Spautin-1 inhibits the growth of diffuse large B-cell lymphoma by inducing mitochondrial damage-mediated PANoptosis and anti-tumor immunity

Abstract

The prognosis of relapsed or refractory diffuse large B-cell lymphoma (DLBCL) is poor. Therefore, searching for new therapeutic agents is particularly important to improve therapeutic efficacy. Targeting the ubiquitin-proteasome system is a potential therapeutic strategy for treating DLBCL. In this study, we investigated the role of the deubiquitase inhibitor Spautin-1 in the treatment of DLBCL. Spautin-1 significantly inhibited the growth of DLBCL, including the growth of cells and transplanted tumors in mice. Notably, Spautin-1 showed enhanced tumor suppression in immune-competent versus immunodeficient mice models, mediated by CD8⁺ T cell infiltration and activation. Mechanistically, Spautin-1 promotes PANoptosis by inducing mitochondrial damage mediated by USP13-ACLY inhibition in DLBCL cells, which in turn induces the release of injury-related molecular patterns and cytokines. Moreover, high USP13 expression in DLBCL is associated with poor prognosis and blocks CD8⁺ T cell infiltration. In summary, Spautin-1 may inhibit the growth of DLBCL cells by promoting mitochondrial damage-mediated PANoptosis and anti-tumor immunity, providing a potential strategy for DLBCL therapy.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-025-04150-9.

Introduction

Diffuse large B-cell lymphoma (DLBCL) is a common lymphohematopoietic malignancy and the most common subtype of non-Hodgkin lymphoma, accounting for 30–40% of adult non-Hodgkin lymphomas [1]. The World Health Organization (WHO) classifies DLBCL into three molecularly distinct subtypes based on cell-of-origin transcriptomic signatures: germinal center B-cell-like (GCB), activated B-cell-like (ABC), and unclassifiable. Crucially, the ABC subtype exhibits a significantly inferior prognosis compared to the GCB subtype owing to its molecular complexity and therapy-resistant phenotypes [2]. The first-line treatment of DLBCL in the past 20 years has been the R-CHOP regimen [3]. First-line induction therapy with R-CHOP can cure approximately 60–70% of patients with DLBCL [3]. However, nearly one-third of patients relapse within a short period post-treatment or even develop tumor progression during treatment, with an extremely poor prognosis. Therefore, it is particularly important to identify new targets to improve the therapeutic effects in DLBCL.

The ubiquitin-proteasome system is a highly specific and selective protein degradation pathway that consists mainly of proteasomes, ubiquitinases, and deubiquitinases (DUBs) [4].Their dysfunction is closely linked to the onset and progression of malignant tumors [5]. The addition of ubiquitin-proteasome inhibitors, such as lenalidomide and bortezomib, to R-CHOP can improve disease control rates in DLBCL [6, 7]. DUBs mediate specific removal of ubiquitin chains from substrate proteins and maintain proteostasis through precise modulation of ubiquitin cycle dynamics [4]. In addition to regulating embryonic development, immune homeostasis, and neural function, DUBs directly modulate core oncogenic pathways including proliferation, angiogenesis, invasiveness and metastasis [8–10]. Therefore, DUB inhibition represents a novel therapeutic strategy for DLBCL treatment.

Spautin-1, a DUB inhibitor of USP10 and USP13, can inhibit tumor progression by modulating multiple biological functions, such as autophagy, apoptosis, glycolytic reprogramming, and DNA damage repair [11–15]. DUBs potentially suppress anti-tumor immunity and targeting DUBs may reverse the immunosuppressive tumor microenvironment [16, 17]. However, the inhibitory effect of Spautin-1 on cell growth and its anti-tumor immune regulation in DLBCL remain unclear.

In this study, we systematically investigated the functional impact of Spautin-1 on DLBCL growth and anti-tumor immunity and its potential molecular mechanism, aiming to clarify its value in DLBCL treatment.

Materials and methods

Chemicals, reagents and antibodies

Spautin-1 (HY-12990), MG-132 (HY-13259), JC-1 (HY-15534), and MitoSOX Red (HY-D1055) were purchased from MedChemExpress (Shanghai, China). Anti-USP13 (16840-1-AP, 1:2000 dilution), anti-CD4 (67786-1-Ig, 1:200 dilution), anti-CD8 (66868-1-Ig, 1:200 dilution), anti-PCNA (60097-1-Ig, 1:200 dilution), anti-calreticulin (27298-1-AP, 1:200 dilution), anti-RIPK1 (29932-1-AP, 1:2000 dilution), anti-RIPK3 (CL488-17563, 1:1000 dilution), anti-MLKL (21066-1-AP, 1:1000 dilution), anti-p-MLKL (82090-2-RR, 1:1000 dilution), anti-Caspase3 (19677-1-AP, 1:1000 dilution), anti-Bcl2 (12789-1-AP, 1:1000 dilution), anti-GSDMD (CL488-20770, 1:1000 dilution), anti-ACLY (15421-1-AP, 1:2000 dilution), anti-GAPDH (10494-1-AP, 1:5000 dilution) and anti-Tubulin (80713-1-RR,1:5000 dilution) were bought from Proteintech Group (Wuhan, China). The TUNEL Cell Apoptosis Detection Kit) was purchased from Servicebio (Wuhan, China). Human (H1554c) and mouse (M0676c) HMGB1 enzyme linked immunosorbent assay (ELISA) Kits were purchased from Elabscience (Wuhan, China). Mouse IFN-γ (EK280HS), IFN-β (EK2236), granzyme B (EK2173), IL-1β (EK201BHS), IL-18 (EK218), and human IL-1β (EK101B) and IL-18 (EK118) ELISA Kits were purchased from MultiSciences (Hangzhou, China). The ROS assay kit (S0033S) was purchased from Beyotime (Shanghai, China). The USP13-overexpressing lentiviral cells were purchased from Obio Technology (Shanghai, China). A DLBCL Tissue Microarray (OD-CT-LyMly02-001) was obtained from the Odo Biotechnology Company (Shanghai, China). Ethical approval was granted by the company's Ethics Committee (ethical number: SHYJS-BC-2310001).

Cell culture

Human B lymphoma cell lines (SU-DHL-4, SU-DHL-6, OCI-Ly3, and OCI-Ly10) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Mouse B lymphoma cell line (A20) was purchased from the Institute of Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China). These cells were cultivated in RPMI-1640 medium (KeyGEN, China) supplemented with 10% foetal bovine serum (Gibco, USA) and cultured in a humidified incubator with 5% CO² at 37 °C.

Cell proliferation analysis

Cell proliferation was quantified via CCK-8 (MedChemExpress, Shanghai, China). The cells were plated in 96-well plates, treated with the specified concentration of drugs for 24 or 48 h, then incubated with CCK-8 solution for 37 ℃ for 2 h, and absorbance measurements at 450 nm were conducted using a microplate reader (Bio-Tek Instruments, USA).

Animal experiment

Female BALB/c nude and wild-type mice (5 weeks old) were obtained from Shanghai Lab. Animal Research Center, with all experimental protocols strictly adhering to the Guidelines for the Care and Use of Laboratory Animals. The research protocol was approved by the Ethics Committee of the Affiliated Huaian No.1 People's Hospital of Nanjing Medical University (ethical number: DW-Y-2024-025-01). Mice were subcutaneously injected with A20 cells (100 μL PBS, 1 × 10⁷ cells). When tumor volume reached 50 mm³, daily intraperitoneal injections of Spautin-1 (20 mg/kg) were administered. Tumor dimensions (longest/shortest axes) were recorded with a precision caliper, and volume was calculated computed via the 0.5 × longest × shortest² formula. Excised tumor specimens were processed including tissue lysis, RNA extraction, and histopathological preparation.

Histological analysis

Tumor tissues were processed through formalin fixation and paraffin embedding. Deparaffinized sections were histochemically stained with hematoxylin and eosin (H&E; Beyotime) using standard protocols, followed by microscopic evaluation (Olympus, Lake Success, USA) of tissue structure and cell morphology.

TUNEL assay

Apoptosis in both in vitro cultured cells and paraffin-embedded specimens was assessed using the TUNEL detection kit. Stained specimens were photographed using a fluorescence microplate reader (Bio-Tek Instruments) and a fluorescence microscope (Olympus).

Immunofluorescence

Following graded ethanol rehydration, tumor sections underwent 0.5% Triton X-100 in PBS permeabilization and 5% BSA blocking. Primary antibody was proceeded overnight at 4 °C prior to fluorophore-conjugated secondary antibody incubation. DAPI nuclear counterstaining (Beyotime) was followed by multispectral fluorescence imaging (Olympus).

Immunohistochemistry (IHC) assay

Paraffin-embedded tumor sections were hydrated with gradient ethanol, accompanied by endogenous peroxidase inactivation and antigen exposure, and stained using an IHC detection kit (Proteintech) according to the manufacturer's instructions. Histological images were acquired at 400-fold magnification using a light microscope (Olympus). A semi-quantitative assessment of the histochemical score of protein expression was conducted using ImageJ software (USA).

ELISA

ELISA kits were used to quantify the protein expression in cellular supernatant or tumor lysates, according to standardized protocols. Absorbance was measured at 450 nm, and the protein expression relative to the protein concentration was calculated for each sample.

Quantitative real-time polymerase chain reaction (qPCR)

Total RNA was extracted from the tissues using TRIzol reagent (TaKaRa, Japan) and reverse transcription was performed. Quantitative analysis was performed using a Real-Time PCR assay system (Roche, California, USA) with SYBR Green Master Mix (MCE, USA). Experimental replicates were conducted in triplicate with relative quantification performed using the 2^−ΔΔCt method. The primer sequences are detailed in Supplementary Table S1.

RNA-seq analysis

Total RNA extraction, library preparation, and RNA sequencing were performed using Applied Protein Technology (Shanghai, China). Each experiment was repeated thrice. The DEGseq algorithm was used to analyse the differentially expressed genes (DEGs) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment were performed.

Transmission electron microscopy (TEM)

SU-DHL-6 cells were treated with Spautin-1, and collected for fixation, dehydration, osmosis, inclusion polymerisation, ultrathin sectioning, and uranium and lead staining. Mitochondrial morphology was observed using a Hitachi HT7700 transmission electron microscope (Tokyo, Japan).

Reactive oxygen species (ROS) detection

Cellular ROS and mitochondrial ROS were quantified using the DCFH-DA Kit and MitoSOX Red Kit respectively, following standardized manufacturer protocols. The relative fluorescence units (RFU) of each sample were determined using a fluorescence microplate reader (Bio-Tek Instruments).

Detection of mitochondrial membrane potential

Mitochondrial membrane potential was quantified via JC-1 probe, with signal acquisition performed on a fluorescence microplate reader (Bio-Tek Instruments).

Membrane calreticulin detection

SU-DHL-6 cells were then incubated with anti-calmodulin and fluorescent secondary antibodies. RFU was detected using Bio-Tek Instruments, and the level relative to the cell content was calculated.

Western blotting

Cellular lysates were prepared using ice-cold RIPA lysis buffer (Beyotime) supplemented with a protease inhibitor mixture (Vicmed, China). Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred onto PVDF membrane (Millipore). Membranes were blocked with 5% milk in TBST and incubated with the antibody at 4°C overnight. Following horseradish peroxidase-conjugated secondary antibody incubation, chemiluminescent signals were detect using an ECL kit (Proteintech).

Bioinformatics analysis

The GEPIA database (http://gepia.cancer-pku.cn/detail.php) was used to analyse USP10 and USP13 expression in the DLBCL-TCGA dataset and normal lymphoid tissues derived from the GTEx dataset [18]. Gene Expression Omnibus (GEO) datasets (https://www.ncbi.nlm.nih.gov/geo/) were used to analyse the expression pattern of USP13 in DLBCL tissues and normal lymphoid tissues, the differential expression of USP13 in ABC and GCB subtypes, and its prognostic implication [19]. X-tile bioinformatics software (version 3.6.1) was used to determine the cutoff value for predicting survival [20].

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 (version 8, San Diego, USA). Experimental data are presented as mean ± standard error of the mean. Group comparisons employed Student's t-test or ANOVA analysis, while variable correlations were determined through linear regression. Statistical significance was defined as P < 0.05.

Results

Spautin-1 inhibits cell growth of DLBCL

We explored the cytostatic action of spautin-1 on DLBCL cell lines (SU-DHL-4, SU-DHL-6, OCI-Ly3, OCI-Ly10, and A20) and found that all five cell lines exhibited a dose- and time-dependent viability reductions upon Spautin-1 treatment (Fig. 1a–e). A mouse DLBCL xenograft model was constructed using A20 cells to evaluate the anti-tumor effect of Spautin-1 in vivo. The growth of tumor xenografts and weights were significantly inhibited by Spautin-1 (Fig. 1f–h). No treatment-related mortality occurred throughout the study duration, with comparable bodyweight trajectories maintained between experimental groups (Fig. 1i). In addition, H&E staining showed that Spautin-1 induced tumor cell shrinkage, fragmentation, and disorganized tissue architecture (Fig. 1j). IHC analysis revealed decreased PCNA expression in the Spautin-1-treated tumors compared to controls (Fig. 1k). These results demonstrate that Spautin-1 could inhibit the growth of DLBCL cells.

Fig. 1.

Spautin-1 inhibits the cell growth of DLBCL both in vitro and in vivo. a–e Cell viability of SU-DHL-4, SU-DHL-6, OCI-LY3, OCI-Ly10, and A20 cell lines detected by a CCK-8 kit after Spautin-1 treatment for the indicated time. Each group of the experiment was replicated three times. f–i The transplanted tumor model of BABL/c nude and BABL/c mice was established using A20 cells, Spautin-1 (20 mg/kg) was injected intraperitoneally from the fifth day. The growth of transplanted tumors in each group f; the volume of the transplanted tumor g; quality of the transplanted tumor h; body weight of mice i. BABL/c mice grafted tumors were sectioned for relevant staining. j H&E staining evaluated tissue morphology. Scale bar, 100 μm. k PCNA staining evaluated tumor proliferation. Scale bar, 100 μm. The bar chart shows the difference in the H-score of PCNA between the two groups. *P < 0.05, **P < 0.01

Spautin-1 promotes anti-tumor immunity by inducing infiltration and activation of CD8⁺ T cells

We further observed that the tumor volume inhibitory effect of Spautin-1 in BALB/c mice was more significant than that in BALB/c nude mice (57.17% vs. 41.79%, Fig. 1d–f), suggesting that Spautin-1 could inhibit tumor growth partly by inducing anti-tumor immunity. Thus, we analyzed the immune cells in transplanted tumors in mice and found that Spautin-1 treatment induced the infiltration of CD3⁺/CD4⁺ and CD8⁺ T cells into the tumor tissues (Fig. 2a, b). Subsequently, we detected the expression of cytotoxic lymphocyte markers IFN-γ and granzyme B in tumor tissues using ELISA, and discovered that the production of both markers was significantly upregulated in tumor tissues treated with Spautin-1 (Fig. 2c, d). In addition, Spautin-1 induced the mRNA expression of CCL5, CXCL9 and CXCL10, which are chemokines for T cells (Fig. 2e–g). The above results demonstrate that Spautin-1 promotes the infiltration and activation of T lymphocytes in tumors and then generate anti-tumor immunity.

Fig. 2.

Effect of Spautin-1 on lymphocyte infiltration and activation in tumor tissues. a, b BABL/c mice grafted tumors were sectioned for CD3⁺/CD4⁺, and CD8⁺ T cells staining; CD3 staining (green), CD4/CD8 staining (red) and DAPI nuclear staining (blue). Scale bar, 100 μm. c, d The protein levels of IFN-γ and granzyme B in tumor tissues were determined using ELISA. e–g The mRNA expression levels of CCL5, CXCL9, and CXCL10 in A20 tumor tissues determined using qPCR. The data represent the mean ± SEM of three mice per group. *P < 0.05, **P < 0.01

Spautin-1 induces PANoptosis of DLBCL cells

To clarify the mechanism by which Spautin-1 regulated tumor growth and anti-tumor immunity, we performed RNA-seq analysis on Spautin-1 treated SU-DHL-6 cells. Comparative transcriptomic profiling delineated intergroup differentially expressed gene (DEG) sets through statistical thresholding (|fold change| > 1, and P < 0.05). There were 1319 downregulated genes and 1734 upregulated genes (Fig. 3a, b). Subsequently, KEGG enrichment analysis indicated that Spautin-1 might regulate apoptosis and necroptosis (Fig. 3c). PANoptosis is an inflammatory programmed cell death with the main characteristics of pyroptosis, apoptosis, and necroptosis, which can suppress tumor progression and mediate anti-tumor immunity [21]. Therefore, we explored the effect of Spautin-1 on PANoptosis in DLBCL cells. We found that Spautin-1 significantly increased TUNEL staining intensity in DLBCL cells (Fig. 3d–h). Spautin-1 elevated the necroptosis-related protein expression of RIPK1, RIPK3, MLKL, and p-MLKL, and the pyroptosis executor GSDMD. Simultaneously, we examined the expression of apoptosis-related proteins and discovered that Caspase 3 increased and Bcl2 decreased in DLBCL cells after Spautin-1 treatment (Fig. 3i, j). Furthermore, Spautin-1 induced an increase in TUNEL-positive cells, enhanced the protein expression of RIPK1, and decreased the protein expression of Bcl2 in the transplanted tumors (Fig. 3k, l). These results demonstrate that Spautin-1 inhibits cell growth by inducing PANoptosis in DLBCL cells.

Fig. 3.

Effect of Spautin-1 on PANoptosis of DLBCL cells. a SU-DHL-6 cells were treated with Spautin-1 (10 μM) for 24 h, and then RNA-seq analysis was performed. Volcano plot showed the distribution of differentially expressed genes between the two groups. b Heatmaps showing the differentially expressed genes of two groups. c KEGG enrichment analysis of differential genes. d–h SU-DHL-4, SU-DHL-6, OCI-Ly3, OCI-Ly10, and A20 cell lines were treated with Spautin-1 (3, 10, and 30 μM) for 24 h. Apoptosis was determined by TUNEL staining and the RFU was detected using a fluorescence microplate reader. i, j SU-DHL-6, and OCI-Ly3 cell lines were treated with Spautin-1 (3, 10, and 30 μM) for 24 h. The PANoptosis-related protein expression of RIPK1, RIPK3, MLKL, p-MLKL, Caspase3, Bcl2, and GSDMD was detected by western blotting. k TUNEL staining evaluated tumor apoptosis, TUNEL staining (green), DAPI nuclear staining (blue). Scale bar, 100 μm. l The protein expression of RIPK1 and Bcl2 in transplanted tumors treated with Spautin-1 was detected using IHC. Scale bar, 100 μm. The bar chart shows the difference in H-scores of RIPK1 and Bcl2 between the two groups. *P < 0.05, **P < 0.01

Spautin-1 induces mitochondrial damage and release of damage-associated molecular patterns (DAMPs) and cytokines release in DLBCL cells

Mitochondria play an important role in orchestrating cell death, and their abnormal dynamics can trigger PANoptosis [21]. Therefore, we investigated whether Spautin-1 has a regulatory effect on mitochondria function in DLBCL cells. TEM demonstrated mitochondrial cristae membrane discontinuities concomitant with a reduction in cristae volume in Spautin-1-treated DLBCL cells (Fig. 4a). Compared with the control group, Spautin-1 elevated the accumulation of total intracellular and mitochondrial ROS in DLBCL cells (Fig. 4b, c). Analysis of mitochondrial membrane potential showed that Spautin-1 induced mitochondrial depolarisation (Fig. 4d). PANoptosis has a considerable effect on the tumor microenvironment and is mainly related to the release of DAMPs and inflammatory cytokines [22, 23]. Then we found that Spautin-1 treatment induced extracellular secretion of HMGB1, IL-1β, and IL-18, along with membrane translocation of calreticulin (Fig. 4e–h). Additionally, Spautin-1 treatment upregulated the expression of ROS, HMGB1, IFN-β, IL-1β, and IL-18 in transplanted tumor tissues (Fig. 4i–m). These results suggest that Spautin-1 promotes PANoptosis via mitochondrial damage, and mediates the release of DAMPs and cytokines in DLBCL cells.

Fig. 4.

Effect of Spautin-1 on the mitochondrial damage and release of DAMPs and cytokines in DLBCL cells. a SU-DHL-6 cells were treated with Spautin-1 (10 μM) for 12 h, mitochondrial morphology was observed using TEM. Scale bar, 5 μm. b SU-DHL-6 cells were treated with Spautin-1 (3, 10, and 30 μM) for 12 h, DCFH-DA staining was used to detect intracellular total ROS, the RFU was detected using a fluorescence microplate reader. c SU-DHL-6 cells were treated with Spautin-1 (3, 10, and 30 μM) for 3 h, MitoSOX staining was used to detect mitochondrial ROS, the RFU was detected using a fluorescence microplate reader. d SU-DHL-6 cells were treated with Spautin-1 (3, 10 and 30 μM) for 3 h, mitochondrial membrane potential was measured using a fluorescence microplate reader after staining with the JC-1 probe. e SU-DHL-6 cells were treated with Spautin-1 (3, 10, and 30 μM) for 24 h, the RFU of membrane-exposed calreticulin was detected using a fluorescence microplate reader. f–h SU-DHL-6 cells were treated with Spautin-1 (3, 10, and 30 μM) for 24 h, the HGMB1, IL-1β, and IL-18 proteins in cell supernatant were determined using ELISA. BABL/c mice graft tumors were used for the following assays: i Fenton reaction was used to detect ROS level; and j–m ELISA was used to detect protein levels of HMGB1, IFN-β, IL-1β, and IL-18. *P < 0.05, **P < 0.01

Upregulated USP13 associates with a poor prognosis and reduced CD8⁺ T cell infiltration in DLBCL

As USP10 and USP13 are drug targets for Spautin-1 [24], we explored the expression of both genes in DLBCL. Bioinformatics analysis showed that USP13 transcript levels, but not USP10 transcripts, were higher in TCGA DLBCL tissue than those in normal lymphoid tissue from GTEx data and were closely related to tumor staging (Fig. 5a–d). GEO dataset analysis identified marked overexpression of USP13 in DLBCL samples versus normal lymphoid tissues (Fig. 5e, f), and the expression of USP13 in the ABC subtype was higher than that in the GCB subtype (Fig. 5g, h). Subsequently, survival analysis revealed that high USP13 expression was closely associated with poor overall survival in DLBCL patients (Fig. 5i, j). Furthermore, CD8⁺ T cell infiltration was reduced in DLBCL tissues with high USP13 expression (Fig. 6a, b). Correlation analysis showed that USP13 expression was significantly negatively correlated with CD8⁺ T cell infiltration (Fig. 6c). These data suggest that USP13 is significantly overexpressed in DLBCL and associated with poor prognosis and low CD8⁺ T cell infiltration.

Fig. 5.

USP13 is overexpressed in DLBCL and negatively correlated with prognosis. a–d The transcription levels of USP10 and USP13 in DLBCL and normal lymph tissues and their correlation with clinical stage were analysed using the GEPIA database, which contains TCGA-DLBCL data and normal lymphoid tissues from GTEx data. e, f GEO (GSE32018, and GSE56315 dataset) analysis of USP13 transcription levels in DLBCL and normal lymph tissues. g, h GEO (GSE23501, and GSE32918 dataset) analysis of USP13 transcription levels in the ABC and GCB subtypes DLBCL. i, j Kaplan–Meier curve was used to explore prognostic significance USP13 in DLBCL using the GSE32918 and GSE53786 dataset. X-tile software was used to determine the cutoff value. *P < 0.05, **P < 0.01

Fig. 6.

USP13 is inversely associated with CD8⁺ T cell infiltration in DLBCL. a USP13 protein (green) and CD8⁺ T cells (red) in DLBCL were measured using immunofluorescence. Scale, 100 μm. b DLBCL tissues were divided into low and high USP13 expression groups by median expression intensity, and CD8⁺ T cell infiltration rates between the two groups were analysed. c Correlation analysis between UP13 expression and CD8⁺ T cell infiltration. *P < 0.05, **P < 0.01

Spautin-1 suppresses ACLY protein expression in DLBCL cells

USP13 regulates ACLY to participate in mitochondrial energy metabolism, leading to decreased ATP synthesis and increased ROS production, resulting in mitochondrial damage [25–27]. We demonstrated that Spautin-1 downregulated the expression of ACLY proteins without inhibiting their mRNA expression (Fig. 7a, b). We then used MG-132 to block ubiquitinated protein degradation and found that MG-132 rescued ACLY downregulation in Spautin-1-treated DLBCL cells (Fig. 7c). Meanwhile, USP13 overexpression rescued the Spautin-1-induced downregulation of ALCY protein in DLBCL cells, effectively reversing subsequent mitochondrial damage, and counteracting cell growth inhibition and apoptosis induction (Fig. 7d–g). In addition, IHC showed that Spautin-1 mediated the reduction of ACLY protein expression in A20 transplanted tumor tissues (Fig. 7h). These data indicate that Spautin-1 induces mitochondrial damage by inhibiting the USP13-ACLY axis, thereby suppressing DLBCL growth.

Fig. 7.

Spautin-1 regulates ACLY protein expression in DLBCL. SU-DHL-6 cells were treated with Spautin-1 (3, 10, and 30 μM) for 24 h. a The protein expression of ACLY was detected using western blotting. b The mRNA expression of ACLY was detected using qPCR. c ACLY protein expression in SU-DHL-6 cells treated with Spautin-1 (10 μM) or/and with MG132 (5 μM) was detected using western blotting. d ACLY protein expression in USP13 overexpression and control SU-DHL-6 cells treated with Spautin-1 (10 μM) was detected using western blotting. e USP13 overexpression and control SU-DHL-6 cells treated with Spautin-1 (10 μM) for 3 h. MitoSOX staining was used to detect mitochondrial ROS; the RFU was detected using a fluorescence microplate reader. f, g USP13 overexpression and control SU-DHL-6 cells treated with Spautin-1 (10 μM) for 24 h. f Cell viability was detected by CCK-8 assay. g Apoptosis was determined by TUNEL staining and the RFU was detected using a fluorescence microplate reader. h The protein expression of ACLY in transplanted tumors treated with Spautin-1 was detected using IHC. Scale bar, 100 μm. The bar chart shows the difference in H-score of ACLY between the two groups. i Graphic illustration of the mechanism by which the deubiquitase inhibitor, Spautin-1, inhibits DLBCL growth. *P < 0.05, **P < 0.01

Discussion

DLBCL is a group of diseases with heterogeneous morphology, genetics, and clinical manifestations [1]. Although initial treatment achieves cure in most patients, refractory cases demonstrate a poor prognosis, with the ABC subtype exhibiting significantly worse clinical outcomes than the GCB subtype [3, 6]. Therefore, there is an urgent need for new therapeutic agents to overcome this challenge and improve the therapeutic efficacy for DLBCL. In this study, as shown in Fig 7i, Spautin-1 inhibits the growth of DLBCL by inducing mitochondrial damage-mediated PANoptosis and anti-tumor immunity.

USP inhibitors have shown promise in targeting cancer pathways, regulating immune responses, and reducing protein aggregation. The candidate drugs, including VLX1570 and b-AP15, have demonstrated preclinical anti-tumor activity in refractory cancers (especially multiple myeloma), and early trials have indicated that they can enhance therapeutic effects [28]. Previous studies have shown that Spautin-1 can induce tumor cell death by degrading mutant TP53/p53 [29], and sensitise the anti-tumor activity of cisplatin by inhibiting GSK3β-ULK1 axis [11]. Our research indicated that Spautin-1 inhibited DLBCL growth both in vivo and in vitro and exerted effects on both GCB and ABC subtypes. Recently, the role of DUBs in the regulation of anti-tumor immunity through various mechanisms has been revealed. USP5, USP7, USP22, and USP9X regulate the stability of PD-L1 proteins, whereas USP8 and USP14 induce T-cell dysfunction, thereby blocking anti-tumor immunity [30–34]. In our study, we found that Spautin-1 partially inhibits DLBCL growth by inducing anti-tumor immunity. CD8⁺ T cells are end-effector cells that play an anti-tumor immune response in the tumor microenvironment [35]. We also found that Spautin-1 treatment increased the infiltration and activity of CD8⁺ T cells in transplanted tumor tissues. The above results indicate that Spautin-1 not only directly inhibits cell growth but also induces the infiltration and activation of CD8⁺ T cells, thereby exerting immune anti-tumor effects. Notably, its efficacy in ABC subtype DLBCL, which is characterised by therapeutic resistance, suggests potential to overcome the current treatment bottlenecks. The observation of recruitment and activation of CD8⁺ T cells suggests that Spautin-1 can be a rational combotherapy candidate to sensitise "immune-cold" DLBCL to anti-PD-1/CTLA-4 regimens.

Spautin-1 is a specific autophagy inhibitor that targets the deubiquitinating enzymes USP10 and USP13 [36]. Bioinformatics analysis demonstrated that USP13, but not USP10, was highly expressed in DLBCL, especially in the ABC subtype, and was associated with inferior overall survival. In addition, elevated USP13 expression exhibited inverse correlation with CD8⁺ T cell infiltration in DLBCL tissues. Overexpression of USP13 blocked cell growth inhibition mediated by Spautin-1. Future investigations should establish whether Spautin-1's anti-tumor effects are mediated through the direct inhibition of USP13 deubiquitinase activity via integrated biochemical, structural, and genetic rescue analyses.

PANoptosis is a unique inflammatory cell death pathway that combines primary features of pyroptosis, apoptosis, and necroptosis [37]. Based on RNA-seq analysis, we found that the differentially expressed genes after Spautin-1 treatment were enriched in the apoptotic and necroptotic pathways. Spautin-1 increased the number of TUNEL-positive DLCBL cells and regulated the expression of pyroptosis-, apoptosis-, and necroptosis-related proteins. When tumor cells undergo pyroptosis and necroptosis, cells release DAMPs and cytokines, such as IL-1β and IL-18, which affect the tumor microenvironment [38, 39]. Among them, DAMPs induce dendritic cell maturation, CD8⁺ T activation, and IFN-γ production [40, 41] , while IL-1β and IL-18 promote the proliferation and differentiation of T cells, and cytokine production [42], thus establishing anti-tumor immunity. Correspondingly, we found that Spautin-1 induced DAMPs and cytokine release in DLBCL cells. Mitochondrial dysfunctions, such as apoptosis and ferroptosis, play a crucial role in the regulation of programmed cell death, such as PANoptosis and ferroptosis [43]. USP13 preserves mitochondrial homeostasis through ACLY stabilization via deubiquitination, whereas USP13 inhibition disrupts this metabolic axis to induce mitochondrial damage [25, 26]. In this study, we found that Spautin-1-mediated ACLY downregulation, which was reversed by USP13 overexpression, triggered mitochondrial damage in DLBCL cells. These data demonstrate that Spautin-1-induced mitochondrial damage drives PANoptosis-executed tumor suppression.

Although our research results indicate that Spautin-1 exerts anti-tumor activity dependent on the induction of PANoptosis, there are still three key limitations that need to be addressed: (1) The mechanism by which Spautin-1 regulates the USP13-ACLY axis has not been thoroughly verified. (2) The expression pattern and prognostic value of USP13 have not yet been verified in clinical samples. Further clinical cohort studies are required to determine its applicability to DLBCL. (3) The therapeutic potential of Spautin-1 as a sensitising agent for chemotherapy/immunotherapy should be explored in the future.

In conclusion, Spautin-1 inhibits DLBCL growth by inducing PANoptosis and mediating anti-tumor immunity. Mechanistically, Spautin-1 blocks the USP13-ACLY axis, thereby causing mitochondrial damage and releasing DAMPs and cytokines, which promotes the infiltration and activation of CD8⁺ T cells in DLBCL. Therefore, targeting DUBs is an alternative strategy for DLBCL treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

BW and YG conceived and supervised the study. JW and YD performed experiments and analyzed data. JW and BW interpreted data and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Funding

This study was supported by National Natural Science Foundation of China (82103318), Huai'an Natural Science Research Program (HAB202205), Innovation key Talent Program of Huai’an First People’s Hospital (ZC202201), Young Innovative Talent Program of Huai’an First People’s Hospital (QC202213), General Program Cultivation Project of Northern Jiangsu Institute of Clinical Medicine, Nanjing Medical University (HAKY202400314), Key Cultivation Project of Northern Jiangsu Institute of Clinical Medicine, Nanjing Medical University (HAKY202400402), and Jiangsu Provincial Medical Key Discipline Cultivation Unit (202233).

Data availability

We confirm we understand the terms of the share upon reasonable request data policy and we will make the data freely available upon reasonable request.

Declarations

Competing interest

The authors declare that they have no conflicts of interest to this work. Graphical abstract was created by Figdraw (www.figdraw.com).

Ethical approval

The use of human tumor specimens was approved by the Ethics Committee of the Odo Biotechnology Company (ethical number: SHYJS-BC-2310001). The animal research protocol was approved by the Ethics Committee of the Affiliated Huaian No.1 People's Hospital of Nanjing Medical University (ethical number: DW-Y-2024-025-01)