated with varying concentrations of small molecules (10–100 μM) for 12 h at 4 °C. Protease was introduced into the mixture at a 1:1000 ratio and then incubated at 37 °C for 10 min. Following this, protease inhibitors were added, and the resultant mixture was placed on ice for 10 min. Finally, SDS buffer was added for subsequent western blot identification.

Molecular docking

First, the two-dimensional structural information of the small molecule F1929-1458 was retrieved from the PubChem database. Subsequently, this information was imported into the Chem Office 20.0 software, where the two-dimensional structure was transformed into a three-dimensional one and saved in the mol2 file format. Then, we chose the high-resolution crystal structure of NFKB1 as the receptor for molecular docking from the RCSB PDB database. We performed pretreatment steps such as water removal and dephosphorylation on the selected protein with the help of PyMOL 2.6.0 software. Energy minimization was performed on F1929-1458 using MOE 2019 and the target protein was preprocessed and its active pocket regions were localized. Finally, the molecular docking program was initiated in the MOE 2019 software environment, and the number of operations was set to 50. We assessed the binding strength of the ligand-receptor based on the binding energy value. Meanwhile, the docking results were displayed visually using PyMOL 2.6.0 software.

Nuclear cytoplasmic separation

The experimental operation was conducted in accordance with the commercial kit (Beyotime, China). Tumor cells were scraped from the dish, and 200 µL of reagent A supplemented with PMSF was added. The sample was vortexed vigorously for 10 s, and then placed in an ice bath for 15 min. Then, 10 µL of reagent B was added and the sample was centrifuged (15,000g, 5 s, 4 °C) to obtain the supernatant. Immediately, the supernatant was transferred into a pre-cooled EP tube. This is the cytoplasmic protein. The remaining supernatant was completely aspirated, and 50 µL of Nuclear Protein-Extracting Reagent was added. The sample was vortexed vigorously for 20 s. Finally, the sample was centrifuged (12,000g, 15 min, 4 °C) to obtain the supernatant. The supernatant was immediately transferred to a pre-cooled EP tube and this is the nucleoprotein.

Quantitative real-time PCR

Total RNA was extracted using Trizol, and then cDNA was generated using a commercial kit (Novoprotein, China). These procedures were followed as directed by the manufacturer. Next, a commercial kit (Novoprotein, China) was used to perform real-time PCR. The primer sequences are presented in Supplemental Table 2.

Enzyme-linked immunosorbent assay

Chemokines CCL5, CXCL9 and CXCL10 in the supernatants were analyzed by mouse RANTES ELISA kit (Lianke Bio, China) according to the manufacturer’s instructions.

Extraction of cell culture supernatant proteins

The extraction of cell culture supernatant protein was performed using the Liquid Sample Protein Extraction Kit (Solarbio, China). In brief, 100 μL of reagent A was added to 1 mL of the liquid sample. The sample was centrifuged (15,000g, 15 min, 4 °C) to obtain the sediment. 200 μL of reagent B was added to the centrifuge tube containing the sediment. The sample was centrifuged (15,000g, 15 min, 4 °C) to remove the supernatant. The precipitate was dissolved with 100 μL of reagent C and mixed thoroughly. The sample was centrifuged (10,000g, 10 min, 4 °C) to collect the supernatant for downstream experiments.

Cellular ATP detection

The following steps were taken to carry out the experiment using the Enhanced ATP Detection Kit (Beyotime, China). In brief, 100 µL of lysis solution was applied to each well of a 24-well plate. The cell lysate was centrifuged (12,000g, 5 min, 4 °C) to obtain the supernatant for further analysis. To create the standard curve, the ATP standard solution was diluted with the lysis solution. The ATP detection reagent was diluted at a ratio of 1:4. 100 µL of ATP detection working fluid was added to the detection well. After 5 min at room temperature, 20 µL of the sample or standard was quickly mixed in the detection well. The ELISA reader was used to measure the chemiluminescence value.

ScRNA-seq data quality control, integration and unsupervised clustering

We utilized Seurat [22] to process the scRNA-seq data and constructed the Seurat object for analysis. We defined and excluded “low-quality” cells from the original Seurat object based on the following criteria: ‘nFeature_RNA’ less than 200, ‘nFeature_RNA’ greater than 8000, and ‘percent.mt’ greater than 20. Subsequently, we merged the data from the F1929-1458 treated group and the control group, performing downstream analysis in accordance with the standard Seurat workflow.

We normalized the expression matrix using the NormalizeData function and identified highly variable genes with the FindVariableFeatures function. We then used the ScaleData function to scale the data matrix and the RunPCA method was used to perform PCA with the parameter features set to VariableFeatures(data). To integrate the data from the two samples, we used RunHarmony [23] with default parameters.

The appropriate number of principal components (pc.num) was determined through an ElbowPlot. We then constructed the shared nearest neighbor graph by utilizing the FindNeighbors function. Finally, we performed unsupervised clustering with the FindClusters function and applied RunTSNE or RunUMAP for dimensionality reduction and data visualization.

Gene ontology (GO) analysis

DEGs were find using FindMarkers function of Seurat package [22] with default parameters, and filtered by p_val < 0.05 and abs(avg_log2 FC) > 0.25. GO analysis was performed on the DEGs using enrichGO function of clusterProfiler package [24] (version 4.9.0.002), with parameters set as: OrgDb = ‘org.Mm.eg.db’, pvalueCutoff = 0.05, qvalueCutoff = 0.05. Important GO pathways were selected based on their GeneRatio_numeric values.

Statistical analysis

All data were analyzed and presented as means ± SEM. Student’s t-test was used to compare two groups, and a one-way ANOVA was used to compare several groups as part of the statistical study. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The term ‘ns’ was used to indicate that a result was not significant.

Results

An in vivo chemical screen identified F1929-1458 as a novel agent for eliminating stem cell tumors

Utilizing the stem cell tumor systems of the Ras^V12-transformed kidney stem cells [11,12,13], we adopted a 96-well format with some modification as previously described [25] and conducted in vivo chemical library screening (Fig. 1A). By screening over 9000 compounds, we identified F1929-1458 (Fig. 1B) that effectively eliminated the stem cell tumors once added to the fly food (Fig. 1C). To investigate the therapeutic effect of F1929-1458 on tumors in mammals, we selected CT26 tumor cells (mouse colorectal cancer cells) that highly express the surface markers CD44 and CD133 of CSCs [3] as a model for in vivo study (Fig. 1D). We found that treatment with the compound led to a downregulation of the expression of CD44 and CD133 (Fig. 1E). This suggests that the compound is capable of selectively influencing colorectal cancer stem cells. In BALB/c mice, we established a subcutaneous graft tumor model of colorectal cancer, and administered control (ddH₂O), F1929-1458 (5 mg/kg, 15 mg/kg) and the positive control (5-FU) to tumor-bearing mice by gavage. In comparison with the control, we found that F1929-1458 treatment dramatically reduced the volume and weight of the transplanted tumors (Fig. 1F–H), without affecting the weight of mice (Fig. 1I). Although 5-FU also demonstrated a good anti-tumor effect (Fig. 1F–H), the mice exhibited a significant decrease in body weight and one of them died at the end of the administration (Fig. 1I). Splenomegaly (enlarged spleen) has been identified as a stereotypical response to chronic systemic immune activation [26]. Hence, we measured spleen weight as an indicator of toxicity. The group treated with F1929-1458 did not exhibit splenomegaly compared with the control group (Fig. 1J, K), indicating no significant change in systemic inflammation. The traditional indicators of systemic inflammatory activation are serum levels of the proinflammatory cytokines TNF-α and IL-6. There were no significant changes in serum IL-6 and TNF-α in the F1929-1458-treated group compared to the control group (Fig. 1L, M). Together, these results indicate that F1929-1458 treatment can restrain stem cell tumor growth both in Drosophila and mammals. Moreover, F1929-1458 at a dose of 5 mg/kg and 15 mg/kg caused no systemic toxic effects in the tumor-bearing mice.

Fig. 1

An in vivo chemical screen identified F1929-1458 as a novel agent for eliminating stem cell tumors. A An in vivo chemical screening platform in Drosophila. B Structure of F1929-1458. C Ras^v12 mutated Drosophila intestinal stem cell tumors treated with DMSO or F1929-1458 (10 µM). Scale bars, 100 µm. D Expression profiles of CD133 and CD44 in CT26 cell lines characterized by flow cytometry. E The expression of CD44 and CD133 in both the group treated with the compound and the untreated group. F–I Negative control (ddH₂O), positive control (5-FU), and 5 mg/kg and 15 mg/kg doses of F1929-1458-treated colorectal cancer graft tumors. (n = 5). The relative tumor volumes (G). The relative tumor weights (H). Body weights of mice (I). J Representative spleens from control (ddH₂O), 5 mg/kg and 15 mg/kg doses of F1929-1458-treated tumor-bearing mice. K The spleen weight. L, M Systemic secretion of cytokines IL-6 (L) and TNF-α (M) in serum characterized by ELISA

The anti-tumor effect of F1929-1458 is mediated by enhancing T cell infiltration

Employing flow cytometry to analyze the infiltrating T cells in the tumors, we found that compared with the control, the infiltration of CD3+ T and CD3+CD8+ T cells in the tumors of the groups treated with F1929-1458 increased significantly. In contrast, the infiltration of CD3+CD8+ T cells in the tumors treated with the positive control, 5-FU, decreased significantly (Fig. 2A–C). In addition, there was a significant increase in the level of T cell activation (Fig. S1 A). Using immunofluorescence techniques, we also observed a significant increase in infiltrating CD3+ T cells in the tumor treated with F1929 −1458 in comparison with the control (Fig. 2D). However, the proportion of CD3+ T cells in peripheral blood mononuclear cells (PBMCs) have no significant difference between treated and untreated mice (Fig. 2E). To explore the involvement of T cells in the anti-tumor impact of F1929-1458, we conducted subcutaneous tumor transplantation experiments in nude mice and showed that F1929-1458 could not reduce the size of tumors in nude mice (Fig. 2F–H). To further functionally verify that the observed adaptive immune response was actually necessary for the anti-tumor effect of F1929-1458 in mice, we treated CT26 tumor-bearing BALB/c mice intraperitoneally with anti-CD3 antibody to deplete T cells after tumor formation (Fig. 2I). As expected, the significant inhibition of CT26 tumor growth by F1929-1458 treatment compared with control was significantly reversed by anti-CD3 antibody treatment (Fig. 2J–K). Colony formation assay and cytotoxicity experiments showed that F1929-1458 did not directly affect the survival of tumor cells and normal cells (Fig. S2 A–D). These data strongly suggest that T cell-dependent immune responses are responsible for the anti-tumor effects of F1929-1458.

Fig. 2

The anti-tumor effect of F1929-1458 is mediated by enhancing T cell infiltration. A–C FACS analysis of CD3+ T cells (B) and CD3+CD8+ T cells (C) in CT26 graft tumors treated with control (ddH₂O), 5 mg/kg and 15 mg/kg doses of F1929-1458 and positive control (5-FU). D Immunofluorescence staining and quantification of CD3+ T cells in CT26 graft tumors treated with control (ddH₂O) or F1929-1458 (n = 5). Scale bars, 25 μm. E The frequency of CD3 + T cells in peripheral blood mononuclear cells (PBMCs) from both treated and untreated mice. F The image of CT26 graft tumors in nude mice treated with control (ddH₂O) or F1929-1458 (n = 8). The relative tumor volumes (G). The relative tumor weights (H). I Neutralization of T cells in wild-type BALB/c mice by using an anti-CD3 antibody. J, K The tumor volumes (J) and the relative tumor weights (K) of CT26 graft tumors with indicated treatments

F1929-1458 treatment remodels the TME with superior anti-tumor activity

We next adopted single-cell RNA sequencing (scRNA-seq) to investigate the effect of F1929-1458 on the tumor immune microenvironment. We isolated 24,711 CD45+ immune cells and CD45− non-immune cells from F1929-1458 treated and untreated CT26 xenografts via FACS. To identify the main components of the tumor immune microenvironment, we performed unsupervised clustering, dividing both the F1929-1458 treated and untreated samples into 37 distinct cell clusters (Fig. 3A). By annotating these cells based on the expression of widely recognized immune and non-immune cell markers, we identified 12 cell types, including T cells, NK cells, dendritic cells (DCs), monocytes, macrophages, and fibroblasts (Fig. 3B, C). We then compared the proportions of immune cell types between the F1929-1458 treated and untreated groups. Notably, the T cell infiltration in the TME significantly increased in the F1929-1458 treated group, while the proportion of tumor-associated macrophages (TAMs) markedly decreased (Fig. 3D). Given that TAMs are known to have immune-regulating functions and facilitate tumor cell invasion [27, 28]. To further elucidate the changes in T cells following F1929-1458 treatment, we applied unsupervised clustering on all T cells and revealed 8 distinct cell clusters (Fig. 3E). Based on the expression of classic T cell function markers, we manually annotated these clusters as follows: CD8_T_Effector cells (expressing cytotoxicity genes), Tregs (expressing immune suppressive genes), T_Cycling cells (expressing cell cycle genes), T_Memory cells (Tcf7+), CD8_T_Naive cells (Ccr7+), NKT cells (Ncr1+), and T_gammadelta cells (Trdc+) (Fig. 3F, G). To profile the functional changes in T cells, we assessed the proportions of T cell subtypes. We discovered a notable increase in the percentage of CD8_T_Effector cells and a decrease in Tregs following F1929-1458 treatment (Fig. 3H). By performing flow cytometry analysis on the immune cells within the tumor, we obtained results that were consistent with those from the single-cell sequencing analysis (Fig. S1B–E). Additionally, the gene ontology (GO) analysis of differentially expressed genes in T cells showed that several pathways related to T cell-mediated anti-tumor immune responses were highly enriched in the F1929-1458 treated group. These pathways included T cell activation, defense response, cell killing, type II interferon production, and response to tumor cells (Fig. 3I). These results indicate that T cells were robustly stimulated by tumor-related factors and exhibited enhanced tumor-killing capabilities after F1929-1458 treatment.

Fig. 3

ScRNA-seq analysis revealing the effects of F1929-1458 on tumor immune microenvironment. A Unsupervised clustering of immune and non-immune cells in scRNA-seq data. B, C Manual cell annotation of immune and non-immune cells using selected cell type markers. D Bar plots showing the proportion changes of different types of immune cells between the F1929-1458 treated group and the control group. E Unsupervised clustering of T cells in scRNA-seq data. F, G Manual cell annotation of T cell subtypes using selected cell type markers. (H) Bar plots showing the proportion changes of T cell subtypes between the F1929-1458 treated group and the control group. (I) GO analysis of upregulated genes in T cells in the F1929-1458 treated group compared to the control group

In addition to T cells, the features and functions of other immune cells, including neutrophils, macrophages, monocytes, and natural killer (NK) cells were markedly altered following F1929-1458 treatment. To investigate the changes in neutrophils, we isolated the previously annotated neutrophil population and performed downstream analyses. Total neutrophils were categorized into four distinct clusters (Fig. S3 A) and further annotated into four cell types based on marker gene expression profiles (Fig. S3B-C). Cell proportion analysis revealed a notable increase in the enrichment of Cxcl10+ neutrophils and a corresponding decrease in Ccl4+ neutrophils in the F1929-treated group (Fig. S3D). According to previous studies, Ccl4+ neutrophils are a major subset of myeloid-derived suppressor cells (MDSCs) within the TME. These cells play a critical role in recruiting immunosuppressive macrophages and suppressing T cell function [29]. In contrast, Cxcl10 is a chemokine important for T cell recruitment [30]. These findings suggest that F1929-1458 treatment reprogrammed the neutrophil population, reducing their immunosuppressive characteristics while enhancing their ability to support T cell-mediated cytotoxicity.

We further investigated the changes in NK cells following F1929-1458 treatment. NK cells were isolated and categorized into six clusters based on downstream analysis (Fig. S4 A). Using the expression levels of selected marker genes, these clusters were annotated into four distinct cell types: Gzma + NK cells, Xcl1 + NK cells, proliferating NK cells, and Isg15 + NK cells (Fig. S4B-C). Notably, Xcl1 + NK cells were enriched in the F1929-1458 treated group (Fig. S4D). Xcl1 is known to promote the activation and migration of Xcr1 + cDC1 s, thereby enhancing cDC1-mediated antigen presentation and T cell activation [31]. To further explore the functional changes, we performed GO enrichment analysis on the upregulated genes in NK cells from the F1929-1458-treated group. This analysis identified several pathways associated with anti-tumor immune responses, including leukocyte cell–cell adhesion, T cell activation, and Ifng production (Fig. S4E). These findings suggest that NK cells contribute to the enhanced anti-tumor immune response observed after F1929-1458 treatment. This is achieved through both the recruitment and activation of cDC1 s and the stimulation of T cell responses.

Macrophages and monocytes are two major cell types in the TME. To investigate their roles in the anti-tumor immune response induced by F1929-1458 treatment, we isolated these cells and grouped them into 11 distinct clusters based on downstream analysis (Fig. S5 A). Based on previously reported marker genes [32, 33], we annotated the clusters into five cell types: Spp1 + TAMs, Cxcl9 + TAMs, Folr2 + TAMs, cycling myeloid cells, and monocytes (Fig. S5B-C). Macrophage polarization in the TME was characterized by the Cxcl9:Spp1 ratio, where a higher proportion of Cxcl9 + TAMs not only stimulated anti-tumor T cell immunity but also indicated an overall anti-tumor microenvironment [32]. Notably, scRNA-seq analyses showed that the proportion of cells with Cxcl9 + TAMs increased significantly in the F1929-1458-treated group, while the proportion of cells with Spp1 + TAMs decreased (Fig. S5D). To further characterize the changes in macrophages and monocytes, we used previously reported gene sets for “M1” pro-inflammatory macrophages and “M2” anti-inflammatory macrophages and quantified their features in the F1929-1458-treated and control groups [34]. Our analysis revealed that macrophages and monocytes in the F1929-1458-treated group exhibited higher expression of “M1” genes, while those in the control group showed elevated expression of “M2” genes (Fig. S5E). Therefore, macrophages and monocytes in the TME had greater abilities to recruit T cells and stimulate anti-tumor immunity.

Overall, these findings suggest that F1929-1458 can reprogram the tumor immune microenvironment by modulating immune cell functions, reducing its immune-suppressive characteristics and enhancing its cytotoxic and cell-killing functions.

F1929-1458 is an agonist of NFKB1

To define the target of F1929-1458 in tumor cells, we tagged F1929-1458 with biotin and performed small molecule pulldown-mass spectrometry experiments (repeated three times), and discovered that there were two candidate proteins, CDC73 and NFKB1 (Fig. 4A). We then purified CDC73 and NFKB1 proteins and performed isothermal titration experiments with small molecules in vitro, and showed that F1929-1458 bound to NFKB1 more strongly than to CDC73 (Fig. 4B). Database analysis using the SuperPred database further predicted that NFKB1 is a better target of F1929-1458 than CDC73 (Fig. S6). The cellular thermal shift assay and drug affinity experiments both demonstrated that F1929-1458 could effectively protect NFKB1 protein from degradation (Fig. 4C, D). Molecular docking further proves that F1929-1458 strongly binds to NFKB1 (Fig. 4E). The energy of the molecular docking is −7.18 kcal/mol, and the compound can enter the target protein’s binding region. The above data indicate that NFKB1 is the target of F1929-1458. Given that NFKB1 is one of the members of the NF-κB transcription factor family[35], We detected that the levels of p65 as well as phosphorylated p65 gradually increased (Fig. 4F) and more p65 and p50 accumulated in the nucleus (Fig. 4G) accompanying the increase of F1929-1458 concentration. Furthermore, NF-κB was reported to inhibit phosphorylation of JNK through MKK7 [36]. Consistent with this, we found that p-JNK was inhibited with the addition of F1929-1458 (Fig. 4H). These data together demonstrate that F1929-1458 is an agonist of the NF-κB signaling pathway by targeting NFKB1 in tumor cells.

Fig. 4

F1929-1458 targets the NFKB1 and activates NF-κB signaling pathway in CT26 cells. A Small molecule pull-down and mass spectrometry analysis. B Isothermal titration calorimetry experiment of CDC73, NFKB1 and F1929-1458. C Cell thermal shift assay to analyze the protective effect of F1929-1458 on NFKB1. D Drug affinity responsive assay to analyze the protective effect of F1929-1458 on NFKB1. E Molecular docking of NFKB1 and F1929-1458. F The protein levels of p50, p-p65, p65 in CT26 cells treated with F1929-1458 were detected by western blotting. G Nucleocytoplasmic separation to determine the nuclear entry of p50 and p65. H The phosphorylated JNK, JNK in CT26 cells treated with F1929-1458 was detected by western blotting

F1929-1458 treated tumors release factors to activate cGAS-STING and inflammasome signaling pathways in DCs

To investigate the reasons for the increased infiltration of T cells within the tumor, we adopted the co-culture of tumor cells and T cells in vitro (Fig. S7 A), and we discovered that F1929-1458 does not promote the migration of T cells towards tumor cells (Fig. S7B). Meanwhile, the mRNA levels of T cell-recruiting chemokines (CCL5, CXCL9, and CXCL10) did not show a significant difference between the control and F1929-1458 treatments (Fig. S7 C). Consistent with the findings from mRNA detection, by ELISA, no obvious increase was noted in the levels of these chemokines (Fig. S7D). These data indicate that F1929-1458 treatment might not directly promote the recruitment of T cells by tumor cells.

The activation of T cells usually requires the presentation of antigenic peptides by antigen-presenting cells and the stimulation of costimulatory factors [37]. DCs are the most prominent antigen-presenting cells in the mammalian immune system [38]. Therefore, we collected the supernatants of tumor cells treated with F1929-1458 or PBS for 24 h and used them to treat DC2.4 and BMDCs (Fig. 5A). Using western blotting, we found that the cGAS-STING signaling pathways were significantly activated in DC2.4 and BMDC after treatment with the supernatant of F1929-1458-treated tumor cells compared with those of PBS-treated tumor cells (Fig. 5B, C). Correspondingly, the mRNA levels of type I interferon IFNα and IFNβ significantly increased (Fig. 5D). In addition, the NLRP3 inflammasome signaling pathways were also significantly activated (Fig. 5E, F), and the transcription levels of interleukin-1β (IL-1β) significantly increased in the DCs (Fig. 5G). Chemokines CCL19/CCL21 secreted by lymph node stromal cells have been reported to act on CCR7 expressed by DCs and promote the migration of DCs to the T cell zone of the secondary lymphoid organs to initiate and regulate T cell-mediated acquired immune responses [39]. The expression of CCR7 in BMDCs and DC2.4 (Fig. 5H), and the expression of costimulatory factors CD80 and MHCII in BMDCs (Fig. 5I) were both upregulated after treatment with the supernatant of F1929-1458-treated tumor cells. Together, these findings suggest that F1929-1458 treatment promotes the release of molecules by tumor cells that stimulate the NLRP3 inflammasome signaling pathway and cGAS-STING signaling pathways in DCs, hence eliciting T cell-mediated anti-tumor immunity.

Fig. 5

F1929-1458 treated tumors release factors to activate cGAS-STING and inflammasome signaling pathways in DCs. A Experimental design for the co-culture system. B, C Western blot analysis of cGAS, phosphorylated and total STING, phosphorylated and total IRF3 in DC2.4 (B) and BMDCs (C) that were co-cultured with conditioned medium. H3 was used as a loading control. D RT-qPCR analysis of the expression of IFNα and IFNβ in DC2.4 that were treated with conditioned medium. E, F Immunoblotting analysis of NLRP3, pro-Caspase-1, c-Caspase1, pro-IL-1β, IL-1β in DC2.4 (E) and BMDCs (F) that were co-cultured with conditioned medium. Actin and H3 were used as loading controls. G RT-qPCR analysis of the expression IL-1β in DC2.4 that were treated with conditioned medium. H RT-qPCR analysis of the expression of CCR7 in DC2.4 and BMDCs. I The expression levels of CD80 and MHC-II in BMDCs treated with conditioned medium were detected by flow cytometry

The tumor released HMGB1-gDNA complex activates the cGAS-STING pathway

To identify factors secreted by tumor cells that activated the cGAS-STING signaling pathway in DCs, we performed transcriptome sequencing, which revealed that DNA damage and DNA damage repair pathways were significantly enriched in tumor cells treated with F1929-1458 (Fig. 6A). In addition, we detected that the DNA level in the supernatant of tumor cell culture was considerably elevated after F1929-1458 treatment (Fig. 6B). As a receptor of cytosolic DNA, cGAS binds to genomic DNA and mitochondrial DNA to activate the downstream STING signal [40]. In addition, F1929-1458 treatment also significantly increased the expression of proteins involved in cellular response to stress: G3BP1, a component of stress granules (Fig. S8 A) and HSP70 (Fig. S8B), which is a molecular chaperone involved in the formation of G3BP1 stress granules (Fig. S8 C) [41, 42]. This phenomenon indicate that F1929-1458 treatment induces cellular stress. Under stress, cells usually release damage-associated molecular patterns (DAMPs) to trigger immune response [43]. Consistent with this speculation, we found that HMGB1, one of the most prominent DAMPs, was abnormally localized in the cytoplasm (Fig. 6C), and its level was dramatically elevated in the supernatant of the F1929-1458-treated tumor cells (Fig. 6D). We purified extracellular vesicles and noticed that HMGB1 was secreted into the extracellular space in the form of micro-vesicles (Fig. 6E). By employing immunofluorescence, we observed that HMGB1 was colocalized with DNA and they were in the same vesicles extracellularly (Fig. 6F). Meanwhile, DNA was not colocalized with Tom20-labeled mitochondria (Fig. 6G). These findings suggest that HMGB1 and genomic DNA first formed a complex in cytoplasm and then were secreted to the supernatant via vesicles. Furthermore, activation of the cGAS-STING pathway in DCs was effectively inhibited after the addition of HMGB1 neutralizing antibody to the supernatant of the F1929-1458-treated tumor cells (Fig. 6H, I). The presented data indicate that the HMGB1 −gDNA complex released by tumor cells upon F1929-1458 treatment activates the cGAS-STING pathway in DCs.

Fig. 6

The tumor-released HMGB1-gDNA complex activates the cGAS-STING pathway. A Pathway enrichment analysis of transcriptome data. B Relative abundance of DNA in the supernatant of CT26 cells. C Immunofluorescence staining of HMGB1 in CT26 cells that were treated with or without F1929-1458. D Western blot analysis of HMGB1 in CT26 cells as well as in the supernatant. E Western blot analysis of HMGB1, CD9, CD63 and Annexin A1 in the micro-vesicles. F Immunofluorescence staining of HMGB1 and DNA in CT26 cells that were treated with or without F1929-1458. Scale bars, 10 μm. G Immunofluorescence staining of DNA and TOM20 in CT26 cells that were treated with F1929-1458. Scale bars, 10 μm. H, I Immunoblotting analysis of the indicated proteins in DC2.4 (G) and BMDCs (H) that were treated with conditioned medium in presence or absence of the anti-HMGB1 antibody

OxLDL and ATP released by tumor cells stimulate the NLRP3 inflammasome signaling pathway in DCs

We further explored molecular candidates released by tumor cells that activated the NLRP3 inflammasome signaling pathway in DCs. Recent studies have reported that ATP and oxidized lipids, as DAMPs, can activate the NLRP3 inflammasome signaling pathway in DCs [16, 44]. Through the analysis of lipidomics, we found that glycerophospholipids were significantly upregulated in tumor cells (Fig. 7A). Moreover, the secretion levels of both OxLDL and ATP by the tumor cells treated with F1929-1458 were also significantly elevated (Fig. 7B, C). We then used OxLDL antibody to neutralize OxLDL and Apyrase to hydrolyze ATP in the supernatant, respectively. We observed that the activation of the NLRP3 inflammasome signaling pathway was significantly downregulated after treatments of either the OxLDL neutralizing antibody or Apyrase (Fig. 7D). Consequently, the release of OxLDL and ATP from tumor cells triggers the activation of the NLRP3 inflammasome signaling pathway within DCs. Furthermore, treatment with JSH-23, the inhibitor of the NF-κB signal transduction pathway, significantly reduced the generation of ATP, OxLDL and HMGB1 in the F1929-1458-treated tumor cells (Fig. 7E–G). The NF-κB Inhibitor JSH-23 can abrogate the anti-tumor effect of F1929-1458 (Fig. 7H–J) and reduce CD3+ T and CD3+CD8+ T cells infiltration (Fig. 7K). It further proves that F1929-1458 exerts its anti-tumor effect by activating the NF-κB signaling pathway. These data together demonstrate that F1929-1458 treatment first induced cellular stress and activated the NF-κB signaling pathway in tumor cells, which then released gDNA-HMGB1, ATP and OxLDL, activating the cGAS-STING and NLRP3 inflammasome signaling pathway in DCs to promote T cell-mediated anti-tumor immunity.

Fig. 7

The tumor-released OxLDL and ATP activates NLRP3 inflammasome pathways in DCs. A Heatmap of different lipid fractions in the treated with or without F1929-1458 CT26 cells. B Western blot analysis of OxLDL in CT26 cells as well as in the supernatant. C Relative abundance of ATP level in CT26 cells supernatant with or without F1929-1458 treatment. D Immunoblotting analysis of the indicated proteins in BMDCs that were cocultured with F1929-1458 treated CT26 cells in presence or absence of both anti-OxLDL antibody and Apyrase. E Relative abundance of ATP level in the supernatant of CT26 cells in the presence or absence of NF-κB inhibitor JSH-23. F, G Western blot analysis of OxLDL and HMGB1 in CT26 cells and their supernatant with or without NF-κB inhibitor JSH-23 treatment. H The tumor images of CT26 allografts with the DMSO, F1929-1458 and simultaneous administration of F1929-1458 and JSH-23 (n = 8). I Tumor volume of CT26 allograft treated with indicated treatments. J Tumor weights of CT26 allograft treated with indicated reagents. K FACS analysis of the percentages of CD3+ T cells, CD3+CD8+ T cells in tumors with the indicated treatments

The anti-tumor activity of F1929-1458 and anti-PD-1 blocking is synergistic

Given that F1929-1458 can enhance the infiltration of T cells and immune checkpoint inhibitors can improve the viability of T cells, the combined use of the two may produce a synergistic effect. We then intraperitoneally injected anti-PD-1 antibody into CT26 xenograft tumor-bearing mice treated and not treated with F1929-1458 (Fig. 8A). When F1929-1458 and anti-PD-1 antibody were combined, the anti-tumor activity was much higher than when either agent was used alone (Fig. 8B–D), and the mice’s body weights were unchanged (Fig. 8E). Further flow cytometry analysis revealed that this combination significantly increased the infiltration of CD3+ and CD3+CD8+ T cells in tumor (Fig. 8F, G). The above results suggest that F1929-1458 and anti-PD-1 antibody have synergistic effects on tumor treatment. Moreover, we also test whether pre-treatment with the F1929-1458 enhances checkpoint inhibitor efficacy (Fig. S9 A). We found that pre-treatment with F1929-1458 could enhance the efficacy of anti-PD-1 therapy (Fig. S9B–D). Pretreatment with the compounds resulted in a significant increase in both infiltration and activation of CD8+ T cells (Fig. S9E–I). In addition, there was a decrease in the proportion of regulatory T cells (Fig. S9 J). Together, these results reveal that F1929-1458 treatment improves tumor sensitivity to anti-PD-1 therapy and that it could be an effective approach for overcoming tumor resistance to anti-PD-1 therapy.

Fig. 8

F1929-1458 and PD-1 blockade have a synergistic anti-tumor effect. A The experimental procedure for the combined use of F1929-1458 and PD-1 antibody. B The tumor images of CT26 allografts with the indicated treatments (n = 8). C Tumor volume of CT26 allograft treated with vehicle or the indicated reagents. D Tumor weights of CT26 allograft treated with vehicle or the indicated reagents. E Body weights of mice subcutaneously inoculated with CT26 cells and the indicated treatments. F, G FACS analysis of the percentages of CD3+ T cells (F), CD3+CD8+ T cells (G) in tumors with the indicated treatments

Discussion

The development of STING agonists in cancer immunotherapy has recently received widespread attention [45]. Like the cGAS-STING pathway, the NF-κB signaling pathway performs a pivotal function in the context of innate immunity in organisms [46]. In this study, we have demonstrated that F1929-1458 has the capacity to bind to NFKB1, thereby activating the NF-κB pathway. It has been proven that the activation of the NF-κB pathway can induce stress responses in tumor cells, which subsequently cause tumor cells to release DAMPs (such as the HMGB1-gDNA complex, ATP, and OxLDL). These DAMPs can respectively activate the cGAS-STING and NLRP3 inflammasome signaling pathways in DCs, generating type I IFNs and IL-1β inflammatory factors. These cytokines enhance T cell-mediated immune response against tumors by encouraging dendritic cell maturation, functional activity, and antigen presentation (Fig. 9).

Fig. 9

Schematic diagram of the mechanism by which F1929-1458 activates anti-tumor immunity. F1929-1458 binds NFKB1 to activate the NF-κB signaling pathway in tumor cells. The activation further induces cellular stress, causing tumor cells to release HMGB1-gDNA complex, ATP, and OxLDL, which in turn activate the cGAS-STING and NLRP3 inflammasome signaling pathways in DCs to produce type I IFNs and IL-1β. These cytokines together promote DCs maturation, activation and antigen presentation that ultimately enhance T cell anti-tumor immunity

Many candidate drugs identified in cell culture systems are inefficacious or toxic in patients due to in vivo physiological conditions or metabolic changes. Using whole-animal tumor models for in vivo screening would be the most straightforward way to find such drugs. However, due to time and expense constraints, such screening in mouse models is currently not feasible. Drosophila has made significant contributions to the field of cancer biology, elucidating the mechanisms of oncogenic signaling pathways such as EGFR, WNT, Notch and Hippo [47]. A bioinformatics-based exploration has revealed that up to 90% of the genes identified by the Cancer Genome Atlas as driving the development of human cancers have direct homologues in Drosophila [48]. These findings offer a robust theoretical foundation for utilizing the fly model in human cancer research, indicating that the fruit fly could play a distinctive and crucial role in dissecting cancer mechanisms and in the pre-screening during drug discovery and development. In this research, we carried out large-scale in vivo chemical screening using a Drosophila stem cell-like tumor model and identify compound F1929-1458, which can activate the systemic response in Drosophila to destroy stem cell-like tumors. By utilizing a mouse subcutaneous colorectal cancer transplantation tumor model enriched with CSCs, we first showed that F1929-1458 was effective in inhibiting tumor growth and enhancing the infiltration of T cells within the tumor.

To achieve an effective anti-cancer immune response, a series of stepwise events called “the Cancer-Immunity Cycle (CIC)’’ must be initiated and allowed to proceed iteratively [49, 50]. However, the immunosuppressive TME makes it difficult to turn on the CIC. The scRNA-seq results show that the infiltrated T cells, neutrophils, macrophages, monocytes and NK cells within the tumors in the F1929-1458 treatment group all develop in an immunostimulatory direction, indicating that F1929-1458 can remodel the TME and render the TME in an immunologically activated state. The onset of immune responses occurs in the T cell regions of secondary lymphoid organs. In these regions, naive T lymphocytes encounter DCs that have acquired antigens either in peripheral tissues or locally [51]. The antigen-presenting DCs in lymph nodes will stimulate naive T cell proliferation and differentiation [52]. Thus, DCs, which function at the interface between immunogenic antigens and T lymphocytes, are the key regulators of cellular immunity. In our work, the DAMPs-mediated activation of DCs is responsible for initiating anti-tumor immune responses.

We noticed that treatment with the compound at a low concentration (5 mg/kg vs. 15 mg/kg) yielded better anti-tumor effects. Similarly, previous literature has reported that administering a low dose of a STING agonist demonstrated better anti-tumor efficacy [53]. We postulate that the following factors might explain this. A high dose potentially leads to the compound interacting with other targets, giving rise to off-target effects and adverse reactions, thus nullifying the therapeutic benefits. Moreover, a relatively high dose could activate feedback loops within the biological system, consequently countering the drug’s actions. Furthermore, in future experiments, we will persist in investigating the underlying causes of this phenomenon. If small-molecule drugs can be effective against various types of tumors, their applicability in clinical practice will be significantly enhanced. In light of this, we administered the F1929-1458 to B16-F10 tumor-bearing mice. Regrettably, no significant differences in both size and weight were observed between the transplanted tumors of the control group and those of the drug-administered group. Correspondingly, there was also no notable increase in T cell infiltration within the melanoma xenografts of the drug-administered group. Given that F1929-1458 was initially screened in a Drosophila stem cell-like tumor system, we speculate that it might be a type of small molecule drug that exhibits higher sensitivity towards CSCs. To verify this speculation, we will subsequently carry out relevant verification work using more types of tumor models, with the aim of further enhancing the universality of F1929-1458 in anti-tumor immunological applications.

Conclusion

Our study has identified a novel NFKB1 agonist that promotes anti-tumor immunity by remodeling the tumor microenvironment and activating dendritic cells in colorectal cancer. Moreover, the combination of F1929-1458 and anti-PD-1 antibody had a synergistic anti-tumor effect. Thus, the novel agonist of the NF-κB pathway may provide a new agent for overcoming resistance to the current anti-tumor immunotherapy.