Introduction

Clofarabine (Clo) is an Food and Drug Administration-approved purine nucleoside analog that has been shown to be effective in treating pediatric patients with acute lymphoblastic leukemia. Numerous clinical studies have demonstrated the clinical efficacy of Clo in patients with leukemia,1 2 myelodysplastic syndrome,3 and lymphoma.4 Moreover, combination therapy with Clo has been shown to provide additional benefits for patients with hematological malignancies.5 6 Clo has exhibited a wide range of antitumor activities in preclinical studies involving solid tumor cell lines such as colorectal, pancreatic, and breast cancer. However, its antitumor mechanism in melanoma and lung cancer, two specific types of solid tumors, remains unclear.7 Given the success of drug repurposing as a strategy for identifying novel uses for approved drugs, exploring the potential of Clo for treating solid tumors beyond its currently approved indications is a promising approach for extending the landscape of solid tumor therapy.8

Clo enters cells through passive diffusion or active transport via nucleoside transporters (equilibrative nucleoside transporters, ENTs and concentrative nucleoside transporters, CNTs), where it is then phosphorylated in a stepwise manner by cytosolic kinases to form the active metabolite Clo triphosphate (Clo-TP).9 This process is essential for the antitumor activity of this drug, as Clo-TP interferes with DNA synthesis and repair by inhibiting ribonucleotide reductase and DNA polymerase activity.10 11 This interference with DNA replication and repair leads to the accumulation of DNA damage within cancer cells. This triggers intracellular surveillance mechanisms, including the activation of the tumor suppressor protein P53. Once activated, P53 acts as a transcription factor, regulating genes involved in cell cycle arrest, DNA repair, and apoptosis. In the presence of DNA damage induced by Clo, P53 activates several downstream processes, including cell cycle arrest and DNA repair mechanisms.12 13

DNA damage has also been shown to activate the stimulator of interferon genes (STING), which triggers an effective antitumor immune response.14 Cyclic GMP-AMP synthase (cGAS) senses damaged double-stranded DNA (dsDNA) and binds to it, subsequently activating STING and promoting downstream type I interferon signaling.15 Additionally, DNA damage can activate alternative non-canonical immune response pathways14 ; for example, the direct binding of the tumor suppressor protein p53 to STING, independent of cGAS, induces NF-κB-dependent transcription of cytokines and augments antitumor immune responses.16 However, research on the relationship between cGAS-independent STING signaling and the antitumor effects of CD8+ T cells is still insufficient.

In this study, we found that Clo induces apoptosis and GSDME-related pyroptosis in melanoma and lung cancer cells via a non-canonical STING activation pathway. Furthermore, NF-κB activation increases the expression and secretion of the chemokines CCL5 and CXCL10 and upregulates the expression of MHC-I molecules on the surface of tumor cells, thereby enhancing antitumor immunity mediated by CD8+ T cells. Our findings support the potential of Clo as a promising therapeutic agent for melanoma and lung cancer and provide a mechanistic rationale for its expanded clinical application.

MethodsCell culture

293 T cells were purchased from Clontech (Mountain View, California, USA). Human-derived melanoma cell A375, mouse-derived melanoma cell line B16F10, human-derived non-small cell lung cancer cell line A549, mouse-derived Lewis lung cancer cell LLC, and human-derived melanocyte cell PIG1 were obtained from the American Type Culture Collection. B16F10 and A549 were cultured in RPMI-1640 medium (Gibco, USA); A375, LLC, and 293T were cultured in high glucose Dulbecco’s Modified Eagle’s Medium (Gibco); PIG1 was cultured in Opti-MEM (Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

Cell cycle and apoptosis assay

Tumor cells were collected after trypsin digestion and resuspended in pre-cooled phosphate-buffered saline and washed. Subsequently, tumor cells were fixed at 4°C for 12 hours using 70% ethanol and then stained with propidium iodide (PI)/RNase staining buffer (BD Pharmingen, USA) to detect the cell cycle. Cell apoptosis was analyzed after cells were incubated with Apoptosis Detection Kit I (BD Pharmingen).

Quantitative reverse transcription-PCR

Cells were collected and total RNA was extracted from cell pellets. Complementary DNAs (cDNAs) were generated using a cDNA synthesis reagent (Yeasen, China) according to the manufacturer’s steps. SYBR Green qPCR Master Mix (Bimake, USA) and Q3 system (Applied Biosystems, USA) were used to conduct quantitative reverse transcription-PCR. Primers used are shown in online supplemental table 1.

Western blotting

Cells were lysed on ice for 0.5 hours using a lysing solution (Beyotime, China) supplemented with protease and phosphatase inhibitors. The protein concentration of the supernatant retained after centrifugation was determined by the bicinchoninic acid assay (BCA) method (Beyotime). Equal amounts of different samples were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred semi-dry to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). It was followed by blocking with 5% non-fat milk dissolved in Tris Buffered Saline with Tween-20 (TBST) and incubation with primary antibodies (GSDME-N-terminal, ab215191, Abcam, USA; BAX, 50 599–2-Ig, Proteintech, USA; cGAS, 29 958–1-AP, Proteintech; STING, 66 680–1-Ig-100, Proteintech; Lamin A/C, 10 298–1-AP, Proteintech; NF-κB1, 14 220–1-AP, Proteintech; GAPDH, 60 004–1-Ig, Proteintech; β-Tubulin, 10 094–1-AP, Proteintech; Cleaved Caspase-3, 9664#, CST, USA; Cleaved Caspase-6, 9761#, CST; Cleaved Caspase-7, 8438#, CST; Cleaved PARP, 5625#, CST; Phospho-IκBα, 9246#, CST; p53, 2527#, CST; Phospho-p53, 9284#, CST; NF-κB p65, 6956#, CST) overnight at 4°C. The next day, membranes were incubated with secondary antibodies (anti-rabbit/anti-mouse, Abclonal, China) for 1 hour at 25°C. Detection was performed using an image analysis system (Bio-Rad, USA).

Animal studies

To verify the antitumor activity of Clo in vivo, B16F10-bearing C57BL/6 mice randomly received one of the following treatments: (1) vehicle; (2) Clo (MCE, USA). To test whether CD8+ T cell inhibition in melanoma reverses the antitumor effects of Clo, B16F10-bearing C57BL/6 mice randomly received one of the following treatments: (1) vehicle+immunoglobulin G2 (IgG2) a (#BE0089, Bio X Cell, USA); (2) Anti-CD8α (A2102, Selleck, USA); (3) Clo+IgG2 a; (4) Clo+Anti-CD8α. Tumors were collected for single-cell preparation for multicolor flow cytometry assay to detect the proportion of different immune cells. Cells were stained with Zombie Aqua Fixable Viability Kit (#423102, BioLegend, USA) and blocked with anti-CD16/32 (#101320, BioLegend). Subsequently, cells were incubated with surface antibodies (APC/Cyanine7 CD45; APC CD3; BV650 TIM-3; PerCP/Cyanine5.5 CD4; Brilliant Violet 421 PD-1; Brilliant Violet 570 CD8a; PE/Cyanine7 LAG-3, BioLegend). Cells were incubated with intracellular antibodies (Alexa Fluor 488 IFN-γ; Alexa Fluor 700 Granzyme B (GZMB), BioLegend) since fixed and permeabilized.

Immunoprecipitation

Protein solutions were obtained by lysing tumor cells with NP-40 lysis buffer (Beyotime) supplemented with protease and phosphatase inhibitors and subsequently incubated with protein A/G-agarose (Beyotime) at 4°C to block non-specific protein binding. The protein solution was incubated with STING or IgG antibody and protein A/G-agarose at 4°C overnight. The beads were subjected to a metal bath after being washed three times with pre-cooled NP-40 buffer to obtain denatured proteins which were loaded on SDS-PAGE for immunoblotting.

ELISA

Supernatants of tumor cells were collected to analyze CXCL10 and CCL5 using ELISA Kits (Elabscience, China) according to the manufacturer’s steps. The standard curve was drawn according to the standard product results to calculate the corresponding sample results.

ChIP assay

Tumor cells with or without Clo treatment for 24 hours were collected and assayed according to the steps of the chromatin immunoprecipitation (ChIP) kit (CST, No. 9003). The steps can be concisely described as cross-linking the proteins of the tumor cells to DNA by 37% formaldehyde first. Nuclei preparation and chromatin digestion were then performed, followed by obtaining 10 µg of chromatin and immunoprecipitation using the corresponding antibodies (NF-κB p65, 6956#, CST; rabbit IgG, #2729S, CST). Chromatin was eluted from the magnetic beads the next day and de-crosslinked. Real-time qPCR was performed after purification of the sample DNA.

CD8+ T cells isolation

Peripheral blood from healthy donors or the spleen of mice was harvested to isolate lymphocytes using the lymphocyte isolation solution (Sigma, USA). Lymphocytes were incubated with CD3 and CD8 antibodies at 4℃ for 30 min. Subsequently, flow cytometry was used to sort CD3+CD8+ T cells. The CD3+CD8+ T cells were cultured with beads which were coupled with anti-CD3/CD28 antibodies to activate CD8+ T cells. Subsequently, the activated CD8+ T cells were used in subsequent CD8+ T cell killing essays, CD8+ T cell chemotaxis experiments, and CD8+ T cell cytotoxicity detection.

Lentiviral infection and siRNA transfection

The plasmids of Sh-mock and Sh-Sting1 were purchased from GeneChem (GeneChem, China). The lentiviral infection process refers to the previous article.17 Gsdme-specific small interfering RNAs (siRNAs) and negative control siRNA were synthesized by Ribobio (Ribobio, China) and were transfected into B16F10 cells for 48 hours using Lipofectamine 2,000 according to the manufacturer’s protocol.

Statistical analysis

Data was expressed as mean±SD and the statistical significance was determined using the Student t-test or one-way analysis of variance as indicated in Prism V.10 (GraphPad Software). Significance was set at p=0.05. The number of asterisks indicated the degree of significance (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

ResultsClo selectively inhibits melanoma and lung cancer cell growth in vitro and in vivo

Recent studies suggest that Clo may hinder the growth of solid tumor cells, but the exact mechanisms, particularly concerning melanoma and lung cancer, remain largely unknown. Thus, further studies are necessary to elucidate Clo’s antitumor mechanisms and to assess its clinical application potential. Such investigations could enhance our understanding of its therapeutic benefits and provide a foundation for novel treatment strategies targeting solid tumors.

In this study, we verified Clo’s inhibitory effects on cancer cells. Our results demonstrated that Clo significantly inhibited tumor cell growth in a time-dependent and dose-dependent manner. The IC50 values for melanoma cells (approximately 60 nM) and lung cancer cells (approximately 411 nM) were significantly lower than that for immortalized melanocytes (15.39 µM), indicating Clo selectively targets tumor cells while sparing normal cells (figure 1A and online supplemental figure S1A). Flow cytometric analysis revealed that Clo significantly induced G2/M arrest and apoptosis in tumor cells (figure 1B,C and online supplemental figure S1 B, C). Additionally, Clo markedly inhibited colony formation in melanoma and lung cancer cells (figure 1D and online supplemental figure S1D). We further validated the tumor-inhibitory effect of Clo in vivo. As shown in figure 1E and F, Clo significantly suppressed tumor growth in B16F10 tumor-bearing mice (figure 1E) without notable effects on body weight (figure 1F), highlighting its potential as a safe and effective treatment for solid tumors.

Figure 1

Clofarabine selectively inhibits the proliferation of melanoma and lung cancer cells. (A) The cell viability of A375, A549, and PIG1 treated with Clo for 24 hours and 48 hours was measured by CCK8 assay, and the IC50 values were obtained by GraphPad Prism. n=6 for A375 and PIG1, n=4 for A549. (B) Flow cytometry was conducted to determine the cell cycle distribution of A375 treated with Clo 30 nM and A549 treated with Clo 1 µM for 24 hours. n=3. (C) Flow cytometry was conducted to determine the apoptosis of A375 and A549 treated with Clo for 48 hours. n=3. (D) The number of colonies of A375 and A549 treated with Clo for 48 hours was measured after crystal violet staining. n=3. (E–F) Schematic diagram of Clo for melanoma treatment. Tumor growth and body weight curves for B16F10 tumor-bearing mice receiving the designated treatments. n=6. The p value was obtained using the unpaired Student’s t-test (B, E), and by multiple comparisons in an ordinary one-way analysis of variance (A, C, D), and the results were presented as the mean±SD. *p<0.05, **p<0.01, ****p<0.0001. Clo, clofarabine; i.g., oral gavage.

The antitumor activity of Clo depends on CD8+ T cells

The above results demonstrated that Clo exhibits tumor-inhibitory effects in B16F10 tumor-bearing immunocompetent C57BL/6J mice. Therefore, we speculate that Clo induces immune-mediated tumor cell killing. To confirm our hypothesis, we developed an in vitro co-culture system comprizing tumor cells and CD8+ T cells (figure 2A). Tumor cells were pretreated with Clo for 24 hours, followed by Clo removal and subsequent co-culture with CD8+ T cells. As shown in figure 2B–C and online supplemental figure 2A,B, Clo pretreatment enhanced CD8+ T-cell chemotaxis and CD8+ T-cell-mediated tumor cell killing (figure 2B,C and online supplemental figure A,B). Flow cytometric analysis further revealed an increase in the proportion of GZMB+CD8+ T cells within the co-culture system (figure 2D,E). These findings indicate that Clo boosts the recruitment, functionality, and cytotoxicity of CD8+ T cells within tumors, suggesting a crucial role for CD8+ T cells in Clo-induced antitumor activity.

Figure 2

Clofarabine enhances the recruitment and cytotoxicity of CD8+ T cells. (A) Diagram of CD8+ T cell isolation and CD8+ T cell-related experiments. (B) The chemotaxis experiment of CD8+ T cells with A549 and B16F10. Clo was removed after treating tumor cells for 24 hours, and the remaining cells were cultured in a complete medium for 24 hours. After collection, the corresponding proportion of supernatants were added to the lower chamber of the transwell plate according to the tumor cell counts. CD8+ T cells (1×10∧5) were placed in the upper chamber, and cells in the lower chamber were collected and counted 48 hours later. In the blank group, the lower chamber was a complete medium. n=3. (C) CD8+ T cell killing assay of A549 and B16F10. After pretreatment with Clo for 24 hours, 1×10∧4 tumor cells and the corresponding CD8+ T cells were cultured for 48 hours. After eliminating the suspended CD8+ T cells, the absorbance of the remaining cells was detected by CCK-8, and the killing ratio was calculated. n=4. (D–E) CD8+ T cell cytotoxicity detection of A549 and B16F10. Clo was removed after treating A549 for 24 hours, and A549 was cultured in a complete medium for 24 hours, and the supernatants were collected. CD8+ T cells were cultured with the corresponding proportion of supernatants according to tumor cell counts and collected after 48 hours. Conduct flow cytometry to detect the proportion of GZMB+CD8+CD3+CD45+ T cells. n=3. (F) Schematic diagram of the melanoma treatment. (G) Tumor growth curves of B16F10-bearing mice with the indicated treatments. n=5. (H–K) Flow cytometry was conducted to measure the proportion of GZMB+CD8+CD3+ T cells/IFNγ+CD8+CD3+ T cells/LAG3+CD8+CD3+ T cells/PD-1+TIM3+CD8+CD3+ T cells in the tumors after the indicated treatments, and the statistical analysis was displayed. n=5. The p value was obtained using the unpaired Student’s t-test (C, D, E), and by multiple comparisons in an ordinary one-way analysis of variance (B, G, H, I, J, K), and the results were presented as the mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. PBMC, peripheral blood mononuclear cell; RBC, red blood cell; Clo, clofarabine; FSC-A, forward scatter area; IgG2a, immunoglobulin G2a; i.p., intraperitoneal; i.g., oral gavage.

Considering the increased cytotoxicity of CD8+ T cells induced by conditioned medium from Clo-treated tumor cells, we assessed the importance of CD8+ T cells to the antitumor activity of Clo. As shown in figure 2F, B16F10 tumor-bearing mice were treated with an anti-CD8a antibody to deplete CD8+ T cells. CD8+ T cells were almost eliminated in the tumor tissues of mice treated with the anti-CD8a antibody (online supplemental figure S2C). The depletion of CD8+ T cells significantly decreased the tumor-inhibitory effect of Clo (figure 2G) without affecting body weight (online supplemental figure S2D). Furthermore, Clo treatment alone significantly enhanced CD8+ T cell functionality (figure 2H,I, online supplemental figure S2E, F) and reduced the proportion of exhausted CD8+ T cells (figure 2J,K and online supplemental figure S2G-J). However, the anti-CD8a antibody reversed these effects (figure 2H-K and online supplemental figure S2E-J), indicating that the inhibitory effect of Clo relies on CD8+ T-cell activity. Notably, the removal of CD8+ T cells did not completely restore the viability of tumor cells treated with Clo, suggesting that in addition to immunogenic cell death, Clo also induces other forms of tumor cell death contributing to its overall antitumor activity.

Clo activates the P53-induced non-canonical STING/NF-κB pathway and induces apoptosis, pyroptosis, and immunogenic cell death

Clo, a nucleoside inhibitor, is known to induce DNA damage, inhibit DNA synthesis, and trigger apoptosis in leukemia cells.9 To investigate its effects on the cell cycle, we examined melanoma and lung cancer cells treated with Clo. G2/M phase arrest was observed, suggesting DNA damage (figure 1B and online supplemental figure S1B). Furthermore, Clo significantly increased the expression of P53 and its phosphorylated form (p-P53), both markers of DNA damage (figure 3A). DNA damage is reported to activate downstream STING signaling through both cGAS-dependent and cGAS-independent pathways via P53 activation.16 18 Based on the results in figure 2, we hypothesize that Clo activates STING immune signaling through P53 activation induced by DNA damage. Here, we found that Clo significantly upregulates the expression of P53, STING, and P50/P65 in melanoma and lung cancer cells (figure 3A and C and online supplemental figure S3A). While these findings confirm that Clo activates the P53-STING-NFκB signaling pathway in tumor cells, it remains unclear whether this activation depends on the classical cGAS-STING pathway. To address this, we examined the expression of cGAS and its direct active product cGAMP in cells. cGAMP is the second messenger required for cGAS to activate STING signaling.19 As shown in figure 3A–B, Clo does not affect the expression of cGAS in tumor cells nor does it increase the levels of cGAMP. These results indicate that Clo activates STING signaling in a cGAS-independent manner. Moreover, we further found that Clo triggered the interaction between P53 and STING, indicating that Clo can directly activate STING-NFκB signaling by promoting P53-STING binding (figure 3D).

Figure 3

Clofarabine activates the P53-induced non-canonical STING/NF-κB pathway and induces apoptosis, pyroptosis, and immunogenic cell death in melanoma and lung cancer cells. (A) Western blotting revealed the protein expression of P53, p-P53, cGAS, STING, p-IkBα, BAX, Cleaved Caspase-3, Cleaved Caspase-6, and Cleaved PARP in lysates collected from A375 and A549 treated with Clo. (B) ELISA was used to analyze the concentration of cGAMP in the lysate of A375 and A549 treated with Clo for 48 hours. n=2. (C) Western blotting revealed the nuclear protein expression of NF-κB p50 and p65 in lysates collected from A375 and A549 cells treated with Clo. (D) Immunoprecipitation was conducted to detect the interaction between P53 and STING. (E) Western blotting revealed the protein expression of GSDME-FL and GSDME-N in lysates collected from A375 and A549 treated with Clo. (F) The images of A375 treated with Clo for 48 hours show PI uptake. The statistical analysis was displayed. n=3. (G) qRT-PCR measurement of CCL5, CXCL10, HLA-A, HLA-B, HLA-C, and BAX mRNA expression in A375 and A549 treated with Clo for 48 hours. n=3. (H) ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of A375 and A549 treated with Clo for 48 hours. n=2. The p value was obtained by multiple comparisons in an ordinary one-way analysis of variance (B, F, G, H), and the results were presented as the mean±SD. ***p<0.001, ****p<0.0001. cGAS, cyclic GMP-AMP synthase; Clo, clofarabine; IgG, immunoglobulin G; PI, propidium iodidemRNA, messenger RNA; qRT-PCR, quantitative reverse transcription-PCR; STING, stimulator of interferon genes.

Consistent with the apoptosis results, Clo dramatically increased the protein levels of BAX, PARP, and Caspase-3/6 in melanoma and lung cancer cells (figure 3A and online supplemental figure S3A). Activated caspase-3 has been shown to activate GSDME20; therefore, we examined the effect of Clo on GSDME activation and cellular pyroptosis. Clo treatment significantly induced GSDME cleavage in melanoma and lung cancer cells (figure 3E and online supplemental figure S3B). During pyroptosis, cell membrane rupture allows PI to enter the cells, resulting in positive PI staining prior to complete cell death. Therefore, PI staining can reflect the occurrence of pyroptosis. Of course, this experiment must exclude the effects of late apoptotic cells. As late apoptotic cells tend to float or loosely adhere to culture dishes, we retained only adherent cells after multiple washes to prevent false positives caused by membrane rupture during late apoptosis. As shown in figure 3F and online supplemental figure 3C, PI staining verified that Clo induced pyroptosis (figure 3F and online supplemental figure S3C). Furthermore, Clo increased the protein and messenger RNA (mRNA) levels of BAX, a key regulator of mitochondrial apoptosis that activates downstream caspases. The above results suggest that Clo may directly regulate mitochondrial apoptosis and downstream cleaved Caspase-3-mediated pyroptosis through BAX (figure 3A-G and online supplemental figure S3A-D).

The cGAS-STING pathway is a key component of innate immune signaling. Its activation enhances tumor immunogenicity by upregulating major histocompatibility complex (MHC)-I expression21 and inducing the activation of cytotoxic T lymphocytes.22 However, it remains unclear whether Clo activates T cell immunogenic killing by increasing the immunogenicity of tumor cells. Therefore, we examined the expression of MHC-I molecules in tumor cells. As expected, Clo upregulated the expression of MHC-I molecules in melanoma and lung cancer cells (figure 3G and online supplemental figure S3D). Additionally, our results showed that Clo exhibited chemotactic effects on T cells and increased their functionality, suggesting the involvement of certain cytokines in Clo-induced immunogenic cell death (figure 2). Therefore, we examined several cytokines related to T-cell function and chemotaxis. Our results showed that Clo increased the expression and secretion of CCL5 and CXCL10 (figure 3G,H and online supplemental figure S3D-F). These cytokines are crucial for T-cell recruitment and functionality within the tumor microenvironment.22 23 The above results indicate that Clo can activate immunogenic cell death through MHC-I signaling and cytokine secretion.

Clo regulates MHC-I/CCL5/CXCL10/BAX expression through the P53-STING-NF-κB pathway

To confirm that Clo regulates tumor cell death and downstream events, including MHC-I/CCL5/CXCL10/BAX expression, through the P53-STING-NFκB signaling pathway, we knocked down P53 or STING in tumor cells (online supplemental figure S4A and B and figure 4. A and B). As shown in online supplemental figure S4C and figure 4C–E, knocking down P53 or STING knockdown significantly reduced the effects of Clo on tumor cell death (online supplemental figure S4C and figure 4C–E). Consistently, knocking down P53 or STING markedly reduced the Clo-induced suppression of mRNA expression for MHC-I molecules, CCL5, CXCL10, and BAX (online supplemental figure S4D and figure 4F). It also decreased the secretion of CCL5 and CXCL10 (online supplemental figure S4E and figure 4G). We further validated the importance of STING in the tumor-suppressive effect of Clo in vivo. Knocking down Sting1 significantly attenuated the suppressive effect of Clo on B16F10 tumor growth (figure 4H). Moreover, knocking down Sting1 significantly diminished Clo’s ability to enhance CD8+ T cell functionality (figure 4I and online supplemental figure S5). Similarly, the addition of an NF-κB inhibitor (JSH-23) partially restored the effects of Clo on tumor cell proliferation, apoptosis, and pyroptosis (figure 5A–C and online supplemental figure S6A-C). Moreover, NF-κB inhibitor treatment partially restored the effect of Clo on inducing the mRNA expression of MHC-I molecules, CCL5, CXCL10, and BAX, as well as the secretion of CCL5 and CXCL10 (figure 5D,E and online supplemental figure S6D-F). Further ChIP assays confirmed that P65 directly bound to the promoters of HLA, CCL5, CXCL10, and BAX and that Clo further increased the binding of P65 to the promoters of these molecules (figure 5F and online supplemental figure S6G). These results confirm that Clo regulates tumor cell death through the P53-STING-NF-κB-BAX signaling pathway and induces immunogenic cell death through the P53-STING-NF-κB-MHC-I/CCL5/CXCL10 signaling pathway.

Figure 4

Clofarabine regulates tumor cell death and downstream MHC I/CCL5/CXCL10/BAX expression through the activation of STING. (A–B) qRT-PCR and western blotting verified the efficiency of Sting1 knockdown in B16F10. (C) The cell viability of B16F10 with or without Sting1 knockdown after Clo 1.5 µM treatment for 48 hours was measured by CCK8 assay. n=4. (D) Flow cytometry was conducted to determine the apoptosis of B16F10 with or without Sting1 knockdown after Clo 1.5 µM treatment for 48 hours. n=3. (E) Western blotting revealed the protein expression of GSDME-FL and GSDME-N in lysates collected from B16F10 with or without Sting1 knockdown after Clo 1.5 µM treatment for 48 hours. (F) qRT-PCR measurement of Sting1, Ccl5, Cxcl10, H2-k1, H2-d1, and Bax mRNA expression in B16F10 with or without Sting1 knockdown after Clo 1.5 µM treatment for 48 hours. n=3. (G) ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of B16F10 with or without Sting1 knockdown after Clo 1.5 µM treatment for 48 hours. n=2. (H) Tumor growth and body weight curves of B16F10-bearing mice with the indicated treatments. n=5. (I) Flow cytometry was used to detect the proportion of GZMB+CD8+CD3+ T cells/IFNγ+CD8+CD3+ T cells/LAG3+CD8+CD3+ T cells/PD-1+TIM3+CD8+CD3+ T cells in the tumors after the indicated treatments, and the statistical analysis was displayed. n=5. The p value was obtained using the unpaired Student’s t-test (C), and by multiple comparisons in an ordinary one-way analysis of variance (A, D, F, G, H, I), and the results were presented as the mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Clo, clofarabine; MHC, major histocompatibility complex; mRNA, messenger RNA; qRT-PCR; quantitative reverse transcription-PCR; STING, stimulator of interferon genes.

Figure 5

Clofarabine regulates tumor cell death and downstream MHC-I/CCL5/CXCL10/BAX expression through NF-κB. (A) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then the cell viability of A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours was measured by CCK8 assay. n=5. (B) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then flow cytometry was conducted to determine the apoptosis of A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours. n=3. (C) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then western blotting revealed the protein expression of GSDME-FL and GSDME-N in lysates collected from A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours. (D–E) Pretreated A375 and A549 with JSH-23 10 µM for 24 hours, then ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of A375 and A549 after Clo treatment alone or treatment with Clo and JSH-23 10 µM for 48 hours. n=2. (F) ChIP assay detects the binding of NF-κB p65 to the CCL5/CXCL10/HLA-B/BAX promoter of A549 after Clo 1.5 µM treatment for 24 hours. n=3. (G) ELISA was used to analyze the concentration of CCL5 and CXCL10 in the supernatants of B16F10 with or without Gsdme knockdown after Clo 1.5 µM treatment for 48 hours. n=2. The p value was obtained by multiple comparisons in an ordinary one-way analysis of variance (A, B, D, E, F, G), and the results were presented as the mean±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ChIP, chromatin immunoprecipitation; Clo, clofarabine; IgG, immunoglobulin G; MHC, major histocompatibility complex; NC, negative control.

According to the results above, Clo induces GSDME-mediated pyroptosis in tumor cells while promoting the secretion of CCL5 and CXCL10 to activate T-cell immune responses. The characteristic of GSDME-mediated pyroptosis involves cell membrane perforation and the release of inflammatory factors. Therefore, we investigated whether the cytokines CCL5 and CXCL10 induced by Clo primarily rely on GSDME-mediated pore formation. Thus, we examined whether knocking down Gsdme in tumor cells affects Clo-induced secretion of CCL5 and CXCL10. As shown in online supplemental figure S6H and figure 5G, partial restoration of Clo-induced secretion of CCL5 and CXCL10 was observed on Gsdme knockdown, indicating that GSDME pores mediate partial secretion of CCL5 and CXCL10 (online supplemental figure S6H and figure 5G). These results further suggest that Clo-induced GSDME-mediated pyroptosis plays an auxiliary role in activating T cell immune cytotoxicity.

Clo increases the cytotoxic activity of CD8+ T cells through the P53-STING-NF-κB signaling pathway

Figure 2 shows that Clo increases the immunogenic function of CD8+ T cells by acting on tumor cells. Furthermore, Clo not only directly induces apoptosis and pyroptosis in melanoma and lung cancer cells through the P53-STING-NF-κB-BAX pathway but also induces the expression of MHC-I molecules and the secretion of chemokines (CCL5 and CXCL10) through the P53-STING-NF-κB pathway. To confirm that Clo increases the cytotoxic activity of CD8+ T cells through the P53-STING-NF-κB signaling pathway, we disrupted this pathway and evaluated CD8+ T-cell functionality. Knockdown of P53-STING or treatment with an NF-κB inhibitor weakened the promoting effect of Clo on CD8+T-cell function, reduced T-cell chemotaxis, and inhibited CD8+T-cell-mediated killing of tumor cells (figure 6A–F and online supplemental figure S7A-E). These results confirm that Clo promotes both CD8+T-cell chemotaxis and functionality via the P53-STING-NF-κB signaling pathway.