ARID1B promotes DNA end resection via MDC1 and co-transactivation of DNA repair genes with c-MYC in small cell lung cancer

ARID1B promotes DNA end resection via MDC1 and co-transactivation of DNA repair genes with c-MYC in small cell lung cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article ARID1B promotes DNA end resection via MDC1 and co-transactivation of DNA repair genes with c-MYC in small cell lung cancer Wenchu Lin, Peng Hou, Gongfeng Li, guozhen cao, Xinhuang Yao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8612664/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract AT-rich interactive domain-containing protein 1B (ARID1B) has been implicated in DNA damage repair, yet its precise molecular mechanisms and contributions to small cell lung cancer (SCLC) pathogenesis remain incompletely defined. In this study, analysis of clinical datasets revealed ARID1B overexpression in SCLC tissues and cell lines compared to normal lung or lung adenocarcinoma counterparts. Functional studies in vitro and in vivo demonstrated that ARID1B depletion significantly impaired SCLC cell proliferation, clonogenicity, and tumor growth while promoting apoptosis. Mechanistically, During DSB response, ARID1B cooperates with the transcription factor c-MYC to co-activate the expression of key DNA damage repair (DDR) genes (e.g., RNF8 and RAD51 ), Concurrently, ARID1B is recruited to DNA double-strand break (DSB) sites, facilitates single-stranded DNA (ssDNA) formation via end resection, and promotes the recruitment of MDC1, a scaffold protein essential for early DDR signaling. Critically, ARID1B deficiency markedly sensitized SCLC cells to DNA-damaging agents, evidenced by enhanced DNA damage persistence, apoptosis, and tumor growth inhibition in vitro and in vivo . This study unveils a dual regulatory role of ARID1B in homologous recombination (HR) repair of DNA double-strand breaks (DSBs) and establishes ARID1B as a key mediator of chemoresistance in SCLC, highlighting its potential as a therapeutic target to overcome treatment resistance. Biological sciences/Cancer/Cancer epidemiology Biological sciences/Molecular biology/DNA damage and repair/Double-strand DNA breaks ARID1B SCLC c-MYC MDC1 DNA end resection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Small cell lung cancer (SCLC) is an aggressive pulmonary malignancy with pronounced neuroendocrine features, characterized by early metastasis and an exceptionally poor prognosis 1 , 2 . First-line treatment relies primarily on cytotoxic chemotherapy, typically combining platinum-based alkylating agents (cisplatin/carboplatin) with topoisomerase inhibitors (such as irinotecan or etoposide) 3 , 4 . However, the rapid development of resistance to conventional therapies significantly contributes to treatment failure and high mortality 5 . Until now, the prognosis of patients with SCLC remains poor, with a dismal five-year survival rate of approximately 5% 6 . Consequently, elucidating novel molecular mechanisms driving SCLC pathogenesis and identifying potential therapeutic targets, are of paramount importance for improving patient outcome. DNA is constantly vulnerable to damage from endogenous and environmental sources 7 , 8 . Among various types of DNA lesion, DNA double-strand breaks (DSB) are particularly severe and potential lethal, as a single unrepaired DSB can trigger cell death. Eukaryotic cells primarily repair DSBs via two pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). HR repair involves a sophisticated cellular process, orchestrated by an integrated signaling cascade involving hierarchically organized sensors, transducers, and effector molecules, including DSB recognition by the MRN complex (Mre11-Rad50-Nbs1), recruitment of mediator proteins (RNF8, RNF168, MDC1), phosphorylation of H2AX (γH2AX), end resection by CtIP to generate 3' single-stranded DNA (ssDNA) overhangs, RPA coating, and subsequent RAD51-mediated strand invasion for homology-directed repair 9 – 13 . The extent of DNA end resection determines the choice of DNA repair pathways. While the critical role of ssDNA in DNA repair is well established, the regulatory mechanisms controlling its generation and their impact on repair efficiency remain incompletely understood. Chromatin remodeling complexes, such as the ATP-dependent SWI/SNF complex, mobilize, destabilize, or restructure nucleosomes, thereby regulating DNA-dependent cellular processes including replication, repair, and transcription 14 – 16 . Growing evidence underscores the pivotal role of specific SWI/SNF subunits in HR regulation. Specifically, inactivation of the core subunit BRG1 impairs homologous recombination (HR) efficiency and reduces RAD51 foci formation 17 ; Additionally, AT-rich interactive domain 1A (ARID1A) accumulates at DSB sites post-damage and facilitates both NHEJ and HR by modulating chromatin organization and accessibility 18 – 20 . ARID1B, a mutually exclusive paralog of ARID1A within the SWI/SNF BAF complex, is implicated in neurodevelopmental disorders and stem cell fate determination 21 . Somatic ARID1B mutations occur in a wide variety of cancers, including neuroblastoma, breast cancer, and pancreatic cancer 22 , 23 , Even though the exact mechanisms by which these mutations drive oncogenesis are still largely unknown. Beyond cancer-associated genetic alterations, loss of ARID1B expression correlates with poor prognosis in several types of cancer including ovarian and colon cancer 24 – 26 , suggesting its potential as a prognostic biomarker. Furthermore, ARID1B is necessary for the survival of ARID1A -deficent cells, positioning it as a potential therapeutic target for ARID1A -mutant cancers. Recent studies indicate that ARID1B deficiency impairs DNA damage response and activates the cGAS-STING Pathway in NSCLC 27 . Depletion of ARID1B also confers sensitivity to radiation, as well as to cisplatin and UV, Evidence suggests it modulates DNA damage response by promoting NHEJ activity 28 , 29 . Despite these advances, the precise roles and regulatory mechanisms of ARID1B in DNA damage response and cancer biology remains poorly defined. A comprehensive understanding of its functions and regulatory networks in DNA repair and cancer progression is crucial for developing safe and efficient therapeutic strategies targeting ARID1B-dependent cancer. Herein, we report elevated ARID1B expression in SCLC tissues compared to normal lung or adjacent noncancerous tissues. Functional studies employing gain- and loss-of-function approaches demonstrate that ARID1B is indispensable for SCLC cell survival, clonogenicity and tumor growth. Mechanistically, biochemical analyses revealed that ARID1B cooperates with the transcription factor c-MYC to co-activate critical DNA damage repair (DDR) genes and is recruited to DSB sites and facilitates ssDNA formation via end resection. Critically, ARID1B deficiency markedly sensitizes SCLC cells to DNA-damaging agents (etoposide, bleomycin, cisplatin) both in vitro and in vivo , evidenced by persistent DNA damage, increased apoptosis, and suppressed tumor growth. These findings provide a rationale for targeting ARID1B in combination with chemotherapy to overcome treatment resistance in ARID1B-dependent SCLC. Results ARID1B is upregulated in SCLC and promotes cell proliferation The SWI/SNF chromatin remodeling complex plays a well-established role in tumorigenesis and cancer progression 15 . As a core subunit, ARID1B has emerged as a significant oncological regulator, prompting our investigation into its tumor-specific expression in SCLC. Pan-cancer analysis of the CCLE database revealed significantly elevated ARID1B mRNA expression in SCLC cell lines compared to lung adenocarcinoma (LUAD) lines, ranking SCLC second highest among 33 tumor types (Fig. 1 A, Supplementary Figure S1 A). Transcriptomic analysis of clinical samples from two datasets confirmed ARID1B upregulation in SCLC tumors relative to adjacent normal tissues (Figs. 1 B-C). Paired sample analysis demonstrated concordant upregulation of ARID1B at both mRNA and protein levels (Figs. 1 D-F, Supplementary Figures S1 B), implicating ARID1B in SCLC pathogenesis. To functionally characterize ARID1B in SCLC, we overexpressed it in DMS273 and SHP77 cell lines (Fig. 1 G and Supplementary Figure S1 C). Colony formation and CellTiter-Glo assays revealed that ectopic ARID1B expression significantly enhanced clonogenic potential and cell viability (Figs. 1 H-I). This proliferative advantage was further corroborated by increased DNA replication activity in EdU incorporation assays (Fig. 1 J). Collectively, these findings provide compelling evidence for the pro-tumorigenic function of ARID1B in SCLC. ARID1B Depletion Suppresses SCLC Growth in vitro and in vivo To further define ARID1B’s functional role, we established stable ARID1B -knockdown DMS273 and H82 cell lines (Fig. 2 A and Supplementary Figure S2 A). CellTiter-Glo and colony formation assays showed that ARID1B depletion significantly reduced cell viability and clonogenic capacity in both lines (Fig. 2 B-C). Correspondingly, EdU incorporation assays revealed a significant inhibition of cellular proliferation (Fig. 2 D). Notably, ARID1B knockdown elicited a pronounced apoptotic response, as demonstrated by the elevated cleavage of PARP (Supplementary Figures S2 B-C). To validate these findings in vivo , we generated subcutaneous xenografts in nu/nu mice (Fig. 2 E). ARID1B knockdown significantly inhibited tumor growth (Figs. 2 F-G) and reduced final tumor mass while maintaining stable body weight parameters (Fig. 2 H, and Supplementary Figure S2 D). Immunohistochemistry (IHC) analysis of ARID1B -deficient tumors showed decreased Ki67 and elevated cleaved-caspase 3 signals (Fig. 2 I), consistent with in vitro observations. These results definitively establish ARID1B as a functionally oncogenic driver in small cell lung cancer (SCLC) tumorigenesis. ARID1B Regulates the DNA Damage Repair To elucidate the molecular mechanisms underlying ARID1B-mediated phenotypes, we performed gene set enrichment analysis (GSEA) of cancer hallmarks 30 . Specimens with higher ARID1B mRNA expression (n = 56) were categorized as ARID1B -high SCLC, others were designated as ARID1B -low SCLC. Hallmark collection analysis identified seven pathways significantly enriched in ARID1B -high SCLC, namely G2M checkpoint, E2F targets, MYC targets V1, spermatogenesis, mitotic spindle, pancreas beta cells and DNA repair (Fig. 3 A). Targeted enrichment analysis of C2 collection further confirmed a strong positive correlation between ARID1B expression and DNA damage repair pathways, particularly homology-directed repair (Figs. 3 B-C and Supplementary Figure S3 A-B). Pearson correlation analysis using a SCLC RNA-seq dataset revealed positive correlation between ARID1B and HR-related genes, including RNF8 and RAD51 (Supplementary Figure S3 C). Consistently. GSEA based on a proteomic dataset ranked DNA repair among the top enriched gene sets (Supplementary Figure S3 B). These findings suggest functional links between ARID1B and DNA repair in SCLC. To assess ARID1B’s direct role in DNA damage response, we evaluated DNA damage levels following ARID1B depletion using comet assay and immunofluorescence (IF) analysis. Comet assay revealed significantly increased DNA damage upon ARID1B knockdown (Figs. 3 D-E). IF quantification of γH2AX foci confirmed elevated DNA damage in ARID1B -deficient SCLC cells, as evidenced by markedly elevation in γH2AX foci (Figs. 3 F-G and Supplementary Figure S3 D). IF microscopy further showed impaired RAD51 foci formation post knockdown (Figs. 3 H-I and Supplementary Figure S3 E). Western blot analysis demonstrated elevated accumulation of γH2AX alongside reduced levels of RAD51 and RNF8 following DNA damage (Fig. 3 J). We hypothesized that ARID1B directly regulates the expression of RAD51 and RNF8 . RT-qPCR analysis of key DNA damage response genes revealed that ARID1B expression levels significantly influenced the transcriptional regulation of DNA damage repair gene (Supplementary Figures S3 F-G). Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis confirmed ARID1B binding to the promoter regions of both genes (Fig. 3 K). Importantly, dual-luciferase reporter assays showed that ARID1B depletion reduced RNF8 promoter activity (Fig. 3 L). Collectively, these data indicate that ARID1B contributes to DSB repair via transcriptional regulation of critical DDR genes. ARID1B Modulates SCLC Progression via c-MYC Previous studies established direct c-MYC binding to promoters of key DNA double-strand break (DSB) repair genes 31 – 33 . Moreover, c-MYC is a direct target of ARID1B-containing SWI/SNF complexes during cell cycle activation 34 . Importantly, Gene Ontology (GO) pathway and GSEA analysis further supported a significant functional association between ARID1B and c-MYC (Figs. 3 A, 4 A and Supplementary Figure S4 A), suggesting that ARID1B may regulate SCLC progression through c-MYC. To investigate this relationship, we first assessed the impact of ARID1B perturbation on c-MYC expression. As depicted in Figs. 4 B, C, ARID1B modulation significantly altered both mRNA and protein levels of c-MYC in DMS273 and H82 cells (Figs. 4 B-C). ChIP-qPCR analysis revealed direct ARID1B binding to the c-MYC promoter, with pronounced enrichment at the c-MYC BS2 site (Fig. 4 D). Importantly, ARID1B knockdown abolished BS2 occupancy (Fig. 4 E and Supplementary Figure S4 B), indicating a functional interaction between ARID1B and c-MYC in transcriptional control. Notably, ectopic c-MYC expression or knockdown partially rescued cell viability and clonogenic defects in ARID1B -manipulated cells (Figs. 4 F-I and Supplementary Figure S4 C). These results provide compelling evidence that ARID1B drives SCLC progression through c-MYC. ARID1B and c-MYC Synergistically Regulate DDR gene Transcription To elucidate cooperative regulation of DDR gene by ARID1B and c-MYC, we performed ChIP and luciferase assays to determine the possible functional interplay. ChIP-qPCR analysis confirmed c-MYC binding to the promoter regions of RNF8 and RAD51 (Fig. 5 A). Luciferase reporter assays showed that c-MYC depletion significantly reduced the promoter activity of c-MYC and Rad51 , compared to scramble controls (Fig. 5 B), phenocopying ARID1B loss. Similarly, pharmacological inhibition of c-MYC with MYCi975 recapitulated this effect (Fig. 5 C). Intriguingly, ARID1B depletion could mimic c-MYC knockdown-mediated suppression of promoter activity of RNF8 and RAD51 (Fig. 5 D and Supplementary Figures. S5A-B), implying functional synergy. Importantly, modulating c-MYC expression partially restored promoter activity in ARID1B -deficient or overexpressing cells (Figs. 5 E-G), indicating that ARID1B and c-MYC function in concert to regulate the expression of key DDR genes. To characterize their interplay, we performed sequential chromatin immunoprecipitation (ChIP-reChIP) analysis to evaluate the co-occupancy of ARID1B and c-MYC at the promoter of co-regulated genes. Indeed, ChIP-reChIP confirmed co-binding of ARID1B and c-MYC at DDR gene promoters, including an autoregulation at the c-MYC locus (Fig. 5 H and Supplementary Figure S5 C). To exclude indirect effect via c-MYC regulation, electrophoretic mobility shift assays (EMSAs) using recombinant c-MYC protein and ARID domain (a characterized DNA-binding module) of ARID1B (Supplementary Figure S5 D-E) demonstrated direct binding of both the ARID domain and c-MYC to the RAD51 promoter. Importantly, Co-incubation enhanced each other’s binding ability to DNA, revealing a synergistic interaction on DNA (Fig. 5 I). These results provide compelling evidence that ARID1B and c-MYC cooperatively activate DDR gene transcription. ARID1B Promotes DNA End Resection at DSB sites via MDC1 Prior studies indicate ARID1B accumulates at the sites of laser micro-radiation (IR)-induced DNA damage 29 . We first analyzed ARID1B spatial distribution relative to γH2AX foci, a canonical marker of double-strand breaks (DSBs). IF staining revealed partial but significant ARID1B co-localization with γH2AX following etoposide or bleomycin treatment (Fig. 6 A). Furthermore, ChIP-PCR using an I-PpoI endonuclease system confirmed that significant ARID1B enrichment at induced DSBs in 28S rDNA and DAB1 loci in HEK293T cells (Fig. 6 B and Supplementary Figure S6 A-C), demonstrating active recruitment of ARID1B to DSB sites. MDC1 orchestrates RNF8/RNF168 recruitment to DSBs, promoting H2A ubiquitination to amplify the damage signal and facilitate HR repair 35 – 37 . Protein-protein interaction (PPI) network analysis was conducted to screen the HR repair-related proteins binding to ARID1B using the STRING database. To further validate the results obtained from the PPI network, we screened the ARID1B-interacting proteins using the BioGRID database and identified a potential interaction between MDC1 and ARID1B 38 .The results showed that MDC1 potentially interact with ARID1B (Fig. 6 C), which was validated by co-immunoprecipitation (Fig. 6 D). IF staining demonstrated ARID1B deficiency impaired MDC1 accumulation at DSB sites (Fig. 6 E), suggesting that requirement of ARID1B at DSB sites for MDC1 recruitment upon DNA damage. The MRN complex composed of dimers of MRE11, RAD50, and NBS1 plays a dual role in homologous recombination (HR)-mediated DSB repair, serving as both a critical end-bridging factor and an essential endonuclease 39 , 40 . To exclude potential transcriptional interference, we examined both mRNA and protein levels of MDC1-associated genes following ARID1B knockdown. The results demonstrated that ARID1B depletion did not reduce the expression of MDC1 at either the mRNA or protein level (Figure. 6F and Supplementary Figure S6 D). Furthermore, ARID1B exhibited no regulatory effect on the early recruitment factor MRE11. Since association of MDC1 facilitates the binding of MRN complex through phosphorylation-dependent interactions with the FHA-BRCT1-BRCT2 domains of NBN 41 , 42 , we therefore sought to determine whether ARID1B modulates DNA end resection via MRN during DSB repair. Notably, phosphorylation of RPA2, a critical determinant of DSB repair pathway choice, was compromised upon ARID1B depletion (Supplementary Figure S6 E-F). To furthermore assess ARID1B's role in DNA end resection during DSB repair, we strategically designed restriction enzyme sites flanking the I-PpoI cleavage locus to generate site-specific DNA damage (Supplementary Figure S6 G). The generation of ssDNA at these sites would confer resistance to restriction enzyme digestion. We detected substantial ssDNA accumulation after I-PpoI-mediated 28S rDNA DSB induction, which was significantly blocked by ARID1B depletion (Fig. 6 G). To provide additional experimental validation, a BrdU incorporation assay coupled with non-denaturing immunofluorescence (IF) staining was employed to detect single-stranded DNA (ssDNA) formation 43 , 44 . ARID1B -deficient cells showed significantly reduced ssDNA generation (Fig. 6 H). During homologous recombination (HR) repair, double-strand breaks (DSBs) undergo a two-step processing mechanism: First, DSB ends are converted into splayed-arm structures, which are subsequently processed by nucleases such as MRE11 or EXO1 to generate 3′ single-stranded overhangs (3′-overhangs),This ssDNA intermediate then serves as an essential molecular scaffold for initiating HR-mediated repair 45 , 46 . Given ARID1B's recruitment to DNA lesions and role in end resection, we hypothesized a direct interaction with damaged DNA. To test this hypothesis, EMSAs using purified ARID domain and DNA substrates were carried out to assess ARID1B association with various forms of DNA. Our results revealed that robust binding to splayed-arm DNA structures and minimal but meaningful affinity for ssDNA (Fig. 6 I). This structure-specific binding preference for replication fork-like intermediates suggests ARID1B directly participates in DNA damage processing. Collectively, these findings elucidate ARID1B’s direct and critical role in DSB site recruitment and end resection, governing the choice of DSB repair pathways and maintaining genomic stability. ARID1B depletion impairs HR repair To assess ARID1B’s impact on HR repair under genotoxic conditions, a series of experiments was performed to systematically analyze key HR components. Western blotting showed that the DNA damage-induced upregulation of RAD51 and RNF8 was markedly impaired in ARID1B -deficient cells exposed to either etoposide or bleomycin, accompanied by elevated γH2AX (Figs. 7 A-B). Quantitative IF revealed significantly reduced p-RPA2 and RAD51 foci formation at 1-hour post-damage in ARID1B -deficient cells (Figs. 7 C-D and Supplementary Figure S7 A-D), thereby establishing ARID1B’s role in efficient HR machinery recruitment. Comet assays showed increased fragmented DNA accumulation (Fig. 7 E and Supplementary Figure S7 E). While IF microscopy showed that while robust γH2AX foci formed at 1-hour post-damage across conditions, ARID1B knockdown cells exhibited persistent γH2AX foci at 4 hours post-damage, indicating defective repair. Similar effects were observed with bleomycin treatment (Figs. 7 F-G and Supplementary Figures S7 F-I). These findings provide compelling evidence that ARID1B is indispensable for an effective homologous recombination-mediated DNA damage response. ARID1B Deficiency Sensitizes SCLC cells to Chemotherapy Given ARID1B's role in HR-mediated repair, we hypothesized its depletion would sensitize SCLC cells to DNA-damaging agents. To test this hypothesis, we exposed SCLC cells to clinically relevant DNA-damaging agents and found that ARID1B deficiency significantly increased sensitivity to clinically relevant chemotherapeutics, as evidenced by reduced cell viability and clonogenic capacity (Figs. 8 A-B and Supplementary Figures S8 A-B). Quantitative apoptosis analysis via flow cytometry with Annexin V-FITC/PI dual staining confirmed that enhanced apoptosis induction in ARID1B -depleted cells treated with etoposide or bleomycin in both DMS273 and H82 cells (Supplementary Figures S8 C-D). To evaluate clinical relevance, we employed a xenograft mouse model to assess the in vivo therapeutic implications of targeting ARID1B. Administration of the etoposide/cisplatin (E/P) regimen 47 resulted in significantly greater tumor growth inhibition and reduced tumor mass in ARID1B -depleted xenografts versus controls (Figs. 8 C-E). Notably, all treatment groups maintained stable body weights throughout the experimental duration, demonstrating favorable treatment tolerance (Supplementary Figure S8 E). IHC analysis of tumor specimens showed baseline elevation of γH2AX signal following E/P treatment, with additional enhancement following ARID1B loss (Fig. 8 F). Consistent with suppressed tumor growth and compromised DNA repair, ARID1B loss led to greater Ki67 downregulation and apoptosis induction than controls. These findings demonstrate that targeting ARID1B potentiates genotoxic therapy efficacy in SCLC, supporting the development of ARID1B-targeted therapeutic interventions as a novel strategy to circumvent chemotherapy resistance in SCLC. Discussion In this study, we provide multiple lines of evidence establishing ARID1B as a multifunctional DNA damage response (DDR) factor that promotes double-strand break (DSB) repair, whose deficiency renders tumors susceptible to chemotherapeutic agents. Mechanistically, we demonstrate that ARID1B not only associates with gene promoters and synergizes with c-MYC to transcriptionally activate key DNA damage repair genes, but also is recruited to DNA lesion sites upon genotoxic stress, where it facilitates MDC1 recruitment and subsequent MRN complex-mediated DNA end resection to generate ssDNA (Fig. 7 H). Consequently, ARID1B deficiency impairs DSB end resection and compromises homologous recombination (HR) repair, creating a targetable vulnerability that can be harnessed to overcome chemoresistance in SCLC. The DNA damage response (DDR) is fundamental to tumor progression and cellular response to DNA-damaging therapies, such as radiation and chemotherapy. Epigenetic dysregulation can transcriptionally silence critical DNA repair components across various cancers 48 . Advances in cancer genomics and understanding of epigenetic-transcriptional interplay, combined with the recognized importance of DNA repair in tumorigenesis, suggesting that epigenetic-targeting therapeutics may offer novel strategies to modulate treatment response by leveraging DNA damage response 49 , 50 . SWI/SNF chromatin remodeling complex modulates DDR in an ATP-dependent and subunit-specific manner. Functionally, they bind to defined genomic loci, disrupts histone-DNA interactions, utilize ATP hydrolysis to reposition nucleosomes, and create accessible chromatin to facilitate recruitment of DNA-binding factors and transcriptional machinery 15 , 51 . Notably, ATPase subunits like BRG1 play critical roles in DNA repair. Recent studies reveal that BRG1 resolves transcription-replication conflicts and employ its ATPase domain to interact with multiple DNA repair proteins, recruiting them to sites of DNA damage sites to promote HR repair 19 , 52 . Within the BAF complex, the ARID1B subunit characterized by an AT-rich interaction domain (ARID) protein but lacking ATPase or bromodomains, plays a distinct role in targeting of the complex 53 – 55 . We demonstrate that ARID1B is frequently overexpressed in SCLC, driving tumor proliferation and survival. Mechanistically, ARID1B subunit plays an indispensable role in HR via dual mechanisms: (1) As a transcriptional co-regulator, it partners with c-MYC to activate key HR genes (e.g. RAD51 and RNF8 ); (2) It is directly recruited to DSBs, facilitates end resection by promoting ssDNA formation. Prior studies have shown that nucleosomes can significantly regulate DNA end resection by acting as physical barriers to the resection machinery 56 , 57 . Nucleosome remodeling complexes such as SWI/SNF or INO80 facilitate CtIP recruitment and end resection initiation 58 , 59 . Additionally, SMARCAD1 and Fun30 likely promote resection not only through their chromatin remodeling activity but also by reducing the local concentration of resection inhibitors such as 53BP1 (or its budding yeast homolog Rad9) 60 , 61 . In line with this observation, we demonstrate for the first time that ARID1B is an indispensable modulator of DNA end resection and homologous recombination (HR) repair. Consequently, ARID1B deficiency cripples HR repair, leading to unresolved DNA damage, apoptosis, and profound sensitization to DNA-damaging agents both in vitro and in vivo . This study defines the role of ARID1B in SCLC pathogenesis and reveals its regulatory network as a chromatin remodeler coordinating DNA repair and transcriptional regulation. While consistent with emerging DDR roles for SWI/SNF subunits, our findings underscore the unique, essential contribution of the non-catalytic ARID1B. The ARID1B/c-MYC cooperation unveils an epigenetic-transcriptional regulatory layer controlling DDR capacity in SCLC. ARID1B overexpression in SCLC and its positive correlation with DDR genes suggest its upregulation may be a key adaptive mechanism underlying the notorious chemoresistance of this malignancy. In summary, this work elucidates the functional role of ARID1B in SCLC and provides novel insights into its regulatory network, highlighting that targeting ARID1B or disrupting its interaction with c-MYC represents a promising strategy to overcome chemoresistance in SCLC. Future studies should explore specific ARID1B inhibitors and their combinatorial efficacy with standard genotoxic therapies. Materials and Methods Cell lines and cell culture Human SCLC cell lines (DMS273, H82, and SHP77) and human embryonic kidney cell line HEK293T were cultured at 37°C in a humidified incubator with 5% CO 2 . SCLC cells were maintained in RPMI1640 medium (Gibco), while HEK293T cells were cultured in DMEM (Gibco) medium. Both media were supplemented with 10% fetal bovine serum (FBS, VivaCell) and penicillin and streptomycin (100X, Servicebio). Microarray, RNA-seq, and genomic datasets and data analysis Standardized gene expression microarray data (GSE149507 and GSE60052) were downloaded from the Gene Expression Omnibus (GEO) database ( http://www.ncbi.nlm.nih.gov/geo ). Additionally, microarray and RNA-seq data for 50 SCLC cell lines, along with RNA-seq data for 33 tumor types, were obtained from the Cancer Cell Line Encyclopedia (CCLE) ( https://portals.broadinstitute.org/ccle/data ). Further, RNA-seq data of 107 SCLC specimens and their matched adjacent non-cancerous tissues were obtained from the Genome Sequence Archive (GSA) (GSA database accession: HRA003419 available at http://bigd.big.ac.cn/gsa-human ). Proteomic data and associated pathological information for 112 SCLC specimens and their corresponding para-cancerous tissues were secured from the OMIX database (OMIX database accession: OMIX002489 accessible at https://ngdc.cncb.ac.cn/omix ). All data sources are publicly available. RNA Extraction and qRT-PCR Total RNA extraction was extracted from 2×10 6 cells using Trizol reagent (TransGen Biotech) according to the manufacturer's protocol. RNA concentration and quality were assessed using a spectrophotometer (Epoch). Reverse transcription was performed using the EvoM-MLV Reverse Transcriptase Kit (Accurate Biology). Quantitative real-time PCR (qRT-PCR) was conducted using SYBR Green Master Mix (Accurate Biology) on a Quantstudio 5 platform (Applied Biosystems). Relative gene expression levels were calculated using the ΔΔCt method with actin as the housekeeping gene. Primer sequences are listed in Supplementary Table S2 . Western blotting Cells were lysed using Western and IP lysis buffer (Beyotime). Proteins were separated by SDS-PAGE and electrotransferred onto PVDF membranes. The membranes were blocked with 5% skim milk (BBI LIFE SCIENCES) for 2 hours, followed by overnight incubation with the primary antibody at 4°C. After the membranes were incubated with the secondary antibody for 1 hour at room temperature. Enhanced chemiluminescence (ECL) reagent was applied to the PVDF membranes, and protein bands were detected using chemiluminescence imaging system (Tanon). Primary antibodies used in the study are listed in Supplementary Table S3 . Colony formation assay DMS273 cells were seeded at 500 cells per well and SHP77 cells at 3,500 cells per well in 6-well plates. Cells were incubated for two weeks to allow colony formation. The clones were washed with 1x PBS, fixed with methanol, and stained with 0.1% crystal violet. Colony formation of H82 cells was assessed using a soft agar assay as previously described 62 . The colonies were photographed and analyzed by Image J Cell viability, apoptosis, and EdU assays Cells designated for drug treatment were exposed to a concentration gradient of cisplatin, etoposide, or bleomycin. After 72 hours of treatment, cell viability was assessed using the CellTiter Glo luminescent assay (Vazyme). Luminescence signals were recorded using a multifunctional microplate reader (Agilent BioTek). Apoptosis rates were measured using the Annexin V-FITC/propidium iodide (PI) assay according to the manufacturer's instructions. Flow cytometric analysis was performed immediately after staining. Data was analyzed using FlowJo software (FlowJo). Cell proliferation was assessed using the EdU assay (Click-iT EdU kit, Thermo Fisher Scientific). Fluorescent images were acquired using a fluorescence microscope. EdU-positive cells were quantified by counting five random fields per sample. Transfection of siRNA and plasmids siRNA and plasmids were transfected into cells using the Effectene Transfection Reagent (Qiagen). Transfection was performed when cells reached approximately 60% confluence. After 48 hours of incubation, cells were harvested for subsequent analysis. Gene silencing efficiency was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). siRNA sequences are provided in Supplementary Table S1 . Lentivirus construction and transduction To generate lentivirus for stable knockdown, shRNA plasmids were co-transfected with packaging plasmids (pMD2.G and psPAX2) into HEK293T cells using Effectene Transfection Reagent (Qiagen) as previously described. For overexpression constructs, the c-MYC gene was amplified by PCR and cloned into the PCMV-3Flag vector. The ARID1B plasmid was obtained from Addgene (#144023). shRNA sequences are detailed in Supplementary Table S1 . Comet assay DNA damage was assessed using comet assay following the manufacturer's instructions. Briefly, cells (1×10⁵ cells/mL) were mixed with molten CometAssay LMAgarose (at 37°C) at a 1:10 (v/v) ratio. The mixture was layered onto comet slides. Cells were then lysed for 30 minutes, followed by immersion in Alkaline Unwinding Solution for 20 minutes at room temperature. Alkaline electrophoresis was performed at 21 V for 30 minutes and visualized using a fluorescence microscope. Immunofluorescence and microscopy For immunofluorescence experiments, cells were seeded onto glass-bottomed confocal dishes and incubated overnight, followed by treatment according to the experimental protocol. Images were acquired using a confocal microscope. The primary antibodies used in the study are listed in Supplementary Table S3 . Chromatin immunoprecipitation (ChIP) A minimum of 2 x 10 7 adherent or suspension cells per sample were crossed-linked with 1% formaldehyde 10 minutes at room temperature. Cross-linking was terminated by adding glycine to a final concentration of 0.125 M, and the cells were incubated on the orbital shaker for an additional 5 minutes. Cells were washed with cold PBS, lysed (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0), and sonicated to shear DNA into fragments ranging from 300 to 500 bp. Chromatin was pre-cleared and immunoprecipitated overnight at 4°C with 2 µg of specific antibody or IgG control. Antibody-chromatin complexes were captured using Protein A/G magnetic beads (Thermo Fisher), washed extensively, and eluted. Cross-links were reversed overnight at 65°C. DNA was then purified using a PCR purification kit (Accurate Biology) and analyzed by qPCR. For the DNA damage site protein recruitment assay, as previously described 63 , HEK293T cells were co-transfected with pBABe-HA-ER-IPpoI and siARID1B, and treated with 1 µM 4-hydroxy-Tamoxifen (4-OHT) for 24 hours. Cells were then collected for ChIP analysis. ChIP primers are listed in the supplementary Table S4 . Dual Luciferase Reporter Assay The Dual-Luciferase Reporter Assay System (Vazyme) was used according to the manufacturer's protocol, HEK293T cells in 12-well plates were co-transfected with PGL4-Ctrl, PGL4-MYC and PGL4-RNF8 reporter vectors. After 48 hours, the culture medium was aspirated and cells were washed with PBS. Cells were lysed by adding passive Cell lysis buffer and incubating for 5 minutes at room temperature with gentle shaking. Cell lysates were centrifuged at 12,000 rpm for 2 minutes at room temperature. A 20 µl aliquot of the supernatant was transferred to a microplate containing 100 µl of Luciferase substrate (Vazyme) and immediately measured using a luminometer; subsequently, freshly prepared Renilla substrate working solution was added to the reaction wells and Renilla luciferase activity was measured immediately. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample. Detecting ssDNA lesions by BrdU incorporation Single-stranded DNA (ssDNA) lesions were detected using BrdU incorporation under non-denaturing conditions as previously described 43 . Briefly, DMS273 cells were pulse-labeled with 10 µM BrdU (BD Biosciences) for 24 h. DNA damage was then induced by treating cells with 40 µM Etopside (ETO) for 1 h. Single-stranded DNA (ssDNA) lesions were detected using BrdU incorporation under non-denaturing conditions as previously described 43 . Fluorescence images were acquired using a high-resolution microscopy system. DNA end-resection assay DNA end resection was quantified by measuring induced single-stranded DNA (ssDNA) formation using the HA-ER-I-PpoI system 44 , 64 . HEK293T cells were co-transfected with the Plasmids pBABe-HA-ER-I-PpoI and siRNA targeting ARID1B (siARID1B). Twenty-four hours post-transfection, 4-hydroxytamoxifen (4-OHT) was added to the culture medium at a final concentration of 1 µM to induce nuclear translocation and I-PpoI endonuclease activity. After an additional 24 hours, cells were collected and genomic DNA was extracted using a DNA extraction kit (Accurate Biology). Purified DNA was then was digested with EcoRI (New England Biolabs) at 37°C for 30 minutes. Subsequently, the digested DNA was purified and analyzed by quantitative PCR (qPCR) using primers flanking the I-PpoI cleavage site. Relative ssDNA levels were calculated using the ΔΔCt method, comparing samples with and without 4-OHT induction and normalized to control siRNA. EMSA assay The plasmids containing either full-length c-MYC or the ARID domain (amino acids 1136–1227) of ARID1B were transformed into Escherichia coli BL21 cells for protein purification. Protein-DNA binding was analyzed by EMSA using the Lightshift chemiluminescent EMSA kit (GS009, Beyotime) according to the manufacturer’s instructions. Briefly, biotin-labeled DNA substrate was incubated with purified ARID protein/c-MYC protein at indicated concentrations in 1× binding buffer at room temperature for 20 min. Protein-DNA complexes were resolved on a 6% non-denaturing polyacrylamide gel electrophoresis (PAGE) in 0.5× TBE buffer at 100 V for 60–90 min at 4°C and subsequently transferred to a positively charged nylon membrane (Beyotime) at 380 mA for 30–45 min. After UV crosslinking (254 nm, 5 min), the membrane was blocked and washed according to standard protocols. Biotin-labeled DNA was then detected using the chemiluminescent substrate provided in the kit. Chemiluminescent signals were captured using an automated chemiluminescence imaging system (Tanon). Oligo primers are listed in the supplementary Table S5 Establishment of xenograft models 2×10 6 DMS273 cells (either transduced with non-targeting control shRNA [SCR] or ARID1B-targeting shRNA [shARID1B#1 or shARID1B#2]) suspended in 100 µL PBS were subcutaneously inoculated into the dorsal flank of 4–5-week-old female BALB/c nude mice. When tumors reached a volume of 100 mm 3 , mice bearing SCR or shARID1B DMS273 cells were randomly divided into control and E/P treatment groups. The E/P group received etoposide (7 mg/kg, i.p.) every other day and cisplatin (3 mg/kg, i.p.) once weekly. No animal mortality occurred during the study. At the experiment endpoint, mice were euthanized, tumors were excised and weighed. Tumor volume was calculated using the formula: Volume (mm³) = (Length × Width²) / 2, where length is the longest diameter and width is the perpendicular diameter. Immunohistochemistry Formalin-fixed, paraffin-embedded (FFPE) tumor tissues were sectioned (4–5 µm). Sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After blocking with 5% BSA, sections were incubated overnight at 4°C with the primary antibodies: Ki-67 (GB121141, Servicebio, 1:200), cleaved caspase-3 (GB11532, Servicebio, 1:200), ARID1B (ab57461, abcam, 1:200), γ-H2AX (GB111841, Servicebio, 1:200), c-MYC (ab32072, abcam, 1:200). IHC staining intensity and percentage of positive cells were independently evaluated and scored by two experienced pathologists blinded to the experimental groups, using a semi-quantitative scoring system as previously reported. Statistical analysis GraphPad Prism 6.0 software was utilized for statistical analysis. Experimental data are presented as the mean ± standard deviation. Intergroup comparisons were conducted using the unpaired two-sided Student's t -test or one-way analysis of variance (ANOVA). Statistical significance was defined as a P < 0.05. Declarations Availability of data and material All data accessed from external sources and prior publications have been referenced in the text and corresponding figure legends. Additional data used and/or analyzed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All mouse experimentations were performed in compliance with the institutional guidelines and the protocol was approved by the Ethics Committee of Longgang District People’s Hospital of Shenzhen(2024067DW). All authors complied with all relevant ethical regulations for animal testing and research. Declaration of competing interest The authors declare no competing interests. Acknowledgements We thank members of the Lin laboratory for critical reading of the manuscript and helpful discussions. Author Contributions Wenchu Lin supervised, and funded the study. Peng Hou, Jiahui Zhang and Gongfeng Li conducted animal experiments. Peng Hou, Guozhen Cao, Jinghan Hua, Xinhuang Yao and Honglin Li performed in vitro cell-based experiments. Peng Hou and Gongfeng Li analyzed RNA-seq data. Peng Hou performed molecular biological experiments. Rui Guo provided platform technical support. Peng Hou, Li Xiang and Wenchu Lin wrote the manuscript. All authors read and approved the article. Funding This study was supported by National Natural Science Foundation of China (Grant Numbers: 82573883, 82302957), Shenzhen Science and Technology Program (Grant Number: JCYJ20250604180129038), Key Medical Technologies R & D Programme of Longang district (Grant Number: LGKCYLWS2023012), A portion of this work was supported by Sanming Project of Medicine in Shenzhen. References Bernhardt, E. B. & Jalal, S. I. Small Cell Lung Cancer. Cancer Treat Res 170, 301–322 (2016). https://doi.org/10.1007/978-3-319-40389-2_14 Taniguchi, H., Sen, T. & Rudin, C. M. Targeted Therapies and Biomarkers in Small Cell Lung Cancer. 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J Biol Chem 294, 7632–7643 (2019). https://doi.org/10.1074/jbc.RA118.005415 Additional Declarations (Not answered) Supplementary Files SupplementaryFigure20101.png Supplementary Figure 2 SupplementaryFigure10101.png Supplementary Figure 1 TableS5PrimersequencesforEMSAassay.docx Table S5 SupplementaryFigure60101.png Supplementary Figure 6 SupplementaryFigure40101.png Supplementary Figure 4 TableS4PrimersequencesforChIPassay.docx Table S4 SupplementaryFigure70101.png Supplementary Figure 7 TableS1shRNAandsiRNAsequencesforgenesilencing.docx Table S1 SupplementaryFigure50101.png Supplementary Figure 5 SupplementaryFigure30101.png Supplementary Figure 3 SuppmentaryFigurelegend.docx Supplementary Figure legend SupplementaryFigure80101.png Supplementary Figure 8 TableS2PrimersequencesforqRTPCR.docx Table S2 TableS3Listofantibodiesusedinthisstudy.docx Table S3 SupplementalMaterialfile.pptx Original Data Files Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 16 Mar, 2026 Review # 2 received at journal 14 Mar, 2026 Review # 1 received at journal 06 Mar, 2026 Reviewer # 2 agreed at journal 28 Feb, 2026 Reviewer # 1 agreed at journal 19 Feb, 2026 Reviewers invited by journal 05 Feb, 2026 Submission checks completed at journal 30 Jan, 2026 First submitted to journal 29 Jan, 2026 Unknown event 16 Jan, 2026 Editor assigned by journal 15 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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China","correspondingAuthor":false,"prefix":"","firstName":"jiahui","middleName":"","lastName":"Zhang","suffix":""},{"id":586368506,"identity":"f6d903b6-29a0-4c22-a67b-24c7fe19dde2","order_by":7,"name":"Jinghan Hua","email":"","orcid":"","institution":"University of Science and Technology of China","correspondingAuthor":false,"prefix":"","firstName":"Jinghan","middleName":"","lastName":"Hua","suffix":""},{"id":586368508,"identity":"67592b4a-21d3-416e-b41d-c616ef369e76","order_by":8,"name":"Rui Guo","email":"","orcid":"","institution":"The Chinese University of Hong Kong, Shenzhen","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Guo","suffix":""},{"id":586368510,"identity":"25ad9bfc-a961-4c00-857e-5bad4aa883d9","order_by":9,"name":"Li Xiang","email":"","orcid":"https://orcid.org/0000-0002-7164-0588","institution":"Longgang District People's Hospital of Shenzhen","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Xiang","suffix":""}],"badges":[],"createdAt":"2026-01-15 17:17:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8612664/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8612664/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102320093,"identity":"de763da6-b7ff-4229-a101-b43bd8972f37","added_by":"auto","created_at":"2026-02-10 13:27:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":954800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated ARID1B expression in SCLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-C) Scatter plots of \u003cem\u003eARID1B\u003c/em\u003e mRNA expression in SCLC cell lines relative to lung adenocarcinoma cell lines (A, CCLE dataset), in SCLC tumor tissues vs. adjacent normal tissues (B, GSE60052), in SCLC blood relative to benign blood (C, exoRBase database 2.0). (mean ± SD, unpaired Student\u0026lt; 0.01; **. (D-E) Paired analysis of\u003cem\u003e ARID1B\u003c/em\u003e mRNA expression in 18 SCLC tumors and matched adjacent normal tissues (D, GSE149507), in 107 SCLC tumors and matched adjacent normal tissues (E, HRA003419). (F) Paired analysis of ARID1B protein level in 112 SCLC tumors and matched adjacent normal tissues (OMIX002489), (paired Student’s \u003cem\u003et\u003c/em\u003e-test). (G) Western blotting of ARID1B in DMS273 and SHP77 SCLC cells upon ectopic \u003cem\u003eARID1B\u003c/em\u003e overexpression, (paired Student’s \u003cem\u003et\u003c/em\u003e-test). (H-I) Analysis of colony-forming ability (H) and cell viability (I) of DMS273 and SHP77 cells with ectopic \u003cem\u003eARID1B\u003c/em\u003e expression. (J) Representative images and quantification of EdU staining in SCLC cells with \u003cem\u003eARID1B\u003c/em\u003e overexpression (mean ± SD; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; unpaired Student’s \u003cem\u003et\u003c/em\u003e-test)\u003c/p\u003e","description":"","filename":"Figure101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/210b1c0b1dc7d321f0432759.png"},{"id":102319991,"identity":"af2753b7-81b3-4c8d-a763-f45f2ef4d69f","added_by":"auto","created_at":"2026-02-10 13:26:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3079286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARID1B Drives Tumor Progression in Small Cell Lung Cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blotting of ARID1B in DMS273 and H82 cells with ARID1B knockdown. (B-C) Clonogenic formation (B) and cell viability (C) assays of DMS273 and H82 cells with \u003cem\u003eARID1B\u003c/em\u003eknockdown. (D)Representative images and quantification of EdU staining in SCLC cells with \u003cem\u003eARID1B\u003c/em\u003eknockdown. (E) Schematic illustration of the experimental design and workflow for the in vivo xenograft study. (F) Representative images of excised xenograft tumors from SCR and \u003cem\u003eARID1B\u003c/em\u003e-knockdown groups at the experimental endpoint. (G)Tumor growth curves of subcutaneous xenografts in different treatment groups. Data are presented as mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01. (H)Tumor weights of DMS273 xenografts from SCR and \u003cem\u003eARID1B\u003c/em\u003e-knockdown groups at the experimental endpoint. Statistical significance was determined by unpaired Student’s t-test (mean ± SEM; ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001. (I) Representative immunohistochemical images of ARID1B, Ki-67, and cleaved caspase-3 in scramble and \u003cem\u003eARID1B\u003c/em\u003e-knockdown xenografts. Scale bar, Histograms depict the average optical density (AOD) quantified from five fields per group (mean ± SD; *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05; ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001; unpaired Student’s \u003cem\u003et\u003c/em\u003e-test. Scale bar, 50 μM.\u003c/p\u003e","description":"","filename":"Figure20101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/b97fc1877969d5d4b6bbdfca.png"},{"id":102319951,"identity":"bb1f2368-83dd-4306-a311-5518f6d624a0","added_by":"auto","created_at":"2026-02-10 13:26:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2009471,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARID1B modulates DNA damage repair machinery in SCLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Bubble plot depicting the top 7 enriched Hallmark gene sets associated with high \u003cem\u003eARID1B\u003c/em\u003e expression in SCLC, as identified by Gene Set Enrichment Analysis (GSEA). (B-C) GSEA highlighting the DNA repair gene sets positively correlated with high \u003cem\u003eARID1B\u003c/em\u003e expression. Gene sets were derived from the hallmark gene sets (B) and Reactome subset of CP (C). (D-E) Comet assay demonstrating increased DNA damage upon \u003cem\u003eARID1B\u003c/em\u003eknockdown in SCLC cells. (D) Representative images of comet tails under scramble and ARID1B-knockdown conditions. Scale bar, 50 μM. (E) Quantification of DNA damage (measured by tail DNA intensity) in SCR versus \u003cem\u003eARID1B\u003c/em\u003e-knockdown cells. Data are presented as mean ± SEM. Statistical significance was determined by unpaired Student’s \u003cem\u003et\u003c/em\u003e-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (F-G) Representative immunofluorescence staining images (G) and quantification (F) of γ-H2AX foci in SCR and \u003cem\u003eARID1B\u003c/em\u003e knockdown SCLC cells. Data represent mean ± SEM; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001 (unpaired Student's \u003cem\u003et\u003c/em\u003e-test). Scale bar, 10 μm. (H-I) Representative RAD51 immunofluorescence staining images (H) and quantification (I) in SCR and \u003cem\u003eARID1B\u003c/em\u003e-knockdown SCLC cells. **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 (unpaired Student's \u003cem\u003et\u003c/em\u003e-test). Scale bar, 10 μm. (J) Western blot analysis of RAD51, RNF8, and γ-H2AX in SCLC cells following \u003cem\u003eARID1B\u003c/em\u003e knockdown or overexpression. β-Actin served as loading control. (K) ChIP-qPCR analysis of \u003cem\u003eRAD51\u003c/em\u003e and \u003cem\u003eRNF8\u003c/em\u003e promoter occupancy in DMS273 cells using IgG as negative control. Data show fold enrichment relative to IgG control (mean ± SD). ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (L) Luciferase reporter assays of RNF8 promoter activity upon \u003cem\u003eARID1B\u003c/em\u003e-knockdown. Firefly luciferase activity was normalized to Renilla luciferase control for transfection efficiency. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure30101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/729a9e157be2bef9ba6c3d38.png"},{"id":102320110,"identity":"120cbe94-740c-4033-881e-3a067805904e","added_by":"auto","created_at":"2026-02-10 13:27:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1375212,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARID1B modulates MYC signaling and cellular proliferation in SCLC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) GSEA demonstrating a strong correlation between \u003cem\u003eARID1B\u003c/em\u003e expression and MYC-related gene sets derived from the C2 collection in SCLC. (B) RT-qPCR analysis of \u003cem\u003ec-MYC\u003c/em\u003e mRNA expression in SCLC cells following \u003cem\u003eARID1B\u003c/em\u003e knockdown or overexpression. (C) Western blot analysis of c-MYC in \u003cem\u003eARID1B\u003c/em\u003e-modulated SCLC cells. β-Actin served as a loading control. (D-E) ChIP-qPCR analysis of \u003cem\u003ec-MYC\u003c/em\u003e promoter occupancy by ARID1B in DMS273 wt (D) and \u003cem\u003eARID1B\u003c/em\u003e knockdown (E) cells. Data represent fold enrichment relative to IgG (control versus \u003cem\u003eARID1B\u003c/em\u003e-knockdown cells. (F-G) Rescue experiment showing cell viability in \u003cem\u003eARID1B\u003c/em\u003e-overexpressing DMS273 cells with concurrent \u003cem\u003ec-MYC\u003c/em\u003eknockdown (F) and in \u003cem\u003eARID1B\u003c/em\u003e knockdown DMS273 cells with \u003cem\u003ec-MYC\u003c/em\u003eoverexpression (G). (H-I) Rescue experiment showing clonogenic potential in ARID1B knockdown DMS273 cells with MYC overexpression (H) and in ARID1B-overexpressing DMS273 cells with concurrent MYC knockdown (I).\u003c/p\u003e","description":"","filename":"Figure40101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/70243e8479e1fc8640c7f174.png"},{"id":102319950,"identity":"0ff5299c-9b37-4370-8301-f3e46520fd53","added_by":"auto","created_at":"2026-02-10 13:26:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":893817,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARID1B cooperates with c-MYC to regulate \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRNF8\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRAD51\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e transcription\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) ChIP-qPCR analysis of c-MYC binding at the \u003cem\u003eRNF8\u003c/em\u003e and \u003cem\u003eRAD51\u003c/em\u003e promoters in DMS273 cells, with IgG as negative control. Data show fold enrichment relative to IgG (mean ± SD, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (B-D) Luciferase reporter assays of \u003cem\u003eRNF8\u003c/em\u003e and \u003cem\u003ec-MYC\u003c/em\u003e promoter activity in 293T cells following \u003cem\u003ec-MYC\u003c/em\u003e knockdown (B), treatment with MYC inhibitor MYCi975 (C), \u003cem\u003eARID1B\u003c/em\u003eknockdown (D). Firefly luciferase activity was normalized to Renilla (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003e(E-G) Rescue experiments of \u003cem\u003eRNF8 \u003c/em\u003eand \u003cem\u003ec-MYC\u003c/em\u003e promoter activity in \u003cem\u003eARID1B\u003c/em\u003e-knockdown cells with\u003cem\u003e c-MYC\u003c/em\u003e overexpression (E), \u003cem\u003eARID1B\u003c/em\u003e-overexpressing cells with \u003cem\u003ec-MYC \u003c/em\u003eknockdown (F), and \u003cem\u003eARID1B\u003c/em\u003e-overexpressing cells after MYCi975 treatment for 24h (G). (H) Sequential ChIP (re-ChIP) analysis demonstrating co-occupancy of ARID1B and c-MYC at the \u003cem\u003eRNF8\u003c/em\u003e and \u003cem\u003eRAD51\u003c/em\u003e promoters in DMS273 cells (fold enrichment relative to IgG, mean ± SD, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (I) Electrophoretic mobility shift assay (EMSA) showing cooperative DNA binding of ARID domain and c-MYC protein to \u003cem\u003eRAD51\u003c/em\u003e promoter. Biotin-labeled DNA probes (40 nM each) were incubated with increasing concentrations of purified ARID domain (0, 0.5, 1, and 2 μg) and c-MYC protein (0, 0.5, 1, and 2 μg).\u003c/p\u003e","description":"","filename":"Figure50101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/2e08e7fd14deaebc280c7517.png"},{"id":102320085,"identity":"17458bcc-3d76-49e4-970c-3b99468b7678","added_by":"auto","created_at":"2026-02-10 13:27:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1870108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eARID1B promotes DNA repair by facilitating DNA End Resection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunofluorescence staining of DMS273 cells treated with Etoposide (40 μM) or Bleomycin (5 μM) for 1 h, co-labeled with anti-ARID1B and anti-γH2AX antibodies. Co-localization coefficients between ARID1B and γH2AX were quantified in at least 50 cells. (B) ChIP-qPCR analysis of ARID1B binding at 28S rDNA with or without I-PpoI-induced DNA double-strand breaks (DSBs) (*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (C) Protein-protein interaction network of ARID1B with HR repair factors predicted by STRING database. Interaction analysis of ARID1B with key DNA damage repair factors (e.g., RPA2, RAD51, RNF8) was performed using the STRING database, with the maximum number of interactors to display set to 20. Edges represent experimentally determined (purple), text-mined (yellow) interactions. (D) Co-immunoprecipitation (Co-IP) assays demonstrating ARID1B-MDC1 interaction. IP samples were resolved by SDS-PAGE and immunoblotted with indicated antibodies. (E) Immunofluorescence staining and quantification of MDC1 and γH2AX in ARID1B-knockdown and control DMS273 cells treated with Etoposide (40 μM, 1 h). Co-localization coefficients were quantified in at least 50 cells (mean ± SEM; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (F) Western blot analysis of \u003cem\u003eMRE11 \u003c/em\u003eand\u003cem\u003e MDC1\u003c/em\u003e protein expression in SCLC cells following \u003cem\u003eARID1B\u003c/em\u003eknockdown. (G) Quantification of single-stranded DNA (ssDNA) at I-PpoI-induced DSBs in HEK293T cells following \u003cem\u003eARID1B\u003c/em\u003e knockdown. qPCR was performed with primers flanking a region 625 bp downstream of the DSB. %ssDNA was calculated as: %ssDNA = 1/ [2^ (ΔCt - 1) + 0.5] × 100. (H) BrdU focus formation assay in ARID1B-knockdown DMS273 cells after etoposide treatment (mean ± SEM; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; unpaired Student’s \u003cem\u003et\u003c/em\u003e-test). (I) Electrophoretic mobility shift assay (EMSA) showing ARID domain binding to ssDNA and splayed-arm DNA. Biotin-labeled DNA substrates (20 nM each) were incubated with increasing concentrations of purified ARID domain (0, 0.5, 1, 1.5, and 2 μg).\u003c/p\u003e","description":"","filename":"Figure60101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/c63c5e709e5b39f3f8d699bb.png"},{"id":102320082,"identity":"0a5b1ac5-ec97-4b5f-9069-1ab2ca9e1576","added_by":"auto","created_at":"2026-02-10 13:27:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2832635,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting ARID1B impairs homologous recombination (HR)-mediated repair\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Western blot analysis showing attenuated induction of c-MYC, RNF8 and RAD51 proteins after etoposide (ETO) (A) or bleomycin (Bleo) (B) treatment (40 μM, 1 h) in \u003cem\u003eARID1B\u003c/em\u003e-knockdown DMS273 cells. β-Actin served as a loading control. (C-D) p-RPA2 (C) and RAD51 (D) foci formation assay in \u003cem\u003eARID1B\u003c/em\u003e-knockdown DMS273 cells treated with ETO (40 μM, 1 h). Foci were quantified in at least 50 cells per condition (mean ± SEM, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (E) Comet assay demonstrating DNA damage in \u003cem\u003eARID1B\u003c/em\u003e-knockdown DMS273 cells. Cells were treated with ETO (40 μM) or Ble (5 μM) for 1 h followed by 4 h recovery. DNA damage was quantified as percentage of tail DNA (\u003cem\u003en\u003c/em\u003e ≥ 50 cells, mean ± SEM, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Representative images shown. (F-G) Representative immunofluorescence staining images (F) and quantification (G) of DNA damage markers in \u003cem\u003eARID1B\u003c/em\u003e-knockdown cells treated with ETO (40 μM) or Bleo (5 μM) for 1 h, followed by 1 h or 4 h recovery. Foci were counted in ≥50 cells (mean ± SEM, *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (H) Mechanistic Model of ARID1B in HR Repair. ARID1B cooperates with c-MYC to transcriptionally regulate DNA repair genes in response to genotoxic insults. Upon double-strand break (DSB) formation, ARID1B is recruited to damaged DNA sites, where it directly binds DNA and facilitates the recruitment of repair machinery proteins (e.g., MDC1, RAD51, RNF8, and MRE11) to promote DNA end resection and subsequent repair processes.\u003c/p\u003e","description":"","filename":"Figure70101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/b56b8ad2df8895d5dd185132.png"},{"id":102319965,"identity":"2e140d08-ed1a-4db0-9e46-1bd5b748be1b","added_by":"auto","created_at":"2026-02-10 13:26:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3045764,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTargeting ARID1B sensitizes SCLC to DNA-damaging agents in vitro and in vivo.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) Cell viability (A) and colony formation (B) assays of \u003cem\u003eARID1B\u003c/em\u003e-deficient SCLC upon 1 hour treatment with etoposide (40 μM). (C) Representative images of excised xenograft tumors from SCR and \u003cem\u003eARID1B\u003c/em\u003e-knockdown groups treated with cisplatin and etoposide. (D) Tumor growth curves of subcutaneous xenografts in different treatment groups. Data are presented as mean ± SEM. **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001. (E) Scatter plots showing tumor weights from xenografts mice. Statistical significance was determined by unpaired Student’s \u003cem\u003et\u003c/em\u003e-test (mean ± SEM; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (F) Histopathological analysis of Cleaved caspase-3 (apoptosis), Ki-67 (proliferation), γH2AX (DNA damage Marker) in SCR and \u003cem\u003eARID1B\u003c/em\u003e-deficient xenografts treated with cisplatin and etoposide. IOD/Area quantified from five fields per group (mean ± SD; *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05; ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; unpaired Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e","description":"","filename":"Figure80101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/975c6b719e72cb791f7fd9f1.png"},{"id":102320095,"identity":"0dc899b0-86b5-4984-ab96-7bc7e10629b7","added_by":"auto","created_at":"2026-02-10 13:27:15","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":596720,"visible":true,"origin":"","legend":"Supplementary Figure 2","description":"","filename":"SupplementaryFigure20101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/16465c1bf48d589629d92390.png"},{"id":102320096,"identity":"37938150-c02e-4773-9833-b6658d540d65","added_by":"auto","created_at":"2026-02-10 13:27:16","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":444986,"visible":true,"origin":"","legend":"Supplementary Figure 1","description":"","filename":"SupplementaryFigure10101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/87fb586b7c11dfade073ce2a.png"},{"id":102320084,"identity":"585d847b-c89a-4944-9cc9-3c6fd09cdce3","added_by":"auto","created_at":"2026-02-10 13:27:11","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17000,"visible":true,"origin":"","legend":"Table S5","description":"","filename":"TableS5PrimersequencesforEMSAassay.docx","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/c5f3f1c68eb39ef46f0d9264.docx"},{"id":102319985,"identity":"0b4bec77-82f4-4de0-b15f-540429f5e6c1","added_by":"auto","created_at":"2026-02-10 13:26:50","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":981083,"visible":true,"origin":"","legend":"Supplementary Figure 6","description":"","filename":"SupplementaryFigure60101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/49692862b90b048ff5c06f20.png"},{"id":102319990,"identity":"be2eeea2-17ec-4a72-9acc-16a004e3fa59","added_by":"auto","created_at":"2026-02-10 13:26:51","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":326391,"visible":true,"origin":"","legend":"Supplementary Figure 4","description":"","filename":"SupplementaryFigure40101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/200c28bf0c3aae475cd64867.png"},{"id":102319992,"identity":"fb875c0c-6afd-4281-8e0e-3892ac40bb8d","added_by":"auto","created_at":"2026-02-10 13:26:52","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":18414,"visible":true,"origin":"","legend":"Table S4","description":"","filename":"TableS4PrimersequencesforChIPassay.docx","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/571018b90cfb35883879af4e.docx"},{"id":102320089,"identity":"02c90cdd-b081-4a2d-83f4-427096477644","added_by":"auto","created_at":"2026-02-10 13:27:12","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":1959615,"visible":true,"origin":"","legend":"Supplementary Figure 7","description":"","filename":"SupplementaryFigure70101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/eab8512381efea406c4353ca.png"},{"id":102319983,"identity":"98b95c77-1dbd-4e97-82f3-f8c1c5741a7c","added_by":"auto","created_at":"2026-02-10 13:26:50","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":16920,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1shRNAandsiRNAsequencesforgenesilencing.docx","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/b5c5f82ceb91a9e087c63c34.docx"},{"id":102319994,"identity":"b30f0a40-31c5-43ec-b35d-cc57b3297148","added_by":"auto","created_at":"2026-02-10 13:26:54","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":740353,"visible":true,"origin":"","legend":"Supplementary Figure 5","description":"","filename":"SupplementaryFigure50101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/f6e31a896a1a97870e336725.png"},{"id":102320098,"identity":"6b8dcc31-8a2a-4898-b0a1-977c98ea1347","added_by":"auto","created_at":"2026-02-10 13:27:17","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1263198,"visible":true,"origin":"","legend":"Supplementary Figure 3","description":"","filename":"SupplementaryFigure30101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/0630c994a7efc0dd1dd63a0c.png"},{"id":102319987,"identity":"28ef8b33-a639-4239-a7b8-88f3b5b56db7","added_by":"auto","created_at":"2026-02-10 13:26:50","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":32431,"visible":true,"origin":"","legend":"Supplementary Figure legend","description":"","filename":"SuppmentaryFigurelegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/7940ec4bb8eaa8dd62725a5d.docx"},{"id":102320097,"identity":"97c418f1-e338-4eeb-b162-ba463879ae90","added_by":"auto","created_at":"2026-02-10 13:27:17","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":988739,"visible":true,"origin":"","legend":"Supplementary Figure 8","description":"","filename":"SupplementaryFigure80101.png","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/a7d91df80640e7b16e06d740.png"},{"id":102320003,"identity":"eb6de8fd-85be-4846-9357-8e88b735201c","added_by":"auto","created_at":"2026-02-10 13:27:01","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":17707,"visible":true,"origin":"","legend":"Table S2","description":"","filename":"TableS2PrimersequencesforqRTPCR.docx","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/da7d320618c7037cf5989382.docx"},{"id":102319999,"identity":"20f8f99a-730a-46d3-9636-13b8baefd894","added_by":"auto","created_at":"2026-02-10 13:26:57","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":20291,"visible":true,"origin":"","legend":"Table S3","description":"","filename":"TableS3Listofantibodiesusedinthisstudy.docx","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/b842695b55e1e903da6d92b3.docx"},{"id":102320100,"identity":"2526afb5-8bb2-45b8-a627-e5a691cd8c37","added_by":"auto","created_at":"2026-02-10 13:27:19","extension":"pptx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":108234594,"visible":true,"origin":"","legend":"Original Data Files","description":"","filename":"SupplementalMaterialfile.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8612664/v1/2eafe3320fbf9c564d71e7e1.pptx"}],"financialInterests":"(Not answered)","formattedTitle":"ARID1B promotes DNA end resection via MDC1 and co-transactivation of DNA repair genes with c-MYC in small cell lung cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSmall cell lung cancer (SCLC) is an aggressive pulmonary malignancy with pronounced neuroendocrine features, characterized by early metastasis and an exceptionally poor prognosis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. First-line treatment relies primarily on cytotoxic chemotherapy, typically combining platinum-based alkylating agents (cisplatin/carboplatin) with topoisomerase inhibitors (such as irinotecan or etoposide)\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, the rapid development of resistance to conventional therapies significantly contributes to treatment failure and high mortality\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Until now, the prognosis of patients with SCLC remains poor, with a dismal five-year survival rate of approximately 5%\u003csup\u003e6\u003c/sup\u003e. Consequently, elucidating novel molecular mechanisms driving SCLC pathogenesis and identifying potential therapeutic targets, are of paramount importance for improving patient outcome.\u003c/p\u003e \u003cp\u003eDNA is constantly vulnerable to damage from endogenous and environmental sources\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Among various types of DNA lesion, DNA double-strand breaks (DSB) are particularly severe and potential lethal, as a single unrepaired DSB can trigger cell death. Eukaryotic cells primarily repair DSBs via two pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). HR repair involves a sophisticated cellular process, orchestrated by an integrated signaling cascade involving hierarchically organized sensors, transducers, and effector molecules, including DSB recognition by the MRN complex (Mre11-Rad50-Nbs1), recruitment of mediator proteins (RNF8, RNF168, MDC1), phosphorylation of H2AX (γH2AX), end resection by CtIP to generate 3' single-stranded DNA (ssDNA) overhangs, RPA coating, and subsequent RAD51-mediated strand invasion for homology-directed repair\u003csup\u003e\u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. The extent of DNA end resection determines the choice of DNA repair pathways. While the critical role of ssDNA in DNA repair is well established, the regulatory mechanisms controlling its generation and their impact on repair efficiency remain incompletely understood.\u003c/p\u003e \u003cp\u003eChromatin remodeling complexes, such as the ATP-dependent SWI/SNF complex, mobilize, destabilize, or restructure nucleosomes, thereby regulating DNA-dependent cellular processes including replication, repair, and transcription\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Growing evidence underscores the pivotal role of specific SWI/SNF subunits in HR regulation. Specifically, inactivation of the core subunit BRG1 impairs homologous recombination (HR) efficiency and reduces RAD51 foci formation\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; Additionally, AT-rich interactive domain 1A (ARID1A) accumulates at DSB sites post-damage and facilitates both NHEJ and HR by modulating chromatin organization and accessibility\u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. ARID1B, a mutually exclusive paralog of ARID1A within the SWI/SNF BAF complex, is implicated in neurodevelopmental disorders and stem cell fate determination\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Somatic \u003cem\u003eARID1B\u003c/em\u003e mutations occur in a wide variety of cancers, including neuroblastoma, breast cancer, and pancreatic cancer\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, Even though the exact mechanisms by which these mutations drive oncogenesis are still largely unknown. Beyond cancer-associated genetic alterations, loss of \u003cem\u003eARID1B\u003c/em\u003e expression correlates with poor prognosis in several types of cancer including ovarian and colon cancer\u003csup\u003e\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, suggesting its potential as a prognostic biomarker. Furthermore, ARID1B is necessary for the survival of \u003cem\u003eARID1A\u003c/em\u003e-deficent cells, positioning it as a potential therapeutic target for \u003cem\u003eARID1A\u003c/em\u003e-mutant cancers. Recent studies indicate that \u003cem\u003eARID1B\u003c/em\u003e deficiency impairs DNA damage response and activates the cGAS-STING Pathway in NSCLC\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Depletion of \u003cem\u003eARID1B\u003c/em\u003e also confers sensitivity to radiation, as well as to cisplatin and UV, Evidence suggests it modulates DNA damage response by promoting NHEJ activity\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Despite these advances, the precise roles and regulatory mechanisms of ARID1B in DNA damage response and cancer biology remains poorly defined. A comprehensive understanding of its functions and regulatory networks in DNA repair and cancer progression is crucial for developing safe and efficient therapeutic strategies targeting ARID1B-dependent cancer.\u003c/p\u003e \u003cp\u003eHerein, we report elevated ARID1B expression in SCLC tissues compared to normal lung or adjacent noncancerous tissues. Functional studies employing gain- and loss-of-function approaches demonstrate that ARID1B is indispensable for SCLC cell survival, clonogenicity and tumor growth. Mechanistically, biochemical analyses revealed that ARID1B cooperates with the transcription factor c-MYC to co-activate critical DNA damage repair (DDR) genes and is recruited to DSB sites and facilitates ssDNA formation via end resection. Critically, \u003cem\u003eARID1B\u003c/em\u003e deficiency markedly sensitizes SCLC cells to DNA-damaging agents (etoposide, bleomycin, cisplatin) both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e, evidenced by persistent DNA damage, increased apoptosis, and suppressed tumor growth. These findings provide a rationale for targeting ARID1B in combination with chemotherapy to overcome treatment resistance in ARID1B-dependent SCLC.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eARID1B is upregulated in SCLC and promotes cell proliferation\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe SWI/SNF chromatin remodeling complex plays a well-established role in tumorigenesis and cancer progression\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. As a core subunit, ARID1B has emerged as a significant oncological regulator, prompting our investigation into its tumor-specific expression in SCLC. Pan-cancer analysis of the CCLE database revealed significantly elevated \u003cem\u003eARID1B\u003c/em\u003e mRNA expression in SCLC cell lines compared to lung adenocarcinoma (LUAD) lines, ranking SCLC second highest among 33 tumor types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Transcriptomic analysis of clinical samples from two datasets confirmed \u003cem\u003eARID1B\u003c/em\u003e upregulation in SCLC tumors relative to adjacent normal tissues (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). Paired sample analysis demonstrated concordant upregulation of ARID1B at both mRNA and protein levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F, Supplementary Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), implicating ARID1B in SCLC pathogenesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo functionally characterize ARID1B in SCLC, we overexpressed it in DMS273 and SHP77 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Colony formation and CellTiter-Glo assays revealed that ectopic \u003cem\u003eARID1B\u003c/em\u003e expression significantly enhanced clonogenic potential and cell viability (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-I). This proliferative advantage was further corroborated by increased DNA replication activity in EdU incorporation assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ). Collectively, these findings provide compelling evidence for the pro-tumorigenic function of ARID1B in SCLC.\u003c/p\u003e \u003cp\u003e \u003cb\u003eARID1B\u003c/b\u003e \u003cb\u003eDepletion Suppresses SCLC Growth\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further define ARID1B\u0026rsquo;s functional role, we established stable \u003cem\u003eARID1B\u003c/em\u003e-knockdown DMS273 and H82 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). CellTiter-Glo and colony formation assays showed that \u003cem\u003eARID1B\u003c/em\u003e depletion significantly reduced cell viability and clonogenic capacity in both lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-C). Correspondingly, EdU incorporation assays revealed a significant inhibition of cellular proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Notably, \u003cem\u003eARID1B\u003c/em\u003e knockdown elicited a pronounced apoptotic response, as demonstrated by the elevated cleavage of PARP (Supplementary Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB-C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo validate these findings \u003cem\u003ein vivo\u003c/em\u003e, we generated subcutaneous xenografts in nu/nu mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). \u003cem\u003eARID1B\u003c/em\u003e knockdown significantly inhibited tumor growth (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-G) and reduced final tumor mass while maintaining stable body weight parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, and Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eD). Immunohistochemistry (IHC) analysis of \u003cem\u003eARID1B\u003c/em\u003e-deficient tumors showed decreased Ki67 and elevated cleaved-caspase 3 signals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI), consistent with \u003cem\u003ein vitro\u003c/em\u003e observations. These results definitively establish ARID1B as a functionally oncogenic driver in small cell lung cancer (SCLC) tumorigenesis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eARID1B Regulates the DNA Damage Repair\u003c/h3\u003e\n\u003cp\u003eTo elucidate the molecular mechanisms underlying ARID1B-mediated phenotypes, we performed gene set enrichment analysis (GSEA) of cancer hallmarks\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Specimens with higher \u003cem\u003eARID1B\u003c/em\u003e mRNA expression (n\u0026thinsp;=\u0026thinsp;56) were categorized as \u003cem\u003eARID1B\u003c/em\u003e-high SCLC, others were designated as \u003cem\u003eARID1B\u003c/em\u003e-low SCLC. Hallmark collection analysis identified seven pathways significantly enriched in \u003cem\u003eARID1B\u003c/em\u003e-high SCLC, namely G2M checkpoint, E2F targets, MYC targets V1, spermatogenesis, mitotic spindle, pancreas beta cells and DNA repair (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Targeted enrichment analysis of C2 collection further confirmed a strong positive correlation between \u003cem\u003eARID1B\u003c/em\u003e expression and DNA damage repair pathways, particularly homology-directed repair (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C and Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA-B). Pearson correlation analysis using a SCLC RNA-seq dataset revealed positive correlation between \u003cem\u003eARID1B\u003c/em\u003e and HR-related genes, including \u003cem\u003eRNF8\u003c/em\u003e and \u003cem\u003eRAD51\u003c/em\u003e (Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC). Consistently. GSEA based on a proteomic dataset ranked DNA repair among the top enriched gene sets (Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB). These findings suggest functional links between ARID1B and DNA repair in SCLC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo assess ARID1B\u0026rsquo;s direct role in DNA damage response, we evaluated DNA damage levels following \u003cem\u003eARID1B\u003c/em\u003e depletion using comet assay and immunofluorescence (IF) analysis. Comet assay revealed significantly increased DNA damage upon \u003cem\u003eARID1B\u003c/em\u003e knockdown (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-E). IF quantification of γH2AX foci confirmed elevated DNA damage in \u003cem\u003eARID1B\u003c/em\u003e-deficient SCLC cells, as evidenced by markedly elevation in γH2AX foci (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-G and Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD). IF microscopy further showed impaired RAD51 foci formation post knockdown (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I and Supplementary Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE). Western blot analysis demonstrated elevated accumulation of γH2AX alongside reduced levels of RAD51 and RNF8 following DNA damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). We hypothesized that ARID1B directly regulates the expression of \u003cem\u003eRAD51\u003c/em\u003e and \u003cem\u003eRNF8\u003c/em\u003e. RT-qPCR analysis of key DNA damage response genes revealed that ARID1B expression levels significantly influenced the transcriptional regulation of DNA damage repair gene (Supplementary Figures \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eF-G). Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis confirmed ARID1B binding to the promoter regions of both genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK). Importantly, dual-luciferase reporter assays showed that \u003cem\u003eARID1B\u003c/em\u003e depletion reduced \u003cem\u003eRNF8\u003c/em\u003e promoter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). Collectively, these data indicate that ARID1B contributes to DSB repair via transcriptional regulation of critical DDR genes.\u003c/p\u003e\n\u003ch3\u003eARID1B Modulates SCLC Progression via c-MYC\u003c/h3\u003e\n\u003cp\u003ePrevious studies established direct c-MYC binding to promoters of key DNA double-strand break (DSB) repair genes\u003csup\u003e\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Moreover, c-MYC is a direct target of ARID1B-containing SWI/SNF complexes during cell cycle activation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Importantly, Gene Ontology (GO) pathway and GSEA analysis further supported a significant functional association between \u003cem\u003eARID1B\u003c/em\u003e and \u003cem\u003ec-MYC\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Supplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA), suggesting that ARID1B may regulate SCLC progression through c-MYC. To investigate this relationship, we first assessed the impact of \u003cem\u003eARID1B\u003c/em\u003e perturbation on c-MYC expression. As depicted in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C, \u003cem\u003eARID1B\u003c/em\u003e modulation significantly altered both mRNA and protein levels of c-MYC in DMS273 and H82 cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). ChIP-qPCR analysis revealed direct ARID1B binding to the \u003cem\u003ec-MYC\u003c/em\u003e promoter, with pronounced enrichment at the \u003cem\u003ec-MYC\u003c/em\u003e BS2 site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Importantly, \u003cem\u003eARID1B\u003c/em\u003e knockdown abolished BS2 occupancy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and Supplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB), indicating a functional interaction between ARID1B and c-MYC in transcriptional control. Notably, ectopic \u003cem\u003ec-MYC\u003c/em\u003e expression or knockdown partially rescued cell viability and clonogenic defects in \u003cem\u003eARID1B\u003c/em\u003e-manipulated cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-I and Supplementary Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eC). These results provide compelling evidence that ARID1B drives SCLC progression through c-MYC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eARID1B and c-MYC Synergistically Regulate DDR gene Transcription\u003c/h3\u003e\n\u003cp\u003eTo elucidate cooperative regulation of DDR gene by ARID1B and c-MYC, we performed ChIP and luciferase assays to determine the possible functional interplay. ChIP-qPCR analysis confirmed c-MYC binding to the promoter regions of \u003cem\u003eRNF8\u003c/em\u003e and \u003cem\u003eRAD51\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Luciferase reporter assays showed that \u003cem\u003ec-MYC\u003c/em\u003e depletion significantly reduced the promoter activity of \u003cem\u003ec-MYC\u003c/em\u003e and \u003cem\u003eRad51\u003c/em\u003e, compared to scramble controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), phenocopying \u003cem\u003eARID1B\u003c/em\u003e loss. Similarly, pharmacological inhibition of c-MYC with MYCi975 recapitulated this effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Intriguingly, \u003cem\u003eARID1B\u003c/em\u003e depletion could mimic \u003cem\u003ec-MYC\u003c/em\u003e knockdown-mediated suppression of promoter activity of \u003cem\u003eRNF8\u003c/em\u003e and \u003cem\u003eRAD51\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and Supplementary Figures. S5A-B), implying functional synergy. Importantly, modulating c-MYC expression partially restored promoter activity in \u003cem\u003eARID1B\u003c/em\u003e-deficient or overexpressing cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-G), indicating that ARID1B and c-MYC function in concert to regulate the expression of key DDR genes. To characterize their interplay, we performed sequential chromatin immunoprecipitation (ChIP-reChIP) analysis to evaluate the co-occupancy of ARID1B and c-MYC at the promoter of co-regulated genes. Indeed, ChIP-reChIP confirmed co-binding of ARID1B and c-MYC at DDR gene promoters, including an autoregulation at the \u003cem\u003ec-MYC\u003c/em\u003e locus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH and Supplementary Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eC). To exclude indirect effect via c-MYC regulation, electrophoretic mobility shift assays (EMSAs) using recombinant c-MYC protein and ARID domain (a characterized DNA-binding module) of ARID1B (Supplementary Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eD-E) demonstrated direct binding of both the ARID domain and c-MYC to the \u003cem\u003eRAD51\u003c/em\u003e promoter. Importantly, Co-incubation enhanced each other\u0026rsquo;s binding ability to DNA, revealing a synergistic interaction on DNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). These results provide compelling evidence that ARID1B and c-MYC cooperatively activate DDR gene transcription.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eARID1B Promotes DNA End Resection at DSB sites via MDC1\u003c/h3\u003e\n\u003cp\u003ePrior studies indicate ARID1B accumulates at the sites of laser micro-radiation (IR)-induced DNA damage\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We first analyzed ARID1B spatial distribution relative to γH2AX foci, a canonical marker of double-strand breaks (DSBs). IF staining revealed partial but significant ARID1B co-localization with γH2AX following etoposide or bleomycin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Furthermore, ChIP-PCR using an I-PpoI endonuclease system confirmed that significant ARID1B enrichment at induced DSBs in 28S rDNA and DAB1 loci in HEK293T cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA-C), demonstrating active recruitment of ARID1B to DSB sites.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMDC1 orchestrates RNF8/RNF168 recruitment to DSBs, promoting H2A ubiquitination to amplify the damage signal and facilitate HR repair\u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Protein-protein interaction (PPI) network analysis was conducted to screen the HR repair-related proteins binding to ARID1B using the STRING database. To further validate the results obtained from the PPI network, we screened the ARID1B-interacting proteins using the BioGRID database and identified a potential interaction between MDC1 and ARID1B\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.The results showed that MDC1 potentially interact with ARID1B (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), which was validated by co-immunoprecipitation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). IF staining demonstrated \u003cem\u003eARID1B\u003c/em\u003e deficiency impaired MDC1 accumulation at DSB sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), suggesting that requirement of ARID1B at DSB sites for MDC1 recruitment upon DNA damage. The MRN complex composed of dimers of MRE11, RAD50, and NBS1 plays a dual role in homologous recombination (HR)-mediated DSB repair, serving as both a critical end-bridging factor and an essential endonuclease\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. To exclude potential transcriptional interference, we examined both mRNA and protein levels of MDC1-associated genes following ARID1B knockdown. The results demonstrated that ARID1B depletion did not reduce the expression of MDC1 at either the mRNA or protein level (Figure. 6F and Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eD). Furthermore, ARID1B exhibited no regulatory effect on the early recruitment factor MRE11. Since association of MDC1 facilitates the binding of MRN complex through phosphorylation-dependent interactions with the FHA-BRCT1-BRCT2 domains of NBN\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, we therefore sought to determine whether ARID1B modulates DNA end resection via MRN during DSB repair. Notably, phosphorylation of RPA2, a critical determinant of DSB repair pathway choice, was compromised upon \u003cem\u003eARID1B\u003c/em\u003e depletion (Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eE-F). To furthermore assess ARID1B's role in DNA end resection during DSB repair, we strategically designed restriction enzyme sites flanking the I-PpoI cleavage locus to generate site-specific DNA damage (Supplementary Figure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eG). The generation of ssDNA at these sites would confer resistance to restriction enzyme digestion. We detected substantial ssDNA accumulation after I-PpoI-mediated 28S rDNA DSB induction, which was significantly blocked by \u003cem\u003eARID1B\u003c/em\u003e depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). To provide additional experimental validation, a BrdU incorporation assay coupled with non-denaturing immunofluorescence (IF) staining was employed to detect single-stranded DNA (ssDNA) formation\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eARID1B\u003c/em\u003e-deficient cells showed significantly reduced ssDNA generation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eDuring homologous recombination (HR) repair, double-strand breaks (DSBs) undergo a two-step processing mechanism: First, DSB ends are converted into splayed-arm structures, which are subsequently processed by nucleases such as MRE11 or EXO1 to generate 3\u0026prime; single-stranded overhangs (3\u0026prime;-overhangs),This ssDNA intermediate then serves as an essential molecular scaffold for initiating HR-mediated repair\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Given ARID1B's recruitment to DNA lesions and role in end resection, we hypothesized a direct interaction with damaged DNA. To test this hypothesis, EMSAs using purified ARID domain and DNA substrates were carried out to assess ARID1B association with various forms of DNA. Our results revealed that robust binding to splayed-arm DNA structures and minimal but meaningful affinity for ssDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). This structure-specific binding preference for replication fork-like intermediates suggests ARID1B directly participates in DNA damage processing. Collectively, these findings elucidate ARID1B\u0026rsquo;s direct and critical role in DSB site recruitment and end resection, governing the choice of DSB repair pathways and maintaining genomic stability.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eARID1B depletion impairs HR repair\u003c/h2\u003e \u003cp\u003eTo assess ARID1B\u0026rsquo;s impact on HR repair under genotoxic conditions, a series of experiments was performed to systematically analyze key HR components. Western blotting showed that the DNA damage-induced upregulation of RAD51 and RNF8 was markedly impaired in \u003cem\u003eARID1B\u003c/em\u003e-deficient cells exposed to either etoposide or bleomycin, accompanied by elevated γH2AX (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B). Quantitative IF revealed significantly reduced p-RPA2 and RAD51 foci formation at 1-hour post-damage in \u003cem\u003eARID1B\u003c/em\u003e-deficient cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-D and Supplementary Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eA-D), thereby establishing ARID1B\u0026rsquo;s role in efficient HR machinery recruitment. Comet assays showed increased fragmented DNA accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE and Supplementary Figure \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eE). While IF microscopy showed that while robust γH2AX foci formed at 1-hour post-damage across conditions, \u003cem\u003eARID1B\u003c/em\u003e knockdown cells exhibited persistent γH2AX foci at 4 hours post-damage, indicating defective repair. Similar effects were observed with bleomycin treatment (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF-G and Supplementary Figures \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eF-I). These findings provide compelling evidence that ARID1B is indispensable for an effective homologous recombination-mediated DNA damage response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eARID1B\u003c/b\u003e \u003cb\u003eDeficiency Sensitizes SCLC cells to Chemotherapy\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven ARID1B's role in HR-mediated repair, we hypothesized its depletion would sensitize SCLC cells to DNA-damaging agents. To test this hypothesis, we exposed SCLC cells to clinically relevant DNA-damaging agents and found that \u003cem\u003eARID1B\u003c/em\u003e deficiency significantly increased sensitivity to clinically relevant chemotherapeutics, as evidenced by reduced cell viability and clonogenic capacity (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-B and Supplementary Figures \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eA-B). Quantitative apoptosis analysis via flow cytometry with Annexin V-FITC/PI dual staining confirmed that enhanced apoptosis induction in \u003cem\u003eARID1B\u003c/em\u003e-depleted cells treated with etoposide or bleomycin in both DMS273 and H82 cells (Supplementary Figures \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eC-D). To evaluate clinical relevance, we employed a xenograft mouse model to assess the \u003cem\u003ein vivo\u003c/em\u003e therapeutic implications of targeting ARID1B. Administration of the etoposide/cisplatin (E/P) regimen\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e resulted in significantly greater tumor growth inhibition and reduced tumor mass in \u003cem\u003eARID1B\u003c/em\u003e-depleted xenografts versus controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC-E). Notably, all treatment groups maintained stable body weights throughout the experimental duration, demonstrating favorable treatment tolerance (Supplementary Figure \u003cspan refid=\"MOESM8\" class=\"InternalRef\"\u003eS8\u003c/span\u003eE). IHC analysis of tumor specimens showed baseline elevation of γH2AX signal following E/P treatment, with additional enhancement following \u003cem\u003eARID1B\u003c/em\u003e loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). Consistent with suppressed tumor growth and compromised DNA repair, \u003cem\u003eARID1B\u003c/em\u003e loss led to greater Ki67 downregulation and apoptosis induction than controls. These findings demonstrate that targeting ARID1B potentiates genotoxic therapy efficacy in SCLC, supporting the development of ARID1B-targeted therapeutic interventions as a novel strategy to circumvent chemotherapy resistance in SCLC.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we provide multiple lines of evidence establishing ARID1B as a multifunctional DNA damage response (DDR) factor that promotes double-strand break (DSB) repair, whose deficiency renders tumors susceptible to chemotherapeutic agents. Mechanistically, we demonstrate that ARID1B not only associates with gene promoters and synergizes with c-MYC to transcriptionally activate key DNA damage repair genes, but also is recruited to DNA lesion sites upon genotoxic stress, where it facilitates MDC1 recruitment and subsequent MRN complex-mediated DNA end resection to generate ssDNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). Consequently, \u003cem\u003eARID1B\u003c/em\u003e deficiency impairs DSB end resection and compromises homologous recombination (HR) repair, creating a targetable vulnerability that can be harnessed to overcome chemoresistance in SCLC.\u003c/p\u003e \u003cp\u003eThe DNA damage response (DDR) is fundamental to tumor progression and cellular response to DNA-damaging therapies, such as radiation and chemotherapy. Epigenetic dysregulation can transcriptionally silence critical DNA repair components across various cancers\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Advances in cancer genomics and understanding of epigenetic-transcriptional interplay, combined with the recognized importance of DNA repair in tumorigenesis, suggesting that epigenetic-targeting therapeutics may offer novel strategies to modulate treatment response by leveraging DNA damage response\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. SWI/SNF chromatin remodeling complex modulates DDR in an ATP-dependent and subunit-specific manner. Functionally, they bind to defined genomic loci, disrupts histone-DNA interactions, utilize ATP hydrolysis to reposition nucleosomes, and create accessible chromatin to facilitate recruitment of DNA-binding factors and transcriptional machinery\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Notably, ATPase subunits like BRG1 play critical roles in DNA repair. Recent studies reveal that BRG1 resolves transcription-replication conflicts and employ its ATPase domain to interact with multiple DNA repair proteins, recruiting them to sites of DNA damage sites to promote HR repair\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWithin the BAF complex, the ARID1B subunit characterized by an AT-rich interaction domain (ARID) protein but lacking ATPase or bromodomains, plays a distinct role in targeting of the complex\u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. We demonstrate that ARID1B is frequently overexpressed in SCLC, driving tumor proliferation and survival. Mechanistically, ARID1B subunit plays an indispensable role in HR via dual mechanisms: (1) As a transcriptional co-regulator, it partners with c-MYC to activate key HR genes (e.g. \u003cem\u003eRAD51\u003c/em\u003e and \u003cem\u003eRNF8\u003c/em\u003e); (2) It is directly recruited to DSBs, facilitates end resection by promoting ssDNA formation. Prior studies have shown that nucleosomes can significantly regulate DNA end resection by acting as physical barriers to the resection machinery\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Nucleosome remodeling complexes such as SWI/SNF or INO80 facilitate CtIP recruitment and end resection initiation\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Additionally, SMARCAD1 and Fun30 likely promote resection not only through their chromatin remodeling activity but also by reducing the local concentration of resection inhibitors such as 53BP1 (or its budding yeast homolog Rad9)\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In line with this observation, we demonstrate for the first time that ARID1B is an indispensable modulator of DNA end resection and homologous recombination (HR) repair. Consequently, \u003cem\u003eARID1B\u003c/em\u003e deficiency cripples HR repair, leading to unresolved DNA damage, apoptosis, and profound sensitization to DNA-damaging agents both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. This study defines the role of ARID1B in SCLC pathogenesis and reveals its regulatory network as a chromatin remodeler coordinating DNA repair and transcriptional regulation.\u003c/p\u003e \u003cp\u003eWhile consistent with emerging DDR roles for SWI/SNF subunits, our findings underscore the unique, essential contribution of the non-catalytic ARID1B. The ARID1B/c-MYC cooperation unveils an epigenetic-transcriptional regulatory layer controlling DDR capacity in SCLC. \u003cem\u003eARID1B\u003c/em\u003e overexpression in SCLC and its positive correlation with DDR genes suggest its upregulation may be a key adaptive mechanism underlying the notorious chemoresistance of this malignancy.\u003c/p\u003e \u003cp\u003eIn summary, this work elucidates the functional role of ARID1B in SCLC and provides novel insights into its regulatory network, highlighting that targeting ARID1B or disrupting its interaction with c-MYC represents a promising strategy to overcome chemoresistance in SCLC. Future studies should explore specific ARID1B inhibitors and their combinatorial efficacy with standard genotoxic therapies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and cell culture\u003c/h2\u003e \u003cp\u003eHuman SCLC cell lines (DMS273, H82, and SHP77) and human embryonic kidney cell line HEK293T were cultured at 37\u0026deg;C in a humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. SCLC cells were maintained in RPMI1640 medium (Gibco), while HEK293T cells were cultured in DMEM (Gibco) medium. Both media were supplemented with 10% fetal bovine serum (FBS, VivaCell) and penicillin and streptomycin (100X, Servicebio).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMicroarray, RNA-seq, and genomic datasets and data analysis\u003c/h2\u003e \u003cp\u003eStandardized gene expression microarray data (GSE149507 and GSE60052) were downloaded from the Gene Expression Omnibus (GEO) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/geo\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov/geo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Additionally, microarray and RNA-seq data for 50 SCLC cell lines, along with RNA-seq data for 33 tumor types, were obtained from the Cancer Cell Line Encyclopedia (CCLE) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://portals.broadinstitute.org/ccle/data\u003c/span\u003e\u003cspan address=\"https://portals.broadinstitute.org/ccle/data\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Further, RNA-seq data of 107 SCLC specimens and their matched adjacent non-cancerous tissues were obtained from the Genome Sequence Archive (GSA) (GSA database accession: HRA003419 available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bigd.big.ac.cn/gsa-human\u003c/span\u003e\u003cspan address=\"http://bigd.big.ac.cn/gsa-human\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Proteomic data and associated pathological information for 112 SCLC specimens and their corresponding para-cancerous tissues were secured from the OMIX database (OMIX database accession: OMIX002489 accessible at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/omix\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/omix\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). All data sources are publicly available.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA Extraction and qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA extraction was extracted from 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells using Trizol reagent (TransGen Biotech) according to the manufacturer's protocol. RNA concentration and quality were assessed using a spectrophotometer (Epoch). Reverse transcription was performed using the EvoM-MLV Reverse Transcriptase Kit (Accurate Biology). Quantitative real-time PCR (qRT-PCR) was conducted using SYBR Green Master Mix (Accurate Biology) on a Quantstudio 5 platform (Applied Biosystems). Relative gene expression levels were calculated using the ΔΔCt method with actin as the housekeeping gene. Primer sequences are listed in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCells were lysed using Western and IP lysis buffer (Beyotime). Proteins were separated by SDS-PAGE and electrotransferred onto PVDF membranes. The membranes were blocked with 5% skim milk (BBI LIFE SCIENCES) for 2 hours, followed by overnight incubation with the primary antibody at 4\u0026deg;C. After the membranes were incubated with the secondary antibody for 1 hour at room temperature. Enhanced chemiluminescence (ECL) reagent was applied to the PVDF membranes, and protein bands were detected using chemiluminescence imaging system (Tanon). Primary antibodies used in the study are listed in Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eDMS273 cells were seeded at 500 cells per well and SHP77 cells at 3,500 cells per well in 6-well plates. Cells were incubated for two weeks to allow colony formation. The clones were washed with 1x PBS, fixed with methanol, and stained with 0.1% crystal violet. Colony formation of H82 cells was assessed using a soft agar assay as previously described\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. The colonies were photographed and analyzed by Image J\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell viability, apoptosis, and EdU assays\u003c/h2\u003e \u003cp\u003eCells designated for drug treatment were exposed to a concentration gradient of cisplatin, etoposide, or bleomycin. After 72 hours of treatment, cell viability was assessed using the CellTiter Glo luminescent assay (Vazyme). Luminescence signals were recorded using a multifunctional microplate reader (Agilent BioTek). Apoptosis rates were measured using the Annexin V-FITC/propidium iodide (PI) assay according to the manufacturer's instructions. Flow cytometric analysis was performed immediately after staining. Data was analyzed using FlowJo software (FlowJo). Cell proliferation was assessed using the EdU assay (Click-iT EdU kit, Thermo Fisher Scientific). Fluorescent images were acquired using a fluorescence microscope. EdU-positive cells were quantified by counting five random fields per sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTransfection of siRNA and plasmids\u003c/h2\u003e \u003cp\u003esiRNA and plasmids were transfected into cells using the Effectene Transfection Reagent (Qiagen). Transfection was performed when cells reached approximately 60% confluence. After 48 hours of incubation, cells were harvested for subsequent analysis. Gene silencing efficiency was evaluated by reverse transcription quantitative polymerase chain reaction (RT-qPCR). siRNA sequences are provided in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLentivirus construction and transduction\u003c/h2\u003e \u003cp\u003eTo generate lentivirus for stable knockdown, shRNA plasmids were co-transfected with packaging plasmids (pMD2.G and psPAX2) into HEK293T cells using Effectene Transfection Reagent (Qiagen) as previously described. For overexpression constructs, the c-MYC gene was amplified by PCR and cloned into the PCMV-3Flag vector. The \u003cem\u003eARID1B\u003c/em\u003e plasmid was obtained from Addgene (#144023). shRNA sequences are detailed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eComet assay\u003c/h2\u003e \u003cp\u003eDNA damage was assessed using comet assay following the manufacturer's instructions. Briefly, cells (1\u0026times;10⁵ cells/mL) were mixed with molten CometAssay LMAgarose (at 37\u0026deg;C) at a 1:10 (v/v) ratio. The mixture was layered onto comet slides. Cells were then lysed for 30 minutes, followed by immersion in Alkaline Unwinding Solution for 20 minutes at room temperature. Alkaline electrophoresis was performed at 21 V for 30 minutes and visualized using a fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence and microscopy\u003c/h2\u003e \u003cp\u003eFor immunofluorescence experiments, cells were seeded onto glass-bottomed confocal dishes and incubated overnight, followed by treatment according to the experimental protocol. Images were acquired using a confocal microscope. The primary antibodies used in the study are listed in Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation (ChIP)\u003c/h2\u003e \u003cp\u003eA minimum of 2 x 10\u003csup\u003e7\u003c/sup\u003e adherent or suspension cells per sample were crossed-linked with 1% formaldehyde 10 minutes at room temperature. Cross-linking was terminated by adding glycine to a final concentration of 0.125 M, and the cells were incubated on the orbital shaker for an additional 5 minutes. Cells were washed with cold PBS, lysed (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0), and sonicated to shear DNA into fragments ranging from 300 to 500 bp. Chromatin was pre-cleared and immunoprecipitated overnight at 4\u0026deg;C with 2 \u0026micro;g of specific antibody or IgG control. Antibody-chromatin complexes were captured using Protein A/G magnetic beads (Thermo Fisher), washed extensively, and eluted. Cross-links were reversed overnight at 65\u0026deg;C. DNA was then purified using a PCR purification kit (Accurate Biology) and analyzed by qPCR. For the DNA damage site protein recruitment assay, as previously described\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, HEK293T cells were co-transfected with pBABe-HA-ER-IPpoI and siARID1B, and treated with 1 \u0026micro;M 4-hydroxy-Tamoxifen (4-OHT) for 24 hours. Cells were then collected for ChIP analysis. ChIP primers are listed in the supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eDual Luciferase Reporter Assay\u003c/h2\u003e \u003cp\u003eThe Dual-Luciferase Reporter Assay System (Vazyme) was used according to the manufacturer's protocol, HEK293T cells in 12-well plates were co-transfected with PGL4-Ctrl, PGL4-MYC and PGL4-RNF8 reporter vectors. After 48 hours, the culture medium was aspirated and cells were washed with PBS. Cells were lysed by adding passive Cell lysis buffer and incubating for 5 minutes at room temperature with gentle shaking. Cell lysates were centrifuged at 12,000 rpm for 2 minutes at room temperature. A 20 \u0026micro;l aliquot of the supernatant was transferred to a microplate containing 100 \u0026micro;l of Luciferase substrate (Vazyme) and immediately measured using a luminometer; subsequently, freshly prepared Renilla substrate working solution was added to the reaction wells and Renilla luciferase activity was measured immediately. Firefly luciferase activity was normalized to Renilla luciferase activity for each sample.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eDetecting ssDNA lesions by BrdU incorporation\u003c/h2\u003e \u003cp\u003eSingle-stranded DNA (ssDNA) lesions were detected using BrdU incorporation under non-denaturing conditions as previously described\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Briefly, DMS273 cells were pulse-labeled with 10 \u0026micro;M BrdU (BD Biosciences) for 24 h. DNA damage was then induced by treating cells with 40 \u0026micro;M Etopside (ETO) for 1 h. Single-stranded DNA (ssDNA) lesions were detected using BrdU incorporation under non-denaturing conditions as previously described\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Fluorescence images were acquired using a high-resolution microscopy system.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eDNA end-resection assay\u003c/h2\u003e \u003cp\u003eDNA end resection was quantified by measuring induced single-stranded DNA (ssDNA) formation using the HA-ER-I-PpoI system\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. HEK293T cells were co-transfected with the Plasmids pBABe-HA-ER-I-PpoI and siRNA targeting \u003cem\u003eARID1B\u003c/em\u003e (siARID1B). Twenty-four hours post-transfection, 4-hydroxytamoxifen (4-OHT) was added to the culture medium at a final concentration of 1 \u0026micro;M to induce nuclear translocation and I-PpoI endonuclease activity. After an additional 24 hours, cells were collected and genomic DNA was extracted using a DNA extraction kit (Accurate Biology). Purified DNA was then was digested with EcoRI (New England Biolabs) at 37\u0026deg;C for 30 minutes. Subsequently, the digested DNA was purified and analyzed by quantitative PCR (qPCR) using primers flanking the I-PpoI cleavage site. Relative ssDNA levels were calculated using the ΔΔCt method, comparing samples with and without 4-OHT induction and normalized to control siRNA.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eEMSA assay\u003c/h2\u003e \u003cp\u003eThe plasmids containing either full-length c-MYC or the ARID domain (amino acids 1136\u0026ndash;1227) of ARID1B were transformed into Escherichia coli BL21 cells for protein purification. Protein-DNA binding was analyzed by EMSA using the Lightshift chemiluminescent EMSA kit (GS009, Beyotime) according to the manufacturer\u0026rsquo;s instructions. Briefly, biotin-labeled DNA substrate was incubated with purified ARID protein/c-MYC protein at indicated concentrations in 1\u0026times; binding buffer at room temperature for 20 min. Protein-DNA complexes were resolved on a 6% non-denaturing polyacrylamide gel electrophoresis (PAGE) in 0.5\u0026times; TBE buffer at 100 V for 60\u0026ndash;90 min at 4\u0026deg;C and subsequently transferred to a positively charged nylon membrane (Beyotime) at 380 mA for 30\u0026ndash;45 min. After UV crosslinking (254 nm, 5 min), the membrane was blocked and washed according to standard protocols. Biotin-labeled DNA was then detected using the chemiluminescent substrate provided in the kit. Chemiluminescent signals were captured using an automated chemiluminescence imaging system (Tanon). Oligo primers are listed in the supplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eEstablishment of xenograft models\u003c/h2\u003e \u003cp\u003e2\u0026times;10\u003csup\u003e6\u003c/sup\u003e DMS273 cells (either transduced with non-targeting control shRNA [SCR] or ARID1B-targeting shRNA [shARID1B#1 or shARID1B#2]) suspended in 100 \u0026micro;L PBS were subcutaneously inoculated into the dorsal flank of 4\u0026ndash;5-week-old female BALB/c nude mice. When tumors reached a volume of 100 mm\u003csup\u003e3\u003c/sup\u003e, mice bearing SCR or shARID1B DMS273 cells were randomly divided into control and E/P treatment groups. The E/P group received etoposide (7 mg/kg, i.p.) every other day and cisplatin (3 mg/kg, i.p.) once weekly. No animal mortality occurred during the study. At the experiment endpoint, mice were euthanized, tumors were excised and weighed. Tumor volume was calculated using the formula: Volume (mm\u0026sup3;) = (Length \u0026times; Width\u0026sup2;) / 2, where length is the longest diameter and width is the perpendicular diameter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eFormalin-fixed, paraffin-embedded (FFPE) tumor tissues were sectioned (4\u0026ndash;5 \u0026micro;m). Sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After blocking with 5% BSA, sections were incubated overnight at 4\u0026deg;C with the primary antibodies: Ki-67 (GB121141, Servicebio, 1:200), cleaved caspase-3 (GB11532, Servicebio, 1:200), ARID1B (ab57461, abcam, 1:200), γ-H2AX (GB111841, Servicebio, 1:200), c-MYC (ab32072, abcam, 1:200). IHC staining intensity and percentage of positive cells were independently evaluated and scored by two experienced pathologists blinded to the experimental groups, using a semi-quantitative scoring system as previously reported.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism 6.0 software was utilized for statistical analysis. Experimental data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Intergroup comparisons were conducted using the unpaired two-sided Student's \u003cem\u003et\u003c/em\u003e-test or one-way analysis of variance (ANOVA). Statistical significance was defined as a \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data accessed from external sources and prior publications have been referenced in the text and corresponding figure legends. Additional data used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e \u003cstrong\u003eand consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll mouse experimentations were performed in compliance with the institutional guidelines and the protocol was approved by the Ethics Committee of Longgang District People’s Hospital of Shenzhen(2024067DW). All authors complied with all relevant ethical regulations for animal testing and research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank members of the Lin laboratory for critical reading of the manuscript and helpful discussions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWenchu Lin supervised, and funded the study. Peng Hou, Jiahui Zhang and Gongfeng Li conducted animal experiments. Peng Hou, Guozhen Cao, Jinghan Hua, Xinhuang Yao and Honglin Li performed in vitro cell-based experiments. Peng Hou and Gongfeng Li analyzed RNA-seq data. Peng Hou performed molecular biological experiments. Rui Guo provided platform technical support. Peng Hou, Li Xiang and Wenchu Lin wrote the manuscript. All authors read and approved the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by National Natural Science Foundation of China (Grant Numbers: 82573883, 82302957), Shenzhen Science and Technology Program (Grant Number: JCYJ20250604180129038), Key Medical Technologies R \u0026amp; D Programme of Longang district (Grant Number: LGKCYLWS2023012), A portion of this work was supported by Sanming Project of Medicine in Shenzhen.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBernhardt, E. B. \u0026amp; Jalal, S. I. 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XPG-related nucleases are hierarchically recruited for double-stranded rDNA break resection. \u003cem\u003eJ Biol Chem\u003c/em\u003e 294, 7632\u0026ndash;7643 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.RA118.005415\u003c/span\u003e\u003cspan address=\"10.1074/jbc.RA118.005415\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ARID1B, SCLC, c-MYC, MDC1, DNA end resection","lastPublishedDoi":"10.21203/rs.3.rs-8612664/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8612664/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAT-rich interactive domain-containing protein 1B (ARID1B) has been implicated in DNA damage repair, yet its precise molecular mechanisms and contributions to small cell lung cancer (SCLC) pathogenesis remain incompletely defined. In this study, analysis of clinical datasets revealed \u003cem\u003eARID1B\u003c/em\u003e overexpression in SCLC tissues and cell lines compared to normal lung or lung adenocarcinoma counterparts. Functional studies \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e demonstrated that \u003cem\u003eARID1B\u003c/em\u003e depletion significantly impaired SCLC cell proliferation, clonogenicity, and tumor growth while promoting apoptosis. Mechanistically, During DSB response, ARID1B cooperates with the transcription factor c-MYC to co-activate the expression of key DNA damage repair (DDR) genes (e.g., \u003cem\u003eRNF8\u003c/em\u003e and \u003cem\u003eRAD51\u003c/em\u003e), Concurrently, ARID1B is recruited to DNA double-strand break (DSB) sites, facilitates single-stranded DNA (ssDNA) formation via end resection, and promotes the recruitment of MDC1, a scaffold protein essential for early DDR signaling. Critically, \u003cem\u003eARID1B\u003c/em\u003e deficiency markedly sensitized SCLC cells to DNA-damaging agents, evidenced by enhanced DNA damage persistence, apoptosis, and tumor growth inhibition \u003cem\u003ein vitro and in vivo\u003c/em\u003e. This study unveils a dual regulatory role of ARID1B in homologous recombination (HR) repair of DNA double-strand breaks (DSBs) and establishes ARID1B as a key mediator of chemoresistance in SCLC, highlighting its potential as a therapeutic target to overcome treatment resistance.\u003c/p\u003e","manuscriptTitle":"ARID1B promotes DNA end resection via MDC1 and co-transactivation of DNA repair genes with c-MYC in small cell lung cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-10 13:25:46","doi":"10.21203/rs.3.rs-8612664/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-16T09:12:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-14T17:20:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-06T14:37:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-28T06:51:57+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-19T11:47:52+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-05T12:54:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-30T13:46:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2026-01-29T12:58:00+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-01-16T16:04:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-15T17:12:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5dfa1273-2a2c-462d-aa37-bb641b5d1903","owner":[],"postedDate":"February 10th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":62387484,"name":"Biological sciences/Cancer/Cancer epidemiology"},{"id":62387485,"name":"Biological sciences/Molecular biology/DNA damage and repair/Double-strand DNA breaks"}],"tags":[],"updatedAt":"2026-03-16T09:16:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-10 13:25:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8612664","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8612664","identity":"rs-8612664","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}