Introduction

The advent of poly(ADP-ribose) polymerase inhibitors (PARPis), such as niraparib and olaparib, has ushered in a seminal era in targeted cancer therapeutics.1–5 The clinical triumph of PARPis lies in their ability to induce synthetic lethality in tumors with homologous recombination deficiency (HRD) caused by mutations in BRCA or other genes involved in homologous recombination repair (HRR), particularly exemplified in ovarian malignancies.3 Remarkably, a growing body of evidence highlighted an additional dimension to their therapeutic prowess. Treatment with PARPis showed an enhancement of immune responses in tumors, encompassing both HRD6 and homologous recombination proficiency (HRP).7 This enhancement was characterized by the conspicuous accumulation of peripheral CD8+ T cells within tumor beds, which was associated with increased levels of chemokines, including CCL5 and CXCL10.6 However, it is not yet completely clear if these infiltrated T cells target the tumors effectively and specifically. To fully grasp the immune-modulating role of PARPis, it is imperative to determine whether and how PARP inhibition orchestrates immune responses that selectively target tumors.

Immunogenic cell death (ICD) is known to instigate immune responses targeting neoantigens subsequent to pharmacologic intervention in tumors.8 9 Recently, there has been increasing interest in a type of ICD called pyroptosis, mainly because of its remarkable ability to accelerate a strong inflammatory cascade.4 10–12 The induction of pyroptosis hinges on the cleavage of gasdermin (GSDM) family proteins and caspase activation. Morphologically, it involves initial cell swelling, gradual emergence of bubble-like protrusions on the cell membrane, and eventual rupture of the cell membrane.12 When cells undergo pyroptosis, they release various inflammatory cytokines, damage-associated molecular patterns, and neoantigens into the tumor microenvironment. This, in turn, reinforces antitumor immune responses.4 10 13–15 For example, granzymes secreted from activated T cells or NK cells could penetrate tumor cells, activate GSDMs, and initiate pyroptosis, thereby boosting the cytotoxicity of lymphocytes.16 17 Also, delivering activated GSDMA3 into tumors through silyl-linked gold nanoparticles18 or employing an oncolytic virus armed with GSDME19 significantly enhanced the antitumor immune response. This heightened immune response renders tumors more susceptible to immune checkpoint blockade (ICB). In light of this, our goal is to figure out the potential of PARP inhibition in inducing pyroptosis, thus reshaping the tumor immune microenvironment (TIME) and amplifying tumor-targeting immune responses. Understanding these mechanisms could open new avenues for improving cancer treatments.

In this study, it was observed that PARP inhibition effectively triggered a type of ICD, pyroptosis, engendering an augmentation of neoantigen-specific T-cell receptor (TCR) clones of tumor-infiltrating T cells and an overall increase in the frequency of intratumoral immune cells engaging in both innate and adaptive immune responses. Further investigations revealed that PARP inhibition activated the NF-κB–TNF–caspase 8 pathway, contributing to the cleavage of GSDMD/E and subsequent pyroptosis. Therefore, our results have identified a novel mechanism by which PARPis exploit the pyroptosis to reprogram the TIME and elicit the tumor-targeting immune responses. These discoveries not only significantly advance the understanding of the pharmacological effect of PARPis but also promote the development of efficacious therapeutic approaches for tumors.

ResultsThe PARP inhibitor (PARPi) triggers tumor-targeting immune responses and tumor ICD

In a recent endeavor, we launched a phase 2 prospective clinical study (NANT, NCT04507841) to assess the therapeutic efficacy of neoadjuvant niraparib monotherapy in newly diagnosed HRD ovarian cancer (OC).20 Within the framework of this large clinical trial, matched specimens before and after neoadjuvant niraparib treatment were obtained and subjected to a highly informative technique of bulk TCR-sequencing (TCR-seq) to understand the impact of PARP inhibition on neoantigen-specific adaptive immune responses. Our findings revealed a significant increase in the proportions of neoantigen-recognized TCR clones following niraparib exposure (figure 1A). Meanwhile, niraparib treatment enhanced the TCR clonal expansion, as evidenced by a reduction in the top 25% clonotypes of the T-cell repertoire (figure 1B,C). To further validate these findings, we established a syngeneic mouse model by engrafting mouse OC ID8 cells into C57BL/6 mice. An analysis of bulk TCR-seq data from these ID8 tumors also showed an enhancement in TCR clonal expansion (online supplemental figure S1A–C) along with a reduction in TCR diversity (online supplemental figure S1D) in response to niraparib exposure. More importantly, T cells infiltrated in niraparib-challenged tumors exhibited a heightened potential for cytotoxicity against ID8 cells (online supplemental figure S1E). These tumor-killing T cells are often primed by antigen-presenting cells such as dendritic cells (DCs) in peripheral lymphatic organs,21 thus we proceeded to evaluate the activation of T cells in spleens. We found that the activated CD69+CD8+ T cells (online supplemental figure S1F,G) and CD137+CD8+ T cells (online supplemental figure S1F,H) were enriched in niraparib-treated spleens. We further assessed the antigen-presentation capacity of DCs within tumor tissues, draining lymph nodes, and spleens (online supplemental figure S1I). A uniform increase in the proportions of DC differentiation (CD11c+MHCIIHigh) and maturation (CD11c+MHCIIHighCD86High) was observed in niraparib-treated tumors (online supplemental figure S1J), draining lymph nodes (online supplemental figure S1K), and spleens (online supplemental figure S1L), compared with untreated counterparts. Collectively, these observations indicate that PARP inhibition augments tumor-targeting immune responses.

Figure 1

The PARP inhibitor induces tumor immunogenic cell death. Neoantigen-recognized T-cell receptor (TCR) clones of tumor-infiltrated lymphocytes before and after niraparib treatment from patients enrolled in NANT ((A) n=11). The frequency of clonotypes in the top 25% of TCR repertoires was quantified ((B) n=11) and shown in pie charts (C). (D) OVCAR8 and (E) primary homologous recombination deficiency (HRD) ovarian cancer (OC) cells were treated with or without olaparib or niraparib for about 3 days and then stained with HMGB1 and calreticulin. Scale bars: 20 µm. (F–H) Clinical OC and triple-negative breast cancer (TNBC) tumors were implanted in immunodeficient NOG mice to generate patient-derived xenograft (PDX) models followed with or without niraparib treatment. Representative images of PDX tumors with immunohistochemical staining of HMGB1 and calreticulin (F). The ratios of HMGB1 cytoplasm-positive cells and calreticulin membrane-positive cells were quantified (G,H). Scale bars: 100 µm, n=8. Mean values±SEM. *P<0.05, **p<0.01, and ***p<0.001 by Student’s t-test in (A,B) and (G,H).

To exclude the possibility that the induction of strengthened immune response was attributed to a direct effect of PARPis on immune cells, we evaluated the cytokine production and apoptosis of T cells, as well as the differentiation and apoptosis of DCs, under olaparib and niraparib administration in vitro. In the case of T cells, the expression level of IFN-γ remained consistent in CD4+ T cells, whereas it declined in CD8+ T cells on exposure to the two PARPis (online supplemental figure S2A,B). Following the PARPis treatment, the frequency of CD8+ T cells producing granzyme B decreased (online supplemental figure S2B). Treatment with niraparib resulted in increased production of IL-2 in both CD4+ and CD8+ T cells, whereas olaparib did not affect IL-2 production (online supplemental figure S2A,B). Additionally, both olaparib and niraparib facilitated the apoptosis of CD4+ and CD8+ T cells (online supplemental figure S2C). As for DCs, the PARPis partially disrupted their differentiation and activation (online supplemental figure S2D), yet exerted minimal influence on DC apoptosis (online supplemental figure S2E). Consequently, these intriguing findings demonstrate that PARPis have a partial restraining effect on T-cell and DC functions in vitro, thereby suggesting that PARPis are unlikely to activate the immune response directly.

To delve deeper into whether ICD played a role in the enhanced DC differentiation and maturation, along with augmented T-cell expansion and cytotoxicity, we examined two ICD biomarkers high-mobility group box 1 (HMGB1) and calreticulin in HRD OC cell line OVCAR8 and HRD primary OC cells. The results showed that PARP inhibition significantly boosted the release of HMGB1 from cell nuclei and the translocation of calreticulin to cell membranes (figure 1D,E). These changes in HMGB1 and calreticulin were also duplicated in HRD OC and triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) tumors using immunohistochemical (IHC) staining (figure 1F–H). Therefore, these compelling findings suggest that PARP inhibition triggers ICD, hinting at the possible mechanism to induce tumor-targeting immune responses.

The PARPi induces an inflammatory TIME

To explore whether PARPis could alter the TIME status, we examined the frequencies of diverse innate and adaptive immune cell populations, including CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ NK cells, CD14+ monocytes, and GZMB+ cytotoxic cells, in the paired human OC specimens before and after treatment with niraparib. Our findings revealed a significant increase in the frequencies of all the tested immune cells following niraparib treatment, as determined by IHC staining (figure 2A–C). On exposure to olaparib and niraparib, ID8 tumors also exhibited enhanced infiltration of immune cells, including CD45+ leukocytes, CD4+ T cells, CD8+ T cells, CD11C+ DCs, NK1.1+ NK cells, and GZMB+ cytotoxic cells, with statistical significance (figure 2D–F,H). Additionally, there was an increased trend in the infiltration of CD14+ monocytes and CD19+ B cells (figure 2D,G). Collectively, these observations suggest that PARP inhibition produces a systemic inflammatory TIME within both clinical OC tumors and ID8 mouse tumors, thereby supporting the hypothesis that PARPis elicit ICD.

Figure 2

The PARP inhibitor promotes infiltration of both innate and adaptive immune cells. (A–C) Paired clinical ovarian cancer (OC) tumors before and after niraparib monotherapy were subjected to immunohistochemical (IHC) staining of CD4, CD8, CD19, CD56, CD14, and granzyme B (GZMB). Representative images of tumor sections before and after niraparib exposure (A). The numbers of indicated cells were counted in a ×20 field of a microscope ((B–C) n=11). Scale bars: 200 µm. (D–H) Olaparib/niraparib (40 mg/kg) was administered to ID8-bearing immunocompetent CD57/B6 mice for about 4 weeks. ID8 tumors were subjected to IHC staining. Representative images of tumor sections with staining CD45, CD4, CD8, NK1.1, CD19, CD11C, CD14, and GZMB (D). The numbers of indicated cells were counted in a ×20 field of a microscope ((E–H) n=5). Scale bars: 200 µm. Mean values±SEM. *P<0.05, **p<0.01, and ***p<0.001 by Student’s t-test in (B–C) and (E–H).

The PARPi induces caspase-dependent and pyroptosis-like cell death in tumor cells

To further characterize the ICD induced by PARPis, we initially assessed the forward scatter (FSC) values across multiple cancer cell lines, including OC (HOC7, SKOV3, A2780) and breast cancer (MDA-MB-231, MDA-MB-468, HCC1937), using flow cytometry in the presence of niraparib. The BRCA gene is wild type (WT) in HOC7, SKOV3, A2780, and MDA-MB-231 cells, whereas it is mutated in MDA-MB-468 and HCC1937 cells. The results showed that both BRCA-WT and BRCA-mutated tumor cells exhibited a consistent augmentation in FSC values in a dose-dependent and time-dependent pattern, indicating cell swelling after PARP inhibition (online supplemental figure S3A). Furthermore, the inhibition of PARP by olaparib and niraparib resulted in membrane rupture in tumor cells, as demonstrated by increased staining of propidium iodide (PI) and leakage of intracellular calcein in HRD OVCAR8 (figure 3A) and HRD primary OC cells (figure 3B) under microscopy. In line with this, olaparib and niraparib treatment increased the population of PI and annexin V double-positive OVCAR8 and A2780 cells (online supplemental figure S3B,C) and potentiated lactate dehydrogenase (LDH) release of these cells (online supplemental figure S3D,E). ICDs with characteristics such as cell swelling and membrane rupture typically include necroptosis, ferroptosis, and pyroptosis.22 23 Thus, to unravel the specific type of ICD elicited by PARPis, the tumor cells were pretreated with a series of death inhibitors in the presence of PARPis. Notably, the percentage of tumor cells positive for both PI and annexin V was dramatically reduced in the presence of Z-VAD-FMK (pan-caspase inhibitor) but was less affected by the incubation with necroptosis inhibitor Necrostatin-1 and ferroptosis inhibitor Ferrostatin-1 (figure 3C,D). Moreover, PARP inhibition induced the formation of bubble-like protrusions on the surface of tumor cells, including HRD primary OC cells and HRD cell lines (OVCAR8, MDA-MB-436, and HCC1937) (figure 3E). Conversely, treatment with Z-VAD-FMK depleted the appearance of these protrusions on PARP-inhibited tumor cells, such as OVCAR8 and A2780 cells (figure 3F,G). Z-VAD-FMK could also abrogate the elevated LDH release induced by PARPis in the two cell lines (figure 3H,I). These results provide evidence that PARP inhibition orchestrates caspase-dependent cell demise imbued with pyroptosis-like characteristics in tumor cells.

Figure 3

PARP inhibition induces a form of cell death similar to pyroptosis. (A,B) OVCAR8 and primary homologous recombination deficiency (HRD) ovarian cancer (OC) cells were treated with or without PARPis (olaparib and niraparib) for about 3 days and then stained with propidium iodide (PI) and calcein. Representative images and quantification of PI+ calcein− OVCAR8 (A) and primary HRD OC cells (B). Scale bars: 100 µm. Quantification of PI+ annexin V+ OVCAR8 (C) and A2780 (D) cells on olaparib and niraparib treatment for about 3 days in the presence and absence of inhibitors, including Necrostatin-1 (Nec-1), Ferrostatin-1 (Fer-1), and Z-VAD-FMK (Z-VAD). (E) Representative images of OVCAR8, primary HRD OC cells, MDA-MB-436, and HCC1937 cells, under different doses of olaparib and niraparib exposure as indicated for 3 days. Representative images of OVCAR8 (F) and A2780 (G) cells under olaparib and niraparib exposure with or without Z-VAD incubation. The lactate dehydrogenase (LDH) levels in the OVCAR8 (H) and A2780 cells (I) culture medium were tested after treatment with olaparib or niraparib in the presence or absence of Z-VAD for 3 days. Mean values±SEM. *P<0.05, **p<0.01, and ***P<0.001 by Student’s t-test in (A–D) and (H,I).

PARP inhibition elicits pyroptosis in tumor cells through the activation of GSDMD and GSDME

Pyroptosis is mediated by the activation of GSDMs, namely GSDMA/B/C/D/E.12 24 To investigate which GSDM member could be activated by PARP inhibition, we initially checked the GSDM mRNA expression profiles in OC cell lines using the Cancer Cell Line Encyclopedia (CCLE) database. We found that the vast majority of these cell lines exhibited higher levels of GSDMD and GSDME than other family members (online supplemental figure S4A). This intriguing finding was further supported by the abundance of GSDMD and GSDME proteins in OVCAR8 and A2780 cells, and both the two GSDMs underwent proteolytic cleavage to their activated forms on treatment with olaparib and niraparib in a dose-dependent manner (figure 4A,B). Consistently, the cleavage of GSDMD and GSDME was also evident in the IHC staining of OC PDX tumors after the niraparib challenge (figure 4C–E). To determine the impact of the activated GSDMs on the antitumor capacity of PARPis, we employed small interfering RNAs (siRNAs) to silence the expression of GSDMA/B/C/D/E (online supplemental figure S5A). As shown in figure 4F,G, only tumor cells with downregulated GSDMD or GSDME displayed a decrease in cell death ratios following exposure to olaparib or niraparib. In addition, the knockdown of GSDMD or GSDME effectively abolished the generation of membrane blebbing (figure 4H) and LDH release (online supplemental figure S5B,C) of OVCAR8 and A2780 cells on the PARPis treatment. These phenomena were also confirmed in MDA-MB-436 TNBC cells (online supplemental figure S5D,E). Thus, the cellular results collectively corroborate that PARPis triggered tumoral pyroptosis.

Figure 4

PARP inhibitors induce pyroptosis by cleaving GSDMD and GSDME. (A,B) Western blotting analyses of GSDMA/B/C/D/E expression and cleavage in OVCAR8 and A2780 cells on olaparib and niraparib treatment at indicated doses. (C–E) Paired clinical ovarian cancer (OC) tumors before and after niraparib monotherapy were subjected to immunohistochemical (IHC) staining of GSDMD-N and GSDME-N. Representation images of IHC staining (C,D). Scale bars: 200 µm. IHC scores of GSDMD-N and GSDME-N were evaluated by two pathologists without patient data ((E) n=5). Quantification of propidium iodide (PI)+ annexin V+ OVCAR8 (F) and A2780 (G) cells on olaparib and niraparib treatment for about 3 days with knocking down GSDMA/B/C/D/E, respectively. (H) Representative images of OVCAR8 and A2780 cells on olaparib and niraparib treatment for 3 days with knocking down GSDMD/E, respectively. (I) The GSDMD/E levels in CRISPR/Cas9-edited ID8 cells were determined by western blotting. (J–L) Wild-type (WT) or Gsdmd/e-deficient ID8 cells were transplanted into immunocompetent C57BL/6 mice with or without niraparib treatment for about 4 weeks (n=6). Volumes of tumors with or without niraparib treatment (J,K). Images of tumors with or without niraparib treatment (L). Mean values±SEM. *P<0.05, **p<0.01, and ***P<0.001 by Student’s t-test in (E–G) and two-way analysis of variance in (J,K).

To test the role of GSDMD and GSDME in tumor suppression by PARPis in vivo, we employed the syngeneic OC mouse model. ID8 cells were genetically engineered using CRISPR/Cas9 to induce knockouts (KOs) of Gsdmd and Gsdme (figure 4I), respectively, and then subcutaneously transplanted into C57BL/6 mice. Once the bearing tumors reached about 100 mm3, oral administration of niraparib was initiated. During 4 weeks of treatment, we monitored the tumor volumes of the WT, Gsdmd KO, and Gsdme KO tumors. The tumor volumes were similar among these three groups without treatment (figure 4J,L); however, the growth of Gsdmd-deficient and Gsdme-deficient tumors was less impacted by niraparib exposure compared with the WT tumors (figure 4K,L). These observations highlight the important role of GSDMD/E and pyroptosis in PARPi-induced tumor suppression.

To understand whether the GSDM protein is responsible for TIME reprogramming during PAPRis treatment, we performed bulk TCR-seq analysis on WT and Gsdme KO ID8 tumors, both with and without niraparib treatment. In comparison with niraparib-treated WT tumors, niraparib-treated Gsdme KO tumors showed a significantly increased frequency of clonotypes in the top 25% of the TCR repertoire (figure 5A,B) and decreased TCR clonality (figure 5C). These findings suggest compromised T-cell clonal expansion within Gsdme-deficient tumors. On the contrary, the TCR diversity was elevated (figure 5D). The deficiency of Gsdme also limited the differentiation and maturation of tumor-infiltrated, lymphatic, and splenic DCs after niraparib treatment (figure 5E–G). Accordingly, the flow cytometry analyses revealed a significant decrease in the proportion of CD3+ T and NK cells in niraparib-treated Gsdme KO tumors compared with niraparib-treated WT tumors (figure 5H). The production of IFN-γ in CD4/8+ T cells and granzyme B in CD8+ T cells was reduced in the absence of Gsdme after niraparib administration (figure 5I,J). However, Gsdme deficiency had a minimal effect on the expression of IFN-γ and granzyme B in NK cells (figure 5I,J). Similarly, Gsdme KO tumors exhibited reduced frequencies of CD4/8+ T cells and GZMB+ cytotoxic cells when compared with WT tumors, as determined by IHC staining (figure 5K,L). Next, we aimed to investigate the critical role of pyroptosis in the established synergistic effect of combining PARPis and ICB for tumor suppression. To this end, we established an orthotopic engraftment model by transplanting WT and Gsdme KO ID8 tumors via intrabursal injection and exposing them to niraparib and/or anti-PD-1 antibodies. As expected, the synergistic effect was attenuated in Gsdme KO ID8 tumors (figure 5M,N). Taken together, these results suggest that PARPis initiate tumor-targeting immune responses by triggering pyroptosis.

Figure 5

Deficiency of GSDME blunts the immune response triggered by PAPR inhibitor in vivo. (A–L) Immunocompetent C57BL/6 mice were transplanted with wild type (WT) or Gsdme-deficient ID8 cells and challenged with niraparib for about 4 weeks. Tumors were harvested and subjected to bulk T-cell receptor (TCR) sequencing and evaluation of immune status. The frequency of T-cell clonotypes in the top 25% of TCR repertoires was shown using pie charts ((A) n=4) and quantified (B). The clonal expansion was evaluated using clonality (C). The TCR diversity was calculated by normalized Shannon diversity entropy (D). The proportion of dendritic cell differentiation (CD11c+MHCIIHigh) and maturation (CD11c+MHCIIHighCD86High) in tumors (E), lymph nodes (F), and spleens (G) was determined by flow cytometry. The proportion of CD3+ T cells and NK cells among CD45+ immune cells in tumors was determined by flow cytometry (H). The production of IFN-γ in tumor-infiltrated CD4/8+ T and NK cells was examined by flow cytometry (I). The expression of granzyme B in tumor-infiltrated CD8+ T and NK cells was evaluated by mean fluorescence intensity (J). Representative images of GSDME, CD4, CD8, and GZMB IHC staining in tumor sections (K) and numbers of indicated immune cells in a ×20 field of a microscope (L). Scale bars: 200 µm. WT or Gsdme-deficient ID8 cells were intrabursally transplanted into C57BL/6 mice and received niraparib and/or anti-PD-1 treatment. Representative bioluminescent images of mice bearing WT and Gsdme-KO ID8 tumors at the endpoint (M). The tumor weight was quantified in each mouse treated with niraparib and/or anti-PD-1 antibodies ((N) n=7). Mean values±SEM. *P<0.05, **p<0.01, and ***p<0.001 by Student’s t-test in (B–J, L, and N).

The cleavage of GSDM proteins induced by PARP inhibition requires the activation of caspase 8

Next, we sought to determine the mechanism underlying the cleavage of GSDM proteins under PARP inhibition. Given that PARPi-induced pyroptosis was shown above to be caspase dependent, we pretreated HRD primary OC, OVCAR8, and A2780 cells with caspase inhibitors. Notably, Z-DEVD-FMK (caspase 3/8 inhibitor), Z-IETD-FMK (caspase 8 inhibitor), and Z-VAD-FMK (pan-caspase inhibitor) significantly weakened the antitumor ability of niraparib, as measured by PI and annexin V staining. However, the caspase 1 inhibitor VX765 did not exhibit a similar effect (figure 6A–C). These results suggest that the activation of caspase 8 is required for the niraparib-induced death. Consistent with this, we found elevated activity of caspase 3 and caspase 8 in PARP-inhibited OC cell lines (OVCAR8 and A2780) (figure 6D) as well as PDX tumors (OC and TNBC) (figure 6E,F). To further investigate whether the cleavage of GSDMD and GSDME is dependent on the activity of caspase 8, we examined the levels of cleaved GSDMD/E in the presence of the caspase inhibitors. Treatment with Z-DEVD-FMK, Z-IETD-FMK, and Z-VAD-FMK sufficiently led to a reduction in the levels of the functional N-terminal domain of GSDMD and GSDME in OVCAR8 and A2780 cells under niraparib exposure (figure 6G). Finally, Z-DEVD-FMK and Z-IETD-FMK, but not VX765, suppressed membrane blebbing (figure 6H,I) and LDH release (online supplemental figure S6A,B) in OVCAR8 and A2780 cells on PARP inhibition by olaparib and niraparib. In summary, these results suggest that caspase 8 mediates GSDMD/E cleavage on PARP inhibition.

Figure 6

Caspase 8 contributes to the pyroptosis induced by PARP inhibition. Ratios of PI+ annexin V+ OVCAR8 (A), A2780 (B), and primary homologous recombination deficiency (HRD) ovarian cancer (OC) tumor cells (C) on niraparib exposure in the presence or absence of caspase inhibitors, including VX765, Z-DEVD-FMK, Z-IETD-FMK, and Z-VAD-FMK. (D) Determination of activated caspase 3 and caspase 8 levels in OVCAR8 and A2780 cells after olaparib and niraparib treatment by flow cytometry. Histological analyses of activated caspase 3 and caspase 8 in OC patient-derived xenograft (PDX) tumors (E) and triple-negative breast cancer (TNBC) PDX tumors (F) with or without niraparib exposure (n=5/6). (G) Western blotting analyses of the cleavage of caspase 8/3 and GSDMD/E in OVCAR8 and A2780 cells on niraparib treatment in the presence or absence of caspase inhibitors, including Z-DEVD-FMK, Z-IETD-FMK, and Z-VAD-FMK. Representative images of OVCAR8 (H) and A2780 cells (I) on niraparib treatment in the presence or absence of caspase inhibitors, including VX765, Z-DEVD-FMK, and Z-IETD-FMK. Mean values±SEM. *P<0.05, **p<0.01, and ***p< 0.001 by Student’s t-test in (A–C) and (E,F).

TNFR signaling contributes to caspase 8-mediated pyroptosis induced by PARP inhibition

Caspase 8, a highly conserved cysteine protease, serves as the principal initiator of the caspase cascade, triggered by the activation of cell surface death receptors, including tumor necrosis factor receptor superfamily member 1 (TNFR1), TNFR superfamily member 10A/B (TRAIL-R1/2), and TNFR superfamily member 6 (FAS).25–28 To identify the stimulus responsible for cell demise and caspase activation, we employed siRNAs to suppress the expression of TNFR1 (genge for TNFR superfamily member 1), DR5 (gene for TRAIL-R2), and FAS (gene for TNFR superfamily member 6) in tumor cells (figure 7A). We discovered that downregulating TNFR1 expression improved the survival of tumor cells on PARP inhibition, whereas reducing DR5 and FAS expression had a negligible effect on tumor cell survival (figure 7B,C). The use of two FDA-approved antibodies, adalimumab and infliximab, blocking the interaction between TNF-α and TNFR1, also effectively restrained the potential of PARPis to induce cell death (figure 7D,E). Moreover, silencing TNFR1 expression led to a significant reduction of caspase 8/3 activity in OVCAR8 (figure 7F,G) and A2780 cells (figure 7H,I). We then investigated the contribution of TNFR signaling to the cleavage of GSDM proteins. It was noted that reducing TNFR1 expression in tumor cells robustly restrained the olaparib-potentiated and niraparib-potentiated activation of GSDMD and GSDME in OVCAR8 and A2780 cells (figure 7J). In line with this, morphological and LDH examination also confirmed the disappearance of membrane blebbing (figure 7K) and a reduction of LDH release (figure 7L,M) in TNFR1-depleted tumor cells in the presence of olaparib and niraparib. Overall, these observations demonstrate the critical role of the TNFR1 signaling pathway in PARP inhibition-induced caspase activation and pyroptosis.

Figure 7

TNFR is located upstream of caspase 8 activation and is responsible for PARP inhibitor-induced pyroptosis. (A) Knocking down TNFR1, DR5, and FAS expression in OVCAR8 cells with siRNAs. Ratios of PI+ annexin V+ OVCAR8 (B) and A2780 cells (C) with silencing TNFR1, DR5, and FAS expression, respectively, on olaparib and niraparib exposure or not. Ratios of PI+ annexin V+ OVCAR8 (D) and A2780 cells (E) on olaparib or niraparib exposure in the presence or absence of TNF-α neutralizing antibodies, including adalimumab and infliximab. Determination of activated caspase 8 and caspase 3 in OVCAR8 (F,G) and A2780 cells (H,I) with downregulating TNFR1 expression on olaparib and niraparib treatment or not by flow cytometry. (J) Western blotting analyses of cleavage of GSDMD/E in OVCAR8 and A2780 cells with downregulating TNFR1 expression on olaparib and niraparib treatment or not. (K) Representative images of OVCAR8 and A2780 cells with downregulating TNFR1 expression on olaparib and niraparib treatment or not. (L,M) Tumor cells with or without TNFR1 knocking down were exposed to olaparib or niraparib for 3 days. The lactate dehydrogenase (LDH) releases in OVCAR8 (L) and A2780 (M) culture medium were tested. Mean values±SEM. *P<0.05, **p<0.01, and ***p<0.001 by Student’s t-test in (B–I) and (L–M).

DNA damage-activated NF-κB contributes to the TNF-α secretion and PARP inhibition-induced pyroptosis

Next, we attempted to unravel the intricate mechanism governing the process of TNFR1/caspase8-associated pyroptosis. To this end, we examined the levels of death-associated ligands upstream of caspase 8, including TNF-α, TRAIL, and FASL, on olaparib or niraparib stimulation in vitro. Of note, TNF-α exhibited an increase at both transcriptional and translational levels in OVCAR8 and A2780 cells, whereas TRAIL and FASL did not (figure 8A,B, online supplemental figure S7A–J). Our RNA-seq analysis also revealed an upregulation of TNF expression in tumor cells treated with niraparib (figure 8C). To confirm this finding, we extended our investigation to OC PDX tumors by IHC staining, where we observed the significant enhancement of TNF-α expression in response to niraparib treatment (figure 8D). On further analysis of the bulk RNA-seq data, we found the enrichment of NF-κB-related genes, which were closely associated with the transcriptional regulation of TNFA, following exposure to niraparib (figure 8E). Consistently, the increased NF-κB transcriptional activity was observed in niraparib-treated OC PDX tumors (figure 8F). To determine the pivotal role of activated NF-κB in mediating TNF-α transcription, we employed siRNA to downregulate RELA (gene for p65) expression in OVACR8 and A2780 cells (online supplemental figure S8A,B) and then evaluated TNF-α expression and secretion in these engineered cells. Knockdown of RELA in the tumor cells elicited a dramatic dampening effect o