Abacavir enhances the efficacy of Doxorubicin via inhibition of histone demethylase KDM5B in breast cancer

Abacavir enhances the efficacy of Doxorubicin via inhibition of histone demethylase KDM5B in breast 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 Abacavir enhances the efficacy of Doxorubicin via inhibition of histone demethylase KDM5B in breast cancer Anmi Jose, Pallavi Kulkarni, Naveena Kumar AN, Nawaz Usman, Gabriel Sunil Rodrigues, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6208718/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Aug, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract KDM5B, a lysine-specific histone demethylase, is widely upregulated in breast cancer. The current study investigated the role of KDM5B in breast cancer and explored the repurposing potential of the antiviral drug abacavir (ABC). The cytotoxic effects and the effect of ABC sensitization on doxorubicin (DOX) efficacy were evaluated using 2-D and 3-D cell culture models. KDM5B expression was elevated in breast cancer tissues compared to normal breast tissues. In vitro studies demonstrated that ABC treatment reduced KDM5B expression in breast cancer cells and increased their sensitivity towards DOX. ABC induced late apoptosis and S phase arrest, while the ABC + DOX combination led to S/G2 phase arrest, late apoptosis, and cell death. Data generated from patient-derived breast tumoroids corroborated the 2-D cell culture-based findings. Additionally, molecular docking studies indicated that active drug metabolite carbovir triphosphate (CBV-TP) could interact with the DNA polymerase β-DNA complex, suggesting its potential mechanism to be incorporated into the DNA synthesis cycle, leading to cell cycle arrest in tumor cells. Our findings highlight the repurposing potential of ABC to target KDM5B in breast cancer. This approach enhanced the efficacy of DOX, which could allow further dose reduction and reduced side effects, offering a promising therapeutic strategy. Biological sciences/Cancer/Breast cancer Biological sciences/Cancer/Cancer therapy Biological sciences/Cancer/Oncogenes Biological sciences/Cancer/Tumour biomarkers Health sciences/Medical research/Pre clinical studies Health sciences/Medical research/Translational research Biological sciences/Cancer Biological sciences/Computational biology and bioinformatics Health sciences/Biomarkers Health sciences/Pathogenesis Breast cancer Carbovir triphosphate Drug repurposing Epigenetic targeting Precision medicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Breast cancer remains the most prevalent cancer among women worldwide, with an estimated 2.2 million new cases and 0.6 million deaths reported in 2022 [ 1 ]. Tumor heterogeneity in breast cancer results in significant variability in pathology, molecular alterations, and tumor microenvironment [ 2 ]. While the development in early diagnosis and treatment modalities has improved overall survival rates of breast cancer, the effective management of breast cancer still faces challenges [ 3 ]. Since breast cancer is influenced by a combination of genetic and epigenetic factors, a detailed understanding of the underlying molecular mechanisms is necessary to improve therapeutic options. Although epigenetic modifications do not cause alteration in DNA sequences, they can significantly influence cancer development at different stages. Hence, targeting epigenetic markers has a substantial role in cancer detection, prevention, and development of targeted therapies [ 4 ]. For example, drugs targeting epigenetic modifying enzymes, such as histone deacetylases (HDACs) and DNA methyltransferases, have received FDA approval [ 5 ]. Furthermore, combining epigenetic targeting drugs (e.g. decitabine, SAHA, etc.) with conventional chemotherapies has also received wide attention [ 6 – 8 ]. KDM5B (Lysine demethylase 5B), or JARID1B, encodes lysine-specific histone demethylase from the Jumanji (JmjC)/ARID domain-containing family of histone demethylases [ 9 ]. This protein plays a pivotal role in the transcriptional repression of genes by demethylating mono-, di-, and trimethylated lysine 4 of histone 3 (H3K4) [ 10 ]. KDM5B is significantly upregulated in various cancers, particularly breast cancer [ 11 , 12 ], and its oncogenic properties make it a promising target for personalized drug therapy [ 13 ]. However, the prognostic significance and drug-targeting potential of KDM5B have not been fully explored. In this context, repurposing existing drugs could be a valuable strategy, owing to the established safety profile, cost-effectiveness, shorter development times, and affordability [ 14 ]. Abacavir (ABC), a nucleoside reverse transcriptase inhibitor (NRTI), is commonly used in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. ABC is generally well tolerated with minimal adverse effects and favorable bioavailability [ 15 ]. Upon cellular uptake, ABC undergoes bioconversion to form the active metabolite carbovir triphosphate (CBV-TP) [ 16 ]. In a previous study, molecular docking analysis identified the key interactions of ABC towards the JmjC domain of KDM5B [ 17 ]. Furthermore, former studies have demonstrated the potential of ABC as an anticancer agent in various cancers [ 18 , 19 ]. In this regard, we conducted a detailed study to evaluate the repurposing potential of ABC by targeting the KDM5B oncogene in breast cancer. KDM5B gene expression was determined in breast cancer patient samples, and the cytotoxic activity and the effect of ABC sensitization on DOX were investigated in 2-D and 3-D cultures. The results from our study demonstrated that ABC could potentiate the cytotoxicity of DOX in breast cancer cells. This approach enhanced the efficacy of DOX, which could allow for further dose reduction leading to lower side effects, thus offering a promising strategy for breast cancer therapy. 2. Results 2.1. KDM5B is over-expressed in breast cancer: Evidence from public databases Comprehensive gene expression profiling of KDM5B across tumor and normal tissues was evaluated using the GEPIA database. The KDM5B gene expression was elevated in most cancers compared to normal tissues, with significant overexpression observed in breast invasive carcinoma, pancreatic adenocarcinoma, and thymoma (Fig. 1 A). Further analysis using UALCAN confirmed that KDM5B gene expression was significantly higher in breast tumors than in normal breast tissues (Fig. 1 B). In addition, RNA-Seq data from the TNMplot database demonstrated increased KDM5B expression in both primary breast tumors and metastatic breast cancer samples compared to normal breast tissue (see Supplementary Fig. S1 online). Among tumor stages, early-stage tumors exhibited slightly higher KDM5B expression than advanced stages, although all tumor stages showed significant upregulation relative to normal tissue (Fig. 1 C). Across all breast cancer subtypes, KDM5B expression was marginally higher in the luminal subtype, followed by HER2 positive and triple negative (Fig. 1 D). Menopausal status also correlated with KDM5B expression. Patients in the premenopausal stage showed significantly higher KDM5B levels compared to those in the perimenopausal, and post-menopausal stages (Fig. 1 E). Additional histopathological and clinical parameters, including patient’s age, race, nodal metastasis, histologic subtypes, and TP53 mutation status, were significantly associated with KDM5B overexpression (see Supplementary Fig. S1 online). Overall survival (OS) in breast cancer patients with KDM5B mRNA expression was analyzed using the GEPIA database (see Supplementary Fig. S2 online). KDM5B expression was not significantly associated with OS in breast cancer patients. 2.2. KDM5B is over-expressed in clinical breast tumor samples The results obtained in databases were validated in clinical tumor samples. Towards this, qPCR was performed on 53 breast cancer and 14 normal breast tissue samples to evaluate KDM5B expression. In line with global data, KDM5B expression was significantly elevated in breast tumors compared to normal breast tissues ( P < 0.0001) (Fig. 2 A). However, no significant differences in KDM5B expression were observed across various clinicopathological factors. Detailed patient characteristics and clinicopathological data in relation to KDM5B mRNA expression are provided in Table 1 . Table 1 Statistical analysis of KDM5B expression levels in clinical breast cancer tissues in relation to clinicopathological characteristics. ER: Estrogen receptor; DCIS: Ductal carcinoma in situ; HER2: Human epidermal growth factor receptor 2; NST: No special type; IQR: Inter quartile range; PR: Progesterone receptor; TNBC: Triple-negative breast cancer. Clinicopathological features Sample size (n) KDM5B mRNA expression Median IQR (Q1, Q3) P value Sample type Tumor 53 5.138 1.610, 9.381 50 26 4.224 1.521, 14.850 Tumor histology Invasive breast carcinoma NST 46 4.599 1.546, 10.36 0.847 Other (DCIS, Mixed, Metaplastic carcinoma, etc) 7 5.809 2.405, 7.803 Menopausal status Pre-menopausal 19 5.767 2.588, 8.594 0.733 Post-menopausal 34 4.402 1.523, 11.190 Pathological grade Grade 1 7 2.702 1.417, 8.185 0.639 Grade 2 19 4.516 1.551, 15.040 Grade 3 27 5.767 1.668, 10.170 TNM stage I 5 2.405 1.365, 5.982 0.576 II 42 5.586 1.625, 11.780 III 4 4.887 2.154, 7.596 IV 2 10.65 1.529, 19.770 Nodal involvement 0 32 5.788 2.617, 10.760 0.150 I 13 2.405 1.116, 11.740 II 3 1.417 0.693, 5.138 III 5 8.192 3.927, 12.040 ER ER+ 36 5.854 2.351, 10.360 0.302 ER- 17 4.287 1.208, 8.662 PR PR+ 26 4.960 2.138, 8.287 0.978 PR- 27 5.138 1.529, 10.950 HER2 HER2+ 12 5.453 2.292, 16.900 0.614 HER2- 40 4.846 1.564, 9.775 Unknown 1 4.161 - Molecular subtypes Luminal A 10 5.784 2.387, 10.210 0.944 Luminal B 25 5.404 1.610, 9.572 TNBC 12 5.048 1.063, 13.230 HER2 enriched 5 4.681 1.525, 12.850 Unknown 1 4.161 - 2.3. Repurposing of the antiviral drug ABC targeting KDM5B, sensitized breast cancer cells to DOX We previously reported the potential of repurposing antiviral drugs targeting the JmjC domain of KDM5B through computational analysis, including molecular docking studies of ABC [ 17 ]. Furthermore, former literatures demonstrate the potential of ABC as an anticancer agent in various cancers [ 18 , 19 ]. To assess its therapeutic efficacy in breast cancer cells, we examined the cytotoxic effects of ABC at concentrations ranging from 0 to 500 µM in MDA-MB-231 and MCF-7 cells. Interestingly, ABC exhibited significant cytotoxicity at clinically relevant concentrations in both cell lines (MDA-MB-231 IC 50 : 289.15 ± 32.25 µM; MCF-7 IC 50 : 281.26 ± 51.59 µM) (see Supplementary Fig. S3 online). In addition, to investigate whether ABC treatment modulates KDM5B expression, we analyzed its levels in ABC-treated cells. Cells were exposed to a low dose of ABC (IC 25 ) for 24 hours, followed by a 3-day media change. Compared to untreated control cells, KDM5B expression was reduced in both cell lines, with a significant decrease observed in ABC-treated MDA-MB-231 cells ( P < 0.01) (Fig. 2 B). Since ABC treatment influenced KDM5B gene expression and KDM5B is known to promote cancer cell proliferation [ 12 ], we further examined whether ABC could enhance the sensitivity of breast cancer cells to DOX treatment. Pre-treatment with a low dose of ABC (IC 25 dose), followed by DOX exposure (0-160 nM), significantly enhanced cytotoxicity, reducing cell proliferation more efficiently than DOX alone (Fig. 2 C & 2 D). In the absence of ABC, MDA-MB-231 cells demonstrated enhanced resistance to DOX (IC 50 = 155.67 ± 17.88 nM), compared to MCF-7 cells (IC 50 = 47.68 ± 17.10 nM). However, in ABC-sensitised cells, the IC 50 of DOX was significantly reduced to 65.03 ± 19.14 nM in MDA-MB-231 cells, representing a 2.4-fold decrease ( P 0.05), demonstrating enhanced cytotoxicity compared to DOX alone (Fig. 2 E). Subsequently, a comparison of three pretreated groups (ABC, DOX, ABC + DOX) with untreated control displayed a significant reduction in colony forming property of breast cancer cells with ABC + DOX treatment, as confirmed by soft agar colony forming assay (Fig. 2 F, and Supplementary Fig. S4 online). The MDA-MB-231 cells showed a greater reduction in colony formation in response to ABC + DOX treatment compared to MCF-7 cells, thereby demonstrating the potential of ABC as a sensitizer for conventional anti-cancer agents for the management of triple-negative breast cancer cells. 2.4. Pre-treatment of ABC followed by DOX enhanced apoptosis and cell cycle arrest in breast cancer cells We investigated the effects of ABC on cell cycle and apoptosis in MDA-MB-231 and MCF-7 cells. Treatment with ABC alone for 72 hours (IC 25 , added every 24 hours) induced S-phase arrest in both cell lines (Fig. 3 ). Combined treatment with ABC (IC 25 for 24 hours) and DOX (IC 25 , for 72 hours) resulted in an arrest at the S/G2 phase. A more evident cell cycle arrest with ABC sensitization was observed in MDA-MB-231 cells compared to MCF-7 cells. In addition, apoptosis study results showed that treatment with ABC alone slightly increased the cell population undergoing late apoptosis in both cell lines, with a slight increase in early-phase apoptosis observed in MCF-7 cells. Combined treatment with ABC (IC 25 for 24 hours) and DOX (IC 25 for 72 hours) significantly increased the percentage of cells in late-phase apoptosis in both cell lines (Fig. 4 ). DNA replication occurs during the S phase, while the G2 phase prepares the cell for mitotic division. Arresting cells in the S/G2 phase indicates disruption of cell cycle progression, effectively inhibiting cell proliferation. Our findings suggest that, as ABC can induce DNA damage, it can lead to transient S/G2-phase arrest and apoptosis. This dual strategy of inducing S/G2 phase arrest and DNA damage represents an effective approach for inhibiting breast cancer cell proliferation and inducing cell death. 2.5. Generation of patient-derived breast cancer organoids Four patient breast cancer samples were obtained under informed consent and used to generate patient-derived breast cancer organoids, as described in the methods section. We observed two types of breast cancer organoid phenotypes, including solid dense (Fig. 5 A, B) and discohesive (Fig. 5 C) [ 20 ]. The solid-dense phenotype was associated with three patients, while the discohesive one was associated with one patient. Both the solid and discohesive organoids showed rapid growth. The discohesive organoids were present in clusters without smooth boundaries. 2.6. ABC enhances the cytotoxic effect of DOX on patient-derived breast cancer organoids We used the antiretroviral nucleoside analog ABC as a single drug and combined it with DOX to determine its role in promoting DOX-mediated cytotoxicity. Based on our in vitro data and other published work, the breast cancer organoids were exposed to 50 µM ABC and 1 µM DOX, respectively [ 18 , 21 ]. We used the bright-field images to analyze the growth rate (diameter) of the organoids treated with ABC, DOX, and their combination and compared them with untreated breast cancer organoid controls (Fig. 5 D). Though the ABC-treated breast cancer organoid looked smaller than the untreated, we did not observe a statistical significance associated with this arm of treatment. DOX showed a substantial decrease in the diameter of breast cancer organoids, suggesting that our model is functional. Moreover, the organoids showed a dark core reminiscent of apoptotic cells. Interestingly, the combination yielded strong inhibition of breast cancer organoid proliferation (diameter), especially in patients 1 (n = 1) and 2 (n = 2) with solid, dense organoid phenotypes. Patient 3 (n = 3) did not significantly differ in the DOX vs ABC + DOX combination, suggesting patient genetic diversity. In patient 4 (n = 4) with discohesive breast cancer organoid phenotype, the data looked interesting, but it is difficult to ascertain the diameter of each organoid conclusively. Hence, we used data from patients 1 and 2 to plot the diameter of the breast cancer organoids post-treatment to evaluate the sensitivity of ABC, DOX, and ABC + DOX compared to untreated (Fig. 6 ). Some representative images used for the data analysis are shown in the Supplementary Fig. S5 online. The data (Fig. 6 ) reveals that ABC, in combination with DOX, can be an effective therapeutic option for some patients. Genetic variability can be at the heart of this observation. Because telomerase activity is a promising therapeutic target in breast cancer, and recent reports establish that ABC is functional against telomerase high medulloblastoma cells, our patient-derived breast cancer organoids can be an interesting strategy for determining the effectiveness of therapeutic regimen based on ABC and DOX [ 22 ]. However, this warrants further investigation. 2.7. Molecular studies reveal CBV-TP interaction with human DNA Polymerase β and DNA Complex The results obtained from this study show that ABC pretreatment enhanced the cytotoxicity of DOX to MCF-7 and MDA-MB-231 cells compared to DOX monotherapy. This was an exciting finding from our study. In this regard, there is previous literature that has demonstrated that nucleoside derivatives exhibit anticancer activity by getting incorporated into the DNA synthesis cycle through the DNA polymerase enzymes, including DNA polymerase β [ 23 – 25 ]. For example, the purine nucleoside cladribine is known to exhibit anticancer activity by inhibiting DNA polymerase enzymes [ 26 ]. It is pertinent to note that these nucleoside derivatives undergo a series of intracellular bioconversions to form the active triphosphate derivatives. In the case of ABC, it undergoes bioconversion to form the active metabolite carbovir triphosphate (CBV-TP, Fig. 7 A) [ 27 ]. Therefore, we investigated the interactions of CBV-TP in the DNA polymerase β-DNA complex using the solved X-ray structure [ 28 ]. The top binding mode of CBV-TP in the DNA polymerase β-DNA complex shows that it was oriented in the catalytic site closer to magnesium atoms and the DNA base pairs, as shown in Fig. 7 B. The planar, bicyclic guanosine ring of CBV-TP underwent multiple π-π stacked interactions with the cytosine ring of the DNA nucleotide cytidine (Fig. 7 C). Both the imidazole and pyrimidine rings of CBV-TP were in contact with the DNA base (distance < 4.2 Å). Furthermore, the triphosphate ester moiety of CBV-TP underwent polar interactions with amino acids Ser180, and Arg183 of DNA polymerase β (distance < 2.0 Å). Also, it underwent a number of electrostatic interactions with the two catalytic magnesium atoms, as shown in Fig. 7 C (distance < 3.5 Å). These molecular docking studies suggest that CBV-TP can interact with the DNA polymerase β-DNA complex and has the potential to get incorporated into the DNA synthesis cycle causing cell cycle arrest in tumor cells. This is further supported by a previous work that demonstrated the ability of CBV-TP to inhibit DNA polymerases [ 29 ]. 3. Discussion Epigenetic modifications, including DNA methylation and histone modifications, play a vital role in cancer development, either by downregulation of tumor suppressor genes or by upregulation of oncogenes [ 30 ]. Targeting these mechanisms depicts a promising approach to develop novel anti-cancer drugs [ 31 ]. Several studies have emphasized the role of combining epigenetic therapies with conventional treatments, such as chemotherapy and radiation therapy. These combinatorial approaches have proven the ability to enhance therapeutic efficacy, improve sensitivity, and reduce the toxicity associated with conventional therapies [ 8 , 32 – 34 ]. The oncogenic significance of KDM5B in breast cancer was confirmed through the analysis of publicly accessible databases. mRNA expression profiles from TCGA datasets revealed significantly higher KDM5B expression levels in breast tumors compared to normal tissues. This finding aligns with our qPCR analysis of 53 breast tumor samples, which confirmed KDM5B overexpression in tumor samples compared to adjacent normal tissues. Our patient cohort exhibited a slightly higher median KDM5B gene expression in luminal subtypes. This is consistent with the established role of KDM5B as a luminal lineage-driving oncogene. Supporting this, Yamamoto et al. demonstrated that high KDM5B activity correlates with poor outcomes in ER + tumors [ 35 ]. However, larger patient cohorts are required in our study to establish a significant statistical association with other clinical parameters. Despite numerous KDM inhibitors being developed in various laboratories, no drugs specifically targeting KDM5B have been approved by the FDA for the management of breast cancer. In this regard, drug repurposing offers an efficient strategy for identifying new therapeutic applications for existing drugs, with fewer time constraints than the traditional drug discovery and development process [ 14 ]. More specifically, anti-viral drugs have been shown to sensitize resistant cancer cells to chemotherapy, thereby increasing the efficacy of other therapeutic approaches. For example, ribavirin has been identified as an EZH2 inhibitor [ 36 ], while acyclovir has shown the ability to inhibit the proliferation and colony-forming properties of breast cancer cells [ 37 ]. In this context, the current study explored ABC's repurposing potential for managing breast cancer. Doxorubicin, an anthracycline derivative that targets topoisomerase II, is widely utilized for various cancers, including breast cancer, carcinomas, sarcomas, and hematological malignancies [ 38 ]. Despite the extensive use of doxorubicin, dose-dependent toxicity, particularly cardiotoxicity, remains a significant concern [ 39 ]. In this regard, several approaches have been explored to reduce these adverse effects, including the combination of antioxidants, advanced drug delivery systems, and prodrug development [ 40 ]. However, some of these approaches have not successfully translated into effective clinical outcomes [ 40 ]. Thus, innovative strategies are necessary to enhance therapeutic outcomes. Towards this, our study highlights, for the first time, that epigenetic targeting of KDM5B oncogene by ABC, when combined with DOX, induces cytotoxic effects within a minimal dose range of DOX and may potentially negate the side effects, including cardiotoxicity. We conducted a series of in vitro experiments to compare the effects of ABC and DOX, both individually as well as in combination, on MCF-7 and MDA-MB-231 breast cancer cell lines. Our results demonstrated that ABC inhibited cell proliferation and induced cytotoxicity. This is in accordance with a previous study showing that ABC causes a dose-dependent decrease in the proliferation rate of medulloblastoma cells [ 22 ]. Furthermore, ABC induced apoptosis by arresting cells in the S phase, consistent with findings reported by Tada et al. [ 18 ]. Our patient-derived breast cancer organoid data suggest that some patients may not be sensitive to this treatment, possibly due to inherent genetic variability. Besides, recent studies have demonstrated the efficacy of ABC against telomerase-high medulloblastoma cells [ 22 ]. Therefore, the patient-derived breast cancer organoids developed in this study may be utilized as a precision medicine approach for deciding the treatment regimen based on ABC for breast cancer patients. However, further investigation is needed to validate our findings. Nucleoside derivatives like ABC exert anti-cancer effects by integrating into the DNA synthesis cycle through DNA polymerase enzymes such as DNA polymerase β [ 23 ]. In cells, ABC is metabolized to its active form, CBV-TP [ 27 ], which is subsequently incorporated into chromosomal DNA by replicative DNA polymerases. This incorporation results in premature termination of DNA replication, replication fork collapse, and the formation of DNA double-strand breaks (DSBs) [ 18 ]. Tada et al. confirmed that therapeutic concentrations of ABC induce DSBs in adult T-cell leukemia (ATL) cells, suggesting that efficient induction of DSBs is the primary mechanism underlying ABC’s cytotoxicity [ 18 ]. Additionally, a study by Rossi et al. also supports the DNA damage induced by ABC and suggests that its antiproliferative effect is accompanied by the inhibition of telomerase activity in medulloblastoma cell lines [ 22 ]. Our computational analysis further supports these findings by demonstrating that CBV-TP interacts with the DNA polymerase β-DNA complex, thereby emphasizing its role in causing cell death by disrupting the DNA synthesis in tumor cells. We also evaluated the expression of KDM5B following ABC treatment and found that ABC reduced its expression, indicating the impact of ABC exposure on KDM5B regulation. This finding is supported by the previous study that explored the repurposing potential of ABC through molecular docking studies, targeting the JmjC domain of KDM5B [ 17 ]. KDM5B also functions as a genome stabilizer and plays a crucial role in the DNA damage response [ 41 ]. Inhibition of KDM5B disrupted the DNA repair mechanism, as demonstrated by Bayo et al., which showed that the small molecule JIB-04 enhanced the sensitivity of lung cancer cells to radiation by inhibiting KDM5B [ 42 ]. In this context, the current study showed increased sensitivity of breast cancer cells towards DOX treatment following ABC pre-treatment. More specifically, our findings suggest that ABC causes DNA damage by being incorporated into the DNA synthesis cycle, leading to the formation of DSB while simultaneously inhibiting KDM5B, which is a critical factor in DNA damage repair. Inhibition of KDM5B exacerbates the accumulation of DNA damage, sensitizing cells to additional DNA-damaging agents such as DOX. Therefore, combining ABC and DOX might amplify the disruption of DNA repair pathways, resulting in enhanced cytotoxicity. These dual actions create a mechanistic synergy, where the inability to repair damaged DNA leads to increased cell death and may also reduce the DOX dosing regimen, potentially lowering its adverse effects. This provides a strong rationale for the combined use of ABC and DOX in breast cancer therapy. Our study provides foundational data, emphasizing the need for further investigation in animal models or patient-derived xenografts to fully assess the therapeutic potential and translate these findings into clinical settings. Furthermore, studies involving larger patient cohorts are essential to establish significant correlations between KDM5B expression and clinical outcomes. A comprehensive proteomic and transcriptomic analysis focusing on the epigenetic regulation of KDM5B and its downstream targets will provide deeper insights into how ABC disrupts KDM5B-mediated oncogenesis. These future studies could pave the way for early-phase clinical trials in breast cancer patients to evaluate the safety, tolerability, and preliminary efficacy of ABC as an adjuvant to DOX. 4. Conclusions The current study highlights the over-expression of KDM5B in human breast tumors compared to non-neoplastic breast tissues in global databases as well as in our patient cohort. In vitro studies showed that ABC treatment reduced KDM5B expression in breast cancer cells and increased their sensitivity towards DOX treatment. ABC induced late apoptosis and S phase arrest, while the ABC + DOX combination led to S/G2 phase arrest, late apoptosis, and cell death. Drug treatment studies in the patient-derived breast tumoroids supported the 2-D cell line-based findings. Additionally, molecular docking studies indicated that CBV-TP could interact with the DNA polymerase β-DNA complex, suggesting its potential mechanism to be incorporated into the DNA synthesis cycle, leading to cell cycle arrest in tumor cells. Our findings highlight the repurposing potential of ABC, a well-established antiviral agent, as a novel therapeutic approach targeting the KDM5B oncogene in breast cancer. This strategy may enhance the efficacy of DOX and could potentially help in reducing its dose, side effects, and drug resistance, thereby offering a promising approach for personalized breast cancer treatment. 5. Methods 5.1. KDM5B gene expression across various cancer types The expression profile of KDM5B across all cancer types was obtained from Gene Expression Profiling Interactive Analysis (GEPIA) [ 43 , 44 ]. The University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) was used to retrieve KDM5B expression data based on various clinical and histopathological factors [ 45 – 47 ]. The TNMplot database was used to evaluate KDM5B expression in breast tumors, normal and metastatic tissues [ 48 , 49 ]. Additionally, overall survival (OS) in breast cancer patients was assessed in relation to KDM5B mRNA expression using GEPIA [ 43 , 44 ]. 5.2. Patient enrolment and sample collection Paired breast tumors and adjacent normal tissues were collected from newly diagnosed breast cancer patients aged 18 to 80 years who underwent breast conservative surgery or modified radical mastectomy at Kasturba Medical College, Manipal, India. Ethical approval for the study was granted by the Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee (IEC: 504/2019), and written informed consent was obtained from all participants. The research was performed in accordance with the Declaration of Helsinki. Clinical samples were collected in RNAprotect reagent (Qiagen, Hilden, Germany) and stored at -80ºC until further use. A certified pathologist characterized all tumor samples, and clinical parameters were retrieved from medical records. 5.3. RNA isolation and cDNA synthesis Total RNA was extracted using the RNeasy Protect Mini Kit (Qiagen, Germany). According to the manufacturer's protocol, an on-column DNase digestion step was performed with the RNAse-Free DNase Set (Qiagen, Germany). The concentration and purity of the RNA were assessed using a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific, USA). A total of 1µg of RNA from each sample was reverse transcribed into cDNA using iScript™ cDNA Synthesis Kit (BioRad, Hercules, CA, USA). 5.4. Real-Time Quantitative Polymerase Chain Reaction (qPCR) After assessing RNA quality and identifying outliers, qPCR was performed on 53 breast tumor samples and 14 unpaired breast normal tissues. The TaqMan gene expression assay kit for KDM5B (Assay ID: Hs00981910_m1, Thermo Fisher Scientific, OH, USA) was used, with beta-actin (Assay ID: Hs99999903_m1, Thermo Fisher Scientific, OH, USA) serving as the internal control. The reactions were performed using the TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific, OH, USA) on a QuantStudio 5 Real-Time PCR (Applied Biosystems, USA) under standard cycling conditions. Ct values were normalized to beta-actin, and relative expression in tumor samples was determined using normal breast tissue samples as the control. The fold change in gene expression was calculated using the 2 − ddCt method [ 50 ]. 5.5. Cell culture Human breast cancer cell lines, MDA-MB-231 (ATCC, Manassas, Virginia) and MCF-7 (ECACC, UK), were cultured at 37 º C, 5% CO 2 in minimum essential media (MEM, Gibco™), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco™), 1% non-essential amino acids (Gibco™) and 1% penicillin/streptomycin (Pen Strep, Gibco™). For subculturing, the media was aspirated, and the cells were washed with PBS (pH 7.4). Cells were passaged using 0.25% Trypsin-EDTA (Thermo Fisher Scientific, OH, USA) for 5 minutes, and the trypsinization was inhibited by adding complete media. The cells were then centrifuged and resuspended in fresh media for subsequent subculture. 5.6. Drug treatment ABC was received from Hetero Labs Limited and Mylan Laboratories Limited, while CBV (> 95% pure) was sourced from Biosynth International, Kentucky, USA. Pharmaceutical-grade DOX was procured from Fresenius Kabi India Pvt. Ltd., India. All compounds were dissolved in sterile water. MDA-MB-231 and MCF-7 cells were seeded into 96 well plates at a density of 5 x 10 3 cells/well. The next day, the cells were treated with ABC and CBV across a concentration range of 0 to 500 µM for 72 hours with daily replenishment to determine the half-maximal inhibitory concentration (IC 50 ) in monotherapy. To assess the influence of ABC on KDM5B mRNA expression, qPCR was performed using cells treated with ABC at its IC 25 concentration for 24 hours. Based on this, for combination therapy, cells were pre-treated with low-dose ABC (IC 25 ) for 24 hours, followed by DOX treatment at concentrations ranging from 0 to 160 nM every 24 hours for three days. For further 2-D in vitro assays, four treatment groups were included: 1. Control (no treatment), 2. ABC-only treatment (IC 25 dose for three days), 3. DOX-only treatment (IC 25 dose for 3 days), and 4. ABC + DOX (ABC pre-treatment at IC 25 dose for 24 hours, followed by DOX treatment at IC 25 dose for three days). 5.7. MTT assay Cell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay. Briefly, MDA-MB-231 and MCF-7 cells (5000 cells/well) were seeded in triplicates in 96-well plates and treated with increasing concentrations of drugs (0 to 500 µM) for 72 hours with daily replenishment. Following treatment, MTT dye (0.5 mg/ml) was added, and cells were incubated for 4 hours. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using an ELISA microplate reader. The IC 50 values were determined using Prism software (GraphPad Software, La Jolla, CA). To evaluate the ability of ABC to enhance DOX sensitivity, MDA-MB-231 and MCF-7 cells were pre-treated with ABC at its IC 25 concentration for 24 hours. Subsequently, cells were exposed to DOX concentrations ranging from 0-160 nM for 72 hours, with daily replenishment. The MTT assay was performed, and the IC 50 values of DOX in ABC-sensitized cells were compared to those in cells treated with DOX alone. 5.8. Soft agar colony forming assay A 24-well plate was prepared with a base layer of 0.6% agar in 2X complete media and allowed to solidify. The top agar layer (0.3%), containing 2000 cells from the respective treatment groups, was seeded onto the solidified base layer and allowed to set. The plates were incubated at 37ºC with 5% CO 2 to promote colony formation. To maintain cell growth, 100 µl of media was added to each well every three days. The assay was completed on day 28, and the colonies were stained with 0.1% crystal violet solution. Stained colonies were then observed and counted [ 51 ]. 5.9. Apoptosis assay The apoptosis study was performed using FITC-Annexin V Apoptosis Detection Kit I (BD Pharmingen™, USA) following the manufacturer's instructions [ 52 ]. Briefly, pretreated cells at a concentration of 1 x 10 6 cells/ml were washed with cold PBS and re-suspended in the 1X binding buffer provided in the kit. A 100 µl aliquot of this cell suspension (1 x 10 5 cells) was stained with 5 µl FITC Annexin V and 5 µl propidium iodide (PI) and then incubated for 15 minutes at room temperature in the dark. 400 µl of 1X binding buffer was added to the stained cells and analyzed for apoptosis using BD FACSAria™ Fusion Flow cytometer (Becton, Dickinson and Company, USA). The results were analyzed using FlowJo™ v10.8 Software (BD Life Sciences, USA). 5.10. Cell cycle analysis Pretreated cells were harvested and fixed in ice-cold 70% ethanol for 30 minutes. After fixation, the cells were rinsed with PBS and incubated with RNase A (100 µg/ml, Thermo Fisher Scientific, USA) for 30 minutes at room temperature. Subsequently, the cells were then stained with PI at a concentration of 50 µg/ml and incubated for an additional 30 minutes at room temperature in the dark. Flow cytometric analysis was performed immediately using the BD FACSAria™ Fusion Flow cytometer (Becton, Dickinson and Company, USA), with a minimum of 10,000 cells collected per sample. Data were analyzed using FlowJo™ v10.8 Software (BD Life Sciences, USA). Appropriate controls, including unstained and single-stained cells, were used to set compensation and gating parameters [ 53 ]. 5.11. 3-D Breast cancer organoid culture Breast cancer tissues were cut into 1–3 mm 3 pieces and processed to isolate viable cells. The tissue was minced and washed with 10 ml of collection medium (Advanced DMEM/F12 with 1X Glutamax, 10 mM HEPES, and antibiotics). The tissue was digested in ~ 5–10 ml breast cancer organoid medium containing 2 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO) and mixed on an orbital shaker at 37°C for 2 hours. The processed tissue suspension was subjected to successive shearing using sterilized glass Pasteur pipettes. After each shearing step, the suspension was strained through a 100 µm filter to retain larger tissue fragments. The fragments were further subjected to additional shearing with a 10 ml collection medium. To the strained suspension, 2% FBS was added, followed by centrifugation at 400 rcf. The resulting pellet was resuspended in a 10 ml collection medium and centrifuged at 400 rcf. If a visible red pellet was observed, erythrocytes were lysed using 2 ml of red blood cell lysis buffer (Cat. No: 11814389001, Roche Diagnostics, Germany) for 5 minutes at room temperature, followed by the addition of 10 ml of collection media and centrifugation at 400 rcf. The final pellet was resuspended in cold growth factor reduced Matrigel (Corning, USA) (1:1 of media and matrigel), and 100 µl of the suspension was plated in transwell chambers. 500 ml of breast cancer organoid medium was added to each well, and plates were incubated at 37ºC, 5% CO 2 . The breast cancer organoid medium contained R-Spondin 3 (Sigma-Aldrich, St. Louis, MO), Neuregulin 1, FGF 7, FGF10, and EGF (Thermo Fisher-PeproTech), along with Rock Inhibitor, HEPES, and N-Acetylcysteine (Sigma-Aldrich, St. Louis, MO) [ 20 ]. The medium was changed every 2 days, and organoids were passaged every 2–4 weeks. For passaging, dispase (Sigma-Aldrich, St. Louis, MO) was added to the transwells and incubated for 2–3 hrs. Organoids were dissociated by incubating in 2 ml of TrypLE Express (Gibco, Thermo Fisher), at 37°C for 3–5 minutes, followed by mechanical shearing with sterilized glass Pasteur pipettes. After adding 10 ml of collection medium, the suspension was centrifuged at 400 rcf, and the resulting pellet was harvested to prepare a single-cell suspension. The cells were seeded at high density and reseeded at reduced density after the first passage. Organoid culture was performed by following Hans Clevers group’s recent protocol [ 20 , 54 ]. Once the diameter of breast cancer organoids reached around 20–30 µm, they were subjected to doses of ABC (50 µM at 24, 48, 72, and 96 hours) and DOX (1 µM at 48, 72, and 96 hours), and a combination of ABC + DOX (24, 48, 72, and 96 hours). The overall shape and dimensions of the breast cancer organoids were analyzed using a brightfield microscope. Three sets of experiments were conducted per patient (n = 3 per patient), and ten representative organoids per condition were measured [ 55 , 56 ]. Organoid sizes were determined using Image J (OpenJDK 13.0.6.) [ 56 , 57 ]. 5.12. In silico molecular studies The binding interactions of CBV-TP in the human DNA polymerase β enzyme in complex with DNA were studied using the computational software Discovery Studio (DS) Structure-Based-Design (BIOVIA Inc, Dassault Systemes, USA). The CBV-TP was built in 3D using the Small Molecules module in the software and was energy minimized over 2000 steps using the Smart Minimizer, distance-dependent dielectrics, and CHARMm force field. The X-ray structure of human DNA polymerase β enzyme in complex with DNA and the nucleotide derivative 2’-deoxyuridine-5’-[(a,b)-imido]triphosphate (dUMPNPP) was obtained from PDB (PDB ID: 2FMS) [ 28 ]. The protein-DNA-nucleotide complex was prepared first, by removing water molecules. The two magnesium atoms (Mg 2+ ) in the catalytic site were retained. The bound nucleotide dUMPNPP was selected, and a binding site sphere of 8 Å was created around the nucleotide dUMPNPP. In the next step, the nucleotide was deleted. Subsequently, the Macromolecules module in the software was used to prepare the protein-DNA complex using the CHARMm force field. Molecular docking was carried out using the LibDock algorithm in DS with the following parameters: 100 hotspots, docking tolerance of 0.25 Å, implicit solvent function, and distance-dependent dielectric constant. The docking poses were energy minimized over 1000 steps using the Smart Minimizer. The CHARMm force field was used during the docking simulation. The docking poses were ranked based on the LibDock score and were analyzed by investigating the polar and nonpolar interactions of CBV-TP with human DNA polymerase β enzyme and DNA. 5.13. Statistical analysis The normality of distribution was tested using the Shapiro-Wilk test. Continuous variables were reported as either the median with an interquartile range or the mean with standard deviation, depending on the normality of the data. Differences in normally distributed data were analyzed using the Student’s t-test or one-way analysis of variance (ANOVA). The Wilcoxon Mann-Whitney test was used to compare the differences in gene expression levels between tumor and normal samples. Overall survival rates were estimated using the Kaplan–Meier method, with the median KDM5B expression level taken as the cutoff for high and low expression groups. All statistical analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software Inc., San Diego, CA, USA), with P < 0.05 considered statistically significant. Declarations Acknowledgments AJ is thankful to the Department of Science and Technology, Govt. of India, for providing the DST-INSPIRE Fellowship [IF190197] and Manipal Academy of Higher Education [MAHE], Manipal, for awarding Dr. TMA Pai fellowship and MAHE contingency fund. M.R. is thankful to the Department of Science & Technology for the DST-FIST grant (DST-FIST TPN: 32196). PK (45/20/2020-PHA/BMS) thanks the Indian Council of Medical Research (ICMR) for providing Senior Research Fellowship. NA acknowledges the SERB POWER grant from the Science and Engineering Research Board for financial support (SPG/2021/004209). The authors thank Manipal Academy of Higher Education [MAHE], Manipal, All India Institute of Medical Sciences [AIIMS] Bhopal and Global Cancer Consortium Interdisciplinary Lab, Manipal School of Life Sciences, Manipal, for providing the facilities to conduct this research. Author contributions AJ, MR, GSR, and GGS designed and supervised the study. AJ acquired the data, performed the analysis and interpretation, and drafted the manuscript. sPPNR and GGS conducted the molecular docking studies, and SV guided with bioinformatics analysis. MP and BB performed the 3-D culture experiments. AJ, PK, NA, and MM coordinated, conducted, and analyzed the 2-D culture experiments. AJ, MR, NK, NU, GSR, and RRD collected, analyzed and interpreted the patient data and critically reviewed the manuscript. All authors read and approved the final manuscript. Competing interests The author(s) declare no competing interests. Data availability All data supporting the findings of this study are available within the paper and its Supplementary Information. Ethics declarations The Ethical approval for the study was granted by the Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee (IEC: 504/2019), and written informed consent was obtained from all participants. The research was performed in accordance with the Declaration of Helsinki. Funding This work was supported by the Department of Science and Technology, Govt. of India INSPIRE Fellowship [IF190197] and the Manipal Academy of Higher Education [MAHE] contingency fund [190600118]. References Bray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 74 , 229–263 (2024). Turashvili, G. & Brogi, E. Tumor Heterogeneity in Breast Cancer. Front. Med. 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The horizontal axis cancer name in red indicates high expression, green indicates low expression, and black indicates no statistical significance; \u003cstrong\u003e(B-E) \u003c/strong\u003eUALCAN analysis of expression profiles of KDM5B in breast cancer across different sample types \u003cstrong\u003e(B)\u003c/strong\u003e, stages \u003cstrong\u003e(C)\u003c/strong\u003e, subclasses \u003cstrong\u003e(D)\u003c/strong\u003e, and menopausal status \u003cstrong\u003e(E)\u003c/strong\u003e. *\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","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6208718/v1/b9453b03eb6f6b03865d5add.png"},{"id":79822479,"identity":"d6b8dd7d-1523-4877-b21b-4fcb05457226","added_by":"auto","created_at":"2025-04-03 09:07:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":495792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e KDM5B mRNA expression levels in normal and breast cancer tissues; \u003cstrong\u003e(B)\u003c/strong\u003e KDM5B mRNA expression following ABC treatment (IC\u003csub\u003e25\u003c/sub\u003e) for 24 hours in MDA-MB-231 and MCF-7 cells; \u003cstrong\u003e(C) \u003c/strong\u003eRepresentative images of cell viability analysis using MTT assay demonstrated enhanced DOX cytotoxicity upon ABC sensitization in MDA-MB-231 and \u003cstrong\u003e(D)\u003c/strong\u003e MCF-7 cells; \u003cstrong\u003e(E) \u003c/strong\u003eBar graphs depicting a significant reduction in the IC\u003csub\u003e50\u003c/sub\u003e of DOX upon ABC sensitization. Data is expressed as the mean ± SD of three independent experiments; \u003cstrong\u003e(F)\u003c/strong\u003e Soft agar colony formation assay with three treatment groups and an untreated control group in MDA-MB-231 and MCF-7 cells. Bar graph data is expressed as the mean ± SD of three independent experiments. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ****\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6208718/v1/e6bfeebc81652391a7af572f.png"},{"id":79824075,"identity":"a1ca47fd-3a88-4f05-bca1-015e0e598fb8","added_by":"auto","created_at":"2025-04-03 09:15:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1203744,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of cell\u003cstrong\u003e \u003c/strong\u003ecycle analysis by flow cytometry showing ABC treatment induced S phase arrest, whereas ABC+DOX had more cells in the S/G2 phase in \u003cstrong\u003e(A)\u003c/strong\u003eMDA-MB-231 and \u003cstrong\u003e(B)\u003c/strong\u003e MCF-7 cells.\u003cstrong\u003e \u003c/strong\u003eBar graph data are expressed as the mean ± SD of three independent experiments. *\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","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6208718/v1/4fd8ce78c97348485cf775e3.png"},{"id":79822483,"identity":"4bc6e7b6-8097-453a-b192-d203328fca79","added_by":"auto","created_at":"2025-04-03 09:07:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":643307,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of Annexin V apoptosis study by flow cytometry showing ABC+DOX treatment-induced late apoptosis in \u003cstrong\u003e(A)\u003c/strong\u003e MDA-MB-231; \u003cstrong\u003e(B)\u003c/strong\u003e MCF-7 cells. Bar graph data are expressed as the mean ± SD of three independent experiments. *\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","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6208718/v1/618839b0a74d54d82a7c0dcb.png"},{"id":79822489,"identity":"163c08c3-fc84-43d3-8e5a-a705830b408d","added_by":"auto","created_at":"2025-04-03 09:07:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1265476,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative bright-field images of patient-derived breast cancer organoid phenotypes. Two types of breast cancer organoids were observed in different patients: (\u003cstrong\u003eA, B)\u003c/strong\u003e Solid-dense and \u003cstrong\u003e(C)\u003c/strong\u003eDiscohesive. \u003cstrong\u003e(D) \u003c/strong\u003eSensitivity of patient-derived breast cancer organoids to Abacavir (ABC), Doxorubicin (DOX), and their combination. Representative bright field images of breast cancer organoids treated with ABC (50 μM) and DOX (1 µM). Scale = 100 µm. Patients 1, 2, 3, and 4 are represented as n = 1, n = 2, n = 3, and n = 4. The lower panel shows zoomed-up images of the discohesive breast cancer organoids.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6208718/v1/cb611993cd22bdc9badc3dcc.png"},{"id":79822496,"identity":"c256af79-816d-4c3c-9ea4-0eb1800ea91b","added_by":"auto","created_at":"2025-04-03 09:07:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":28846,"visible":true,"origin":"","legend":"\u003cp\u003eGraph representing the diameter of breast cancer organoids from patients 1 and 2 treated with ABC, DOX, and ABC+DOX and compared with untreated. Data are presented as mean ± standard deviation. Data from patients 1 and 2 are used for plotting the graph. Each experiment was done in triplicates, and 10 spheres per condition were used to measure diameter size. ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6208718/v1/d6c57a8bd6526acc054838e0.png"},{"id":79824079,"identity":"34f93796-8c3c-4fb9-915f-2f150ade0a10","added_by":"auto","created_at":"2025-04-03 09:15:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1859038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Abacavir (ABC) is a prodrug that undergoes intracellular bioconversion to form abacavir monophosphate (ABC-MP), carbovir monophosphate (CBV-MP) and the active metabolite carbovir triphosphate (CBV-TP). \u003cstrong\u003e(B)\u003c/strong\u003e Interactions of CBV-TP (ball and stick cartoon) with human DNA polymerase β (ribbon diagram) and DNA substrate complex; \u003cstrong\u003e(C)\u003c/strong\u003e Close-up view of CBV-TP interactions with DNA polymerase β and DNA with key interacting residues highlighted.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6208718/v1/beefaa9e9a0b7b1de9f051ed.png"},{"id":88814154,"identity":"be434b43-1c9f-4d7a-9d72-0dc2a3b8f1f0","added_by":"auto","created_at":"2025-08-11 16:07:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7935301,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6208718/v1/b7b2fed4-6463-42bf-8527-fbcc8ff3907a.pdf"},{"id":79822485,"identity":"f14afed1-135e-4f40-9c66-3df710ad2fff","added_by":"auto","created_at":"2025-04-03 09:07:36","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4083038,"visible":true,"origin":"","legend":"","description":"","filename":"SupplimentaryScientificReports.docx","url":"https://assets-eu.researchsquare.com/files/rs-6208718/v1/98fd34bedc0160caeb0408fe.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Abacavir enhances the efficacy of Doxorubicin via inhibition of histone demethylase KDM5B in breast cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast cancer remains the most prevalent cancer among women worldwide, with an estimated 2.2\u0026nbsp;million new cases and 0.6\u0026nbsp;million deaths reported in 2022 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Tumor heterogeneity in breast cancer results in significant variability in pathology, molecular alterations, and tumor microenvironment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While the development in early diagnosis and treatment modalities has improved overall survival rates of breast cancer, the effective management of breast cancer still faces challenges [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Since breast cancer is influenced by a combination of genetic and epigenetic factors, a detailed understanding of the underlying molecular mechanisms is necessary to improve therapeutic options. Although epigenetic modifications do not cause alteration in DNA sequences, they can significantly influence cancer development at different stages. Hence, targeting epigenetic markers has a substantial role in cancer detection, prevention, and development of targeted therapies [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For example, drugs targeting epigenetic modifying enzymes, such as histone deacetylases (HDACs) and DNA methyltransferases, have received FDA approval [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, combining epigenetic targeting drugs (e.g. decitabine, SAHA, etc.) with conventional chemotherapies has also received wide attention [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eKDM5B (Lysine demethylase 5B), or JARID1B, encodes lysine-specific histone demethylase from the Jumanji (JmjC)/ARID domain-containing family of histone demethylases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. This protein plays a pivotal role in the transcriptional repression of genes by demethylating mono-, di-, and trimethylated lysine 4 of histone 3 (H3K4) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. KDM5B is significantly upregulated in various cancers, particularly breast cancer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and its oncogenic properties make it a promising target for personalized drug therapy [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the prognostic significance and drug-targeting potential of KDM5B have not been fully explored. In this context, repurposing existing drugs could be a valuable strategy, owing to the established safety profile, cost-effectiveness, shorter development times, and affordability [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAbacavir (ABC), a nucleoside reverse transcriptase inhibitor (NRTI), is commonly used in the treatment of human immunodeficiency virus type 1 (HIV-1) infection. ABC is generally well tolerated with minimal adverse effects and favorable bioavailability [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Upon cellular uptake, ABC undergoes bioconversion to form the active metabolite carbovir triphosphate (CBV-TP) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In a previous study, molecular docking analysis identified the key interactions of ABC towards the JmjC domain of KDM5B [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, former studies have demonstrated the potential of ABC as an anticancer agent in various cancers [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In this regard, we conducted a detailed study to evaluate the repurposing potential of ABC by targeting the KDM5B oncogene in breast cancer. KDM5B gene expression was determined in breast cancer patient samples, and the cytotoxic activity and the effect of ABC sensitization on DOX were investigated in 2-D and 3-D cultures. The results from our study demonstrated that ABC could potentiate the cytotoxicity of DOX in breast cancer cells. This approach enhanced the efficacy of DOX, which could allow for further dose reduction leading to lower side effects, thus offering a promising strategy for breast cancer therapy.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. KDM5B is over-expressed in breast cancer: Evidence from public databases\u003c/h2\u003e \u003cp\u003eComprehensive gene expression profiling of KDM5B across tumor and normal tissues was evaluated using the GEPIA database. The KDM5B gene expression was elevated in most cancers compared to normal tissues, with significant overexpression observed in breast invasive carcinoma, pancreatic adenocarcinoma, and thymoma (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Further analysis using UALCAN confirmed that KDM5B gene expression was significantly higher in breast tumors than in normal breast tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, RNA-Seq data from the TNMplot database demonstrated increased KDM5B expression in both primary breast tumors and metastatic breast cancer samples compared to normal breast tissue (see Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online). Among tumor stages, early-stage tumors exhibited slightly higher KDM5B expression than advanced stages, although all tumor stages showed significant upregulation relative to normal tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Across all breast cancer subtypes, KDM5B expression was marginally higher in the luminal subtype, followed by HER2 positive and triple negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Menopausal status also correlated with KDM5B expression. Patients in the premenopausal stage showed significantly higher KDM5B levels compared to those in the perimenopausal, and post-menopausal stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Additional histopathological and clinical parameters, including patient\u0026rsquo;s age, race, nodal metastasis, histologic subtypes, and TP53 mutation status, were significantly associated with KDM5B overexpression (see Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e online). Overall survival (OS) in breast cancer patients with KDM5B mRNA expression was analyzed using the GEPIA database (see Supplementary Fig. S2 online). KDM5B expression was not significantly associated with OS in breast cancer patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. KDM5B is over-expressed in clinical breast tumor samples\u003c/h2\u003e \u003cp\u003eThe results obtained in databases were validated in clinical tumor samples. Towards this, qPCR was performed on 53 breast cancer and 14 normal breast tissue samples to evaluate KDM5B expression. In line with global data, KDM5B expression was significantly elevated in breast tumors compared to normal breast tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, no significant differences in KDM5B expression were observed across various clinicopathological factors. Detailed patient characteristics and clinicopathological data in relation to KDM5B mRNA expression are provided in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical analysis of KDM5B expression levels in clinical breast cancer tissues in relation to clinicopathological characteristics. ER: Estrogen receptor; DCIS: Ductal carcinoma in situ; HER2: Human epidermal growth factor receptor 2; NST: No special type; IQR: Inter quartile range; PR: Progesterone receptor; TNBC: Triple-negative breast cancer.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClinicopathological features\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample size (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eKDM5B mRNA expression\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedian\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIQR (Q1, Q3)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.610, 9.381\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003e\u0026lt;\u0026thinsp;0.0001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.740\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.557,1.556\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge (years)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.588, 8.594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.414\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.521, 14.850\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTumor histology\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInvasive breast carcinoma NST\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.599\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.546, 10.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.847\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOther (DCIS, Mixed, Metaplastic carcinoma, etc)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.809\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.405, 7.803\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMenopausal status\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePre-menopausal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.588, 8.594\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.733\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePost-menopausal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.402\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.523, 11.190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePathological grade\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrade 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.702\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.417, 8.185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.639\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrade 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.551, 15.040\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGrade 3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.668, 10.170\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTNM stage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.365, 5.982\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.576\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.586\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.625, 11.780\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.887\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.154, 7.596\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.529, 19.770\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNodal involvement\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.788\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.617, 10.760\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e0.150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2.405\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.116, 11.740\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.417\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.693, 5.138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIII\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.927, 12.040\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eER\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eER+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.854\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.351, 10.360\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.302\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eER-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.208, 8.662\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePR+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.138, 8.287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.978\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePR-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.138\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.529, 10.950\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHER2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHER2+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.453\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.292, 16.900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.614\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHER2-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.564, 9.775\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMolecular subtypes\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuminal A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.784\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.387, 10.210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e0.944\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLuminal B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.610, 9.572\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTNBC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.063, 13.230\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHER2 enriched\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.525, 12.850\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnknown\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4.161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Repurposing of the antiviral drug ABC targeting KDM5B, sensitized breast cancer cells to DOX\u003c/h2\u003e \u003cp\u003eWe previously reported the potential of repurposing antiviral drugs targeting the JmjC domain of KDM5B through computational analysis, including molecular docking studies of ABC [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Furthermore, former literatures demonstrate the potential of ABC as an anticancer agent in various cancers [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To assess its therapeutic efficacy in breast cancer cells, we examined the cytotoxic effects of ABC at concentrations ranging from 0 to 500 \u0026micro;M in MDA-MB-231 and MCF-7 cells. Interestingly, ABC exhibited significant cytotoxicity at clinically relevant concentrations in both cell lines (MDA-MB-231 IC\u003csub\u003e50\u003c/sub\u003e: 289.15\u0026thinsp;\u0026plusmn;\u0026thinsp;32.25 \u0026micro;M; MCF-7 IC\u003csub\u003e50\u003c/sub\u003e: 281.26\u0026thinsp;\u0026plusmn;\u0026thinsp;51.59 \u0026micro;M) (see Supplementary Fig. S3 online).\u003c/p\u003e \u003cp\u003eIn addition, to investigate whether ABC treatment modulates KDM5B expression, we analyzed its levels in ABC-treated cells. Cells were exposed to a low dose of ABC (IC\u003csub\u003e25\u003c/sub\u003e) for 24 hours, followed by a 3-day media change. Compared to untreated control cells, KDM5B expression was reduced in both cell lines, with a significant decrease observed in ABC-treated MDA-MB-231 cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eSince ABC treatment influenced KDM5B gene expression and KDM5B is known to promote cancer cell proliferation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], we further examined whether ABC could enhance the sensitivity of breast cancer cells to DOX treatment. Pre-treatment with a low dose of ABC (IC\u003csub\u003e25\u003c/sub\u003e dose), followed by DOX exposure (0-160 nM), significantly enhanced cytotoxicity, reducing cell proliferation more efficiently than DOX alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC \u0026amp; \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). In the absence of ABC, MDA-MB-231 cells demonstrated enhanced resistance to DOX (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;155.67\u0026thinsp;\u0026plusmn;\u0026thinsp;17.88 nM), compared to MCF-7 cells (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;47.68\u0026thinsp;\u0026plusmn;\u0026thinsp;17.10 nM). However, in ABC-sensitised cells, the IC\u003csub\u003e50\u003c/sub\u003e of DOX was significantly reduced to 65.03\u0026thinsp;\u0026plusmn;\u0026thinsp;19.14 nM in MDA-MB-231 cells, representing a 2.4-fold decrease (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and to 20.45\u0026thinsp;\u0026plusmn;\u0026thinsp;11.91 nM in MCF-7 cells indicating a 2.3-fold reduction (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), demonstrating enhanced cytotoxicity compared to DOX alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Subsequently, a comparison of three pretreated groups (ABC, DOX, ABC\u0026thinsp;+\u0026thinsp;DOX) with untreated control displayed a significant reduction in colony forming property of breast cancer cells with ABC\u0026thinsp;+\u0026thinsp;DOX treatment, as confirmed by soft agar colony forming assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, and Supplementary Fig. S4 online). The MDA-MB-231 cells showed a greater reduction in colony formation in response to ABC\u0026thinsp;+\u0026thinsp;DOX treatment compared to MCF-7 cells, thereby demonstrating the potential of ABC as a sensitizer for conventional anti-cancer agents for the management of triple-negative breast cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e2.4. Pre-treatment of ABC followed by DOX enhanced apoptosis and cell cycle arrest in breast cancer cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe investigated the effects of ABC on cell cycle and apoptosis in MDA-MB-231 and MCF-7 cells. Treatment with ABC alone for 72 hours (IC\u003csub\u003e25\u003c/sub\u003e, added every 24 hours) induced S-phase arrest in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Combined treatment with ABC (IC\u003csub\u003e25\u003c/sub\u003e for 24 hours) and DOX (IC\u003csub\u003e25\u003c/sub\u003e, for 72 hours) resulted in an arrest at the S/G2 phase. A more evident cell cycle arrest with ABC sensitization was observed in MDA-MB-231 cells compared to MCF-7 cells. In addition, apoptosis study results showed that treatment with ABC alone slightly increased the cell population undergoing late apoptosis in both cell lines, with a slight increase in early-phase apoptosis observed in MCF-7 cells. Combined treatment with ABC (IC\u003csub\u003e25\u003c/sub\u003e for 24 hours) and DOX (IC\u003csub\u003e25\u003c/sub\u003e for 72 hours) significantly increased the percentage of cells in late-phase apoptosis in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDNA replication occurs during the S phase, while the G2 phase prepares the cell for mitotic division. Arresting cells in the S/G2 phase indicates disruption of cell cycle progression, effectively inhibiting cell proliferation. Our findings suggest that, as ABC can induce DNA damage, it can lead to transient S/G2-phase arrest and apoptosis. This dual strategy of inducing S/G2 phase arrest and DNA damage represents an effective approach for inhibiting breast cancer cell proliferation and inducing cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Generation of patient-derived breast cancer organoids\u003c/h2\u003e \u003cp\u003eFour patient breast cancer samples were obtained under informed consent and used to generate patient-derived breast cancer organoids, as described in the methods section. We observed two types of breast cancer organoid phenotypes, including solid dense (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B) and discohesive (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The solid-dense phenotype was associated with three patients, while the discohesive one was associated with one patient. Both the solid and discohesive organoids showed rapid growth. The discohesive organoids were present in clusters without smooth boundaries.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.6. ABC enhances the cytotoxic effect of DOX on patient-derived breast cancer organoids\u003c/h2\u003e \u003cp\u003eWe used the antiretroviral nucleoside analog ABC as a single drug and combined it with DOX to determine its role in promoting DOX-mediated cytotoxicity. Based on our \u003cem\u003ein vitro\u003c/em\u003e data and other published work, the breast cancer organoids were exposed to 50 \u0026micro;M ABC and 1 \u0026micro;M DOX, respectively [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. We used the bright-field images to analyze the growth rate (diameter) of the organoids treated with ABC, DOX, and their combination and compared them with untreated breast cancer organoid controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Though the ABC-treated breast cancer organoid looked smaller than the untreated, we did not observe a statistical significance associated with this arm of treatment. DOX showed a substantial decrease in the diameter of breast cancer organoids, suggesting that our model is functional. Moreover, the organoids showed a dark core reminiscent of apoptotic cells.\u003c/p\u003e \u003cp\u003eInterestingly, the combination yielded strong inhibition of breast cancer organoid proliferation (diameter), especially in patients 1 (n\u0026thinsp;=\u0026thinsp;1) and 2 (n\u0026thinsp;=\u0026thinsp;2) with solid, dense organoid phenotypes. Patient 3 (n\u0026thinsp;=\u0026thinsp;3) did not significantly differ in the DOX vs ABC\u0026thinsp;+\u0026thinsp;DOX combination, suggesting patient genetic diversity. In patient 4 (n\u0026thinsp;=\u0026thinsp;4) with discohesive breast cancer organoid phenotype, the data looked interesting, but it is difficult to ascertain the diameter of each organoid conclusively. Hence, we used data from patients 1 and 2 to plot the diameter of the breast cancer organoids post-treatment to evaluate the sensitivity of ABC, DOX, and ABC\u0026thinsp;+\u0026thinsp;DOX compared to untreated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Some representative images used for the data analysis are shown in the Supplementary Fig. S5 online. The data (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) reveals that ABC, in combination with DOX, can be an effective therapeutic option for some patients. Genetic variability can be at the heart of this observation. Because telomerase activity is a promising therapeutic target in breast cancer, and recent reports establish that ABC is functional against telomerase high medulloblastoma cells, our patient-derived breast cancer organoids can be an interesting strategy for determining the effectiveness of therapeutic regimen based on ABC and DOX [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, this warrants further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Molecular studies reveal CBV-TP interaction with human DNA Polymerase β and DNA Complex\u003c/h2\u003e \u003cp\u003eThe results obtained from this study show that ABC pretreatment enhanced the cytotoxicity of DOX to MCF-7 and MDA-MB-231 cells compared to DOX monotherapy. This was an exciting finding from our study. In this regard, there is previous literature that has demonstrated that nucleoside derivatives exhibit anticancer activity by getting incorporated into the DNA synthesis cycle through the DNA polymerase enzymes, including DNA polymerase β [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. For example, the purine nucleoside cladribine is known to exhibit anticancer activity by inhibiting DNA polymerase enzymes [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. It is pertinent to note that these nucleoside derivatives undergo a series of intracellular bioconversions to form the active triphosphate derivatives. In the case of ABC, it undergoes bioconversion to form the active metabolite carbovir triphosphate (CBV-TP, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, we investigated the interactions of CBV-TP in the DNA polymerase β-DNA complex using the solved X-ray structure [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe top binding mode of CBV-TP in the DNA polymerase β-DNA complex shows that it was oriented in the catalytic site closer to magnesium atoms and the DNA base pairs, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB. The planar, bicyclic guanosine ring of CBV-TP underwent multiple π-π stacked interactions with the cytosine ring of the DNA nucleotide cytidine (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Both the imidazole and pyrimidine rings of CBV-TP were in contact with the DNA base (distance\u0026thinsp;\u0026lt;\u0026thinsp;4.2 \u0026Aring;). Furthermore, the triphosphate ester moiety of CBV-TP underwent polar interactions with amino acids Ser180, and Arg183 of DNA polymerase β (distance\u0026thinsp;\u0026lt;\u0026thinsp;2.0 \u0026Aring;). Also, it underwent a number of electrostatic interactions with the two catalytic magnesium atoms, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC (distance\u0026thinsp;\u0026lt;\u0026thinsp;3.5 \u0026Aring;). These molecular docking studies suggest that CBV-TP can interact with the DNA polymerase β-DNA complex and has the potential to get incorporated into the DNA synthesis cycle causing cell cycle arrest in tumor cells. This is further supported by a previous work that demonstrated the ability of CBV-TP to inhibit DNA polymerases [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eEpigenetic modifications, including DNA methylation and histone modifications, play a vital role in cancer development, either by downregulation of tumor suppressor genes or by upregulation of oncogenes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Targeting these mechanisms depicts a promising approach to develop novel anti-cancer drugs [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Several studies have emphasized the role of combining epigenetic therapies with conventional treatments, such as chemotherapy and radiation therapy. These combinatorial approaches have proven the ability to enhance therapeutic efficacy, improve sensitivity, and reduce the toxicity associated with conventional therapies [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe oncogenic significance of KDM5B in breast cancer was confirmed through the analysis of publicly accessible databases. mRNA expression profiles from TCGA datasets revealed significantly higher KDM5B expression levels in breast tumors compared to normal tissues. This finding aligns with our qPCR analysis of 53 breast tumor samples, which confirmed KDM5B overexpression in tumor samples compared to adjacent normal tissues. Our patient cohort exhibited a slightly higher median KDM5B gene expression in luminal subtypes. This is consistent with the established role of KDM5B as a luminal lineage-driving oncogene. Supporting this, Yamamoto et al. demonstrated that high KDM5B activity correlates with poor outcomes in ER\u0026thinsp;+\u0026thinsp;tumors [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, larger patient cohorts are required in our study to establish a significant statistical association with other clinical parameters.\u003c/p\u003e \u003cp\u003eDespite numerous KDM inhibitors being developed in various laboratories, no drugs specifically targeting KDM5B have been approved by the FDA for the management of breast cancer. In this regard, drug repurposing offers an efficient strategy for identifying new therapeutic applications for existing drugs, with fewer time constraints than the traditional drug discovery and development process [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. More specifically, anti-viral drugs have been shown to sensitize resistant cancer cells to chemotherapy, thereby increasing the efficacy of other therapeutic approaches. For example, ribavirin has been identified as an EZH2 inhibitor [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], while acyclovir has shown the ability to inhibit the proliferation and colony-forming properties of breast cancer cells [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this context, the current study explored ABC's repurposing potential for managing breast cancer.\u003c/p\u003e \u003cp\u003eDoxorubicin, an anthracycline derivative that targets topoisomerase II, is widely utilized for various cancers, including breast cancer, carcinomas, sarcomas, and hematological malignancies [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Despite the extensive use of doxorubicin, dose-dependent toxicity, particularly cardiotoxicity, remains a significant concern [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In this regard, several approaches have been explored to reduce these adverse effects, including the combination of antioxidants, advanced drug delivery systems, and prodrug development [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, some of these approaches have not successfully translated into effective clinical outcomes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thus, innovative strategies are necessary to enhance therapeutic outcomes. Towards this, our study highlights, for the first time, that epigenetic targeting of KDM5B oncogene by ABC, when combined with DOX, induces cytotoxic effects within a minimal dose range of DOX and may potentially negate the side effects, including cardiotoxicity.\u003c/p\u003e \u003cp\u003eWe conducted a series of \u003cem\u003ein vitro\u003c/em\u003e experiments to compare the effects of ABC and DOX, both individually as well as in combination, on MCF-7 and MDA-MB-231 breast cancer cell lines. Our results demonstrated that ABC inhibited cell proliferation and induced cytotoxicity. This is in accordance with a previous study showing that ABC causes a dose-dependent decrease in the proliferation rate of medulloblastoma cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, ABC induced apoptosis by arresting cells in the S phase, consistent with findings reported by Tada et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Our patient-derived breast cancer organoid data suggest that some patients may not be sensitive to this treatment, possibly due to inherent genetic variability. Besides, recent studies have demonstrated the efficacy of ABC against telomerase-high medulloblastoma cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Therefore, the patient-derived breast cancer organoids developed in this study may be utilized as a precision medicine approach for deciding the treatment regimen based on ABC for breast cancer patients. However, further investigation is needed to validate our findings.\u003c/p\u003e \u003cp\u003eNucleoside derivatives like ABC exert anti-cancer effects by integrating into the DNA synthesis cycle through DNA polymerase enzymes such as DNA polymerase β [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In cells, ABC is metabolized to its active form, CBV-TP [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], which is subsequently incorporated into chromosomal DNA by replicative DNA polymerases. This incorporation results in premature termination of DNA replication, replication fork collapse, and the formation of DNA double-strand breaks (DSBs) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Tada et al. confirmed that therapeutic concentrations of ABC induce DSBs in adult T-cell leukemia (ATL) cells, suggesting that efficient induction of DSBs is the primary mechanism underlying ABC\u0026rsquo;s cytotoxicity [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, a study by Rossi et al. also supports the DNA damage induced by ABC and suggests that its antiproliferative effect is accompanied by the inhibition of telomerase activity in medulloblastoma cell lines [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our computational analysis further supports these findings by demonstrating that CBV-TP interacts with the DNA polymerase β-DNA complex, thereby emphasizing its role in causing cell death by disrupting the DNA synthesis in tumor cells.\u003c/p\u003e \u003cp\u003eWe also evaluated the expression of KDM5B following ABC treatment and found that ABC reduced its expression, indicating the impact of ABC exposure on KDM5B regulation. This finding is supported by the previous study that explored the repurposing potential of ABC through molecular docking studies, targeting the JmjC domain of KDM5B [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. KDM5B also functions as a genome stabilizer and plays a crucial role in the DNA damage response [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Inhibition of KDM5B disrupted the DNA repair mechanism, as demonstrated by Bayo et al., which showed that the small molecule JIB-04 enhanced the sensitivity of lung cancer cells to radiation by inhibiting KDM5B [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, the current study showed increased sensitivity of breast cancer cells towards DOX treatment following ABC pre-treatment. More specifically, our findings suggest that ABC causes DNA damage by being incorporated into the DNA synthesis cycle, leading to the formation of DSB while simultaneously inhibiting KDM5B, which is a critical factor in DNA damage repair. Inhibition of KDM5B exacerbates the accumulation of DNA damage, sensitizing cells to additional DNA-damaging agents such as DOX. Therefore, combining ABC and DOX might amplify the disruption of DNA repair pathways, resulting in enhanced cytotoxicity. These dual actions create a mechanistic synergy, where the inability to repair damaged DNA leads to increased cell death and may also reduce the DOX dosing regimen, potentially lowering its adverse effects. This provides a strong rationale for the combined use of ABC and DOX in breast cancer therapy.\u003c/p\u003e \u003cp\u003eOur study provides foundational data, emphasizing the need for further investigation in animal models or patient-derived xenografts to fully assess the therapeutic potential and translate these findings into clinical settings. Furthermore, studies involving larger patient cohorts are essential to establish significant correlations between KDM5B expression and clinical outcomes. A comprehensive proteomic and transcriptomic analysis focusing on the epigenetic regulation of KDM5B and its downstream targets will provide deeper insights into how ABC disrupts KDM5B-mediated oncogenesis. These future studies could pave the way for early-phase clinical trials in breast cancer patients to evaluate the safety, tolerability, and preliminary efficacy of ABC as an adjuvant to DOX.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eThe current study highlights the over-expression of KDM5B in human breast tumors compared to non-neoplastic breast tissues in global databases as well as in our patient cohort. \u003cem\u003eIn vitro\u003c/em\u003e studies showed that ABC treatment reduced KDM5B expression in breast cancer cells and increased their sensitivity towards DOX treatment. ABC induced late apoptosis and S phase arrest, while the ABC\u0026thinsp;+\u0026thinsp;DOX combination led to S/G2 phase arrest, late apoptosis, and cell death. Drug treatment studies in the patient-derived breast tumoroids supported the 2-D cell line-based findings. Additionally, molecular docking studies indicated that CBV-TP could interact with the DNA polymerase β-DNA complex, suggesting its potential mechanism to be incorporated into the DNA synthesis cycle, leading to cell cycle arrest in tumor cells. Our findings highlight the repurposing potential of ABC, a well-established antiviral agent, as a novel therapeutic approach targeting the KDM5B oncogene in breast cancer. This strategy may enhance the efficacy of DOX and could potentially help in reducing its dose, side effects, and drug resistance, thereby offering a promising approach for personalized breast cancer treatment.\u003c/p\u003e"},{"header":"5. Methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e5.1. KDM5B gene expression across various cancer types\u003c/h2\u003e \u003cp\u003eThe expression profile of KDM5B across all cancer types was obtained from Gene Expression Profiling Interactive Analysis (GEPIA) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The University of Alabama at Birmingham Cancer Data Analysis Portal (UALCAN) was used to retrieve KDM5B expression data based on various clinical and histopathological factors [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The TNMplot database was used to evaluate KDM5B expression in breast tumors, normal and metastatic tissues [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Additionally, overall survival (OS) in breast cancer patients was assessed in relation to KDM5B mRNA expression using GEPIA [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5.2. Patient enrolment and sample collection\u003c/h2\u003e \u003cp\u003ePaired breast tumors and adjacent normal tissues were collected from newly diagnosed breast cancer patients aged 18 to 80 years who underwent breast conservative surgery or modified radical mastectomy at Kasturba Medical College, Manipal, India. Ethical approval for the study was granted by the Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee (IEC: 504/2019), and written informed consent was obtained from all participants. The research was performed in accordance with the Declaration of Helsinki. Clinical samples were collected in RNAprotect reagent (Qiagen, Hilden, Germany) and stored at -80\u0026ordm;C until further use. A certified pathologist characterized all tumor samples, and clinical parameters were retrieved from medical records.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e5.3. RNA isolation and cDNA synthesis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted using the RNeasy Protect Mini Kit (Qiagen, Germany). According to the manufacturer's protocol, an on-column DNase digestion step was performed with the RNAse-Free DNase Set (Qiagen, Germany). The concentration and purity of the RNA were assessed using a NanoDrop\u0026reg; ND-1000 spectrophotometer (Thermo Scientific, USA). A total of 1\u0026micro;g of RNA from each sample was reverse transcribed into cDNA using iScript\u0026trade; cDNA Synthesis Kit (BioRad, Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5.4. Real-Time Quantitative Polymerase Chain Reaction (qPCR)\u003c/h2\u003e \u003cp\u003eAfter assessing RNA quality and identifying outliers, qPCR was performed on 53 breast tumor samples and 14 unpaired breast normal tissues. The TaqMan gene expression assay kit for KDM5B (Assay ID: Hs00981910_m1, Thermo Fisher Scientific, OH, USA) was used, with beta-actin (Assay ID: Hs99999903_m1, Thermo Fisher Scientific, OH, USA) serving as the internal control. The reactions were performed using the TaqMan\u0026reg; Fast Advanced Master Mix (Thermo Fisher Scientific, OH, USA) on a QuantStudio 5 Real-Time PCR (Applied Biosystems, USA) under standard cycling conditions. Ct values were normalized to beta-actin, and relative expression in tumor samples was determined using normal breast tissue samples as the control. The fold change in gene expression was calculated using the 2\u003csup\u003e\u0026minus;\u0026thinsp;ddCt\u003c/sup\u003e method [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.5. Cell culture\u003c/h2\u003e \u003cp\u003eHuman breast cancer cell lines, MDA-MB-231 (ATCC, Manassas, Virginia) and MCF-7 (ECACC, UK), were cultured at 37\u003csup\u003e\u0026ordm;\u003c/sup\u003eC, 5% CO\u003csub\u003e2\u003c/sub\u003e in minimum essential media (MEM, Gibco\u0026trade;), supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco\u0026trade;), 1% non-essential amino acids (Gibco\u0026trade;) and 1% penicillin/streptomycin (Pen Strep, Gibco\u0026trade;). For subculturing, the media was aspirated, and the cells were washed with PBS (pH 7.4). Cells were passaged using 0.25% Trypsin-EDTA (Thermo Fisher Scientific, OH, USA) for 5 minutes, and the trypsinization was inhibited by adding complete media. The cells were then centrifuged and resuspended in fresh media for subsequent subculture.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e5.6. Drug treatment\u003c/h2\u003e \u003cp\u003eABC was received from Hetero Labs Limited and Mylan Laboratories Limited, while CBV (\u0026gt;\u0026thinsp;95% pure) was sourced from Biosynth International, Kentucky, USA. Pharmaceutical-grade DOX was procured from Fresenius Kabi India Pvt. Ltd., India. All compounds were dissolved in sterile water.\u003c/p\u003e \u003cp\u003eMDA-MB-231 and MCF-7 cells were seeded into 96 well plates at a density of 5 x 10\u003csup\u003e3\u003c/sup\u003e cells/well. The next day, the cells were treated with ABC and CBV across a concentration range of 0 to 500 \u0026micro;M for 72 hours with daily replenishment to determine the half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) in monotherapy. To assess the influence of ABC on KDM5B mRNA expression, qPCR was performed using cells treated with ABC at its IC\u003csub\u003e25\u003c/sub\u003e concentration for 24 hours. Based on this, for combination therapy, cells were pre-treated with low-dose ABC (IC\u003csub\u003e25\u003c/sub\u003e) for 24 hours, followed by DOX treatment at concentrations ranging from 0 to 160 nM every 24 hours for three days. For further 2-D \u003cem\u003ein vitro\u003c/em\u003e assays, four treatment groups were included: 1. Control (no treatment), 2. ABC-only treatment (IC\u003csub\u003e25\u003c/sub\u003e dose for three days), 3. DOX-only treatment (IC\u003csub\u003e25\u003c/sub\u003e dose for 3 days), and 4. ABC\u0026thinsp;+\u0026thinsp;DOX (ABC pre-treatment at IC\u003csub\u003e25\u003c/sub\u003e dose for 24 hours, followed by DOX treatment at IC\u003csub\u003e25\u003c/sub\u003e dose for three days).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e5.7. MTT assay\u003c/h2\u003e \u003cp\u003eCell viability was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide) assay. Briefly, MDA-MB-231 and MCF-7 cells (5000 cells/well) were seeded in triplicates in 96-well plates and treated with increasing concentrations of drugs (0 to 500 \u0026micro;M) for 72 hours with daily replenishment. Following treatment, MTT dye (0.5 mg/ml) was added, and cells were incubated for 4 hours. Formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm using an ELISA microplate reader. The IC\u003csub\u003e50\u003c/sub\u003e values were determined using Prism software (GraphPad Software, La Jolla, CA).\u003c/p\u003e \u003cp\u003eTo evaluate the ability of ABC to enhance DOX sensitivity, MDA-MB-231 and MCF-7 cells were pre-treated with ABC at its IC\u003csub\u003e25\u003c/sub\u003e concentration for 24 hours. Subsequently, cells were exposed to DOX concentrations ranging from 0-160 nM for 72 hours, with daily replenishment. The MTT assay was performed, and the IC\u003csub\u003e50\u003c/sub\u003e values of DOX in ABC-sensitized cells were compared to those in cells treated with DOX alone.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e5.8. Soft agar colony forming assay\u003c/h2\u003e \u003cp\u003eA 24-well plate was prepared with a base layer of 0.6% agar in 2X complete media and allowed to solidify. The top agar layer (0.3%), containing 2000 cells from the respective treatment groups, was seeded onto the solidified base layer and allowed to set. The plates were incubated at 37\u0026ordm;C with 5% CO\u003csub\u003e2\u003c/sub\u003e to promote colony formation. To maintain cell growth, 100 \u0026micro;l of media was added to each well every three days. The assay was completed on day 28, and the colonies were stained with 0.1% crystal violet solution. Stained colonies were then observed and counted [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e5.9. Apoptosis assay\u003c/h2\u003e \u003cp\u003eThe apoptosis study was performed using FITC-Annexin V Apoptosis Detection Kit I (BD Pharmingen\u0026trade;, USA) following the manufacturer's instructions [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Briefly, pretreated cells at a concentration of 1 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml were washed with cold PBS and re-suspended in the 1X binding buffer provided in the kit. A 100 \u0026micro;l aliquot of this cell suspension (1 x 10\u003csup\u003e5\u003c/sup\u003e cells) was stained with 5 \u0026micro;l FITC Annexin V and 5 \u0026micro;l propidium iodide (PI) and then incubated for 15 minutes at room temperature in the dark. 400 \u0026micro;l of 1X binding buffer was added to the stained cells and analyzed for apoptosis using BD FACSAria\u0026trade; Fusion Flow cytometer (Becton, Dickinson and Company, USA). The results were analyzed using FlowJo\u0026trade; v10.8 Software (BD Life Sciences, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e5.10. Cell cycle analysis\u003c/h2\u003e \u003cp\u003ePretreated cells were harvested and fixed in ice-cold 70% ethanol for 30 minutes. After fixation, the cells were rinsed with PBS and incubated with RNase A (100 \u0026micro;g/ml, Thermo Fisher Scientific, USA) for 30 minutes at room temperature. Subsequently, the cells were then stained with PI at a concentration of 50 \u0026micro;g/ml and incubated for an additional 30 minutes at room temperature in the dark. Flow cytometric analysis was performed immediately using the BD FACSAria\u0026trade; Fusion Flow cytometer (Becton, Dickinson and Company, USA), with a minimum of 10,000 cells collected per sample. Data were analyzed using FlowJo\u0026trade; v10.8 Software (BD Life Sciences, USA). Appropriate controls, including unstained and single-stained cells, were used to set compensation and gating parameters [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e5.11. 3-D Breast cancer organoid culture\u003c/h2\u003e \u003cp\u003eBreast cancer tissues were cut into 1\u0026ndash;3 mm\u003csup\u003e3\u003c/sup\u003e pieces and processed to isolate viable cells. The tissue was minced and washed with 10 ml of collection medium (Advanced DMEM/F12 with 1X Glutamax, 10 mM HEPES, and antibiotics). The tissue was digested in ~\u0026thinsp;5\u0026ndash;10 ml breast cancer organoid medium containing 2 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO) and mixed on an orbital shaker at 37\u0026deg;C for 2 hours. The processed tissue suspension was subjected to successive shearing using sterilized glass Pasteur pipettes. After each shearing step, the suspension was strained through a 100 \u0026micro;m filter to retain larger tissue fragments. The fragments were further subjected to additional shearing with a 10 ml collection medium. To the strained suspension, 2% FBS was added, followed by centrifugation at 400 rcf. The resulting pellet was resuspended in a 10 ml collection medium and centrifuged at 400 rcf. If a visible red pellet was observed, erythrocytes were lysed using 2 ml of red blood cell lysis buffer (Cat. No: 11814389001, Roche Diagnostics, Germany) for 5 minutes at room temperature, followed by the addition of 10 ml of collection media and centrifugation at 400 rcf.\u003c/p\u003e \u003cp\u003eThe final pellet was resuspended in cold growth factor reduced Matrigel (Corning, USA) (1:1 of media and matrigel), and 100 \u0026micro;l of the suspension was plated in transwell chambers. 500 ml of breast cancer organoid medium was added to each well, and plates were incubated at 37\u0026ordm;C, 5% CO\u003csub\u003e2\u003c/sub\u003e. The breast cancer organoid medium contained R-Spondin 3 (Sigma-Aldrich, St. Louis, MO), Neuregulin 1, FGF 7, FGF10, and EGF (Thermo Fisher-PeproTech), along with Rock Inhibitor, HEPES, and N-Acetylcysteine (Sigma-Aldrich, St. Louis, MO) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The medium was changed every 2 days, and organoids were passaged every 2\u0026ndash;4 weeks. For passaging, dispase (Sigma-Aldrich, St. Louis, MO) was added to the transwells and incubated for 2\u0026ndash;3 hrs. Organoids were dissociated by incubating in 2 ml of TrypLE Express (Gibco, Thermo Fisher), at 37\u0026deg;C for 3\u0026ndash;5 minutes, followed by mechanical shearing with sterilized glass Pasteur pipettes. After adding 10 ml of collection medium, the suspension was centrifuged at 400 rcf, and the resulting pellet was harvested to prepare a single-cell suspension. The cells were seeded at high density and reseeded at reduced density after the first passage. Organoid culture was performed by following Hans Clevers group\u0026rsquo;s recent protocol [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Once the diameter of breast cancer organoids reached around 20\u0026ndash;30 \u0026micro;m, they were subjected to doses of ABC (50 \u0026micro;M at 24, 48, 72, and 96 hours) and DOX (1 \u0026micro;M at 48, 72, and 96 hours), and a combination of ABC\u0026thinsp;+\u0026thinsp;DOX (24, 48, 72, and 96 hours). The overall shape and dimensions of the breast cancer organoids were analyzed using a brightfield microscope. Three sets of experiments were conducted per patient (n\u0026thinsp;=\u0026thinsp;3 per patient), and ten representative organoids per condition were measured [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Organoid sizes were determined using Image J (OpenJDK 13.0.6.) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e5.12. \u003cem\u003eIn silico\u003c/em\u003e molecular studies\u003c/h2\u003e \u003cp\u003eThe binding interactions of CBV-TP in the human DNA polymerase β enzyme in complex with DNA were studied using the computational software Discovery Studio (DS) Structure-Based-Design (BIOVIA Inc, Dassault Systemes, USA). The CBV-TP was built in 3D using the Small Molecules module in the software and was energy minimized over 2000 steps using the Smart Minimizer, distance-dependent dielectrics, and CHARMm force field. The X-ray structure of human DNA polymerase β enzyme in complex with DNA and the nucleotide derivative 2\u0026rsquo;-deoxyuridine-5\u0026rsquo;-[(a,b)-imido]triphosphate (dUMPNPP) was obtained from PDB (PDB ID: 2FMS) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The protein-DNA-nucleotide complex was prepared first, by removing water molecules. The two magnesium atoms (Mg\u003csup\u003e2+\u003c/sup\u003e) in the catalytic site were retained. The bound nucleotide dUMPNPP was selected, and a binding site sphere of 8 \u0026Aring; was created around the nucleotide dUMPNPP. In the next step, the nucleotide was deleted. Subsequently, the Macromolecules module in the software was used to prepare the protein-DNA complex using the CHARMm force field. Molecular docking was carried out using the LibDock algorithm in DS with the following parameters: 100 hotspots, docking tolerance of 0.25 \u0026Aring;, implicit solvent function, and distance-dependent dielectric constant. The docking poses were energy minimized over 1000 steps using the Smart Minimizer. The CHARMm force field was used during the docking simulation. The docking poses were ranked based on the LibDock score and were analyzed by investigating the polar and nonpolar interactions of CBV-TP with human DNA polymerase β enzyme and DNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e5.13. Statistical analysis\u003c/h2\u003e \u003cp\u003eThe normality of distribution was tested using the Shapiro-Wilk test. Continuous variables were reported as either the median with an interquartile range or the mean with standard deviation, depending on the normality of the data. Differences in normally distributed data were analyzed using the Student\u0026rsquo;s t-test or one-way analysis of variance (ANOVA). The Wilcoxon Mann-Whitney test was used to compare the differences in gene expression levels between tumor and normal samples. Overall survival rates were estimated using the Kaplan\u0026ndash;Meier method, with the median KDM5B expression level taken as the cutoff for high and low expression groups. All statistical analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software Inc., San Diego, CA, USA), with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAJ is thankful to the Department of Science and Technology, Govt. of India, for providing the DST-INSPIRE Fellowship [IF190197] and Manipal Academy of Higher Education [MAHE], Manipal, for awarding Dr. TMA Pai fellowship and MAHE contingency fund. M.R. is thankful to the Department of Science \u0026amp; Technology for the DST-FIST grant (DST-FIST TPN: 32196). PK (45/20/2020-PHA/BMS) thanks the Indian Council of Medical Research (ICMR) for providing Senior Research Fellowship. NA acknowledges the SERB POWER grant from the Science and Engineering Research Board for financial support (SPG/2021/004209). The authors thank Manipal Academy of Higher Education [MAHE], Manipal, All India Institute of Medical Sciences [AIIMS] Bhopal and Global Cancer Consortium Interdisciplinary Lab, Manipal School of Life Sciences, Manipal, for providing the facilities to conduct this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAJ, MR, GSR, and GGS designed and supervised the study. AJ acquired the data, performed the analysis and interpretation, and drafted the manuscript. sPPNR and GGS conducted the molecular docking studies, and SV guided with bioinformatics analysis. MP and BB performed the 3-D culture experiments. AJ, PK, NA, and MM coordinated, conducted, and analyzed the 2-D culture experiments. AJ, MR, NK, NU, GSR, and RRD collected, analyzed and interpreted the patient data and critically reviewed the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and its Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ethical approval for the study was granted by the Kasturba Medical College and Kasturba Hospital Institutional Ethics Committee (IEC: 504/2019), and written informed consent was obtained from all participants. The research was performed in accordance with the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Department of Science and Technology, Govt. of India INSPIRE Fellowship [IF190197] and the Manipal Academy of Higher Education [MAHE] contingency fund [190600118].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray, F. et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA Cancer J. Clin.\u003c/em\u003e \u003cb\u003e74\u003c/b\u003e, 229\u0026ndash;263 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurashvili, G. \u0026amp; Brogi, E. Tumor Heterogeneity in Breast Cancer. \u003cem\u003eFront. Med. 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Development of a Three-Dimensional Bioengineering Technology to Generate Lung Tissue for Personalized Disease Modeling. \u003cem\u003eStem Cells Transl Med.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 622\u0026ndash;633 (2017).\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, Carbovir triphosphate, Drug repurposing, Epigenetic targeting, Precision medicine","lastPublishedDoi":"10.21203/rs.3.rs-6208718/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6208718/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKDM5B, a lysine-specific histone demethylase, is widely upregulated in breast cancer. The current study investigated the role of KDM5B in breast cancer and explored the repurposing potential of the antiviral drug abacavir (ABC). The cytotoxic effects and the effect of ABC sensitization on doxorubicin (DOX) efficacy were evaluated using 2-D and 3-D cell culture models. KDM5B expression was elevated in breast cancer tissues compared to normal breast tissues. \u003cem\u003eIn vitro\u003c/em\u003e studies demonstrated that ABC treatment reduced KDM5B expression in breast cancer cells and increased their sensitivity towards DOX. ABC induced late apoptosis and S phase arrest, while the ABC\u0026thinsp;+\u0026thinsp;DOX combination led to S/G2 phase arrest, late apoptosis, and cell death. Data generated from patient-derived breast tumoroids corroborated the 2-D cell culture-based findings. Additionally, molecular docking studies indicated that active drug metabolite carbovir triphosphate (CBV-TP) could interact with the DNA polymerase β-DNA complex, suggesting its potential mechanism to be incorporated into the DNA synthesis cycle, leading to cell cycle arrest in tumor cells. Our findings highlight the repurposing potential of ABC to target KDM5B in breast cancer. This approach enhanced the efficacy of DOX, which could allow further dose reduction and reduced side effects, offering a promising therapeutic strategy.\u003c/p\u003e","manuscriptTitle":"Abacavir enhances the efficacy of Doxorubicin via inhibition of histone demethylase KDM5B in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-03 09:07:31","doi":"10.21203/rs.3.rs-6208718/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-07T18:57:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-07T07:43:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-02T19:09:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"287276935209515264992493798457422531172","date":"2025-03-25T08:30:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8047319611884558669117680383606106552","date":"2025-03-23T07:46:28+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-22T22:27:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-22T22:09:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-19T03:58:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-17T09:42:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-03-12T05:11:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af0f6fd2-a70e-4a2c-b5c5-4543995c0565","owner":[],"postedDate":"April 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":46223281,"name":"Biological sciences/Cancer/Breast cancer"},{"id":46223282,"name":"Biological sciences/Cancer/Cancer therapy"},{"id":46223283,"name":"Biological sciences/Cancer/Oncogenes"},{"id":46223284,"name":"Biological sciences/Cancer/Tumour biomarkers"},{"id":46223285,"name":"Health sciences/Medical research/Pre clinical studies"},{"id":46223287,"name":"Health sciences/Medical research/Translational research"},{"id":46223289,"name":"Biological sciences/Cancer"},{"id":46223291,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":46223293,"name":"Health sciences/Biomarkers"},{"id":46223295,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-08-11T16:01:39+00:00","versionOfRecord":{"articleIdentity":"rs-6208718","link":"https://doi.org/10.1038/s41598-025-13845-z","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-08-05 15:57:31","publishedOnDateReadable":"August 5th, 2025"},"versionCreatedAt":"2025-04-03 09:07:31","video":"","vorDoi":"10.1038/s41598-025-13845-z","vorDoiUrl":"https://doi.org/10.1038/s41598-025-13845-z","workflowStages":[]},"version":"v1","identity":"rs-6208718","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6208718","identity":"rs-6208718","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}