Senotherapeutic potential against xeroderma pigmentosum

Abstract

Background

Xeroderma pigmentosum (XP) is an inherited photoaging syndrome caused by mutations in genes involved in the nucleotide excision repair (NER) pathway. XP patients exhibit hypersensitivity to ultraviolet (UV) radiation, leading to accelerated skin aging and requiring lifelong sun avoidance. In this study, we aimed to investigate UV-induced cellular senescence in XP melanocytes and explore potential senotherapeutic strategies.

Methods

Melanocytes differentiated from iPSCs derived from an XP-A patient and a healthy individual, respectively, were used to induce senescence. Senescence was assessed by SA-β-gal and γH2AX staining. The drug screening tested a panel of 12 senolytic agents and 15 senomorphic agents for their ability to target senescent XP-iMCs. The effects of these agents were evaluated through cell viability assays and SPiDER-βGal staining followed by flow cytometry. RNA sequencing was performed to analyze transcriptional changes induced by senomorphic treatments.

Results

We demonstrate that UV-induced DNA damage triggers cellular senescence and up-regulates senescence-associated secretory phenotype (SASP) genes in melanocytes derived from an XP patient. The drug screening identified JAK inhibitors and curcuminoids as promising senomorphic agents. In addition, two classes of senolytic agents, BCL-2-like protein inhibitors and HSP90 inhibitors, effectively eliminate senescent melanocytes. Further analysis demonstrates that senomorphic treatment effectively counteracts senescence and reduces SASP gene expression in XP-derived melanocytes. Moreover, genes in senescence-related pathways, including the cytokine-cytokine receptor interaction, JAK/STAT, and PI3K/AKT pathways, which are activated in senescent cells, are down-regulated by senomorphic treatment.

Conclusions

This study highlights a potential senotherapeutic strategy for XP, which may help alleviate photoaging symptoms in XP patients.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13287-026-05227-7.

Introduction

Xeroderma pigmentosum (XP) is an autosomal recessive disorder caused by mutations in the XPA−XPG genes, which encode proteins involved in the nucleotide excision repair (NER) pathway. These mutations lead to the classification of XP into subtypes, from XP-A to XP-G [1]. In addition, the XP variant (XP-V) subtype is associated with a mutation in the XPV gene encoding DNA polymerase η, which is required for DNA replication through pyrimidine dimers [1]. There are notable geographic differences in the incidence and subtype distribution of XP. In Western countries, XP occurs in approximately 2 to 4 cases per 1 million individuals, with XP-C being the most prevalent subtype [2, 3]. In contrast, the incidence in Japan is significantly higher, ranging from 10 to 25 cases per 1 million individuals, which is approximately 10 times the rate observed in Western populations [4]. Notably, more than half of XP patients in Japan belong to the XP-A subtype, primarily due to a founder mutation in the XPA gene [g.15148G > C (c.390-1G > C)]. This mutation results in aberrant splicing of the XPA transcript, leading to negligible expression of the mutant gene [5, 6].

XP patients exhibit extreme sensitivity to ultraviolet (UV) radiation and accelerated photoaging. Clinical manifestations of XP include the development of freckle-like, pigmented or depigmented maculae and an increased susceptibility to skin cancers in sun-exposed areas [1, 7, 8]. The underlying cause of XP is a deficiency in the NER pathway, which is responsible for repairing and removing UV-induced DNA photolesions such as cyclobutane pyrimidine dimers and 6−4 pyrimidine-pyrimidone photoproducts. In the absence of effective DNA repair, these lesions accumulate, leading to genomic mutations and a significantly increased risk of carcinogenesis [9]. This study focuses on the impact of UV-B exposure, specifically within the 280–315 nm wavelength range, which accounts for the majority of UV-induced DNA lesions in the epidermis [9]. XP patients, particularly those under the age of 20, have a significantly higher incidence of skin cancer. They exhibit a 2,000-fold increase in melanoma cases and a 10,000-fold increase in non-melanoma skin cancers compared to non-XP individuals [8, 10–12]. In addition to cutaneous manifestations, 20–55% of XP patients also exhibit neurological dysfunction due to central and peripheral neurodegeneration [1, 7]. Despite the severity of XP symptoms, no radical cure exists. Symptomatic management, including sun protection and early detection and excision of skin cancer lesions, remains the primary clinical intervention [7]. Among the XP subtypes, XP-A patients exhibit the most severe cutaneous and neurological symptoms due to their severely impaired DNA repair capacity, highlighting the critical need for novel therapeutic strategies [8, 12].

XP results from defects in the NER pathway, leading to an accumulation of DNA damage following UV irradiation [13]. Since DNA damage can trigger cellular senescence, a state of stable cell-cycle arrest [14], the premature aging symptoms observed in XP patients, particularly in the skin, may be attributed to accelerated senescence induced by UV exposure. Consequently, targeting cellular senescence represents a promising therapeutic approach for the treatment of XP. In recent years, accumulating evidence has linked cellular senescence to a variety of human diseases, establishing it as a potential therapeutic target [15, 16]. Clinical interventions aimed at modulating senescence are referred to as senotherapy. Senotherapeutic agents are categorized into two groups: senolytic agents, which selectively eliminate senescent cells via apoptosis, and senomorphic agents, which inhibit the production of senescence-associated secretory phenotype (SASP) factors [15, 16]. Currently, the precise role of cellular senescence in the etiology of XP, and, more importantly, the potential effectiveness of senotherapeutic strategies for treating XP, remains unclear.

Senolytic agents, such as ABT-263 (inhibitor of BCL-2-like proteins), 17-DMAG (HSP90 inhibitor), and digoxin (Na⁺/K⁺ ATPase pump inhibitor), have been shown to eliminate senescent cells, highlighting the therapeutic potential for various diseases such as cancer, pulmonary fibrosis, and atherosclerosis [17–19]. BCL-2-like proteins (e.g., BCL-2, BCL-W, BCL-XL, and MCL-1) are up-regulated in senescent cells and promote resistance to apoptosis. ABT-263 specifically inhibits these BCL-2-like proteins, thereby inducing apoptosis in senescent cells [20]. While the effects of senolytic agents are preferentially restricted to senescent cells, ABT-263 is known to cause platelet toxicity, raising safety concerns for its clinical application [21–23].

Senomorphic agents suppress the production of SASP factors, thereby inhibiting the propagation of senescent cells by blocking both autocrine and paracrine senescence [15, 16]. Notably, senomorphic agents such as metformin, rapamycin, and baricitinib target key SASP regulators, including NF-κB, mTOR, and JAK, respectively, leading to a reduction in SASP factor production [24–26]. In preclinical models, these senomorphic agents have shown efficacy in treating various diseases, including certain cancers, age-related dysfunctions, and progeria syndromes [24–28]. Moreover, the natural product curcumin and its analog EF24 also exhibit senomorphic properties by suppressing NF-κB, suggesting their potential for treating various senescence-related diseases [29–31]. Collectively, these studies indicate that senotherapeutic strategies employing senolytic and senomorphic agents could be effective for aging-related syndromes, although their efficacy in XP remains undetermined. In this study, we screened for effective senolytic and senomorphic agents and propose senomorphic agents, JAK inhibitors (baricitinib and upadacitinib) and curcuminoids (curcumin and EF24), as promising candidates for potential therapeutic strategies against XP.

Materials and methods

Senotherapeutic drug screening agents

The following agents were applied for senolytic drug screening: ABT-263 (Selleck, S1001), A-1331852 (Selleck, S7801), A-1155463 (Selleck, S7800), S63845 (MedchemExpress, HY-100741), BPTES (Selleck, S7753), RSL3 (Selleck, S8155), digoxin (Sigma, D6003), ouabain (Sigma, O3125), 17-DMAG (Selleck, S1142), ganetespib (Selleck, S1159), panobinostat (Selleck, S1030), and romidepsin (MedchemExpress, HY-15149).

In addition, the following agents were applied for senomorphic drug screening: A485 (Selleck, S8740), acetylcysteine (Selleck, S1623), curcumin (Selleck, S1848), EF24 (MedchemExpress, HY-119272), metformin (Selleck, S5958), apremilast (Selleck, S8034), difamilast (MedchemExpress, HY-109085), abrocitinib (Selleck, S8765), baricitinib (Selleck, S2851), upadacitinib (Selleck, S8162), IFN alpha-IFNAR-IN-1 (Selleck, E2982), VBIT-4 (Selleck, S3544), rapamycin (Selleck, S1039), dasatinib (Selleck, S1021), and quercetin (Selleck, S2391).

Senescence-associated (SA)-β-gal staining

SA-β-gal staining of melanocytes was performed as previously described [32]. Briefly, cells were seeded on cover glasses and fixed with PBS containing 2% formaldehyde and 0.2% glutaraldehyde for 5 min at room temperature. After two washes with PBS, the cells were stained overnight at 37 ℃ in a non-CO₂ incubator (EYELA, SLI-700) using X-gal staining solution [150 mM NaCl, 2 mM MgCl₂, 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, 40 mM Na₂HPO₄ pH 6.0, and 1 mg/mL X-gal (Sigma, B9146)]. Following staining, the cells were washed with PBS and water. Images were captured using an upright microscope (ZEISS, Primostar 3) with a 10× lens.

Additional methods

Details of cell viability assay, SA-β-gal quantification, colony formation assay, western blotting, immunofluorescence, RT-qPCR, and RNA sequencing are described in the Supplementary Materials and Methods.

Results

Senescence induction of XP-derived melanocytes (XP-iMCs) by UV irradiation and cisplatin treatment

Since melanocytes, rather than keratinocytes, are more prone to become senescent in aged skin [33, 34], we hypothesized that melanocytes from XP patients would have an increased tendency to undergo senescence upon DNA damage due to their deficiency in the NER pathway. This heightened susceptibility could contribute to the accelerated skin aging observed in XP patients. To test this hypothesis, we examined the response of XP patient-derived melanocytes to DNA damage. In our preparatory study, we established a system in which induced pluripotent stem cells (iPSCs) derived from an XP-A patient and a healthy control individual were differentiated into melanocytes, referred to as XP-iMCs and HC-iMCs, respectively [8]. We employed these iPSC-derived melanocytes after verifying their physiological and functional identity, as evidenced by their characteristic morphology and the expression of the melanocyte markers MITF and SOX10 [8, 35] (Supplementary Figure S1).

To investigate senescence induction of XP-iMCs, XP-iMCs and HC-iMCs (as a control) were irradiated with UV or treated with cisplatin (Fig. 1a). Cisplatin was used as an additional senescence inducer because it induces intrastrand DNA crosslinks, which are repaired by the NER pathway in the same manner as UV-induced pyrimidine dimers (Supplementary Figure S2; Supplementary Results). We first performed immunofluorescence staining for the DNA damage marker γH2AX and observed stronger γH2AX staining in UV-irradiated XP-iMCs compared to unirradiated cells (Fig. 1b). In contrast, HC-iMCs showed a minimal UV-dependent increase in γH2AX staining. Similar results were observed in cisplatin-treated cells (Fig. 1c). These results indicate that both UV- and cisplatin-induced DNA lesions are not efficiently repaired in XP-iMCs due to their NER deficiency, leading to the accumulation of DNA damage.

Fig. 1.

Senescence induction of XP-iMCs by UV and cisplatin. a Schematic representation of the experimental procedure. HC-iMCs and XP-iMCs are melanocytes differentiated from HC-iPSCs and XP-iPSCs, respectively, derived from a healthy control individual and xeroderma pigmentosum patient (Supplementary Materials and Methods). Cells were analyzed one day after exposure to 150 J/m² UV or 6 days after treatment with 1 µM cisplatin, as shown in (b-i). b Effect of UV irradiation on HC-iMCs and XP-iMCs. DNA damage marker γH2AX (green) was visualized by immunofluorescence microscopy and is shown alongside DAPI-stained nuclei (blue). Scale bar = 200 μm. c Effect of cisplatin treatment on HC-iMCs and XP-iMCs. Scale bar = 100 μm. d Senescence-associated β-galactosidase (SA-β-gal) staining of XP-iMCs with or without UV irradiation. Scale bar = 200 μm. e Percentages of SA-β-gal-positive XP-iMCs with or without UV irradiation. SA-β-gal staining images, as exemplified in (d), were used for quantification. f SA-β-gal staining of XP-iMCs treated or untreated with cisplatin. Scale bar = 200 μm. g Percentages of SA-β-gal-positive XP-iMCs treated or untreated with cisplatin. SA-β-gal-positive cells were quantified as described in Supplementary Figure S2f. h Up-regulation of SASP genes in XP-iMCs irradiated with UV. RT-qPCR was performed to assess the expression of the indicated SASP genes. Expression levels in non-irradiated XP-iMCs were set to 1. i Up-regulation of SASP genes in XP-iMCs treated with cisplatin. In e, g, h, and i, data are represented as means and standard deviations (n = 3). Statistical significance was assessed using a two-sided Student’s t-test.

We next investigated whether accumulated DNA damage in XP-iMCs triggers cellular senescence. To do so, we monitored senescence-associated β-galactosidase (SA-β-gal) activity, a typical senescence marker, in XP-iMCs [32]. We observed that both UV irradiation and cisplatin treatment significantly increased the number of SA-β-gal-positive XP-iMCs (Fig. 1d-g). In addition, we performed RT-qPCR to assess the expression of SASP factors in XP-iMCs. We found that mRNA levels of certain SASP factors were more abundant in XP-iMCs treated with UV or cisplatin compared to untreated cells (Fig. 1h, i). Taken together, these results indicate that DNA damage induced by UV or cisplatin accumulates in XP-iMCs and triggers cellular senescence, accompanied by the expression of SASP factors.

As described above, both UV and cisplatin induce cellular senescence of XP-iMCs, which may contribute to aging phenotypes in the skin of XP patients. Conversely, senotherapeutic strategies employing senolytic or senomorphic agents could offer novel clinical interventions for treating XP. To explore this possibility, we established a drug screening system using XP-iMCs. A key challenge in this screening process was the uneven effects of UV irradiation, as factors such as cell positioning in culture dishes may influence UV exposure. Alternatively, cisplatin-induced DNA lesions and damage can be precisely controlled by adjusting the cisplatin concentration in the culture medium, ensuring a uniform effect on all cells, regardless of their position in culture dishes. This is reflected by the results that cisplatin treatment induced a higher proportion of SA-β-gal-positive cells than UV irradiation (Fig. 1e, g). Therefore, we selected cisplatin as a senescence inducer for senotherapeutic drug screening in this study.

BCL-2-like protein inhibitors and HSP90 inhibitors as effective senolytic agents

Senotherapeutic agents can be categorized into senolytic agents, which selectively eliminate senescent cells, and senomorphic agents, which inhibit the production of SASP factors [15]. Our initial goal was to identify potential senolytic agents capable of eliminating cisplatin-induced senescent XP-iMCs. To this end, we reviewed recent literature and selected 12 senolytic agents based on their reported effects on senescent cells and mechanisms of action [16] (Supplementary Table S1). In the screening, we performed a sequential strategy, in which senescence was first induced with cisplatin, followed by senolytic treatment (Fig. 2a). This approach was designed to exploit the heightened susceptibility of senescent melanocytes to senolytic agents compared to non-senescent melanocytes. The cell viability assay revealed that senolytic agents had a negligible effect on HC-iMCs with cisplatin treatment (Fig. 2b), but cisplatin-treated XP-iMCs were more sensitive to two categories of senolytic agents: BCL-2-like protein inhibitors (ABT-263, A-1331852, A-1155463, and S63845) and HSP90 inhibitors (17-DMAG and ganetespib) (Fig. 2c). These findings are consistent with previous reports showing that BCL-2-like protein inhibitors exhibit senolytic effects on senescent melanocytes [34, 36]. Previous studies have also reported that ABT-263 exhibits senolytic effects on senescent melanocytes [34, 36]. In addition, HSP90 inhibitors have demonstrated senolytic effects on a cell line from a mouse model of a human progeroid syndrome that is hyper-sensitive to DNA damage, further supporting the validity of our screening system [17]. Taken together, these results indicate that senolytic agents targeting the BCL-2-like proteins or HSP90 selectively eliminate senescent XP-iMCs. Although certain senolytic agents appear effective in eliminating senescent XP-iMCs, melanocytes are more vulnerable to senolytic agents compared to keratinocytes (Supplementary Figure S3; Supplementary Results). This increased sensitivity toward melanocytes was also observed in a previous study [34]. This raises concerns about the dose optimization of senolytic agents for XP treatment, specifically targeting senescent melanocytes while sparing non-senescent melanocytes. Therefore, identifying an alternative group of senotherapeutic candidates, such as senomorphic agents, could potentially be beneficial for therapeutic strategies against XP.

Fig. 2.

Screening of effective senolytic agents targeting cisplatin-induced senescent XP-iMCs. a Schematic representation of the senolytic screening strategy. HC-iMCs and XP-iMCs were first treated with 1 µM cisplatin for 6 days to induce senescence, followed by a 3-day treatment with candidate senolytic agents. Cell viability was measured afterward. b Cell viability of HC-iMCs treated or untreated with cisplatin, assessed after 3 days of culture with the indicated senolytic agents. c Cell viability of XP-iMCs treated or untreated with cisplatin, assessed after 3 days of culture with the indicated senolytic agents. In b and c, data are represented as means and standard deviations (n = 3). Statistical significance was assessed using a two-sided Student’s t-test. No significant difference was observed between cisplatin-treated and untreated cells in (b)

JAK inhibitors and curcuminoids as effective senomorphic agents against cisplatin-treated XP-iMCs

We next sought to identify effective senomorphic agents that inhibit the accumulation of senescent XP-iMCs treated with cisplatin. For this purpose, 15 senomorphic agents were chosen based on previous reports of their effects on senescent cells and their mechanisms of action [15, 16] (Supplementary Table S2). XP-iMCs were simultaneously treated with cisplatin and senomorphic agents, as depicted in the schematic procedure (Fig. 3a). We predicted that simultaneous treatment would suppress SASP factor production, thereby limiting paracrine senescence. In the screening procedure, SPiDER-βGal staining combined with flow cytometric analysis was employed, allowing for a more objective estimation of the senescent cell population (Fig. 3b; Supplementary Figure S2f; Supplementary Results). We observed that simultaneous treatment with cisplatin and the senomorphic agents, baricitinib, EF24, curcumin, and upadacitinib, significantly reduced the senescent cell population of XP-iMCs compared to cisplatin treatment alone (Fig. 3b-d; Supplementary Figure S4; Supplementary Results). Since baricitinib and upadacitinib are JAK inhibitors [37], and EF24 and curcumin are classified as curcuminoids [38, 39], our results suggest that JAK inhibitors and curcuminoids are effective senomorphic agents against cisplatin-treated XP-iMCs.

Fig. 3.

Screening for effective senomorphic agents against cisplatin-induced senescent XP-iMCs. a Schematic representation of the senomorphic screening procedure. XP-iMCs were simultaneously treated with cisplatin and senomorphic agents for 6 days, followed by SPiDER-βGal staining and FACS analysis. b Reduction of cisplatin-induced senescent cell populations by senomorphic treatment. The percentages of the two populations, SA-β-gal-positive senescent and negative non-senescent cells, were estimated as described in Supplementary Figure S2f. c Identification of effective senomorphic agents against cisplatin-treated XP-iMCs. The percentages of SA-β-gal-positive senescent cells after the treatment with the indicated senomorphic agents were estimated as described in (b). The additional results from different agent concentrations are shown in Supplementary Figure S4a. Data are represented as means from three biological replicates. Statistical significance was assessed using one-way ANOVA and Tukey’s test. d Effect of senomorphic agents on cisplatin-treated XP-iMCs. XP-iMCs were treated with cisplatin or simultaneously with cisplatin and the indicated senomorphic agents, followed by SA-β-gal staining. Untreated cells serve as a control. Scale bar = 200 μm. e GSEA of the cytokine-cytokine receptor interaction pathway, comparing expression profiles from XP-iMCs with and without cisplatin treatment (left) and XP-iMCs treated with cisplatin + upadacitinib to that treated with cisplatin alone (right). The normalized Enrichment Score (NES) quantifies the enrichment of a gene set within a ranked gene list, normalized for gene set size. f Heatmap depicting expression alterations of SASP genes between the indicated conditions. SASP genes commonly up-regulated by either cisplatin treatment or UV irradiation were selected (Supplementary Figure S9d). Genes are clustered based on expression similarities, with red and blue indicating increased and reduced expression, respectively, between the five paired conditions compared. Asterisks indicate genes significantly up-regulated by cisplatin or down-regulated by senomorphic treatment

To further examine SASP gene expression after treatment with senomorphic agents, we performed RNA-seq with samples subjected to the same treatment conditions (Fig. 3a; Supplementary Figure S5; Supplementary Results). Gene Set Enrichment Analysis (GSEA) revealed that the cytokine-cytokine receptor interaction pathway, which contains many SASP genes, was activated in cisplatin-treated XP-iMCs (Fig. 3e). In addition, genes in SASP-regulating pathways, including JAK/STAT and PI3K/AKT signaling, were up-regulated by cisplatin treatment (Supplementary Table S3). This result aligns with the observation that dual treatment with cisplatin and the JAK inhibitors (baricitinib and upadacitinib) reduces the senescent cell population compared to cisplatin treatment alone (Fig. 3b–d). Furthermore, GSEA revealed that genes in the cytokine-cytokine receptor interaction pathway tended to be down-regulated by dual treatment with cisplatin and the senomorphic agents (Fig. 3e; Supplementary Table S3). Expression of many SASP genes, which were up-regulated by cisplatin, was significantly decreased by cisplatin + senomorphic dual treatment, although SASP gene responses varied between the JAK inhibitors (baricitinib and upadacitinib) and curcuminoids (EF24 and curcumin) (Fig. 3f). These results demonstrate that the JAK inhibitors and curcuminoids can effectively inhibit the accumulation of senescent XP-iMCs induced by cisplatin treatment, thus holding potential as therapeutic agents for XP.

To further investigate whether SASP factors secreted by senescent XP-iMCs can induce paracrine senescence, we cultured fresh XP-iMCs with conditioned media collected from cisplatin-induced senescent XP-iMCs or untreated XP-iMCs as a control (Supplementary Figure S6a). Flow cytometric analysis revealed that SA-β-gal activity was significantly increased in XP-iMCs cultured with the conditioned medium from senescent cells (Supplementary Figure S6b). This result suggests that SASP factors secreted by senescent melanocytes can promote paracrine senescence.

JAK inhibitors and curcuminoids as effective senomorphic agents against UV-irradiated XP-iMCs

Our goal in this study was to determine whether the senomorphic agents identified through cisplatin-based screening are also effective on UV-irradiated XP-iMCs. Our hypothesis was that if the same genes were activated or repressed by cisplatin treatment and UV irradiation, we could expect similar effects from the senomorphic agents. We conducted gene expression profiling analysis of XP-iMCs, with and without UV irradiation, using HC-iMCs as a control, and we found that UV irradiation and cisplatin treatment activate shared target genes in senescence-related pathways (Supplementary Figure S7; Supplementary Results).

Since UV irradiation and cisplatin treatment affect shared target genes, we next investigated whether the senomorphic agents identified in cisplatin-based screening are also effective on UV-irradiated XP-iMCs. To assess the effectiveness of senomorphic agents against UV-treated XP-iMCs, we combined a relatively low dose of UV irradiation (40 J/m²) with senomorphic treatment (Fig. 4a; Supplementary Figure S8; Supplementary Results). After 3 and 6 days of treatment with senomorphic agents, the percentages of SA-β-gal-positive cells were significantly reduced compared to untreated XP-iMCs (Fig. 4b, c). These results demonstrate that the senomorphic agents identified through cisplatin-based screening can also suppress UV-induced senescence.

Fig. 4.

Effects of senomorphic agents on UV-irradiated XP-iMCs. a Schematic representation of the experimental procedure using XP-iMCs simultaneously treated with UV and senomorphic agents. b SA-β-gal staining of UV-irradiated XP-iMCs treated with the indicated senomorphic agents. UV-irradiated XP-iMCs without senomorphic treatment are shown as a control. Scale bar = 200 μm. c Quantification of SA-β-gal-positive XP-iMCs irradiated with UV, with and without senomorphic treatment. SA-β-gal staining images, as exemplified in b, were quantified. Data are represented as means and standard deviations (n = 3). Statistical significance was assessed using one-way ANOVA and Tukey’s test. d GSEA of the cytokine-cytokine receptor interaction pathway, comparing expression profiles from XP-iMCs with UV irradiation at day 6 to the non-treatment control (left) and XP-iMCs with UV irradiation + upadacitinib at day 6 to that with UV irradiation alone at day 6 (right). e Heatmap depicting expression alterations of SASP genes between the indicated conditions. SASP genes commonly up-regulated by either cisplatin treatment or UV exposure were selected (Supplementary Figure S9d). Genes are clustered based on expression similarities, with red and blue indicating increased and reduced expression, respectively, between the ten paired conditions compared. Asterisks indicate genes significantly up-regulated by cisplatin or down-regulated by senomorphic treatment.

To understand how senomorphic agents modulate SASP expression in UV-irradiated XP-iMCs, we performed RNA-seq with samples subjected to the same treatment conditions (Fig. 4a; Supplementary Figure S9; Supplementary Results). GSEA revealed that the cytokine-cytokine receptor interaction pathway was activated in UV-irradiated XP-iMCs at day 3 and day 6 (Fig. 4d; Supplementary Table S3). The JAK/STAT and PI3K/AKT signaling pathways were also activated in UV-irradiated XP-iMCs, suggesting their roles in regulating SASP expression in XP-iMCs (Supplementary Table S3). Moreover, genes in the cytokine-cytokine receptor interaction pathway, as well as the JAK/STAT or PI3K/AKT signaling pathways, were down-regulated by senomorphic treatment (Fig. 4d; Supplementary Table S3). Some SASP genes that were up-regulated by UV were significantly decreased by senomorphic treatment (Fig. 4e). These results suggest that senomorphic agents can also suppress senescence-related genes in UV-irradiated XP-iMCs.

Discussion

In this study, we demonstrated that UV irradiation induces senescence of XP-iMCs, which are melanocytes derived from an XP patient. Additionally, we developed a cisplatin-based drug screening system to identify candidates for potential senotherapeutic strategies against XP. Cisplatin was selected to mimic UV-induced senescence due to its high reproducibility and ease of dose adjustment. Through this screening, we first identified two groups of effective senolytic agents against cisplatin-treated XP-iMCs: BCL-2-like protein inhibitors (ABT-263, A-1331852, A-1155463, and S63845) and HSP90 inhibitors (17-DMAG and ganetespib). However, we also observed that melanocytes are inherently more vulnerable to senolytic agents than keratinocytes, even in the absence of cisplatin treatment. This finding led us to prioritize senomorphic agents as a potentially safer option for the treatment of XP, although optimizing the dosage of senolytic agents may still hold therapeutic potential.

Senomorphic agents have recently been shown to extend lifespan and reduce age-related inflammation [15, 27]. Among the senomorphic agents we tested, the JAK inhibitors baricitinib and upadacitinib effectively reduced the percentages of senescent XP-iMCs and SASP gene expression. Expression profiling further revealed that the cytokine-cytokine receptor interaction, JAK/STAT, and PI3K/AKT pathways are activated by both UV irradiation and cisplatin treatment. This finding aligns with a previous report indicating that activation of the JAK/STAT pathway is a common hallmark of age-related diseases [26]. Moreover, coactivation of the JAK/STAT and PI3K/AKT pathways has been observed under various conditions, including oxidative stress, antiviral response, and carcinogenesis [40]. Based on these results, we propose that activation of the cytokine-cytokine receptor interaction, JAK/STAT, and PI3K/AKT pathways may play a role in the etiology of skin symptoms of XP. Additionally, curcuminoids (curcumin and its analog EF24) were also found to suppress senescence-related pathways in the drug screening. While the mechanisms of curcuminoids are complex, previous studies have shown that curcuminoids can inhibit NF-κB signaling, which functions downstream of the PI3K/AKT pathway, and exert anti-tumor effects [31, 41]. On the other hand, several JAK inhibitors have already been approved by the FDA for treating certain diseases, such as rheumatoid arthritis and atopic dermatitis [37], making them promising candidates for repurposing in potential therapeutic strategies against XP.

One of the limitations of this study is that the XP-iMCs and HC-iMCs were each derived from a single donor. Therefore, validation using cells from additional XP patients and healthy control donors will be required in the future. In addition, our conventional 2D melanocyte monoculture model does not allow adequate evaluation of therapeutic effects on non-melanoma skin cancers, which account for the majority of skin cancers in patients with XP [10]. Future studies using advanced 3D skin models containing both melanocytes and keratinocytes could determine whether senotherapeutic interventions provide therapeutic benefits across multiple skin cell types, including keratinocytes, through both direct effects and senescence-associated paracrine signaling.

Conclusion

In this study, we employed cisplatin as a senescence inducer to mimic UV irradiation. Our findings indicate that both cisplatin treatment and UV irradiation activate similar senescence-related pathways, including the cytokine-cytokine receptor interaction, JAK/STAT, and PI3K/AKT pathways, while up-regulating multiple shared SASP genes. Moreover, agents identified through cisplatin-based drug screening significantly suppressed senescence of UV-irradiated melanocytes. These results suggest that cisplatin treatment successfully recapitulates the effects of UV irradiation in our screening system at least in part. Therefore, this cisplatin-based approach holds promise for large-scale drug screening to identify potential senotherapeutic agents against XP. Finally, further validation of these findings using cells from additional donors and other XP subtypes is warranted.

Abbreviations

GSEA

Gene Set Enrichment Analysis

iPSCs

induced pluripotent stem cells

NER

nucleotide excision repair

SA-β-gal

senescence-associated β-galactosidase

SASP

senescence-associated secretory phenotype

UV

ultraviolet

XP

xeroderma pigmentosum

Author contributions

X.W., M.N., A.Y., S.H., and S.O. performed the experiments. X.W., C.Y.C., and H.T. analyzed the RNA-seq data. X.W. and S.O. assisted with project supervision. T.F. and K.N. conceived and designed the study and oversaw the overall direction. All authors contributed to writing and reviewing the manuscript.

Funding

This work was supported by the Japan Agency for Medical Research and Development (AMED) under Grant Numbers 24ym0126809j0003, 24ama221535h0001, and 25ama221535h0002, KOSÉ Cosmetology Research Foundation, Atsushi Kukita Award from the Japanese Society of Pigment Cell Research, the Naito Foundation, Takeda Science Foundation, the Nakatomi Foundation, the Ichiro Kanehara Foundation, The POLA R&M Grant for Vitiligo Research, the Grant for Joint Research Program of the Institute for Genetic Medicine, Hokkaido University (to T.F.), Japan Society for the Promotion of Science (JSPS) KAKENHI grant JP20K23376, Takeda Science Foundation, the Mitsubishi Foundation, the Photo-excitonix Project in Hokkaido University (to K.N.), Japanese Government (MEXT) Scholarship, Japan Science and Technology Agency (JST) SPRING grant JPMJSP2119, JSPS Research Fellowship for Young Scientists (DC2, PD), and JSPS KAKENHI grant JP25KJ0515 (to X.W.).

Data availability

The RNA-seq data were deposited in the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo/) with accession number GSE293695. All relevant data supporting the findings of this study are available from the authors upon request.

Declarations

Ethics approval and consent to participate

Title of the approved project: Study on the generation of human disease-specific iPS cells and their application in disease analysis. Name of the institutional approval committee or unit: Medical Ethics Committee of Kobe University Graduate School of Medicine. The approval number: 1722. Date of approval: 2015/2/2. Normal diploid fibroblasts from human skin (TIG-120; JCRB0542) and fibroblasts from xeroderma pigmentosum patient cells (XP3OS; JCRB0303) were obtained from the Japanese Collection of Research Bioresources Cell Bank [42, 43]. Normal human epidermal melanocytes (NHEM) were obtained from Kurabo Industries Ltd. The original sources have verified that proper ethical approval was secured for the cell collection.

Consent for publication

Not applicable.

Competing interests

Takeshi Fukumoto has received payments for lectures from Eli Lilly Japan K.K., AbbVie and Pfizer Japan Inc. All other authors state no Conflict of Interest.