Cellular senescence-associated gene IFI16 promotes HMOX1-dependent evasion of ferroptosis and radioresistance in glioblastoma

Zhou, Yuchuan; Zeng, Liang; Cai, Linbo; Zheng, Wang; Liu, Xinglong; Xiao, Yuqi; Jin, Xiaoya; Bai, Yang; Lai, Mingyao; Li, Hainan; Jiang, Hua; Hu, Songling; Pan, Yan; Zhang, Jianghong; Shao, Chunlin

doi:10.1038/s41467-025-56456-y

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Published: 31 January 2025

Cellular senescence-associated gene IFI16 promotes HMOX1-dependent evasion of ferroptosis and radioresistance in glioblastoma

Yuchuan Zhou¹^na1,
Liang Zeng¹^na1,
Linbo Cai²^na1,
Wang Zheng¹,
Xinglong Liu¹,
Yuqi Xiao¹,
Xiaoya Jin¹,
Yang Bai¹,
Mingyao Lai²,
Hainan Li²,
Hua Jiang³,
Songling Hu⁴,
Yan Pan ORCID: orcid.org/0000-0003-4307-3616¹,
Jianghong Zhang ORCID: orcid.org/0000-0003-4884-6411¹ &
…
Chunlin Shao ORCID: orcid.org/0000-0001-9336-9912¹

Nature Communications volume 16, Article number: 1212 (2025) Cite this article

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Abstract

Glioblastoma multiforme (GBM) remains a therapeutic challenge due to its aggressive nature and recurrence. This study establishes a radioresistant GBM cell model through repeated irradiation and observes a cellular senescence-like phenotype in these cells. Comprehensive genomic and transcriptomic analyses identify IFI16 as a central regulator of this phenotype and contributes to radioresistance. IFI16 activates HMOX1 transcription thereby attenuating ferroptosis by reducing lipid peroxidation, ROS production, and intracellular Fe²⁺ content following irradiation. Furthermore, IFI16 interacts with the transcription factors JUND and SP1 through its pyrin domain, robustly facilitating HMOX1 expression, further inhibiting ferroptosis and enhancing radioresistance in GBM. Notably, glyburide, a sulfonylurea compound, effectively disrupts IFI16 function and enhances ferroptosis and radiosensitivity. By targeting the pyrin domain of IFI16, glyburide emerges as a potential therapeutic agent against GBM radioresistance. These findings underscore the central role of IFI16 in GBM radioresistance and offer promising avenues to improve GBM treatment.

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Introduction

Glioblastoma multiforme (GBM), a WHO grade IV brain malignancy, remains a formidable challenge in oncology, with a 5-year survival rate of only 7.2% due to its aggressive nature and therapeutic resistance^1,2. Despite advancements in treatment modalities such as surgery, radiotherapy, and chemotherapy, GBM exhibits a propensity for recurrence due to its cellular heterogeneity and evolution during treatment³. Recent investigations have highlighted the evolutionary trajectory of gliomas during treatment and showed a trend from nascent to old tumors during therapy^4,5, suggesting potential cellular senescence during GBM treatment.

Traditionally, cellular senescence is considered an anti-tumor facet with positive treatment outcomes⁶. However, emerging evidence indicates that radiotherapy and chemotherapy induce the accumulation of senescent cells in tumors^7,8. Moreover, these senescent tumor cells may promote relapse, metastasis, and drug resistance through the senescence-associated secretory phenotype (SASP)⁹. Gamma-interferon-inducible protein 16 (IFI16), recognized as an innate immune sensor for intracellular DNA¹⁰, has been implicated in cellular senescence¹¹. IFI16 triggers IFN-β production and inflammasome activation in a STING-dependent manner, initiating proinflammatory cytokine release and potentially driving cellular senescence¹². Despite the documented correlation of increased IFI16 expression with poor prognosis in various tumors^13,14,15, the precise mechanism of IFI16’s impact on tumor development remains elusive. This study elucidated whether IFI16 contributes to programmed cell death (PCD), particularly ferroptosis, in GBM.

Ferroptosis, a newly recognized PCD that depends on iron-dependent lipid peroxidation accumulation^16,17, has been suggested to be the most enriched PCD process in GBM¹⁸. Heme oxygenase-1 (HMOX1), a stress-responsive enzyme that traditionally protects against PCD¹⁹, associates with drug resistance and the increased invasive capacity in GBM^20,21. However, due to its ability to mediate the production of unstable ferrous ions, the relationship between HMOX1 and ferroptosis remains controversial²². Consequently, the precise role of HMOX1 in regulating ferroptosis in the context of GBM remains a mystery.

In this work, we establish a radioresistant GBM cell model and identify IFI16 as a key gene associated with cellular senescence and radiosensitivity. We demonstrate that IFI16 activates HMOX1 by interacting with JunD proto-oncogene (JUND) and Sp1 transcription factor (SP1), enhancing ferroptosis inhibition and radioresistance. Furthermore, we show that glyburide binds to IFI16’s pyrin domain and act as a potent radiosensitizer both in vitro and in vivo, offering a promising therapeutic strategy for GBM.

Results

Radioresistant GBM cells exhibit a cellular senescence-like phenotype

As detailed in our previous study²³, we established two radioresistant GBM cell lines, U251R and Ln229R, by exposing GBM cells to fractional irradiation (Fig. 1a). These cell lines exhibited sensitivity enhancement ratios (SER) of 0.64 and 0.67 compared to their parental U251 and Ln229 counterparts, respectively (Fig. 1b, c). In these cells, heterochromatin was reorganized and evidenced by the increased expression of heterochromatin protein 1 (HP1) and decreased level of H3K27ac²⁴ (Fig. 1d). Additionally, cell cycle arrest-related proteins p21 and p16 were upregulated (Fig. 1d). Morphologically, the radioresistant cells were elongated, displaying a higher length-to-width ratio (Fig. 1e).

Furthermore, U251R and Ln229R cells exhibited elevated β-galactosidase activity, a well-established indicator of cellular senescence (Fig. 1f). These radioresistant GBM cells also exhibited a marked increase in SASP factors, such as cytokines, chemokines, and matrix metalloproteinase (Fig. 1g), other hallmarks of cellular senescence²⁵. Specifically, the secretions of CCL5, IL-6, MMP-9, and IFN-β were elevated (Fig. 1h). A decrease of Ki67-positive area was observed in the radioresistant cells, indicating a reduced proliferative capacity (Fig. 1i).

Activation of immune response signals typically results in heightened release of SASP factors⁹. Gene set enrichment analysis (GSEA) of RNA-seq data from U251R versus U251 cells showed a conspicuous upregulation of the immune system process in the radioresistant GBM cells (Fig. 1j). Furthermore, Gene Ontology (GO) analysis highlighted significant enrichment of differentially expressed genes (DEGs) linked to the immune system process (Fig. 1k).

IFI16 promotes a cellular senescence-like phenotype in GBM

Genome accessibility alterations play a pivotal role in cellular senescence²⁶. Thus, we performed ATAC-seq analyses on U251R and U251 cells to identify the key gene alterations associated with the cellular senescence-like phenotype. The results elucidated a higher peak count frequency in U251 compared to U251R (Supplementary Fig. 1a). Additionally, the peak analysis underscored a higher chromatin accessibility of the promoter region in U251 cells compared to U251R cells (Supplementary Fig. 1b). Further exploration via GSEA analysis of the different peaks demonstrated the heightened chromatin accessibility for the genes associated with inflammatory response in U251R cells (Supplementary Fig. 1c). Using DESseq2 for ATAC-seq peak analysis and subsequent integration with RNA-seq data, 647 DEGs were identified (Fig. 2a). Protein interaction network analysis of these DEGs highlighted the “defense response to virus” pathway (Supplementary Fig. 1d), with IFI16 positioned as a central regulator in this network. Elevated IFI16 expression in radioresistant GBM cells was confirmed through both ATAC-seq and Western blot analysis (Fig. 2b, c). Additionally, IFI16 was linked to the pathways associated with SASP (Fig. 2d).

**Fig. 2: IFI16 is a critical gene in regulating cellular senescence-like phenotype in GBM.**

To further validate the critical role of IFI16 in promoting the cellular senescence-like phenotype, we strategically knocked-down IFI16 by transferring shIFI16 into U251R cells, followed by comprehensive RNA-seq and GSEA analyses, and found the downregulation of the immune system process (Fig. 2e). IFI16 knockdown led to decreased β-galactosidase activity (Fig. 2f), reduced expression of HP1, p21, and p16, increased H3K27ac level (Fig. 2g), and increased Ki67-positive cells that indicated the enhancement of cell proliferative ability (Fig. 2h). Moreover, the SASP-related inflammatory factors were remarkably downregulated in the shIFI16-transfected radioresistant cells (Fig. 2i, j).

Conversely, overexpression of IFI16 in U251 and Ln229 cells led to increased expressions of HP1, p21, and p16, and decreased H3K27ac level (Supplementary Fig. 2a). This was accompanied by upregulation of SASP-related inflammatory factors (Supplementary Fig. 2b) and a reduction in Ki67-positive area (Supplementary Fig. 2c, d). Reintroduction of IFI16 into IFI16 knockdown cells restored HP1, p21, and p16 expressions, reduced H3K27ac level (Supplementary Fig. 2e), and decreased Ki67-positive area (Supplementary Fig. 2f, g).

To further investigate IFI16’s role in promoting a cellular senescence-like phenotype, we treated GBM cells with temozolomide (TMZ) and doxorubicin (DOX), both established senescence inducers^27,28. These treatments resulted in a marked decrease in Ki67 expression (Supplementary Fig. 3a, b) and increased the expressions of IFI16, HP1, p21, and p16, alongside a reduction in H3K27ac level (Supplementary Fig. 3c). Knockdown of IFI16 in these cells led to increased Ki67 expression (Supplementary Fig. 3d, e) and reduced levels of HP1, p21, and p16, while increased H3K27ac (Supplementary Fig. 3f). Together, these findings highlight IFI16 as a critical regulator of the cellular senescence-like phenotype in GBM.

IFI16 contributes to radioresistance through inhibition of ferroptosis in GBM cells

Utilizing the GlioVis clinical database²⁹, we revealed a remarkable association between elevated IFI16 expression and poor prognosis in GBM (Fig. 3a), and IFI16 was also found to be upregulated in the recurrent GBM (Fig. 3b). In addition, a gradual increase of IFI16 expression was congruent with the ascending grades of GBM (Fig. 3c). The colony formation assay showed that shIFI16 increased the radiosensitivity of U251R and Ln229R cells (Fig. 3d), while overexpression of IFI16 resulted in radioresistance of U251 and Ln229 cells (Fig. 3e). In vivo experiments also demonstrated that the growth of xenograft tumor was significantly inhibited by shIFI16 (Fig. 3f, h) but enhanced by IFI16 overexpression after irradiation (Fig. 3g, h).

**Fig. 3: IFI16 contributes to the radioresistance of GBM.**

To elucidate the predominant mode of radiation-induced cell death, we treated the IFI16 knockdown cells of U251R and Ln229R with different agents including iron chelator (DFO), inhibitors targeting ferroptosis (Fer-1), necroptosis (Nec-1), apoptosis (Z-VAD) or autophagy (3-MA), followed by exposure to 6 Gy irradiation. It was found that Fer-1 and DFO rescued the growth inhibition after irradiation, whereas Nec-1, Z-VAD, and 3-MA had no significant effect on these IFI16 knockdown cells. (Fig. 4a). Furthermore, Fer-1 treatment rescued the radiosensitization effect of shIFI16 (Fig. 4b, c). These findings indicate that radiosensitization in shIFI16 cells is likely associated with increased ferroptosis, as only the ferroptosis inhibitor effectively counteracted the radiosensitization. Subsequently, we analyzed some key indicators of ferroptosis, including the disruption of iron homeostasis, accumulation of reactive oxygen species (ROS), and formation of lipid peroxide^16,17. Glutathione peroxidase 4 (GPX4), a crucial enzyme that mitigates ferroptosis by reducing lipid hydroperoxides¹⁷, showed significantly higher expression in the irradiated U251R and Ln229R cells compared to their parental counterparts, along with reduced levels of lipid peroxidation, ROS, and intracellular Fe²⁺ (Supplementary Fig. 4a–d). IFI16 knockdown led to decreased GPX4 expression, particularly after irradiation (Fig. 4d), and correspondingly increased levels of lipid peroxides, ROS, and intracellular Fe²⁺ (Fig. 4e–g).

**Fig. 4: IFI16 as a key regulator in the suppression of ferroptosis following irradiation.**

Conversely, IFI16 overexpression reduced lipid peroxidation, ROS, and intracellular Fe²⁺ levels while increasing GPX4 expression in the irradiated GBM cells (Supplementary Fig. 4e–h). Moreover, reintroducing IFI16 into IFI16 knockdown cells reversed the increase in lipid peroxidation, ROS, and Fe²⁺ levels, and reinstated GPX4 expression (Fig. 4h–k). These results suggest that IFI16 may potentially contribute to the radioresistance of GBM cells by inhibiting ferroptosis.

IFI16 inhibits ferroptosis in a HMOX1-dependent manner

To understand how IFI16 mitigates ferroptosis, we performed a comprehensive analysis integrating RNA-seq of GBM cells with the FerrDb ferroptosis database (http://www.zhounan.org/ferrdb/), which provided 11 overlapping genes (Fig. 5a). Notably, five of these genes, as the suppressors of ferroptosis, were significantly upregulated in U251R cells and decreased in IFI16 knockdown cells (Supplementary Fig. 5a). Intriguingly, within these genes, HMOX1 stood out as the sole gene categorized both as a suppressor and driver of ferroptosis in the FerrDb database (Fig. 5a). RT-qPCR analysis verified the upregulation of HMOX1 in two radioresistant GBM cells (Supplementary Fig. 5b), and this upregulation was attenuated by shIFI16 (Supplementary Fig. 5c), emphasizing the critical role of HMOX1 in IFI16-mediated ferroptosis in GBM cells.

**Fig. 5: IFI16 regulates GBM ferroptosis in a HMOX1-dependent manner.**

Moreover, the elevated HMOX1 expression correlated with an unfavorable prognosis in GBM (Supplementary Fig. 5d), and HMOX1 was significantly upregulated in the recurrent tumor and gradually increased with ascending GBM grade (Supplementary Fig. 5e, f). It was found that siRNA HMOX1 (siHMOX1) increased the radiosensitivity (Supplementary Fig. 6a), whereas overexpression of HMOX1 promoted radioresistance of GBM cells (Supplementary Fig. 6b). Moreover, siHMOX1 correlated with the decrease of GPX4 expression and the increases of lipid peroxide, ROS, and intracellular Fe²⁺ in the GBM cells after irradiation (Supplementary Fig. 6c–f), while HMOX1 overexpression had opposite effects on the GBM cells (Supplementary Fig. 6g–j). Accordingly, HMOX1 inhibited radiation-induced ferroptosis in the GBM cells.

The role of HMOX1 in ferroptosis remains contentious, as evidenced by its dual classification as both a suppressor and a driver of ferroptosis in the FerrDb database (Fig. 5a). Although our experiments supported the inhibitory role of HMOX1 in ferroptosis, further investigations are essential to elucidate whether the conditions existed for HMOX1-promoted ferroptosis. Given the consistent correlation between HMOX1-induced ferroptosis and the activation of heme metabolism³⁰, we investigated the dynamics of heme metabolism in the GBM cells. Notably, the radioresistant GBM cells exhibited low expression levels of heme synthesis genes UROS, CPOX, and PPOX, except HMBS in these cells (Supplementary Fig. 7a). Intriguingly, manipulating the expression of HMOX1, whether through knockdown or overexpression, had no significant effect on the heme metabolism level in the GBM cells (Supplementary Fig. 7b, c). In addition, a lower level of PPOX and UROS significantly correlated with poorer prognosis in GBM, implying that the heme metabolism is impaired in this malignancy (Supplementary Fig. 7d, e). Moreover, the protein expression HMOX1 was highly expressed in the radioresistant GBM cells (Supplementary Fig. 7f), but it was downregulated by shIFI16 and upregulated by IFI16 overexpression (Supplementary Fig. 7g, h).

In GBM cells treated with TMZ and DOX, HMOX1 and GPX4 expression levels were significantly elevated. However, these increases were suppressed by IFI16 knockdown (Supplementary Fig. 8a). Moreover, knocking down IFI16 led to further increases in lipid peroxidation, ROS levels, and intracellular Fe²⁺ levels following irradiation in these cells (Supplementary Fig. 8b–d).

The immunohistochemical assay of clinical GBM samples also showed that the expressions of IFI16, HMOX1 GPX4, and p21 had high levels in the recurrent GBM tissues in comparison of primary GBM (Fig. 5b). Analysis of the GlioVis database identified a positive correlation between IFI16 and HMOX1 (Fig. 5c). Overexpression of HMOX1 partially reversed the decrease of GPX4 level induced by shIFI16 in both nonirradiated and irradiated GBM cells (Fig. 5d), and it attenuated the increases of lipid peroxidation, ROS and intracellular Fe²⁺ content induced by shIFI16 after irradiation (Fig. 5e–g). Crucially, overexpression of HMOX1 effectively counteracted the increased radiosensitivity caused by IFI16 knockdown (Fig. 5h, i). These observations further underscored the ability of IFI16 in inducing HMOX1 expression and thereby reducing ferroptosis in the irradiated GBM cells.

IFI16 promotes HMOX1 elevation by interacting with JUND and SP1

Given the nuclear localization of IFI16¹¹ and its predominant distribution in the nucleus following irradiation (Supplementary Fig. 9a, b), we speculate that IFI16 plays its regulation role in HMOX1 expression through some specific transcription factors. To unravel this intricate mechanism, we performed motif analysis on the ATAC-seq data. The top 5 scoring motifs corresponded to FOSL2::JUND, CTCF, MSANTD3, PH0114.1, and SOX4 (Fig. 6a and Supplementary Table 1). Interestingly, the JUND binding motif (JBM) was located within the promoter region of HMOX1 (Fig. 6b). Nevertheless, ATAC-seq analysis indicated a seemingly paradoxical finding i.e., chromatin accessibility at the transcription start site of HMOX1 in the radioresistant U251R cells was lower than that in U251 cells (Fig. 6c), in contrast to the uniform elevation of IFI16 expression in both RNA-seq and ATAC-seq analyses about U251R cells (Fig. 6d). This difference prompted us to hypothesize the existence of a transcriptional repressor in the parental GBM cells, imposing constraints on HMOX1 expression.

**Fig. 6: IFI16 promotes HMOX1 transcription by interacting with JUND and SP1.**

Coincidentally, the literatures support the interaction of IFI16 with the transcriptional repressor SP1^31,32, known for its collaboration with HDAC1 in transcriptional repression^33,34. Besides, the HMOX1 promoter harbors both JBM and SP1 binding motifs (SBM) (Fig. 6b). These findings gave rise to a hypothesis: the intriguing coexistence of elevated RNA level and reduced chromatin accessibility of HMOX1 in radioresistant GBM cells may intricately link to the dynamic interplay of IFI16, JUND and SP1. Specifically, IFI16 facilitates transcription of HMOX1 through JUND while counteracting transcriptional repression mediated by SP1. This hypothesis was verified by further experiments. Compared to the GBM parental cells, the radioresistant GBM cells exhibited high levels of JUND and H3K27ac in the JUND binding site (p1 region) (Fig. 6e), but had low levels of SP1 and HDAC1 along with increased H3K27ac in the SP1 binding site (p2 region) (Fig. 6f). IFI16 knockdown decreased JUND signal in the p1 region (Fig. 6g) and increased SP1 and HDAC1 signals in the p2 region (Fig. 6h), accompanied with decreased H3K27ac in both p1 and p2 regions (Fig. 6g, h). Conversely, IFI16 overexpression increased JUND and H3K27ac signals in the p1 region (Fig. 6i), and it decreased SP1 and HDAC1 signals and increased H3K27ac signals in the p2 region (Fig. 6j).

To understand how IFI16, JUND, and SP1 regulate HMOX1 expression in GBM cells, we generated a series of mutations of JBM and SBM in the HMOX1 promoter fused with a luciferase reporter and then examined their responses to the co-transfected IFI16, JUND, and SP1 expression vectors in U251 cells (Fig. 6k). It was found that transfection of JUND activated the wild-type HMOX1 promoter luciferase reporter (pGL-HMOX1-WT), while SP1 transfection exerted an inhibitory effect. However, IFI16 transfection countered the inhibitory impact of SP1 on pGL-HMOX1-WT. Notably, the luciferase transcriptional activity of the JBM-deleted HMOX1 promoter (pGL-HMOX1-ΔJBM) was significantly lower than that of pGL-HMOX1-WT, and neither overexpressed JUND nor IFI16 affected the activity of pGL-HMOX1-ΔJBM (Fig. 6k).

Conversely, the luciferase plasmids with SBM-deleted HMOX1 promoter (pGL-HMOX1-ΔSBM) exhibited the activity comparable to pGL-HMOX1-WT. SP1 transfection did not affect luciferase transcriptional activity in the pGL-HMOX1-ΔSBM group (Fig. 6k). However, JUND and IFI16 overexpression significantly increased the luciferase transcriptional activity in pGL-HMOX1-ΔSBM. These luciferase reporter assays demonstrated that the HMOX1 expression was repressed by SP1 but promoted by JUND, and IFI16 reduced the suppression effect of SP1 on HMOX1 expression while maintained the ability of JUND in promoting HMOX1 expression.

Subsequent studies of the effects of IFI16, JUND, and SP1 overexpression on HMOX1 in U251 and Ln229 cells confirmed our hypothesis. Transfection of JUND and IFI16 overexpression vectors amplified HMOX1 mRNA and protein expressions, while SP1 overexpression inhibited HMOX1 (Fig. 6l, m). Notably, the overexpression of IFI16 protected the transcriptional role of JUND and counteracted the repressive influence of SP1, ultimately promoting HMOX1 expression. These outcomes were congruent with luciferase reporter assay (Fig. 6k), underscoring the intricate interplay among IFI16, JUND and SP1 in modulating HMOX1 expression in GBM cells.

IFI6 interacts with JUND through its pyrin domain

In light of well-studied IFI16-SP1 interaction^31,32, we turned to exploring the direct interaction between IFI16 and JUND in GBM cells by co-immunoprecipitation (Co-IP) (Fig. 7a). Our findings indicated that the binding between IFI16 and JUND occurred independently of DNA (Fig. 7b). To delineate the interaction regions between IFI16 and JUND, we divided the IFI16 protein into five distinct domains: amino acids 1–88, 89–188, 189–389, 390–562, and 563–789 (Fig. 7c, upper pane). Truncation plasmids of a, b, c, d, and e were accordingly constructed, each tagged with a 3 × Flag at C-terminus. Remarkably, only the plasmid a represented the pyrin truncation where JUND interacts with IFI16 (Fig. 7c, lower pane). Likewise, to identify the structural domain of JUND interacting with IFI16, five truncation plasmids of a, c, ab, ac, and bc were generated based on the structural domains of JUND (Fig. 7d, upper pane). It was found that IFI16 interacted with the amino acid sequence at 1–127 of JUND (Fig. 7d, lower pane). These findings were further confirmed by the rigid protein-protein docking model between IFI16 and JUND, with the interface primarily in the pyrin domain of IFI16 and the 1–127 amino acid region of JUND (Fig. 7e). Hydrogen bonds could be formed at amino acid residues such as Asp-19: Asp-85, Tyr-20: Ser-55, and Arg-40: Thr-51, confirming the stability of the interaction between IFI16 and JUND.

**Fig. 7: IFI16 interacts with JUND through its pyrin domain.**

Comparison of the amino acid sequences of IFI16 pyrin region to NLRP3, ASC, and MEFV (proteins with a pyrin domain) revealed the consistent presence of lysine (Lys, K) and leucine (Leu, L) among different proteins (Fig. 7f and Supplementary Table 2). Hypothesizing that these residues are critical in the secondary structure of protein, we created an IFI16 mutant plasmid (IFI16^KL-AA) in which Lys-26 and Leu-29 were both mutated to alanine (Ala, A) (Fig. 7g). It was confirmed that IFI16^WT interacted with JUND and SP1, while the mutant IFI16^KL-AA failed to precipitate JUND and SP1, highlighting the importance of the pyrin domain in the regulation of JUND and SP1 functions by IFI16 (Fig. 7h).

Consistent with expectation, the mutant IFI16^KL-AA had no effect on enhancing the radioresistance of GBM cells (Supplementary Fig. 10a) and had no impact on radiation-induced ferroptosis in these cells (Supplementary Fig. 10b–e). These findings further rule out the impact of conventional factors on IFI16-mediated ferroptosis.

Literature indicates that IFI16 has four main isoforms, generated through alternative splicing of its mRNA (Supplementary Fig. 11a)³⁵. In the radioresistant GBM cells, IFI16 expression was elevated (Supplementary Fig. 11b), with IFI16-A being the predominant isoform, followed by IFI16-B (Supplementary Fig. 11c). In contrast, IFI16-C and IFI16-B2 were less common, and their expressions did not significantly differ between radioresistant and parental cells (Supplementary Fig. 11d). Additionally, it is known that the regulatory activity of IFI16 can be negatively modulated by other PYHIN family members, such as AIM2³⁶. To explore this, we assessed AIM2 expression and found it had no significant changes in the radioresistant cells, IFI16 knockdown cells, or IFI16 overexpression cells (Supplementary Fig. 11e–g).

Glyburide enhances the radiosensitivity and ferroptosis by directly binding to the pyrin domain of IFI16

Since glyburide, an inhibitor of NLRP3^37,38, is capable of inhibiting the activation of inflammasomes containing pyrin domain³⁹, it was speculated that glyburide might also modulate IFI16 that also carries this domain (Fig. 7c). Autodock Vina predictive analysis indicated that glyburide molecule could form hydrogen bonds with the specific residues (Asp-19, Lys-26, and Lys-45) of the pyrin domain in IFI16 (Fig. 8a). Following protein extraction from U251 cells, we conducted drug affinity responsive target stability (DARTS) assays. It was found that, when cell lysates were treated with glyburide, the degradation of IFI16 by pronase was impeded (Fig. 8b). However, glyburide had no observable impact on the degradation of IFI16 with a mutated pyrin domain (IFI16^KL-AA) (Supplementary Fig. 12a), and it specifically bound only to the truncated form a of IFI16 (Supplementary Fig. 12b), not to other truncated forms (Supplementary Fig. 12c–f). These results highlight the selective binding of glyburide to the pyrin domain of IFI16.

**Fig. 8: Glyburide sensitizes GBM cells to radiotherapy.**

We delved deeper into the impact of glyburide on the radiosensitivity and ferroptosis and found that glyburide treatment increased the radiosensitivity (Supplementary Fig. 13a), elevated the levels of lipid peroxides, ROS and intracellular Fe²⁺ in the radioresistance GBM cells after irradiation (Supplementary Fig. 13b–d), and reduced the expression of GPX4 (Supplementary Fig. 13e). However, in the IFI16 knockdown GBM cells, glyburide failed to enhance the radiosensitivity (Supplementary Fig. 14a) and it had no effects on GPX4 expression, lipid peroxides, ROS, and intracellular Fe²⁺ content with or without irradiation (Supplementary Fig. 14b–g).

To ascertain the radiosensitive effect of glyburide in vivo, we administered glyburide in an orthotopic U251R xenograft model (Fig. 8c). Mice bearing U251R tumors exhibited a median survival of 26 days that was not improved by glyburide (Fig. 8d). Tumor irradiation alone improved the median survival to 39 days, while the combination of radiotherapy and glyburide significantly prolonged the survival to 60 days of 60% mice (Fig. 8d). In addition, a weight gain was observed in the combination group, contrasting with malnutrition and eventual cachexia of other three groups due to tumor development (Fig, 8e, f). MRI scans at 20 days after cell implantation in situ showed that the average tumor volume in the combination group was significantly smaller than those of control and irradiation alone groups (Fig. 8g, h). Brain specimen images also confirmed a substantial tumor elimination following to the above combinational treatment (Fig. 8g). Immunohistochemical analysis revealed that the levels of IFI16, HMOX1, and GPX4 were decreased in brain tumors duo to the glyburide treatment (Fig. 8i).

Discussion

Radiation is a well-known inducer of cellular senescence⁴⁰, and our study confirmed a cellular senescence-like phenotype in radioresistant GBM cell lines developed through fractionated irradiation, compared to their parental counterparts (Fig. 1). While cellular senescence can promote tumor cell survival by enhancing resistance to radiotherapy⁴¹. Therefore, identification of key genes that drive senescence in GBM cells may provide valuable insights into mechanisms underlying radioresistance.

Through comprehensive genomic and transcriptomic analyses, we identified IFI16 as a critical driver of the cellular senescence-like phenotype in GBM (Fig. 2). IFI16 interacts with STING in response to DNA damage, promoting p53-mediated transcriptional activation and cell cycle arrest³⁶. Notably, the association of IFI16 with poor prognosis and radioresistance of GBM underscores the clinical relevance of IFI16 (Fig. 3). Although silencing IFI16 expression can reduce GBM recurrence⁴², the survival advantage of cells with elevated IFI16 expression after irradiation could not be solely attributed to cellular senescence. Hence, we applied various cell death inhibitors and found that IFI16 overexpression attenuated ferroptosis following irradiation (Fig. 4).

Notably, IFI16 may not directly modulate ferroptosis, prompting further analysis of the FerrDb ferroptosis database. Results suggested that IFI16’s effect on ferroptosis may be mediated through HMOX1 expression (Supplementary Fig. 5). The role of HMOX1 in ferroptosis remains controversial. Some studies suggest that HMOX1 may inhibit ferroptosis by promoting carbon monoxide metabolism, which triggers an antioxidant response^43,44. Evidence also indicates that both HMOX1 and GPX4 are involved in the cellular antioxidative stress response⁴⁵. Conversely, other reports proposed that HMOX1 contributed to the degradation of heme, potentially promoting ferroptosis by elevating intracellular labile iron levels^46,47. Despite prevalent perceptions positioning HMOX1 as a facilitator of ferroptosis²², our investigation offers distinctive insights. As a characteristic of cellular senescence⁴⁸, the suppressed heme metabolism was observed in the radioresistant GBM cells (Supplementary Fig. 7a), which was not influenced by HMOX1 (Supplementary Fig. 7b, c). These observations positioned HMOX1 as a potent inhibitor of ferroptosis in radioresistant GBM cells, consistent with the correlation between elevated HMOX1 level and poor prognosis in GBM^20,21.

While the prevailing research has predominantly linked IFI16 to the activation of cytoplasmic proteins such as NF-κB and STING¹², it is crucial to pay attention to the primary localization of IFI16 in nucleus⁴⁹ and thus to investigate the potential role of IFI16 in transcriptional regulation¹¹. Previous studies have shown that IFI16 has an ability to bind to transcription factors and influence transcriptional processes⁵⁰. Our results uncovered the direct binding interactions between IFI16 and transcription factors JUND and SP1. IFI16-JUND interaction facilitated the transcriptional promotion of JUND, whereas the IFI16-SP1 interaction alleviated the inhibitory effect of SP1 (Fig. 6). Consequently, IFI16 emerged as a pivotal mediator in enhancing HMOX1 transcription. Although the intricate mechanisms underlying IFI16’s participation in the transcriptional regulation remain elusive, our results pioneered a paradigm by proposing the direct involvement of IFI16 in this intricate orchestration of gene transcription.

Our study highlights IFI16, GPX4, and HMOX1 as potential biomarkers of GBM radioresistance, with elevated levels in the recurrent GBM (Fig. 5). We hypothesize that these increases are induced by irradiation and ultimately contribute to GBM recurrence. Additionally, our findings suggest a process like “natural selection” during radiotherapy, where cells with higher IFI16 expression are more prone to adopt a cellular senescence-like phenotype, thus gaining a survival advantage and becoming dominant within the tumor population. It was demonstrated that the ROS level was increased while ferroptosis was reduced in the radioresistant GBM cells after irradiation (Supplementary Fig. 4a–d), indicating an adaptive response to the treatment.

IFI16 appears central to this process. Elevated IFI16 expression enhances the production of SASP, thereby facilitating the establishment of cellular senescence^11,12. This phenotype may further upregulate IFI16 through a positive feedback loop, thereby inhibiting ferroptosis and reinforcing radioresistance after irradiation. Our data indicate that, despite diverse pathways leading to cellular senescence, IFI16 consistently plays a key role in suppressing ferroptosis within senescent cells (Supplementary Fig. 8).

Furthermore, our studies underscored the critical role of the pyrin domain within IFI16, as mutations in this domain consistently impaired its functionality (Fig. 7e–g and Supplementary Fig. 10). Although IFI16 has four isoforms, the identical pyrin domain across all isoforms suggests that alternative splicing may not significantly impact IFI16-mediated radioresistance (Supplementary Fig. 11a).

Cellular senescence has gained considerable attention in cancer therapy due to its dual role in tumor progression and treatment response. A key focus is the development of senolytics, aimed at selectively targeting and clearing senescent tumor cells^42,51,52. Although the anti-aging effects of some sulfonylurea drugs have been demonstrated⁵³, the potential of glyburide, a well-known sulfonylurea, had not been fully explored. We demonstrated that glyburide, known for its inhibition of inflammasome activation via the pyrin domain^37,38,39, effectively suppressed IFI16 activity, thereby promoting ferroptosis and enhancing the radiosensitivity of GBM cells (Fig. 8 and Supplementary Fig. 13). Although some studies have mentioned the radioprotective effects of glyburide⁵⁴, growing evidence supports its potential as an anti-cancer agent^55,56.

We acknowledge the limitation to fully capturing the post-irradiation dynamics of IFI16, HMOX1, and GPX4 expressions in the orthotopic model. Specifically, when the IFI16 level was elevated after IR treatment alone, the increases in HMOX1 and GPX4 were not consistently significant (Fig. 8g). This discrepancy may arise from differences in species-specific expression, as IFI16 is present exclusively in human cells⁵⁷, whereas GPX4 and HMOX1 are broadly expressed in both mouse and human cells. Consequently, the expression of IFI16 reflects human tumor cell dynamics, while GPX4 and HMOX1 levels may be influenced by broader systemic cellular responses in the mouse model. Future studies using humanized mouse models or patient-derived xenografts may offer a more precise understanding of these dynamics. Despite this limitation, our findings have demonstrated the combination effects of radiation and glyburide on IFI16, GPX4, and HMOX1 expressions, supporting the potential of glyburide as a radiosensitizer of GBM.

In summary, our study provides insights into the initiation of a cellular senescence-like phenotype induced by fractionated radiotherapy. Through comprehensive genomic and transcriptomic analyses, we identified IFI16 as a central driver of radioresistance, promoting a senescence-like state and suppressing ferroptosis by activating HMOX1 transcription in GBM cells. Given the compromised heme metabolism in GBM, HMOX1 exhibited an antioxidant response and acted as a suppressor of ferroptosis. Besides, IFI16 served as a guardian for JUND transcription promoter, countering the inhibitory influence of transcription repressor SP1, ultimately facilitating HMOX1 expression (Fig. 9). Our findings suggest that glyburide may serve as a potential radiosensitizer by targeting the pyrin domain of IFI16, promoting ferroptosis and thereby increasing tumor cell sensitivity to radiotherapy. Conclusively, these findings deepened the understanding of the nature of GBM radioresistance and unveiled an innovative therapeutic strategy with potential clinical implication.

**Fig. 9: The mechanistic diagram of IFI16 driving HMOX1-dependent ferroptosis inhibition and radioresistance in GBM.**

Methods

Ethics statement

All animal studies complied with the ARRIVE guidelines and was approved by the Animal Welfare and Ethics Committee of Fudan University. At no point did we exceed the approved limits for tumor size/burden in our animal experiments. All experimental procedures involving clinical tissues were conducted in accordance with the ethical standards set forth by the World Medical Association’s Declaration of Helsinki. Informed written consent was obtained from all participants enrolled in the human studies, and ethical approval was obtained from the Institutional Review Board of Guangdong Sanjiu Brain Hospital.

Cell culture and irradiation

GBM cell lines, U251 and Ln229, were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). To develop radioresistant U251R and Ln229R cells, U251 and Ln229 cells were exposed to 2 Gy X-rays per day for 30 fractions, with a total dose of 60 Gy, according to our previous study^23,58. To maintain the radioresistant phenotype, U251R and Ln229R cells were subjected to an additional 6 Gy of X-ray irradiation every 4 weeks. These cells were cultured in DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in 5% CO₂. Cell lines were routinely tested for mycoplasma every three months. Cells in log-phase were exposed to X-ray at a dose rate of 1 Gy/min (X-RAD 320, PXI Inc., North Branford, CT, USA; 12 mA, 2 mm aluminum filtration) at room temperature.

Colony formation assay

The radiosensitivities of U251, U251R, Ln229, and Ln229R cells were evaluated by a cell colony formation assay. Once the cells adhered, they were exposed to X-rays with a dose of 0, 2, 4, 6, and 8 Gy. About two weeks after irradiation, cell colonies were fixed, stained, and counted. Each cell sample was examined three times. A single-hit multitarget model was used to fit the cell survival curve using GraphPad Prism 8 software (GraphPad Software, LLC, San Diego, USA). The sensitization enhancement ratio (SER) was calculated at the survival fraction (SF) of 37%. Statistical significance was assessed using two-way ANOVA. The assay was performed in at least three independent experiments.

Western blotting assay

Cellular proteins were extracted with RIPA buffer (Beyotime Biotechnology, Shanghai, China) or Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. An equivalent amount of protein was subjected to SDS-PAGE on a gel and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% nonfat milk in Tris-buffered saline/Tween 0.05% (TBST) for 2 h and then incubated with primary antibodies overnight at 4 °C. After TBST washing, the membrane was incubated with secondary antibodies (#A0208 and #A0216, 1:5000, Beyotime Biotechnology, Shanghai, China). Proteins on the membrane were detected using an ECL kit (Millipore, St. Louis, MO, USA). The primary antibodies used for WB are listed in Supplementary Table 3.

Morphological characteristics detection

A total of 2 × 10⁵ cells were seeded in a 6-well plate and incubated overnight. Cells were then washed with PBS and fixed with 4% formaldehyde for 10 min. Following three additional PBS washes, the cells were imaged using an Olympus microscope (Tokyo, Japan). The length-width ratio was analyzed semi-quantitatively using ImageJ software from three independent experiments. For each biological replicate, images were randomly selected from at least five fields of view.

β-Galactosidase staining assay

β-Galactosidase activity was detected using a Senescence β-Galactosidase Staining Kit (Beyotime Biotechnology, Shanghai, China). Cells were seeded in complete media in 6-well plates. After 48 h, the cells were fixed and incubated overnight at 37 °C in a dry incubator without CO₂ with the β-galactosidase staining solution. The cell images were then observed under a microscope and analyzed with ImageJ software. For each biological replicate, images were randomly selected from at least five fields of view. The assay was conducted in triplicate.

RNA extraction and RT‑qPCR

The RNA expression levels of genes were assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Total cellular RNA was extracted using Trizol reagent (Invitrogen, San Diego, CA, USA) and reverse transcribed into cDNA using a FastKing RT Kit (with gDNase) (Tiangen Biotechnology, Beijing, China). RT-qPCR was performed on the Bio-Rad CFX Opus 96 platform using SuperReal PreMix Plus (SYBR Green) (Tiangen Biotechnology, Beijing, China). ACTB was used as an endogenous control gene. The primers used for the RT-qPCR assay are listed in Supplementary Table 4.

Immunofluorescence (IF) staining

Following cell adhesion or at various time points post-irradiation, cells were washed with PBS, fixed with 4% formaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 (Beyotime Biotechnology, P0096) for 10 min. Cells were then blocked for 1 h in 0.1% PBS-Tween containing 1% BSA, 10% normal goat serum, and 0.3 M glycine (QuickBlock assay, Beyotime Biotechnology, P0260) to prevent nonspecific protein interactions. Subsequently, cells were incubated with the primary antibody (anti-Ki67 or anti-IFI16) for 12 h, followed by an Alexa Fluor 555-conjugated secondary antibody for 1 h at 4 °C. Nuclear staining was performed using DAPI at a concentration of 1.43 μM. Fluorescence images were captured using the ImageXpress Micro 4 screening system (Molecular Devices, San Jose, CA, USA). Quantification of fluorescence spots per cell was carried out using the Granularity Analysis function of the ImageXpress software. Details of the antibodies used are listed in Supplementary Table 3. The assay was performed in triplicate.

Detection of secretory phenotypes

A total of 2 × 10⁵ cells were seeded in a 6-well plate and incubated overnight. Following incubation, 2 ml of fresh cell culture medium was added to the plate. After 24 h, the culture medium was collected for analysis. The concentrations cytokines (CCL5, IL-6, MMP-9, and IFN-β) in the medium were quantitatively measured using a flow cytometry bead-based assay (ABplex Human Chemokine 4-Plex Assay Kit, Abclonal, RK04381) according to the manufacturer’s instructions.

RNA-seq

RNA sequencing was performed by HaploX Biotechnology (Shenzhen, China). Trizol reagent was used to prepare total RNA samples. The NEBNext Ultra mRNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) was used for RNA library construction. The molarity of the library was measured using the KAPA Library Quant Kit universal qPCR Mix (Illumina, San Diego, CA, USA). The library was sequenced using NovaSeq 6000 (Illumina, San Diego, CA, USA). The Bioconductor package limma (v3.36.0, https://bioconductor.org/packages/limma) was used to analyze the differential gene expressions (DEGs), and the Bioconductor package clusterProfiler (v3.18.1, https://bioconductor.org/packages/clusterProfiler/) was used for Gene Ontology (GO) term enrichment analysis and gene set enrichment analysis (GSEA).

ATAC-seq

A total of 5000 cells were resuspended in cold PBS and used for ATAC-seq analysis following the manufacturer’s protocol (Active motif Inc., Shanghai, China). Chromatin was extracted and processed for Tn5-mediated labeling and adapter incorporation using the Nextera DNA Sample Preparation Kit (Illumina, San Diego, CA). The quality of the library was evaluated using a DNA-based fluorometric assay (Thermo Fisher Scientific, Waltham, MA). The library sequencing was performed on Illumina HiSeq2500. Unmapped reads were trimmed using Trim Galore (v0.6.6). Alignment files were sorted and indexed using Bowtie2 software (v2.2.5, http://bowtie-bio.sourceforge.net/bowtie2/index.shtml) and Samtools software (v1.10, http://github.com/samtools/samtools). Peaks were defined using MACS2 software (v2.1.4, https://pypi.python.org/pypi/MACS2). Peak annotation, comparison, and visualization were performed using the Bioconductor package ChIPseeker (v1.26.2, https://bioconductor.org/packages/ChIPseeker/). Fold changes were reported using DESseq2 analysis of the raw data. Motif analysis was performed using HOMER (v4.8 http://homer.ucsd.edu/).

Construction of knockdown cell lines

The lentivirus containing IFI16 interference (shIFI16) was purchased from Hanbio Biotechnology (Shanghai, China). Their negative control (shNC) had random sequences. Briefly, cells were infected with the lentivirus for 24 h according to the manufacturer’s instructions. When U251R and Ln229R cells grew stably in a medium containing 3 µg/ml blasticidin and puromycin to exclude any off-targeted cells, the culture medium was replaced with DMEM for cell culture for another week.

Plasmid construction

A plasmid carrying IFI16, HMOX1, JUND, and SP1 genes in the pCDH vector (Genechem, GV657) was generated. Subsequently, GBM cells were transiently transfected with the respective plasmids using Lipofectamine 3000 (Invitrogen, L3000008). An empty vector of pCDH was employed as a negative control.

Drug treatment

To induce senescence, U251 and Ln229 cells were cultured in DMEM containing either 0.5 μM doxorubicin (Dox) or 100 μM temozolomide (TMZ) for two weeks. After this period, the medium was replaced with a maintenance dose of 0.1 μM Dox or 20 μM TMZ. In preparation for irradiation, cells in log-phase were treated with 50 µM Glyburide, 5 μM Fer-1, 500 nM DFO, 1 μM Nec-1, 5 μM Z-VAD, 25 μM 3-MA, or 5 mM NAC for 24 h prior to 6 Gy irradiation. All these drugs were purchased from MedChemExpress (Shanghai, China). The control group was treated with DMSO (0.1%).

Database description

The open-source clinical data of GBM was accessed, analyzed, and visualized using the GlioVis database (https://gliovis.bioinfo.cnio.es/).

Xenograft tumor mouse model

Six-week-old male nude mice (BALB/C-nu/nu) were obtained from the Shanghai Laboratory Animal Research Center. Mice were housed in a controlled environment with a 12 h light/dark cycle (7:00 AM to 7:00 PM), a temperature of 20–22 °C, and humidity of 40–60%. They had ad libitum access to food and water and were housed in individually ventilated cages with appropriate bedding. Subcutaneous injection of 5 × 10⁶ cells was performed on the right flanks of nude mice. Once the average tumor volume reached approximately 100 mm³, half of the mice received local irradiation at a dose of 15 Gy, with the rest of the body shielded using a lead block to ensure that only the tumor site was exposed. Tumor size was measured using a caliper every two days and calculated by using the modified ellipse formula (volume = length × width²/2). Several days after irradiation, the mice were euthanized, and tumor samples were collected. The maximum tumor burden of 1500 mm³ did not exceed the ethical limits set by the institutional review board.

Cell proliferation assay

Cell proliferation was measured using the CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan). Briefly, cells were seeded in 96-well plates at the appropriate concentration and cultured at 37 °C. When cells adhered, CCK-8 working buffer was added to the 96-well plates and incubated at 37 °C for 2 h. The optical density at 450 nm was measured using a microplate reader (Tecan Infinite M200 Pro, Männedorf, Switzerland) to evaluate cell proliferation rate. The CCK-8 assay was repeated three times with three replicates for each cell sample.

Lipid peroxidation assay

C11-BODIPY was used to detect the lipid peroxidation of cells according to the manufacturer’s protocol. Briefly, at 6 h of irradiation, cells were resuspended in 500 μl PBS containing 20 mM C11-BODIPY 581/591 (RM02821, ABclonal, Wuhan, China) and incubated for 1 h at 37 °C in a cell culture incubator. The signals from both non-oxidized C11 (wavelength > 580 nm) and oxidized C11 (wavelength 505–550 nm) were monitored. Flow cytometry (Beckman CytoFLEX, CA, USA) was employed to assess the lipid peroxidation. The data were normalized to control and shown as the relative level of lipid peroxidation.

Intracellular ROS measurement

Intracellular ROS was detected by a cell-permeable molecular probe of 2′,7′-dichlorofluorescein diacetate (DCFH-DA) after 1 h of 6 Gy irradiation. Cells were incubated in serum-free medium containing 3 μM DCFH-DA (Beyotime, Shanghai, China) for 20 min at 37 °C and then washed with PBS twice to remove any residual DCFH-DA. DCF fluorescence intensity was immediately detected by an automatic microplate spectrophotometer (Synergy H1, BioTek Instruments, Vermont, USA) with an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The relative level of ROS in the irradiated cells was normalized to the control without irradiation.

Intracellular Fe²⁺ content measurement

The level of intracellular Fe²⁺ ions was measured with a Ferro-orange kit (Dojingo, Molecular Technologies Inc., Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were irradiated with 6 Gy of X-rays, incubated in serum-free medium for 4 h, and stained with 1 μM Ferro-orange in HBSS for 30 min at 37 °C. Then the fluorescence absorbance of culture was immediately detected in an automatic microplate spectrophotometer (Synergy H1, BioTek Instruments, Vermont, USA) with an excitation wavelength of 543 nm and an emission wavelength of 580 nm.

Patient samples

Thirty-seven specimens of GBM were procured from the patients underwent surgical resection or guided brain biopsy at the Guangdong Sanjiu Brain Hospital in China. Detailed clinical profiles of these patients are provided in Supplementary Table 5. Disease progression and prognostic evaluations were conducted using pre- and post-chemotherapy MRI scans, following the Response Assessment in Neuro-Oncology (RANO) criteria. Recurrent GBM (19 specimens) were characterized by disease recurrence within one year of systemic treatment. Conversely, primary GBM (18 specimens) displayed no evidence of recurrence within one year of systemic treatment. Informed consents were obtained from all subjects.

Immunohistochemistry (IHC) assay

GBM specimens were fixed overnight in 10% neutral buffered formalin and then transferred to 70% ethanol, embedded, and sectioned. Next, the specimens were stained using primary antibody and counterstained using hematoxylin. IHC images were obtained using a microscope at 400× magnification. The random fields of IHC staining were analyzed semi-quantitatively from three independent experiments using ImageJ software. The analysis index was the average optical density (AOD; AOD = Integrated optical density (IOD)/Area). Relevant antibodies are listed in Supplementary Table 3.

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed utilizing an EZ-Magna ChIP A/G kit (Millipore, Darmstadt, Germany) according to the manufacturer’s guidelines. Briefly, cell lysates were prepared and sonicated to obtain a mixture of DNA fragments associated with the specific antibody (Relevant antibodies was listed in Supplementary Table 3) and subjected to immunoprecipitation using protein A/G agarose (Santa Cruz Biotechnology, Shanghai, China). Anti-normal rabbit IgG was used as a negative control. The protein-DNA complex was then eluted using low-salt buffer. The solid matrix was washed to remove unbound chromatin and reduce the background. The protein-DNA complex was then incubated at 65 °C for 4 h to release DNA, followed by DNA purification after removing residual protein and salts. The purified DNA was used as a template for RT-qPCR assay.

Luciferase reporter assay

pGL3-basic vectors, including wild-type sequence of HMOX1 promoter, JBM deletion of HMOX1 promoter, and SBM deletion of HMOX1 promoter, were purchased from Genechem (Shanghai, China). Cells were seeded in triplicate in 24-well plates and allowed to settle for 24 h. Indicated plasmids and pRL-TK Renilla plasmids were transfected into the cells using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Forty-eight hours after transfection, the firefly and renilla luciferase activities were assessed using a Dual Luciferase Reporter Gene Assay Kit (Yeasen Biotechnology, Shanghai, China) according to the manufacturer’s instructions.

Co-immunoprecipitation (Co-IP) assay

Whole cell lysates (WCL) were collected and centrifuged at 10,000 × g for 10 min at 4 °C (Beyotime Biotechnology). Then, 1 ml of supernatant was incubated with primary antibodies for 16 h, followed by the addition of 20 μl fresh protein A/G agarose and incubated overnight at 4 °C. Samples were centrifuged, washed four times with immunoprecipitation buffer, and fractionated by SDS-PAGE, followed by Western blot analysis. Relevant antibodies are listed in Supplementary Table 3.

Molecular docking analysis

Rigid protein-protein docking was performed to investigate the relationship between IFI16 and JUND using GRAMM-X (http://gramm.compbio.ku.edu/). Autodock Vina (version 4.2.6) was used to investigate the interaction between glyburide and IFI16. The protein structural domains of IFI16 and JUND were obtained from the Protein Data Bank PDB database (http://www.rcsb.org/). PDBePISA (https://www.ebi.ac.uk/pdbe/pisa/) was used to investigate the interaction between IFI16 and JUND (The results of molecular docking were listed in Supplementary Table 6). Pymol (version 2.4) was used for further visual analysis.

Drug affinity responsive target stability (DARTS) assay

The DARTS assay was performed according to the protocol⁵⁹. Briefly, cells were lysed with M-PER lysis buffer. BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to measure the protein concentration of cell lysates. 3 μl glyburide at the final concentration of 100 μM was added to the lysates and incubated for 1 h at 4 °C, and 3 μl DMSO was used for the negative control. Samples were then incubated with 1 μg/mL Pronase (Cat. no. 10165921001, Roche, Mannheim, Germany) or distilled water for indicated times. Stop digestion of the aliquot by adding 20 × Protease inhibitor solution (Cat. no. 11836153001, Roche, Mannheim, Germany). The sample was added to 5 × loading buffer and incubated at 95 °C for 10 min, and ready for western blotting assay.

Orthotopic xenograft model

A total of 20 five-week-old male BALB/c nude mice (18–20 × g) were used for establishing the orthotopic xenograft model. U251R cells (1 × 10⁶) in a volume of 3 μl were stereotactically injected into the right striatum⁶⁰. Tumor volume was assessed by MRI on days 10 and 20 post-injection. On day 10 after tumor cell implantation, mice were randomly divided into four groups: saline + sham-irradiation (Ctrl), glyburide + sham-irradiation (Gly), saline + irradiation (Ctrl+IR), and combined irradiation and glyburide (Gly+IR). A single dose of 15 Gy irradiation was administered to the brain on day 10 post-implantation, with lead shielding to protect the rest of the body. For Gly+IR or Gly groups, a dose of 1 mg/kg glyburide was administered intragastrically 2 h before irradiation or shielding irradiation, while equal volumes of saline were given to Ctrl or Ctrl+IR groups. After irradiation, mice were weighed every three days and maintained until death, or a maximum of 60 days, and their survival was also counted. For brain sampling, mice were anesthetized with ketamine and xylazine before intracardiac perfusion with PBS, followed by 4% PFA-PBS. Brains were collected and fixed with 4% PFA-PBS for 12 h, followed by HE and immunohistochemical staining. This maximum orthotopic tumor burden of 150 mm³ did not exceed the ethical limits set by the institutional review board.

Magnetic resonance imaging (MRI)

Mice were imaged by a 3.0 T MRI scanner (United Imaging, Shanghai China) with uExceed R002 software. T2 weighted images were acquired with a fast spin echo sequence, the parameters were: TR = 3871 ms, TE = 126.88 ms, field of view = 25 × 25 mm, Matrix 128 × 128, voxel size = 0.2 × 0.2 × 1.0, 15 sagittal slices, average = 10, and scan time = 5:53 min.

Statistics & Reproducibility

GraphPad Prism software (GraphPad Software, Inc.) was used to perform statistical analyses. Student’s t-test or ANOVA was used for group comparison. Overall survival curves were plotted via the Kaplan–Meier method and compared by the log-rank test. Bars and errors represent the mean ± standard deviation (SD) of repeated measurements. Unless otherwise stated, the experiments were performed independently in triplicate with the sample number “n” indicated in the figure legends. Statistical significance was defined as a p‐value of <0.05.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq and ATAC-seq data are available on GEO database at GSE274090. The remaining data are available within the Article, Supplementary Information or Source Data file. Source data are provided with this paper.

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Acknowledgements

We appreciate Dr. Feizhen Wu (Institute of Biomedical Science, Fudan University) for his valuable advice in the bioinformatic analyses, and appreciate the Medical Science Data Center of Shanghai Medical Collage for the support and assistance in the bioinformatic analysis. This study was funded by the National Natural Science Foundation of China (Nos. 12235004 (c), 32171235 (c), 12175044 (j), 12375338 (yp), and 82203964 (s)).

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These authors contributed equally: Yuchuan Zhou, Liang Zeng, Linbo Cai.

Authors and Affiliations

Institute of Radiation Medicine, Shanghai Medical College, Fudan University, Shanghai, China
Yuchuan Zhou, Liang Zeng, Wang Zheng, Xinglong Liu, Yuqi Xiao, Xiaoya Jin, Yang Bai, Yan Pan, Jianghong Zhang & Chunlin Shao
Department of Neuro-Oncology, Guangdong Sanjiu Brain Hospital, Guangzhou, China
Linbo Cai, Mingyao Lai & Hainan Li
School of Biomedical Engineering, Shanghai Tech University, Shanghai, China
Hua Jiang
Department of Preventive Dentistry, Shanghai Stomatological Hospital & School of Stomatology, Shanghai Medical College, Fudan University, Shanghai, China
Songling Hu

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Yuchuan Zhou
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Liang Zeng
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Linbo Cai
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Wang Zheng
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Xinglong Liu
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Yuqi Xiao
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Xiaoya Jin
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Yang Bai
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Mingyao Lai
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Hainan Li
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Hua Jiang
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Songling Hu
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Yan Pan
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Jianghong Zhang
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Chunlin Shao
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Contributions

Y.Z. and C.S. designed the study; Y.Z., L.Z., W.Z., and H.J. performed the experiments and prepared the figures; L.C., X.L., Y.X., M.L., H.L., and X.J. analyzed the data; Y.P. and J.Z. guided the experiments; Y.B. and S.H. assisted the experiments. Y.Z. drafted the manuscript; C.S. supervised the study and revised the manuscript; S.H., Y.P., J.Z., and C.S. acquired the funding. All authors reviewed and approved the manuscript.

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Correspondence to Yan Pan, Jianghong Zhang or Chunlin Shao.

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Zhou, Y., Zeng, L., Cai, L. et al. Cellular senescence-associated gene IFI16 promotes HMOX1-dependent evasion of ferroptosis and radioresistance in glioblastoma. Nat Commun 16, 1212 (2025). https://doi.org/10.1038/s41467-025-56456-y

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Received: 29 February 2024
Accepted: 20 January 2025
Published: 31 January 2025
DOI: https://doi.org/10.1038/s41467-025-56456-y

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