Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2024 Feb 26.
Published in final edited form as: Biochem Biophys Res Commun. 2023 Jan 20;646:78–85. doi: 10.1016/j.bbrc.2023.01.055

PARP1 Inhibitors Induce Pyroptosis via Caspase 3-mediated Gasdermin E Cleavage

Chiho Kim 1,3, Xu-Dong Wang 1,3, Seoyeon Jang 2, Yonghao Yu 1,*
PMCID: PMC9933147  NIHMSID: NIHMS1870536  PMID: 36706709

Abstract

The identification of PARP1 as a therapeutic target for BRCA1/2-deficient cells has led to a paradigm shift for the treatment of human malignancies with BRCA1/2 mutations. However, our understanding of the mechanism of action of PARP1 inhibitors (PARPi) is still evolving. It is being increasingly appreciated that the immunomodulatory function of PARPi is a critical contributor of the anti-tumor effects of these compounds. Here, we identify a novel cell death effector pathway for PARPi where PARPi induces inflammatory pyroptosis that is mediated by caspase 3-dependent cleavage of GSDME. Caspase 3 is activated upon PARPi treatment which directly cleaves GSDME and, subsequently induces pyroptosis. Genetic and pharmacological experiments show that the presence of the PARP1 protein with uncompromised DNA binding capability is required for PARPi-induced pyroptosis, suggesting that PARP1 trapping is a key driver of this phenomenon. Importantly, we show that PARPi-induced GSDME cleavage and pyroptosis occurred only in the BRCA1-deficient cells, but not in those reconstituted with BRCA1 wild-type (WT). These findings suggest that pyroptosis could be a novel aspect of the immunomodulatory function of PARPi. Our studies could also offer new insights to the potential biomarkers and therapeutic strategies to achieve better anti-tumor effects of PARPi for BRCA-deficient tumors with low GSDME expression.

Keywords: Poly-ADP-ribose-polymerase 1 (PARP1), PARP1 inhibitors (PARPi), Gasdermin E (GSDME), Pyroptosis, Caspase 3, BRCA gene

1. Introduction

BRCA1 and BRCA2 are tumor suppressor genes that are frequently mutated in breast and ovarian cancers [1]. The identification of PARP1 as a therapeutic target for BRCA1/2-deficient human malignancies has led to a paradigm shift for the treatment and management of these diseases [2–4]. Poly-ADP-ribose-polymerase 1 (PARP1) is an enzyme that is critically involved in DNA damage response (DDR). PARP1 functions as a sensor protein for genotoxic stimuli. In response to DNA damage signals, PARP1 is rapidly recruited to, and binds to DNA strand breaks [5]. This binding event dramatically stimulates its enzymatic activity, which then results in the synthesis of many Poly-ADP-ribosylated (PARylated) proteins that recruit and assemble a protein machinery (e.g., XRCC1) to mediate the repair of DNA strand breaks [5–7].

BRCA1/2-mutated tumors are deficient for DNA double-strand break (DSB) repair mechanisms, and they are selectively killed by PARP1 inhibitors (PARPi) via a mechanism known as synthetic lethality [4]. Indeed, four PARPi (olaparib, niraparib, rucaparib and talazoparib) have been approved by the FDA to treat various human malignancies with BRCA1/2 mutations (i.e., breast, ovarian, prostate and pancreatic cancers). Despite these progresses, results from recent clinical studies showed that a significant fraction of the BRCAmut tumors do not respond to PARPi (i.e., intrinsic PARPi resistance) [8]. These data point to the critical need to better understand the mechanism of action for PARPi, and to improve PARPi-based therapies to achieve a more complete therapeutic response in BRCA-deficient cancers.

Recent studies have provided evidence pointing to an important crosstalk between tumor DNA damage and the response from the immune system, during the treatment of cancers. Indeed, it is being increasingly appreciated that PARPi have immunomodulatory functions, and the therapeutic effect of PARPi in cancer can be ascribed, at least in part, to their ability to regulate innate immune signaling (e.g., via the cGAS-STING pathway) [9–14]. However, other aspects of the immunomodulatory functions of PARPi are poorly understood. Pyroptosis represents a lytic form of programmed, necrotic cell death that is tightly associated with inflammation [15, 16]. Pyroptosis is mediated by several members of the gasdermin superfamily that is composed of gasdermin A/B/C/D (GSDMA/B/C/D), gasdermin E (GSDME, also referred to as DFNA5), and DFNB59 (Pejvakin, PJVK) [15]. These proteins mainly consist of two conserved domains, the N-terminal pore-forming domain and the C-terminal autoinhibitory domain. Among the various gasdermins, GSDME is known to be cleaved by caspases [17, 18]. This proteolytic cleavage allows the translocation of the N-terminal fragment of GSDME and then its oligomerization in the cellular membrane. Consequently, the oligomerized N-terminal fragment of GSDME triggers pyroptosis by binding to cell membrane lipids and forming pores. This then allows the release of inflammatory molecules (e.g., interleukin (IL)-1β and IL-18) and other cellular contents to initiate pyroptosis [18, 19].

In this study, we identify a novel cell death effector pathway for PARPi where PARPi induces pyroptosis via a mechanism that is mediated by caspase 3-dependent cleavage of GSDME. Caspase 3 is activated upon PARPi treatment, which directly cleaves GSDME at the D270 site. Genetic and pharmacological experiments show that the presence of the PARP1 protein with uncompromised DNA binding capability is required for PARPi-induced pyroptosis, suggesting that PARP1 trapping is a key driver of this phenomenon. Importantly, we showed that PARPi-induced GSDME cleavage and pyroptosis occurred only in the BRCA1-deficient cells, but not in those reconstituted with wild-type BRCA1. These results provide evidence that the ability to promote pyroptosis could represent a novel aspect of the immunomodulatory function of PARPi in BRCA-mutated tumors.

2. Materials and methods

2.1. Cell culture and chemicals

All cell lines used in this study were purchased from ATCC and were cultured according to the directions from ATCC. Human cervical carcinoma (HeLa) cells were maintained in the high-glucose Dulbecco’s Modified Eagle’s Medium (DMEM; MilliporeSigma) supplemented with 10% Fetal Bovine Serum (FBS; MilliporeSigma) at 37°C in 5% CO2. UWB1.289 (UWB1) and UWB1.289+BRCA1 (UWB1+BRCA1) cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 (MilliporeSigma) and MEGM bullet kit (1:1; Lonza) with 3% FBS at 37°C in 5% CO2. All cells were found to be mycoplasma-free using the e-Myco kit (Boca Scientific). Talazoparib (#S7048) and z-VAD-FMK (z-VAD; #S7023) were all purchased from Selleck. Rucaparib and iRucaparib-AP6 were synthesized in our lab. The concentration and time of each chemical compound treatment are indicated in the figure legends.

2.2. Antibodies

Antibodies against the following proteins were used. Cell Signaling Technology: PARP1 (#9542), γH2AX (#9718), cleaved caspase 3 (Asp175, #9661L); Santa Cruz Biotechnology: GAPDH (#sc-32233); Abcam: GSDME (ab215191); MilliporeSigma: Flag (#F7425).

2.3. Plasmids

Flag-tagged PAPR1 WT (PARP1-Flag; #111575) was purchased from Addgene. The Flag-tagged PARP1 R138C mutant was generated by the site-directed mutagenesis Kit (Agilent, La Jolla, CA, USA) according to the manufacturer’s instructions [9]. 3XFlag-HA-GSDME WT and D270A were kind gifts from Dr. Feng Shao (National Institute of Biological Sciences, China). Plasmids were subjected to DNA sequencing for verification.

2.4. Real-time quantitative polymerase chain reaction (qRT-PCR, qPCR)

The mRNA extraction was performed using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. Subsequently, cDNA was prepared by reverse transcription using the SuperScript III Reverse Transcriptase kit (Thermo Fisher Scientific) with random oligo(dT) primers (MilliporeSigma). The qPCR was performed on a CFX384 Touch Real-Time PCR Detection System using the 2X Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) [9] and analyzed using the primer sets listed. IL-1β forward, 5’-CCACAGACCTTCCAGGAGAATG-3’ and reverse, 5’-GTGCAGTTCAGTGATCGTACAGG; GAPDH forward, 5’-TGCACCACCAACTGCTTAGC-3’ and reverse, 5’-GGCATGGACTGTGGTCATGAG-3’.

2.5. LDH release assay

Pyroptosis with membrane lysis was quantified by assaying the activity of LDH released into cell culture supernatants using the CytoTox96 LDH release kit (Promega) according to the manufacturer’s protocol. The LDH activity in the culture supernatant was expressed as a percentage of total LDH in the cell lysate.

2.6. IL-1β detection

Cells were treated with talazoparib (1 μM for 72 h). Supernatants were harvested, and the level of human IL-1β was directly detected using the IL-1β human ELISA kit (Invitrogen) according to the manufacturer’s instructions.

2.7. Immunoblot analysis

Cells were harvested and lysed in the 1% SDS lysis buffer (10 mM HEPES, pH 7.0, 2 mM MgCl2, 20 U/mL universal nucleases). Total cellular protein concentrations were measured using the BCA assay (Thermo Fisher Scientific). The lysates were mixed with the 4x reducing buffer (60 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 14.4 mM 2-mercaptoethanol, 0.1% bromophenol blue). Equal amounts of the protein samples were subjected to SDS-PAGE with the corresponding antibodies [9]. Proteins were developed using enhanced chemiluminescence exposed on an autoradiograph film using standard methods as previously described [20]. The relative band intensity was measured using the Image J imaging software.

2.8. SYTOX green uptake assay

For talazoparib-induced cell death, cells were seeded in 96-well plates overnight and treated with 1 μM talazoparib for 72 h in the presence of 2.5 μM SYTOX green (Thermo Fisher Scientific). Then, fluorescence images were observed under an LSM 510 META confocal laser scanning microscope equipped with epifluorescence and an LSM digital image analyzer (Carl Zeiss, Zana, Germany).

2.9. Statistical analysis

Statistical analyses including unpaired Student’s t-tests, one- and two-way ANOVA were performed using the GraphPad Prism software (v9.2.0). Data were calculated as mean ± SEM or SD. The following indications of significance were used throughout the manuscript: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S, not significant.

3. Results and discussion

3.1. PARPi treatment promotes pyroptosis by regulating GSDME cleavage

It has recently been reported that PARPi generate cytosolic double-stranded DNA (dsDNA) that induces the activation of cGAS-STING signaling [9, 12–14]. However, it is unclear whether PARPi could also modulate anti-tumor immune response via additional mechanisms. Here, we evaluated whether PARPi treatment could regulate pyroptosis, which is a lytic form of inflammatory cell death. As shown in Fig. 1A, we first found that talazoparib (a potent PARPi) treatment indeed resulted in the cleavage of GSDME (i.e., generation of the GSDME N-terminal fragment), which is a hallmark of pyroptosis (Fig. 1A). Because the GSDME N-terminal fragment is known to perforate membranes [18, 19, 21], we performed the SYTOX green (SYTOXG) staining assay to characterize the cell membrane integrity in PARPi-treated cells. We observed that talazoparib treatment led to a profound level of SYTOXG internalization, suggesting that PARPi treatment could impair plasma membrane integrity (Fig. 1B). A key feature of cells undergoing pyroptosis is the release of the large cytosolic tetrameric complex, lactate dehydrogenase (LDH) and also interleukin (IL)-1β. Consistent with this, we observed dramatically increased levels of LDH and IL-1β in the culture media from talazoparib-treated cells, compared to controls (Fig. 1C and 1D). These results suggest that PARPi treatment could induce the cleavage of GSDME and the subsequent pyroptotic cell death.

Figure 1. PARPi treatment induces the cleavage of GSDME to promote pyroptosis.

Figure 1.

Open in a new tab

(A) The cleavage of GSDME in HeLa cells treated with talazoparib (1 μM) in a time-dependent manner. Top, whole cell lysates were probed using indicated antibodies. Bottom, the graph shows the quantification of GSDME cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. *p < 0.05, ****p < 0.0001. (B) SYTOX green staining assay in HeLa cells treated with or without talazoparib (1 μM for 72 h). Scale bars represent 100 μm. (C) LDH release assay in HeLa cells treated with or without talazoparib (1 μM for 72 h). LDH release into the culture media was assessed and values were presented as means ± SEM from three biological replicates. Significance was determined with unpaired Student’s t-test. ****p < 0.0001. (D) Release of IL-1β in HeLa cells treated with or without talazoparib (1 μM for 72 h). IL-1β released into the culture media was assessed and values were presented as means ± SEM from three biological replicates. Significance was determined with unpaired Student’s t-test. ***p < 0.001.

3.2. PARP1 trapping mediates PARPi-dependent GSDME cleavage and pyroptosis

All FDA-approved PARPi (i.e., olaparib, niraparib, rucaparib and talazoparib) are known to possess two activities, i.e., PARP1 inhibition and PARP1 trapping [9]. Here, we sought to dissect the contribution of these two activities in the PARPi-mediated GSDME cleavage and pyroptosis. We found that talazoparib treatment significantly induced the cleavage of GSDME only in PARP1 wild-type (WT) cells. In contrast, talazoparib treatment failed to elicit GSDME cleavage in PARP1 knock-out (KO) cells (Fig. 2A). Consistently, talazoparib treatment induced significant SYTOXG internalization and subsequently, the release of LDH and IL-1β into the culture media in PARP1 WT cells, but not in PARP1 KO cells (Fig. 2B to 2D). These results suggest that the loss of the PARP1 enzymatic activity per se was not sufficient to drive GSDME cleavage and the induction of pyroptosis. Furthermore, PARP1 deletion also prevented the ability of PARPi to induce pyroptosis. These results suggest that the presence of the PARP1 protein and the subsequent PARP1 trapping are likely required for PARPi-induced GSDME cleavage and pyroptosis.

Figure 2. PARP1 trapping mediates PARPi-dependent GSDME cleavage and pyroptosis.

Figure 2.

Open in a new tab

(A) Cleavage of GSDME in PARP1 WT and KO HeLa cells treated with or without talazoparib (1 μM for 72 h). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of GSDME cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ****p < 0.0001, N.S., not significant. (B) SYTOX green staining assay in PARP1 WT and KO HeLa cells treated with or without talazoparib (1 μM for 72 h). Scale bars represent 100 μm. (C) LDH release assay in HeLa cells treated with or without talazoparib (1 μM for 72 h). LDH release into the culture media was assessed and values were presented as means ± SEM from three biological replicates. Significance was determined with two-way ANOVA. ****p < 0.0001, N.S., not significant. (D) Release of IL-1β in HeLa cells treated with or without talazoparib (1 μM for 72 h). IL-1β released into the culture media was assessed and values were presented as means ± SEM from three biological replicates. Significance was determined with two-way ANOVA. ***p < 0.001, N.S., not significant. (E) The cleavage of GSDME in HeLa cells treated with either Rucaparib or iRucaparib-AP6 (1 μM for 72 h). Left, whole cell lysates were probed using the indicated antibodies. Right, the graph shows the quantification of GSDME cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ***p < 0.001, ****p < 0.0001. (F) The cleavage of GSDME in HeLa PARP1 KO cells (expressing either PARP1 WT or the R138C mutant) that were treated with or without Talazoparib (1 μM for 72 h). Left, whole cell lysates were probed using indicated antibodies. Right, the graph shows the quantification of GSDME cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ****p < 0.0001, N.S., not significant.

We also employed a pharmacological approach to further test this hypothesis. Using the PROTAC approach, we recently developed a potent PARP1 degrader (i.e., iRucaparib-AP6) [22]. By degrading the PARP1 protein, this compound blocked the enzymatic activity of PARP1 without inducing its trapping [22]. Consistent with the aforementioned hypothesis, we found that the treatment of the PARPi (Rucaparib), but not the PARP1 degrader (iRucaparib-AP6) led to dramatic GSDME cleavage (Fig. 2E). Finally, to explore the role of PARP1 trapping in mediating GSDME cleavage, we employed a PARP1 mutant (R138C) that shows significantly decreased DNA binding capability, and therefore is unable to be trapped on chromatin upon the treatment of PARPi [23]. Using the PARP1 KO cells [9], we first reconstituted these cells with either PARP1 WT or the PARP1 R138C mutant. These PARP1 proteins (WT or the R138C mutant) were expressed at similar levels. We treated these cells with talazoparib, and observed that talazoparib treatment was able to induce GSDME cleavage in cells expressing PARP1 WT, but not the PARP1 R138C mutant (Fig. 2F). These results again suggest that the presence of PARP1 with uncompromised trapping capability is required for PARPi-induced GSDME cleavage.

3.3. PARPi induces GSDME cleavage and pyroptosis via activated caspase 3

It has been reported that GSDME cleavage could be mediated by several mechanisms, including that by activated caspase 3 [18, 19]. Importantly, caspase 3-mediated cleavage of GSDME results in a switch in the cell death types from apoptosis (usually considered to be non-inflammatory) to pyroptosis (inflammatory cell death) [17, 18]. Indeed, we observed that talazoparib treatment induces the activation of caspase 3. Again, PARPi-induced caspase 3 activation is likely driven by PARP1 trapping, because this was only observed in PARP1 WT cells, but not PARP1 KO cells (Fig. 3A). To investigate whether PARPi-induced GSDME cleavage is mediated by caspase 3, we used a pan-caspase inhibitor z-VAD. We found that co-treatment of cells with talazoparib and z-VAD completely blocked PARPi-induced GSDME cleavage (Fig. 3B).

Figure 3. PARPi induces GSDME cleavage and pyroptosis via activated caspase 3.

Figure 3.

Open in a new tab

(A) The activation of caspase 3 in PARP1 WT and KO HeLa cells treated with or without talazoparib (1 μM for 72 h). Top, whole cell lysates were probed using the indicated antibodies. Bottom, the graph shows the quantification of caspase 3 cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ****p < 0.0001, N.S., not significant. (B) The cleavage of GSDME in HeLa cells treated with talazoparib (1 μM for 72 h) in the presence or absence of z-VAD (50 μM). Top, whole cell lysates were probed using indicated antibodies. Bottom, the graph shows the quantification of GSDME cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ****p < 0.0001. (C) The cleavage of GSDME in HeLa cells expressing either GSDME WT or D270A mutant that were treated with or without Talazoparib (1 μM for 72 h). Left, whole cell lysates were probed using indicated antibodies. Right, the graph shows the quantification of GSDME cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ****p < 0.0001, N.S., not significant. (D) SYTOX green staining assay in HeLa cells treated with talazoparib (1 μM for 72 h) in the presence or absence of z-VAD (50 μM). Scale bars represent 100 μm. (E) LDH release assay in HeLa cells treated with talazoparib (1 μM for 72 h) in the presence or absence of z-VAD (50 μM). LDH released in the culture media was assessed and values were presented as means ± SEM from three biological replicates. Significance was determined with one-way ANOVA. ***p < 0.001, ****p < 0.0001. (D) Release of IL-1β in HeLa cells treated with talazoparib (1 μM for 72 h) in the presence or absence of z-VAD (50 μM). IL-1β release in the culture media was assessed and values were presented as means ± SEM from three biological replicates. Significance was determined with one-way ANOVA. ***p < 0.001, ****p < 0.0001.

It has been shown in several recent studies that GSDME had a potential caspase 3 cleavage site at Asp270 (i.e., DMPD(270)) [17, 18]. We examined whether GSDME is also cleaved at the same site (D270) by caspase 3 under PARPi-treated conditions. We ectopically expressed in HeLa cells either 3XFlag-HA-GSDME WT or the non-cleavable mutant of GSDME (D270A). We found that talazoparib treatment of these cells resulted in the GSDME cleavage in the GSDME WT, but not in the GSDME D270A mutant (Fig. 3C). These results suggest that PARPi treatment resulted in caspase 3 activation, which then cleaved GSDME at the D270 site. We then investigated the role of caspase 3 in PARPi-induced pyroptosis. As expected, talazoparib treatment resulted in a profound level of SYTOXG internalization and also the release of LDH and IL-1β into the culture media. However, co-treatment of cells by talazoparib and z-VAD completely blocked SYTOXG internalization and the release of LDH and IL-1β into the culture media (Fig. 3D to 3F). These results demonstrate that PARPi induces the cleavage of GSDME and subsequent pyroptosis via activated caspase 3.

3.4. PARPi selectively induces GSDME cleavage and pyroptosis in BRCA-deficient cells

It has been previously shown that cells with BRCA1/2 mutations are selectively killed by PARPi via a mechanism known as synthetic lethality [4]. More recent studies have also provided compelling evidence pointing to immunomodulatory functions as a key contributor of the anti-cancer effects of PARPi [14, 24, 25]. However, it is unclear whether BRCA1/2 mutations could affect the ability of PARPi to regulate GSDME cleavage and pyroptosis. Indeed, treatment of UWB1 cells (a BRCA1-mutated ovarian cancer cell line) with talazoparib resulted in robust caspase 3 activation, and subsequently GSDME cleavage (Fig. 4A and 4C). Similar results were obtained using a different PARPi (rucaparib) (Fig. 4B). Again, PARP1 trapping likely mediated these effects because the treatment of UWB1 cells by the PARP1 degrader (i.e., iRucaparib-AP6) failed to elicit GSDME cleavage (Fig. 4B). GSDME cleavage under these conditions was mediated by caspase 3 because it was completely blocked by a pan-caspase inhibitor (i.e., z-VAD) (Fig. 4C). Caspase 3 inhibition by the co-treatment of z-VAD also completely blocked the release of LDH into the culture media (Fig. 4D). These results again suggest a pathway whereby PARPi cause apoptotic cell death in UWB1 cells, and activated caspase 3 under these conditions then mediates GSDME cleavage, potentially converting cells into pyroptosis.

Figure 4. PARPi selectively induces GSDME cleavage and pyroptosis in BRCA-deficient cells.

Figure 4.

Open in a new tab

(A) The cleavage of GSDME in UWB1 and UWB1+BRCA1 cells treated with or without talazoparib (1 μM for 72 h). Left, whole cell lysates were probed using the indicated antibodies. Right, the graph shows the quantification of GSDME cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with two-way ANOVA. ****p < 0.0001, N.S., not significant. (B) The cleavage of GSDME in UWB1 cells treated with either Rucaparib or iRucaparib-AP6 (1 μM for 72 h). Left, whole cell lysates were probed using indicated antibodies. Right, the graph shows the quantification of GSDME cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ****p < 0.0001. (C) The cleavage of GSDME in UWB1 cells treated with talazoparib (1 μM for 72 h) in the presence or absence of z-VAD (50 μM). Left, whole cell lysates were probed using indicated antibodies. Right, the graph shows the quantification of GSDME cleavage. Values were presented as means ± SD from three biological replicates. Significance was determined with one-way ANOVA. ****p < 0.0001. (D) LDH release assay in HeLa cells treated with talazoparib (1 μM for 72 h) in the presence or absence of z-VAD (50 μM). LDH release into the culture media was assessed and values were presented as means ± SEM from three biological replicates. Significance was determined with one-way ANOVA. ***p < 0.001, ****p < 0.0001. (E) SYTOX green staining assay in UWB1 and UWB1+BRCA1 cells treated with or without talazoparib (1 μM for 72 h). Scale bars represent 100 μm. (F) LDH release assay in UWB1 and UWB1+BRCA1 treated with or without talazoparib (1 μM for 72 h). LDH release into the culture media was assessed and values were presented as means ± SEM from three biological replicates. Significance was determined with two-way ANOVA. ****p < 0.0001, N.S., not significant. (G) Release of IL-1β in UWB1 and UWB1+BRCA1 treated with or without talazoparib (1 μM for 72 h). IL-1β release into the culture media was assessed and values were presented as means ± SEM from three biological replicates. Significance was determined with two-way ANOVA. ***p < 0.001, N.S., not significant.

Next, we repeated the abovementioned experiments using the UWB1 cells that were reconstituted with BRCA1 WT (i.e., UWB1+BRCA1 cells). Intriguingly, we found that talazoparib treatment failed to trigger GSDME cleavage in UWB1+BRCA1 cells (Fig. 4A). Accordingly, talazoparib treatment also did not cause SYTOXG internalization or the release of LDH and IL-1β into the culture media (Fig. 4E to 4G). Taken together, these results demonstrate that the mutation status of the BRCA gene is a critical determinant for PARPi-mediated GSDME cleavage and the subsequent pyroptosis.

It is important to note that despite the tremendous success of PARPi in the clinic, a significant fraction of the cancer patients with BRCA mutations fail to respond to PARPi (i.e., intrinsic resistance) [8]. In this context, GSDME could function as a tumor suppressor, the loss of which could render PARPi unable to induce pyroptosis and its associated anti-tumor inflammatory response [18, 19]. The expression of GSDME in tumors could therefore be a potential biomarker that predicts the induction of pyroptosis in tumors and the subsequent therapeutic response to PARPi. The ability of PARPi to induce pyroptosis represents an important aspect of the immunomodulatory function of PARPi, and this warrants future studies using clinically relevant samples. Finally, recent studies suggest that DNA methylation inhibitors (e.g., decitabine) could induce the expression of GSDME [26, 27]. Considering that decitabine has already been approved by the FDA (for the treatment of leukemia), the combination of PARPi and decitabine are worth exploring and testing for BRCA-deficient tumors with low GSDME expression.

In conclusion, our findings suggest that the immunomodulatory functions of PARPi could be mediated by multiple mechanisms. In addition to the modulation of the cGAS-STING pathway [9, 12–14], we report in this study that the treatment of PARPi induces the activation of caspase 3 and subsequently the cleavage of GSDME at D270. This then triggers the potential conversion of the cell death mechanisms from apoptosis to pyroptosis, as evident by the release of LDH and pro-inflammatory cytokines (e.g., IL-1β) into the extracellular space. Our findings suggest that the induction of pyroptosis could be associated with the immunomodulatory function of PARPi. Importantly, the PARPi-mediated pyroptosis was observed in BRCA1-deficient, but not in BRCA1-proficient cells. Tumors with high expression of GSDME could potentially respond better to PARPi, because of enhanced PARPi-induced pyroptosis, and the resulting immune response. In this regard, it would be interesting to determine the level of GSDME expression in tumor samples, and assess whether GSDME expression could serve as a potential biomarker to predict the therapeutic response to PARPi. Finally, our study warrants the future study to test the combination of PARPi with agents that induce GSDME expression (e.g., decitabine) to achieve better anti-cancer effects for BRCA-deficient tumors with low GSDME expression.

Highlights.

  1. PARPi induces pyroptosis via the cleavage of GSDME

  2. PARP1 trapping is required for PARPi-mediated GSDME cleavage and pyroptosis

  3. PARPi induces the cleavage of GSDME and pyroptosis via activated caspase 3

  4. BRCA mutation status determines PARPi-mediated pyroptosis

Acknowledgements

We thank for Dr. Feng Shao for providing the expression construct for 3XFlag-HA-GSDME WT and D270A. This work was supported by NIH (R21 CA261018, R35 GM134883 and R01 NS122533) to Y.Y.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Declaration of competing interest

A patent application on the PARP1 degraders and the technologies described herein has been filed by L. You, L. Han, S. Wang, Y. Yu and C. Chen. Y. Yu is a co-founder and shareholder of ProteoValent Therapeutics.

References

  • [1].Welcsh PL, King MC, BRCA1 and BRCA2 and the genetics of breast and ovarian cancer, Human molecular genetics, 10 (2001) 705–713. [DOI] [PubMed] [Google Scholar]
  • [2].Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A, Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy, Nature, 434 (2005) 917–921. [DOI] [PubMed] [Google Scholar]
  • [3].Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T, Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase, Nature, 434 (2005) 913–917. [DOI] [PubMed] [Google Scholar]
  • [4].Lord CJ, Ashworth A, PARP inhibitors: Synthetic lethality in the clinic, Science (New York, N.Y.), 355 (2017) 1152–1158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Ray Chaudhuri A, Nussenzweig A, The multifaceted roles of PARP1 in DNA repair and chromatin remodelling, Nature reviews. Molecular cell biology, 18 (2017) 610–621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [6].Gibson BA, Kraus WL, New insights into the molecular and cellular functions of poly(ADP-ribose) and PARPs, Nature reviews. Molecular cell biology, 13 (2012) 411–424. [DOI] [PubMed] [Google Scholar]
  • [7].Liu C, Vyas A, Kassab MA, Singh AK, Yu X, The role of poly ADP-ribosylation in the first wave of DNA damage response, Nucleic acids research, 45 (2017) 8129–8141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [8].Pessetto ZY, Yan Y, Bessho T, Natarajan A, Inhibition of BRCT(BRCA1)-phosphoprotein interaction enhances the cytotoxic effect of olaparib in breast cancer cells: a proof of concept study for synthetic lethal therapeutic option, Breast cancer research and treatment, 134 (2012) 511–517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Kim C, Wang XD, Yu Y, PARP1 inhibitors trigger innate immunity via PARP1 trapping-induced DNA damage response, eLife, 9 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Franzese O, Graziani G, Role of PARP Inhibitors in Cancer Immunotherapy: Potential Friends to Immune Activating Molecules and Foes to Immune Checkpoints, Cancers, 14 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Zhu H, Tang YD, Zhan G, Su C, Zheng C, The Critical Role of PARPs in Regulating Innate Immune Responses, Frontiers in immunology, 12 (2021) 712556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Ding L, Kim HJ, Wang Q, Kearns M, Jiang T, Ohlson CE, Li BB, Xie S, Liu JF, Stover EH, Howitt BE, Bronson RT, Lazo S, Roberts TM, Freeman GJ, Konstantinopoulos PA, Matulonis UA, Zhao JJ, PARP Inhibition Elicits STING-Dependent Antitumor Immunity in Brca1-Deficient Ovarian Cancer, Cell reports, 25 (2018) 2972–2980.e2975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Shen J, Zhao W, Ju Z, Wang L, Peng Y, Labrie M, Yap TA, Mills GB, Peng G, PARPi Triggers the STING-Dependent Immune Response and Enhances the Therapeutic Efficacy of Immune Checkpoint Blockade Independent of BRCAness, Cancer research, 79 (2019) 311–319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Pantelidou C, Sonzogni O, De Oliveria Taveira M, Mehta AK, Kothari A, Wang D, Visal T, Li MK, Pinto J, Castrillon JA, Cheney EM, Bouwman P, Jonkers J, Rottenberg S, Guerriero JL, Wulf GM, Shapiro GI, PARP Inhibitor Efficacy Depends on CD8(+) T-cell Recruitment via Intratumoral STING Pathway Activation in BRCA-Deficient Models of Triple-Negative Breast Cancer, Cancer discovery, 9 (2019) 722–737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Yu P, Zhang X, Liu N, Tang L, Peng C, Chen X, Pyroptosis: mechanisms and diseases, Signal transduction and targeted therapy, 6 (2021) 128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Man SM, Karki R, Kanneganti TD, Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases, Immunological reviews, 277 (2017) 61–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Rogers C, Fernandes-Alnemri T, Mayes L, Alnemri D, Cingolani G, Alnemri ES, Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death, Nature communications, 8 (2017) 14128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Wang Y, Gao W, Shi X, Ding J, Liu W, He H, Wang K, Shao F, Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin, Nature, 547 (2017) 99–103. [DOI] [PubMed] [Google Scholar]
  • [19].Zhang Z, Zhang Y, Xia S, Kong Q, Li S, Liu X, Junqueira C, Meza-Sosa KF, Mok TMY, Ansara J, Sengupta S, Yao Y, Wu H, Lieberman J, Gasdermin E suppresses tumour growth by activating anti-tumour immunity, Nature, 579 (2020) 415–420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Kim C, Yun N, Lee J, Youdim MB, Ju C, Kim WK, Han PL, Oh YJ, Phosphorylation of CHIP at Ser20 by Cdk5 promotes tAIF-mediated neuronal death, Cell death and differentiation, 23 (2016) 333–346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Rogers C, Erkes DA, Nardone A, Aplin AE, Fernandes-Alnemri T, Alnemri ES, Gasdermin pores permeabilize mitochondria to augment caspase-3 activation during apoptosis and inflammasome activation, Nature communications, 10 (2019) 1689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [22].Wang S, Han L, Han J, Li P, Ding Q, Zhang QJ, Liu ZP, Chen C, Yu Y, Uncoupling of PARP1 trapping and inhibition using selective PARP1 degradation, Nature chemical biology, 15 (2019) 1223–1231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [23].Herzog M, Puddu F, Coates J, Geisler N, Forment JV, Jackson SP, Detection of functional protein domains by unbiased genome-wide forward genetic screening, Scientific reports, 8 (2018) 6161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [24].Jiao S, Xia W, Yamaguchi H, Wei Y, Chen MK, Hsu JM, Hsu JL, Yu WH, Du Y, Lee HH, Li CW, Chou CK, Lim SO, Chang SS, Litton J, Arun B, Hortobagyi GN, Hung MC, PARP Inhibitor Upregulates PD-L1 Expression and Enhances Cancer-Associated Immunosuppression, Clinical cancer research : an official journal of the American Association for Cancer Research, 23 (2017) 3711–3720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Reisländer T, Lombardi EP, Groelly FJ, Miar A, Porru M, Di Vito S, Wright B, Lockstone H, Biroccio A, Harris A, Londoño-Vallejo A, Tarsounas M, BRCA2 abrogation triggers innate immune responses potentiated by treatment with PARP inhibitors, Nature communications, 10 (2019) 3143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [26].Kim MS, Chang X, Yamashita K, Nagpal JK, Baek JH, Wu G, Trink B, Ratovitski EA, Mori M, Sidransky D, Aberrant promoter methylation and tumor suppressive activity of the DFNA5 gene in colorectal carcinoma, Oncogene, 27 (2008) 3624–3634. [DOI] [PubMed] [Google Scholar]
  • [27].Akino K, Toyota M, Suzuki H, Imai T, Maruyama R, Kusano M, Nishikawa N, Watanabe Y, Sasaki Y, Abe T, Yamamoto E, Tarasawa I, Sonoda T, Mori M, Imai K, Shinomura Y, Tokino T, Identification of DFNA5 as a target of epigenetic inactivation in gastric cancer, Cancer science, 98 (2007) 88–95. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES