TLK1 as a Druggable Target in TMZ-Resistant Glioblastoma: Evaluating Small Molecule as a Promising Drug Candidate

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Temozolomide (TMZ), a widely used alkylating chemotherapeutic in Glioblastoma therapy, often encounters resistance, necessitating the investigation of the underlying mechanisms of TMZ-acquired resistance. To study TMZ resistance, a cell-based model system was generated by intermittently exposing glioblastoma cells to increasing concentrations and time of TMZ over six months. The survival response of cells at higher concentrations confirmed TMZ-resistant cells, which exhibited a phenotypic shift toward a mesenchymal-like state, with decreased epithelial traits, indicating mesenchymal-epithelial transition (MET). This transition likely facilitates the stabilization and clonal growth of TMZ-resistant cells. Subsequent analysis revealed elevated expression of TLK1, a DNA repair protein, thus reinforcing its potential involvement in mechanisms associated with acquired resistance. To explore the therapeutic aspect of TLK1 inhibition, we utilized an in-house developed TLK1 inhibitor, J54. The inhibition of TLK1 in TMZ-resistant cells enhanced cytotoxicity, indicating TLK1 as a potential target to combat TMZ resistance. Moreover, TLK1 inhibition reduced cell migration and invasion, implying its role in promoting metastasis. In conclusion, our study sheds light on the role of TLK1 in the context of TMZ resistance, highlighting its potential as a valuable target for therapeutic intervention. Biological sciences/Chemical biology/Kinases Biological sciences/Chemical biology/Small molecules Biological sciences/Chemical biology/Target validation Biological sciences/Cancer Blood-Brain-Barrier glioblastoma temozolomide phenothiazine tousled-like kinase-1 DNA damage response. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Acquired resistance to cancer treatments is a formidable challenge, particularly in glioblastoma (GBM) therapy. The treatment option available for GBM is very limited, and resistance to the available treatment regimen (temozolomide (TMZ) and radiation therapy) is a major concern. Standard GBM treatment involves maximal safe tumor excision followed by radiotherapy and/or TMZ. 1 Unfortunately, combined radiotherapy and TMZ extend survival to 5 years in less than 5% of patients. 2 Nevertheless, these therapies often relapse and the tumour become invariably resistant to radiation/TMZ regimen. Due to very few therapeutic options available for GBM, TMZ-resistant GBM patients have limited options. Therapeutic resistance to radiation and TMZ is, therefore, a major obstacle in improving treatments for patients with GBM. 3 Understanding the mechanisms of resistance can help improve treatment efficacy and facilitate the development and implementation of novel interventions to improve overall therapy for GBM. As per the current understanding in the field, three primary modes of TMZ resistance have been proposed. 4 Firstly, De-silencing of the direct repair (DR) enzymes (MGMT and ALKBH), which reverse TMZ-induced O6-methylguanine (O6MG), increased expression of DR enzyme can contribute towards TMZ resistance. Secondly, Loss of the mismatch repair (MMR): The mismatch repair introduces continuous single-strand DNA break in order to repair the O6MG introduced by TMZ. Therefore, loss of MMR will compromise the generation of ssDNA and the formation of DSB, thus, preventing the GBM cell from apoptosis. Finally, the up-regulating the DNA double-strand repair (DSB) pathway: The eventual cause of lethality due to TMZ is due to an increased number of DSB formations. Therefore, augmented DSB proteins contribute towards TMZ resistance, particularly in cells with compromised MGMT expression. 5 , 6 Increasing bodies of evidence have suggested that elevated DSB repair majorly contributes to TMZ resistance in cells where MGMT expression is compromised. 7 , 8 , 5 DDR pathway aberrations predispose cells to cancerous growth and make them excessively dependent on other parallel pathways for survival. Interestingly, GBM is widely reported to have defects in the DDR pathway proteins apart from epigenetic MGMT silencing, which can also serve as biomarker/s for TMZ- & radio-resistance. Many DSB repair proteins have been explored as a therapeutic target in glioblastoma therapy in the last few years. A few of the DSB repair protein inhibitors are in clinical trials. However, one of the major limitations of many drug candidates are poor blood-brain barrier permeability. Therefore there is an increasing need to identify novel targets and inhibitors to improve the clinical outcome of therapeutic-resistant GBM. Tousled-like kinase 1 (TLK1) is one of the critical kinases whose role has been implicated in DSB repair and radio-resistance. 9 , 10 , 11 At the molecular level, depletion of TLK1 promotes cell death through the generation of DSBs. 12 Its expression is frequently up-regulated in many human cancers, including GBM. Recent studies of TLK1 in GBM cells and xenograft models have indicated an important role of this protein in cell survival. 13 We further have demonstrated that elevated TLK1 levels facilitate cellular repair of DNA damage induced by temozolomide. Inhibition of TLK1 using J54 results in the effective killing of GBM cells 14 . The previous study in breast and prostate cancer has suggested that an elevated level of TLK and its isoform TLK1b contributes to chemo- and radio-resistance. 15 , 9 , 11 Based on these findings, we hypothesize that TLK1, a key player in DNA repair, may facilitate TMZ resistance in GBM by repairing TMZ-induced DSBs. Thus, inhibiting TLK1 in GBM could sensitize cancer cells to TMZ treatment, potentially improving survival outcomes for patients with TMZ-resistant GBM. This study aims to assess the therapeutic potential of J54, an in-house synthesized TLK1 inhibitor, for improving the treatment response in temozolomide (TMZ)-resistant glioblastoma (GBM). We intend to explore the chemical biology of TLK1 to enhance therapeutic outcomes for these resistant GBM cells. We will employ the phenothiazine-based TLK1 inhibitor J54, developed in-house, which we previously demonstrated to exhibit cytotoxic effects against GBM cells. 14 Phenothiazines, known for their use as antipsychotic drugs, can cross the blood-brain barrier (BBB) and were identified as first-generation TLK inhibitors by our lab and others. 16 , 17 Overall, through this study, we hope to improve the therapeutic outcome of TMZ-resistant glioblastoma cells and anticipate approaches for developing novel treatments for patients with GBM. Materials and methods Cell lines LN229 and LN18 cell lines were procured from National Cell Repository at National Centre For Cell Science, Pune, India. The cell lines were grown in DMEM with 10%FBS. LN229 TMZ-R cells were obtained by subjecting the LN229 cells to intermittent exposure to increasing concentrations of TMZ (ranging from 50 µM to 200 µM), with intervening periods of non-exposure. Cytotoxicity assay The cytotoxicity assay was conducted using the Cell Titer Glo (CTG, Promega Corporation) as previously described 14 . In brief, viable cells were counted using a hemocytometer after staining the cells with trypan blue. Approximately 2000 cells were seeded in 96-well white plates, and 24 hours after seeding, the respective treatments were administered. After 72 hours of treatment, the appropriate amount of CTG reagent was added to the plates, and the plates were subjected to shaking for 15 minutes to ensure proper mixing. Subsequently, luminescence measurements were recorded by Envision multimode plate reader. The results were analyzed as a percentage of viable cells after treatment, relative to the untreated cells, based on a total of three independent replicates. Clonogenic treatment Around 1000 cells of each respective cell line were plated in a 12-well plate and exposed to corresponding treatment groups for a duration of 5 days. Subsequently, the cultures were incubated for approximately two weeks to allow cell colony formation from the surviving fraction of cells. Following the incubation period, the cell colonies were fixed and stained using a 0.5% crystal violet solution, and the resulting colony with more than 50 cells were counted under a microscope. Immunocytochemistry The respective cells were cultured on 18 mm coverslips in 12-well plates at a density of approximately 50,000 cells per well. The plates were then incubated in a humidified CO2 incubator maintained at 37°C. To evaluate the effect of J54 on γH2AX, the cells were treated with 50 µM J54 for 72 hours. Following the treatment period, the cells were fixed with 4% paraformaldehyde, permeabilized with 100% methanol, and subsequently blocked with 5% FBS + 0.3% Triton X-100 in 1X PBS. Next, the cells were incubated with the γH2AX primary antibody (1: 500, #2577, CST) overnight at 4°C, followed by the incubation with the Anti-rabbit IgG Alexa Fluor-488-labeled secondary antibody (1:500, #4412, CST) for 2 hours at room temperature. The cells were mounted using a fluoroshield mounting medium containing DAPI (#ab104139, Abcam). The cells were imaged under a Confocal Laser Scanning Microscopy. Image quantification and processing were performed using the ImageJ software. Western Blot Western Blot LN229 TMZ-R cells were subjected to respective treatments. For the treated samples, the cells were collected and lysed 72 hours after the incubation period. The cellular lysates were obtained using 1X RIPA buffer. Subsequently, the protein bands were resolved on 8% acrylamide gels and subsequently transferred onto PVDF membranes with a pore size of 0.22 µm. The membranes were initially blocked using a 5% skimmed milk solution and subsequently incubated with primary antibodies: E-Cadherin (1:1,000, #14472), Vimentin (1:1,000, #5741), β-Catenin (1:1,000, #8480), N-Cadherin (1:1,000, #3116), β-actin (1:1,000, #SC-47778), TLK1 (1:1,000, #GTX102891), Phospho-RAD9-S328 (1:1,000, #PA5-12670), Phospho-Nek1-T141, Nek1(1:1,000, #sc-398813) overnight at a temperature of 4°C. Following this, the membranes were exposed to secondary antibodies: Anti-mouse IgG, (1:2,500, #7076), Anti-rabbit IgG (1:2,500, #7074), conjugated with HRP for duration of 2 hours at room temperature. The protein bands were then visualized by subjecting the blots to ECL substrates, and the imaging process was carried out using the Bio-Rad ChemiDoc Imaging System and the analysis was done using Image lab software (Biorad). This image shown in the figure panel is cropped sections from the original blot. Full-length uncropped versions of the gel (or blot) are provided in Supplementary file. Wound healing assay The cells were cultured as a monolayer and were pretreated with 25 µM J54 for 36 hrs prior to creating the wound. A wound was made using a micropipette tip. The confluency of the cells is nearly 90% at this point. Subsequently, the cells were incubated in media containing J54 for 24 hours. The wound area was imaged at both time = 0 hours and 24 hours after the wound creation, and the area under the wound was compared. The quantification and processing of the wound area and images were done using the ImageJ software (n = 3, independent replicates). Transwell invasion assay The transwell chambers with an 8-micron pore size (Corning Incorporated, Corning, NY, USA) were pre-coated with 1% geltrex (Thermo Fisher Scientific Inc.) in serum-free media for duration of 1 hour. Afterwards, the cells in serum-free media, along with their respective treatments, were seeded into the transwell chambers that had been pre-coated with geltrex. In the lower chamber, complete media containing 10% FBS was added as a chemo-attractant to promote cell invasion. After a 72-hour incubation period, the cells that had invaded through the geltrex and attached to the lower chamber of the plate were stained using Hoechst. Subsequently, the number of invaded cells was quantified by counting them under the microscopic field (n = 3, independent replicates). Live/Dead staining Approximately 10,000 cells were plated in a black, transparent-bottom plate. After 24 hours of seeding, the respective treatments were applied to the cells. Following a 72-hour treatment period, the media was removed, and the cells were stained with 2.5 µg/ml propidium iodide and Hoechst before being incubated at 37°C in a humidified incubator with 5% CO2 for 15 min. Fluorescence measurements were then recorded using an Envision multimode plate reader for quantitative analysis. Additionally, images were captured using a Nikon inverted fluorescent microscope. Results and Discussion Generation and validation of TMZ-resistant glioblastoma cell line Due to the absence of an existing model system for investigating temozolomide-acquired resistance, an approach was adopted to generate temozolomide-resistant glioblastoma cell lines. Clinically, patients with methylated promoters of the O-6-Methylguanine-DNA Methyltransferase (MGMT) gene display improved responsiveness to temozolomide (TMZ) treatment in comparison to patients with MGMT expression 18 , 19 . Consequently, the LN229 cell line, characterized by MGMT deficiency, was chosen for the generation of temozolomide-resistant cells. The LN229 cells were subjected to intermittent treatment with progressively increasing doses of TMZ over a duration exceeding 6 months. The objective was to identify the cell population that acquired resistance to temozolomide (TMZ-R) during this extended period. In the initial treatment cycle, LN229 cells were exposed to a dose of 50 µM TMZ for 5 days, and subsequently, the surviving fraction of cells was allowed to grow for nearly 3 weeks. The surviving cells underwent a second cycle of TMZ treatment with a dosage of 100 µM for 12 days. Afterwards, these cells were cultivated in TMZ-free media for 18 days. Following this, the surviving cells were exposed to media containing 150 µM TMZ for three weeks. Afterwards, the cells were grown 1 week off TMZ. Further, the surviving cells were continually cultured in TMZ containing media with a dosage of 200 µM for an additional 13 weeks. Subsequently, the TMZ-R cells were maintained in 100 µM TMZ for further experiments. The cells gradually adapted to grow in the presence of TMZ. In the first cycle of TMZ exposure major fraction of cells died. However, with gradual and intermittent increases in TMZ concentration, the fraction of cell death gradually reduced, and the cells started to grow as a colony. Afterwards, the responsiveness towards TMZ-sensitive (TMZ-S) and TMZ-resistant (TMZ-R) cells was assessed by performing the clonogenic assay. Additionally, to check if the TMZ-R cells can revert back to TMZ-sensitive phenotype, the cells were grown in the absence of TMZ for few passages, and the responsiveness of the cells was checked using a clonogenic assay. If the cell has acquired resistance, then there will be a similar response of both the cell population, and they will respond to TMZ better compared to TMZ-S cells. As observed in Fig. 1 , the LN229 TMZ-R cells and TMZ-R cells cultured without TMZ for few passages displayed significantly enhanced cell viability and proliferation in comparison to TMZ-S cells. Furthermore, no significant difference was observed in the responsiveness to TMZ between the TMZ-R cells and TMZ-R cells grown without TMZ. These results strongly suggest that the cells have indeed acquired resistance to TMZ and do not revert to a sensitive phenotype even when cultured in TMZ-free media. Additionally, LN18 cells were taken as an external control, as it is a MGMT proficient cell and can grow in the presence of TMZ. Characterization of TMZ-resistant glioblastoma cell line In LN229 cells, prolonged administration of TMZ led to acquired resistance to the drug and was accompanied by a change in cell morphology. LN229 TMZ-R displayed a relatively elongated, flattened and spread cellular appearance (Fig. 2 A). These morphological changes have been previously reported in other studies exploring the effects of prolonged TMZ treatment. There is a strong correlation between changes in the expression of Epithelial-Mesenchymal (E-M) markers and changes in cellular shape and behavior, resulting in aggressive cancer cell morphologies due to acquired drug resistance. 20 , 21 Deregulation in E-M markers is characterized by the change in expression of epithelial markers (such as E-cadherin) and mesenchymal markers (such as N-cadherin, Vimentin). These changes in marker expression are often accompanied by alterations in cellular morphology and migratory and invasive capabilities. Therefore we next explored whether the change in cellular morphology of TMZ-R cells is also accompanied by the deregulation of the Epithelial-Mesenchymal (E-M) markers. As observed in Fig. 2 B, there was an increase in the expression of E-cadherin and a decrease in the expression of N-cadherin, Vimentin and β-catenine. This suggests that the cells are undergoing Mesenchymal-Epithelial Transition (MET). Emerging evidence indicates that MET-driven drug resistance may contribute to reduced sensitivity to various chemotherapeutic agents and targeted therapies in cancer cells. 22 , 23 During MET, cells undergo morphological changes, and mesenchymal markers are down-regulated, while epithelial markers are up-regulated. This process results in the restoration of cell-cell adhesion. The exact correlation underlying MET-mediated drug resistance is not well understood. Furthermore, MET may potentially offer cancer cells a survival advantage, allowing them to tolerate the cytotoxic effects of chemotherapy and grow clonally. 24 Overall, this may contribute to treatment failure. To determine the protein expression status TLK1/NEK1 DDR axis in temozolomide-resistant and sensitive glioblastoma cell line : A previous study revealed that TLK1 is significantly up-regulated in Glioblastoma, and its knockdown and inhibition (reported elsewhere) were found to strongly decrease the viability of glioblastoma cells. The up-regulation of DNA repair proteins, including TLK1, facilitates the efficient repair of drug-induced DNA damage, leading to resistance against DNA-damaging drugs like TMZ. To investigate the potential role of TLK1 overexpression in TMZ-acquired resistance in glioblastoma, firstly, the expression and activity of TLK1 were examined. As shown in Fig. 3 A, the expression of TLK1/1B and p-Nek1 was up-regulated in TMZ-resistant cells, and inhibiting TLK1 may sensitize these resistant cells to TMZ. To explore this further, cell viability analysis and clonogenic assays were performed using J54, a previously characterized in-house potent TLK1 inhibitor 17 , 14 . J54 belongs to the phenothiazine class of small molecules, which are known to cross the blood-brain barrier (BBB). Furthermore, our previous study identified J54 as a promising drug candidate for GBM, with a high likelihood of crossing the blood-brain barrier (BBB) 14 . Therefore, this study investigated the therapeutic potential of J54 in sensitizing TMZ-acquired resistant cells. Figure 3 B-C demonstrates that J54 exhibited a dose-dependent reduction in cell viability and clonogenic potential. However, no significant change was observed when TMZ was exposed to the TMZ-resistant cells; in contrast, LN229 (TMZ sensitive) cells showed responsiveness to TMZ. These findings suggest that TLK1 overexpression may play a role in the development of TMZ-acquired resistance in glioblastoma cells. Moreover, the inhibition of TLK1 with the potent in-house developed TLK1 inhibitor, J54, showed promise in sensitizing TMZ-resistant cells, thereby providing potential therapeutic implications for addressing drug resistance in glioblastoma treatment. Inhibition of TLK1 results in accumulation of DNA strand break in GBM Cells In this study, we aimed to investigate the impact of J54 on the TLK1 signaling pathway in Glioblastoma (GBM). To assess this effect, we performed an immunoblot assay to analyze the phosphorylation status of downstream substrates of TLK1 in response to cellular treatment. Specifically, we focused on Rad9 (S328) and Nek1 (T141), which are known to be phosphorylated by TLK1/1b in the context of DNA damage response and repair processes. As expected, the results demonstrated a dose-dependent reduction in the phosphorylation of Rad9 and Nek1 with an increase in the concentration of J54 (Fig. 4 A). This observation indicates that J54 effectively inhibits the cellular activity of TLK1 kinase, thereby disrupting the downstream signaling events involved in DNA damage response and repair. By attenuating the phosphorylation of Rad9 and Nek1, J54 may impair the proper functioning of DNA repair mechanisms mediated via TLK1. In the process of acquiring resistance to TMZ through prolonged exposure, cancer cells experience an overall increased burden of DNA strand breaks. Consequently, inhibiting TLK1 further compromises the repair of DNA strand breaks, resulting in the accumulation of unrepaired DNA damage that may be lethal for cell survival. To assess this effect, the level of γH2AX, a marker of DNA strand break, was examined. As observed in Fig. 4 B, a significant increase in the level of γH2AX was observed in response to J54 treatment, indicating the substantial accumulation of DNA breaks in the TMZ-R cells. The findings highlight the potential of J54 as a therapeutic agent that disrupts the DNA repair process mediated by TLK1, leading to increased DNA damage accumulation in cancer cells. Inhibition of TLK1 results in the cell death of GBM Cells The impairment of DNA repair processes could result in the accumulation of DNA strand breaks, ultimately triggering cell death and contributing to the inhibition of tumour growth. To confirm this, the J54-treated cells were stained with propidium Iodide (PI) and Hoechst-33342. The staining technique using PI and Hoechst-33342 allows for the differentiation between live and dead cells. PI is a cell impermeable dye that can selectively stain dead cells, while Hoechst-33342 can stain all the cells. Upon examination, the J54-treated cells exhibited a significantly higher fraction of cells that stained positively for PI (Fig. 5 ). This overall confirms that the J54-treated cells result in the accumulation of DNA strand breaks due to TLK1 inhibition that leads to cell death. These findings contribute to the growing understanding of the molecular mechanisms underlying the therapeutic effects of J54 and its potential as a targeted therapy in cancer treatment. By disrupting DNA repair processes and promoting the accumulation of DNA strand breaks, J54 offers a promising approach to induce cell death and inhibit tumour growth. This may hold promise for sensitizing TMZ-resistant GBM cells, thereby opening the window for therapeutic interventions. Further investigation into the precise mechanisms underlying the interaction between TLK1, DNA repair pathways, and acquired drug resistance is crucial for fully understanding the therapeutic implications and potential of J54 as a targeted therapy in cancer treatment. In conclusion, inhibition of TLK1 in cancer cells can disrupt the repair of DNA strand breaks, leading to DNA damage accumulation and potential cell death. Targeting TLK1 as a therapeutic approach holds promise in sensitizing cancer cells to DNA-damaging treatments and may offer new strategies to overcome drug resistance and improve cancer treatment outcomes. However, further investigation is needed to fully understand the role of TLK1 in DNA repair and its therapeutic potential in cancer therapy. Inhibition of TLK1 results in Reduced Cell migration and Invasive Capacity of GBM Cells Recent research has also identified TLK1 as a key kinase that plays a crucial role in promoting cancer cell motility and invasiveness. Inhibition of TLK1 can have significant effects on cell migration and invasion in cancer cells. TLK1 has been linked to the regulation of actin cytoskeleton dynamics, which is essential for cell migration and invasion. By promoting actin filament stability and remodeling, TLK1 facilitates the formation of cellular protrusions, such as lamellipodia and filopodia, necessary for cell movement. Consequently, cancer cells treated with TLK1 inhibitors may exhibit decreased migratory capacity, limiting their ability to invade surrounding tissues and metastasize to distant sites 25 , 26 . To investigate this further, wound healing and transwell invasion assays were conducted. The wound healing assay allows the observation of cell migration in vitro, where a wound is created in a cell monolayer, and the closure of the wound over time indicates cell migration capacity. On the other hand, the transwell invasion assay assesses the ability of cells to invade through an extracellular matrix barrier, implying the invasive potential of cancer cells in vivo. As depicted in Fig. 6 , treatment with J54 resulted in a substantial decrease in the migratory and invasive abilities of TMZ-resistant cells. Overall, these findings emphasize the therapeutic significance of targeting TLK1 as a strategy to potentially impede cancer cell migration and invasion. By inhibiting the kinase activity of TLK1, it may be possible to hinder the processes that promote cell motility, invasiveness, and metastasis, ultimately suppressing cancer progression. Conclusion Glioblastoma multiforme is a particularly challenging form of brain cancer, and the use of temozolomide has shown marginal progress in improving patient outcomes. This chemotherapeutic agent has become a cornerstone of treatment protocols. However, there is a growing concern regarding the development of TMZ acquired resistance, which might lead to therapeutic failure. Conventionally, the resistance to TMZ has been closely associated with the expression MGMT enzyme. This enzyme is known to repair the DNA damage inflicted by TMZ, thereby reducing the effectiveness of the drug. Yet, recent research has unveiled a more complex landscape. Interestingly, the potential involvement of the mismatch repair (MMR) complex is associated with TMZ-acquired resistance. Furthermore, emerging evidence suggests that proteins responsible for repairing double-strand breaks (DSBs) in DNA may also be a major contributor in TMZ resistance. The augmented activity of these DSB repair proteins could potentially enhance the ability of the cancer cells to overcome the damage inflicted by TMZ, rendering the treatment less effective. TLK1, a DNA repair enzyme, was identified as one of the highly up-regulated kinase in glioblastoma using genetic screening. The overexpression of TLK1 in TMZ-resistant cells is likely to trigger the DNA damage response (DDR) cascade, leading to an augmented DNA repair process. In our study, we found that the introduction of TLK1 inhibitor, J54, hampers the DDR pathway, leading to the accumulation of DNA damage and double-strand breaks (DSBs), ultimately culminating in cell death in TMZ resistance-acquired cells. The findings from this study suggest that J54 holds immense promise as a potential therapeutic agent for GBM, as it targets TLK1, an important kinase involved in DNA damage response and repair. Further investigation into the precise mechanisms underlying the role of TLK1 in drug resistance and the potential of J54 as a sensitizing agent is warranted for the development of effective therapeutic strategies. The development of targeted therapies that interfere with the TLK1 signaling pathway may offer new avenues for the treatment of TMZ-acquired resistance GBM cells. Declarations Conflict of Interest: The authors disclose no conflicts. Author Contribution The project was conceptualized by BP and SK, with BP conducting the investigation. BP performed all the experiments. Data analysis was carried out by SK and BP. Acknowledgement The authors extend their sincere gratitude to Prof. Benedetti for generously providing the pNek1 antibody. 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Cancers (Basel) ; 14 (23). (2022). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Nov, 2024 Reviews received at journal 01 Nov, 2024 Reviewers agreed at journal 18 Oct, 2024 Reviewers agreed at journal 16 Oct, 2024 Reviewers invited by journal 16 Oct, 2024 Editor assigned by journal 16 Oct, 2024 Editor invited by journal 14 Oct, 2024 Submission checks completed at journal 10 Oct, 2024 First submitted to journal 09 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5229526","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":373696139,"identity":"aff60ecf-7b6b-4599-b70a-be4085f58f13","order_by":0,"name":"Sivapriya Kirubakaran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYFACxgYGhgM2PGwwPhsetcha0njYYEqJ0AICBw4TrZSBwVz6cJvEjzPnZfjk2689YKixY+CTbsCvxbIvsU2y58ZtoMN4yg0YjiUzsMkcwK/F4AxjmwTPB7CWNAkGtgMMbBIJhLVI/vlwDqrlH5FapHluHABqYT8mwdhGhBbLHsZma5kzyUAtOWwSiX1ABiEt5jzsD2++OWZnL998/JnEh292cvIzCDmMgYFFAsLkMWAAKubBrx6ihfkDhMn+gKDqUTAKRsEoGJkAAKGxOL35P3bJAAAAAElFTkSuQmCC","orcid":"","institution":"Indian Institute of Technology Gandhinagar","correspondingAuthor":true,"prefix":"","firstName":"Sivapriya","middleName":"","lastName":"Kirubakaran","suffix":""},{"id":373696141,"identity":"7954821a-50e5-4105-b35c-c63643310894","order_by":1,"name":"Bhanu Priya","email":"","orcid":"","institution":"Indian Institute of Technology Gandhinagar","correspondingAuthor":false,"prefix":"","firstName":"Bhanu","middleName":"","lastName":"Priya","suffix":""}],"badges":[],"createdAt":"2024-10-09 05:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5229526/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5229526/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-86599-3","type":"published","date":"2025-04-26T15:57:16+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":69429542,"identity":"e6dfb647-72d2-4089-984d-9bed6964adb1","added_by":"auto","created_at":"2024-11-20 09:20:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":99214,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Overview of generation of TMZ-resistant GBM Cells in this study. LN229 and LN18 cells were intermittently treated with increasing concentrations of TMZ (50-200 µM). (B) A clonogenic assay was performed to assess if the LN229 cells grown intermittently in an increasing TMZ media have acquired resistance, LN18 cells were used as control. (C) Quantification of colony formation assay.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5229526/v1/fd4ce2e8dc349426f0ffb328.jpg"},{"id":69430550,"identity":"8d0f8e7d-f286-4660-96cc-1bb80b2405f0","added_by":"auto","created_at":"2024-11-20 09:28:10","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74607,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Morphology of LN229 and LN229-TMZ-R cells observed under confocal and bright-field microscope. Cells are stained with DAPI and β-catenine. Quantification of cell length shows a two-fold increase in cell length. (B) Immunoblot images and quantification of E-M markers show an increase in epithelial markers and a decrease in mesenchymal markers. Statistical significance was determined by student’s t-test, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.0001 compared to the control group (n =3, independent replicates).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5229526/v1/9b2857c541914199400177ab.jpg"},{"id":69429539,"identity":"c3905df9-95c4-4f74-afef-1fcd7f010c64","added_by":"auto","created_at":"2024-11-20 09:20:10","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":94508,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Western blot analysis shows an increase in the level of TLK1 and its splice variant TLK1b along with p-Nek1 in LN229 TMZ-R compared to LN229 TMZ-S cells. Statistical significance was determined by student’s t-test, *p \u0026lt; 0.05, ***p \u0026lt; 0.001, compared to the control group (n =2, independent replicates). (B) LN229 TMZ-R cell showing a decrease in cell viability in response to J54. On the other hand, no significant cytotoxicity was observed against TMZ, even at 100μM TMZ concentration. Statistical significance was determined by student’s t-test, **p \u0026lt; 0.01 compared to the control group (n =3, independent replicates). (C) Representative images of cell colonies formed in untreated control and cells treated with increasing concentrations of J54 compared. Statistical significance was determined one-way ANOVA, ****p \u0026lt; 0.0001, compared to the untreated control (n =3, independent replicates).\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5229526/v1/b4b567cdc4ca2946c3366c09.jpg"},{"id":69429544,"identity":"fdf01be4-8fa2-4166-9290-b8eabd61b5a9","added_by":"auto","created_at":"2024-11-20 09:20:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76010,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Western blot analysis demonstrating a dose-dependent decrease in the phosphorylation levels of TLK1 substrate, namely, NEK1 (p-NEK1) and RAD9 (p-RAD9) in response to J54 treatment. Statistical significance was determined by one-way ANOVA, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, compared to the untreated control (n =3, independent replicates). (B) Immunocytochemistry analysis demonstrating an increase in the γH2AX levels in response to J54. Statistical significance was determined one-way ANOVA, **p \u0026lt; 0.01, compared to the untreated control (n =3, independent replicates).\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5229526/v1/55e333600b75900e8d9f6199.jpg"},{"id":69429541,"identity":"70f2293d-9337-4f4f-bf1c-8bf4861d678f","added_by":"auto","created_at":"2024-11-20 09:20:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":69222,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of live and dead staining using propidium iodide (PI) and Hoechst stain in untreated control cells and J54 treated cells. Cells treated with J54 demonstrate increased cell death as evidenced by the presence of red-stained dead cells (PI positive), which are significantly lower in the untreated control. Quantification of cell death reveals a significant increase in the percentage of dead cells in response to J54 treatment compared to the untreated control. Statistical significance was determined by Student's t-test, ***p \u0026lt; 0.001, compared to the control group (n =3, independent replicates).\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5229526/v1/daeb37a7ec7452cbf4a753ce.jpg"},{"id":69430753,"identity":"3fa1358b-6b4f-4c29-8640-a85378acfbdf","added_by":"auto","created_at":"2024-11-20 09:36:10","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":98233,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The Figure shows the results of the wound healing assay performed on cell monolayers treated with J54. Representative images at t=0 and t=24 hours reveal the inhibition of cell migration by J54. The wound gap closure is delayed in the presence of the J54, indicating its anti-migratory effect. (B) Transwell Invasion Assay demonstrating the anti-invasive property of J54. The upper chamber was coated with a geltrex, and cells treated with J54 were placed inside. J54 exhibited a notable reduction in cell invasion through the geltrex, as indicated by the reduced number of invaded cells in the lower chamber. Statistical significance was determined by student’s t-test, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, compared to the control group (n =3, independent replicates).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5229526/v1/7eca0e1d1daafb4b933812b1.jpg"},{"id":81569567,"identity":"14893eb3-96a1-4091-9378-0b715dffc968","added_by":"auto","created_at":"2025-04-28 16:07:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1188975,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5229526/v1/ca43a00f-5186-497f-a6ed-62d91aad2bd4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"TLK1 as a Druggable Target in TMZ-Resistant Glioblastoma: Evaluating Small Molecule as a Promising Drug Candidate","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAcquired resistance to cancer treatments is a formidable challenge, particularly in glioblastoma (GBM) therapy. The treatment option available for GBM is very limited, and resistance to the available treatment regimen (temozolomide (TMZ) and radiation therapy) is a major concern. Standard GBM treatment involves maximal safe tumor excision followed by radiotherapy and/or TMZ.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Unfortunately, combined radiotherapy and TMZ extend survival to 5 years in less than 5% of patients.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e Nevertheless, these therapies often relapse and the tumour become invariably resistant to radiation/TMZ regimen. Due to very few therapeutic options available for GBM, TMZ-resistant GBM patients have limited options. Therapeutic resistance to radiation and TMZ is, therefore, a major obstacle in improving treatments for patients with GBM.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Understanding the mechanisms of resistance can help improve treatment efficacy and facilitate the development and implementation of novel interventions to improve overall therapy for GBM.\u003c/p\u003e \u003cp\u003eAs per the current understanding in the field, three primary modes of TMZ resistance have been proposed.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Firstly, De-silencing of the direct repair (DR) enzymes (MGMT and ALKBH), which reverse TMZ-induced O6-methylguanine (O6MG), increased expression of DR enzyme can contribute towards TMZ resistance. Secondly, Loss of the mismatch repair (MMR): The mismatch repair introduces continuous single-strand DNA break in order to repair the O6MG introduced by TMZ. Therefore, loss of MMR will compromise the generation of ssDNA and the formation of DSB, thus, preventing the GBM cell from apoptosis. Finally, the up-regulating the DNA double-strand repair (DSB) pathway: The eventual cause of lethality due to TMZ is due to an increased number of DSB formations. Therefore, augmented DSB proteins contribute towards TMZ resistance, particularly in cells with compromised MGMT expression.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIncreasing bodies of evidence have suggested that elevated DSB repair majorly contributes to TMZ resistance in cells where MGMT expression is compromised.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e DDR pathway aberrations predispose cells to cancerous growth and make them excessively dependent on other parallel pathways for survival. Interestingly, GBM is widely reported to have defects in the DDR pathway proteins apart from epigenetic MGMT silencing, which can also serve as biomarker/s for TMZ- \u0026amp; radio-resistance. Many DSB repair proteins have been explored as a therapeutic target in glioblastoma therapy in the last few years. A few of the DSB repair protein inhibitors are in clinical trials. However, one of the major limitations of many drug candidates are poor blood-brain barrier permeability. Therefore there is an increasing need to identify novel targets and inhibitors to improve the clinical outcome of therapeutic-resistant GBM.\u003c/p\u003e \u003cp\u003eTousled-like kinase 1 (TLK1) is one of the critical kinases whose role has been implicated in DSB repair and radio-resistance.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e At the molecular level, depletion of TLK1 promotes cell death through the generation of DSBs.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Its expression is frequently up-regulated in many human cancers, including GBM. Recent studies of TLK1 in GBM cells and xenograft models have indicated an important role of this protein in cell survival.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e We further have demonstrated that elevated TLK1 levels facilitate cellular repair of DNA damage induced by temozolomide. Inhibition of TLK1 using J54 results in the effective killing of GBM cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The previous study in breast and prostate cancer has suggested that an elevated level of TLK and its isoform TLK1b contributes to chemo- and radio-resistance.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Based on these findings, we hypothesize that TLK1, a key player in DNA repair, may facilitate TMZ resistance in GBM by repairing TMZ-induced DSBs. Thus, inhibiting TLK1 in GBM could sensitize cancer cells to TMZ treatment, potentially improving survival outcomes for patients with TMZ-resistant GBM.\u003c/p\u003e \u003cp\u003eThis study aims to assess the therapeutic potential of J54, an in-house synthesized TLK1 inhibitor, for improving the treatment response in temozolomide (TMZ)-resistant glioblastoma (GBM). We intend to explore the chemical biology of TLK1 to enhance therapeutic outcomes for these resistant GBM cells. We will employ the phenothiazine-based TLK1 inhibitor J54, developed in-house, which we previously demonstrated to exhibit cytotoxic effects against GBM cells.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Phenothiazines, known for their use as antipsychotic drugs, can cross the blood-brain barrier (BBB) and were identified as first-generation TLK inhibitors by our lab and others.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Overall, through this study, we hope to improve the therapeutic outcome of TMZ-resistant glioblastoma cells and anticipate approaches for developing novel treatments for patients with GBM.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines\u003c/h2\u003e \u003cp\u003eLN229 and LN18 cell lines were procured from National Cell Repository at National Centre For Cell Science, Pune, India. The cell lines were grown in DMEM with 10%FBS. LN229 TMZ-R cells were obtained by subjecting the LN229 cells to intermittent exposure to increasing concentrations of TMZ (ranging from 50 \u0026micro;M to 200 \u0026micro;M), with intervening periods of non-exposure.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCytotoxicity assay\u003c/h3\u003e\n\u003cp\u003eThe cytotoxicity assay was conducted using the Cell Titer Glo (CTG, Promega Corporation) as previously described\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. In brief, viable cells were counted using a hemocytometer after staining the cells with trypan blue. Approximately 2000 cells were seeded in 96-well white plates, and 24 hours after seeding, the respective treatments were administered. After 72 hours of treatment, the appropriate amount of CTG reagent was added to the plates, and the plates were subjected to shaking for 15 minutes to ensure proper mixing. Subsequently, luminescence measurements were recorded by Envision multimode plate reader. The results were analyzed as a percentage of viable cells after treatment, relative to the untreated cells, based on a total of three independent replicates.\u003c/p\u003e\n\u003ch3\u003eClonogenic treatment\u003c/h3\u003e\n\u003cp\u003eAround 1000 cells of each respective cell line were plated in a 12-well plate and exposed to corresponding treatment groups for a duration of 5 days. Subsequently, the cultures were incubated for approximately two weeks to allow cell colony formation from the surviving fraction of cells. Following the incubation period, the cell colonies were fixed and stained using a 0.5% crystal violet solution, and the resulting colony with more than 50 cells were counted under a microscope.\u003c/p\u003e\n\u003ch3\u003eImmunocytochemistry\u003c/h3\u003e\n\u003cp\u003eThe respective cells were cultured on 18 mm coverslips in 12-well plates at a density of approximately 50,000 cells per well. The plates were then incubated in a humidified CO2 incubator maintained at 37\u0026deg;C. To evaluate the effect of J54 on γH2AX, the cells were treated with 50 \u0026micro;M J54 for 72 hours. Following the treatment period, the cells were fixed with 4% paraformaldehyde, permeabilized with 100% methanol, and subsequently blocked with 5% FBS\u0026thinsp;+\u0026thinsp;0.3% Triton X-100 in 1X PBS. Next, the cells were incubated with the γH2AX primary antibody (1: 500, #2577, CST) overnight at 4\u0026deg;C, followed by the incubation with the Anti-rabbit IgG Alexa Fluor-488-labeled secondary antibody (1:500, #4412, CST) for 2 hours at room temperature. The cells were mounted using a fluoroshield mounting medium containing DAPI (#ab104139, Abcam). The cells were imaged under a Confocal Laser Scanning Microscopy. Image quantification and processing were performed using the ImageJ software.\u003c/p\u003e\n\u003ch3\u003eWestern Blot\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eWestern Blot\u003c/div\u003e \u003cp\u003eLN229 TMZ-R cells were subjected to respective treatments. For the treated samples, the cells were collected and lysed 72 hours after the incubation period. The cellular lysates were obtained using 1X RIPA buffer. Subsequently, the protein bands were resolved on 8% acrylamide gels and subsequently transferred onto PVDF membranes with a pore size of 0.22 \u0026micro;m. The membranes were initially blocked using a 5% skimmed milk solution and subsequently incubated with primary antibodies: E-Cadherin (1:1,000, #14472), Vimentin (1:1,000, #5741), β-Catenin (1:1,000, #8480), N-Cadherin (1:1,000, #3116), β-actin (1:1,000, #SC-47778), TLK1 (1:1,000, #GTX102891), Phospho-RAD9-S328 (1:1,000, #PA5-12670), Phospho-Nek1-T141, Nek1(1:1,000, #sc-398813) overnight at a temperature of 4\u0026deg;C. Following this, the membranes were exposed to secondary antibodies: Anti-mouse IgG, (1:2,500, #7076), Anti-rabbit IgG (1:2,500, #7074), conjugated with HRP for duration of 2 hours at room temperature. The protein bands were then visualized by subjecting the blots to ECL substrates, and the imaging process was carried out using the Bio-Rad ChemiDoc Imaging System and the analysis was done using Image lab software (Biorad). This image shown in the figure panel is cropped sections from the original blot. Full-length uncropped versions of the gel (or blot) are provided in Supplementary file.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWound healing assay\u003c/h2\u003e \u003cp\u003eThe cells were cultured as a monolayer and were pretreated with 25 \u0026micro;M J54 for 36 hrs prior to creating the wound. A wound was made using a micropipette tip. The confluency of the cells is nearly 90% at this point. Subsequently, the cells were incubated in media containing J54 for 24 hours. The wound area was imaged at both time\u0026thinsp;=\u0026thinsp;0 hours and 24 hours after the wound creation, and the area under the wound was compared. The quantification and processing of the wound area and images were done using the ImageJ software (n\u0026thinsp;=\u0026thinsp;3, independent replicates).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTranswell invasion assay\u003c/h3\u003e\n\u003cp\u003eThe transwell chambers with an 8-micron pore size (Corning Incorporated, Corning, NY, USA) were pre-coated with 1% geltrex (Thermo Fisher Scientific Inc.) in serum-free media for duration of 1 hour. Afterwards, the cells in serum-free media, along with their respective treatments, were seeded into the transwell chambers that had been pre-coated with geltrex. In the lower chamber, complete media containing 10% FBS was added as a chemo-attractant to promote cell invasion. After a 72-hour incubation period, the cells that had invaded through the geltrex and attached to the lower chamber of the plate were stained using Hoechst. Subsequently, the number of invaded cells was quantified by counting them under the microscopic field (n\u0026thinsp;=\u0026thinsp;3, independent replicates).\u003c/p\u003e\n\u003ch3\u003eLive/Dead staining\u003c/h3\u003e\n\u003cp\u003eApproximately 10,000 cells were plated in a black, transparent-bottom plate. After 24 hours of seeding, the respective treatments were applied to the cells. Following a 72-hour treatment period, the media was removed, and the cells were stained with 2.5 \u0026micro;g/ml propidium iodide and Hoechst before being incubated at 37\u0026deg;C in a humidified incubator with 5% CO2 for 15 min. Fluorescence measurements were then recorded using an Envision multimode plate reader for quantitative analysis. Additionally, images were captured using a Nikon inverted fluorescent microscope.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGeneration and validation of TMZ-resistant glioblastoma cell line\u003c/h2\u003e \u003cp\u003eDue to the absence of an existing model system for investigating temozolomide-acquired resistance, an approach was adopted to generate temozolomide-resistant glioblastoma cell lines. Clinically, patients with methylated promoters of the O-6-Methylguanine-DNA Methyltransferase (MGMT) gene display improved responsiveness to temozolomide (TMZ) treatment in comparison to patients with MGMT expression\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Consequently, the LN229 cell line, characterized by MGMT deficiency, was chosen for the generation of temozolomide-resistant cells.\u003c/p\u003e \u003cp\u003eThe LN229 cells were subjected to intermittent treatment with progressively increasing doses of TMZ over a duration exceeding 6 months. The objective was to identify the cell population that acquired resistance to temozolomide (TMZ-R) during this extended period. In the initial treatment cycle, LN229 cells were exposed to a dose of 50 \u0026micro;M TMZ for 5 days, and subsequently, the surviving fraction of cells was allowed to grow for nearly 3 weeks. The surviving cells underwent a second cycle of TMZ treatment with a dosage of 100 \u0026micro;M for 12 days. Afterwards, these cells were cultivated in TMZ-free media for 18 days. Following this, the surviving cells were exposed to media containing 150 \u0026micro;M TMZ for three weeks. Afterwards, the cells were grown 1 week off TMZ. Further, the surviving cells were continually cultured in TMZ containing media with a dosage of 200 \u0026micro;M for an additional 13 weeks. Subsequently, the TMZ-R cells were maintained in 100 \u0026micro;M TMZ for further experiments.\u003c/p\u003e \u003cp\u003eThe cells gradually adapted to grow in the presence of TMZ. In the first cycle of TMZ exposure major fraction of cells died. However, with gradual and intermittent increases in TMZ concentration, the fraction of cell death gradually reduced, and the cells started to grow as a colony. Afterwards, the responsiveness towards TMZ-sensitive (TMZ-S) and TMZ-resistant (TMZ-R) cells was assessed by performing the clonogenic assay. Additionally, to check if the TMZ-R cells can revert back to TMZ-sensitive phenotype, the cells were grown in the absence of TMZ for few passages, and the responsiveness of the cells was checked using a clonogenic assay. If the cell has acquired resistance, then there will be a similar response of both the cell population, and they will respond to TMZ better compared to TMZ-S cells. As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the LN229 TMZ-R cells and TMZ-R cells cultured without TMZ for few passages displayed significantly enhanced cell viability and proliferation in comparison to TMZ-S cells. Furthermore, no significant difference was observed in the responsiveness to TMZ between the TMZ-R cells and TMZ-R cells grown without TMZ. These results strongly suggest that the cells have indeed acquired resistance to TMZ and do not revert to a sensitive phenotype even when cultured in TMZ-free media. Additionally, LN18 cells were taken as an external control, as it is a MGMT proficient cell and can grow in the presence of TMZ.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of TMZ-resistant glioblastoma cell line\u003c/h2\u003e \u003cp\u003eIn LN229 cells, prolonged administration of TMZ led to acquired resistance to the drug and was accompanied by a change in cell morphology. LN229 TMZ-R displayed a relatively elongated, flattened and spread cellular appearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These morphological changes have been previously reported in other studies exploring the effects of prolonged TMZ treatment. There is a strong correlation between changes in the expression of Epithelial-Mesenchymal (E-M) markers and changes in cellular shape and behavior, resulting in aggressive cancer cell morphologies due to acquired drug resistance.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e Deregulation in E-M markers is characterized by the change in expression of epithelial markers (such as E-cadherin) and mesenchymal markers (such as N-cadherin, Vimentin). These changes in marker expression are often accompanied by alterations in cellular morphology and migratory and invasive capabilities. Therefore we next explored whether the change in cellular morphology of TMZ-R cells is also accompanied by the deregulation of the Epithelial-Mesenchymal (E-M) markers. As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, there was an increase in the expression of E-cadherin and a decrease in the expression of N-cadherin, Vimentin and β-catenine. This suggests that the cells are undergoing Mesenchymal-Epithelial Transition (MET). Emerging evidence indicates that MET-driven drug resistance may contribute to reduced sensitivity to various chemotherapeutic agents and targeted therapies in cancer cells.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e During MET, cells undergo morphological changes, and mesenchymal markers are down-regulated, while epithelial markers are up-regulated. This process results in the restoration of cell-cell adhesion. The exact correlation underlying MET-mediated drug resistance is not well understood. Furthermore, MET may potentially offer cancer cells a survival advantage, allowing them to tolerate the cytotoxic effects of chemotherapy and grow clonally.\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Overall, this may contribute to treatment failure.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTo determine the protein expression status TLK1/NEK1 DDR axis in temozolomide-resistant and sensitive glioblastoma cell line\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eA previous study revealed that TLK1 is significantly up-regulated in Glioblastoma, and its knockdown and inhibition (reported elsewhere) were found to strongly decrease the viability of glioblastoma cells. The up-regulation of DNA repair proteins, including TLK1, facilitates the efficient repair of drug-induced DNA damage, leading to resistance against DNA-damaging drugs like TMZ. To investigate the potential role of TLK1 overexpression in TMZ-acquired resistance in glioblastoma, firstly, the expression and activity of TLK1 were examined. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, the expression of TLK1/1B and p-Nek1 was up-regulated in TMZ-resistant cells, and inhibiting TLK1 may sensitize these resistant cells to TMZ. To explore this further, cell viability analysis and clonogenic assays were performed using J54, a previously characterized in-house potent TLK1 inhibitor\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. J54 belongs to the phenothiazine class of small molecules, which are known to cross the blood-brain barrier (BBB). Furthermore, our previous study identified J54 as a promising drug candidate for GBM, with a high likelihood of crossing the blood-brain barrier (BBB)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Therefore, this study investigated the therapeutic potential of J54 in sensitizing TMZ-acquired resistant cells. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C demonstrates that J54 exhibited a dose-dependent reduction in cell viability and clonogenic potential. However, no significant change was observed when TMZ was exposed to the TMZ-resistant cells; in contrast, LN229 (TMZ sensitive) cells showed responsiveness to TMZ. These findings suggest that TLK1 overexpression may play a role in the development of TMZ-acquired resistance in glioblastoma cells. Moreover, the inhibition of TLK1 with the potent in-house developed TLK1 inhibitor, J54, showed promise in sensitizing TMZ-resistant cells, thereby providing potential therapeutic implications for addressing drug resistance in glioblastoma treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of TLK1 results in accumulation of DNA strand break in GBM Cells\u003c/h2\u003e \u003cp\u003eIn this study, we aimed to investigate the impact of J54 on the TLK1 signaling pathway in Glioblastoma (GBM). To assess this effect, we performed an immunoblot assay to analyze the phosphorylation status of downstream substrates of TLK1 in response to cellular treatment. Specifically, we focused on Rad9 (S328) and Nek1 (T141), which are known to be phosphorylated by TLK1/1b in the context of DNA damage response and repair processes. As expected, the results demonstrated a dose-dependent reduction in the phosphorylation of Rad9 and Nek1 with an increase in the concentration of J54 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). This observation indicates that J54 effectively inhibits the cellular activity of TLK1 kinase, thereby disrupting the downstream signaling events involved in DNA damage response and repair.\u003c/p\u003e \u003cp\u003eBy attenuating the phosphorylation of Rad9 and Nek1, J54 may impair the proper functioning of DNA repair mechanisms mediated via TLK1. In the process of acquiring resistance to TMZ through prolonged exposure, cancer cells experience an overall increased burden of DNA strand breaks. Consequently, inhibiting TLK1 further compromises the repair of DNA strand breaks, resulting in the accumulation of unrepaired DNA damage that may be lethal for cell survival. To assess this effect, the level of γH2AX, a marker of DNA strand break, was examined. As observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, a significant increase in the level of γH2AX was observed in response to J54 treatment, indicating the substantial accumulation of DNA breaks in the TMZ-R cells. The findings highlight the potential of J54 as a therapeutic agent that disrupts the DNA repair process mediated by TLK1, leading to increased DNA damage accumulation in cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of TLK1 results in the cell death of GBM Cells\u003c/h2\u003e \u003cp\u003eThe impairment of DNA repair processes could result in the accumulation of DNA strand breaks, ultimately triggering cell death and contributing to the inhibition of tumour growth. To confirm this, the J54-treated cells were stained with propidium Iodide (PI) and Hoechst-33342. The staining technique using PI and Hoechst-33342 allows for the differentiation between live and dead cells. PI is a cell impermeable dye that can selectively stain dead cells, while Hoechst-33342 can stain all the cells. Upon examination, the J54-treated cells exhibited a significantly higher fraction of cells that stained positively for PI (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This overall confirms that the J54-treated cells result in the accumulation of DNA strand breaks due to TLK1 inhibition that leads to cell death.\u003c/p\u003e \u003cp\u003eThese findings contribute to the growing understanding of the molecular mechanisms underlying the therapeutic effects of J54 and its potential as a targeted therapy in cancer treatment. By disrupting DNA repair processes and promoting the accumulation of DNA strand breaks, J54 offers a promising approach to induce cell death and inhibit tumour growth. This may hold promise for sensitizing TMZ-resistant GBM cells, thereby opening the window for therapeutic interventions. Further investigation into the precise mechanisms underlying the interaction between TLK1, DNA repair pathways, and acquired drug resistance is crucial for fully understanding the therapeutic implications and potential of J54 as a targeted therapy in cancer treatment. In conclusion, inhibition of TLK1 in cancer cells can disrupt the repair of DNA strand breaks, leading to DNA damage accumulation and potential cell death. Targeting TLK1 as a therapeutic approach holds promise in sensitizing cancer cells to DNA-damaging treatments and may offer new strategies to overcome drug resistance and improve cancer treatment outcomes. However, further investigation is needed to fully understand the role of TLK1 in DNA repair and its therapeutic potential in cancer therapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of TLK1 results in Reduced Cell migration and Invasive Capacity of GBM Cells\u003c/h2\u003e \u003cp\u003eRecent research has also identified TLK1 as a key kinase that plays a crucial role in promoting cancer cell motility and invasiveness. Inhibition of TLK1 can have significant effects on cell migration and invasion in cancer cells. TLK1 has been linked to the regulation of actin cytoskeleton dynamics, which is essential for cell migration and invasion. By promoting actin filament stability and remodeling, TLK1 facilitates the formation of cellular protrusions, such as lamellipodia and filopodia, necessary for cell movement. Consequently, cancer cells treated with TLK1 inhibitors may exhibit decreased migratory capacity, limiting their ability to invade surrounding tissues and metastasize to distant sites\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. To investigate this further, wound healing and transwell invasion assays were conducted. The wound healing assay allows the observation of cell migration in vitro, where a wound is created in a cell monolayer, and the closure of the wound over time indicates cell migration capacity. On the other hand, the transwell invasion assay assesses the ability of cells to invade through an extracellular matrix barrier, implying the invasive potential of cancer cells in vivo. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, treatment with J54 resulted in a substantial decrease in the migratory and invasive abilities of TMZ-resistant cells. Overall, these findings emphasize the therapeutic significance of targeting TLK1 as a strategy to potentially impede cancer cell migration and invasion. By inhibiting the kinase activity of TLK1, it may be possible to hinder the processes that promote cell motility, invasiveness, and metastasis, ultimately suppressing cancer progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eGlioblastoma multiforme is a particularly challenging form of brain cancer, and the use of temozolomide has shown marginal progress in improving patient outcomes. This chemotherapeutic agent has become a cornerstone of treatment protocols. However, there is a growing concern regarding the development of TMZ acquired resistance, which might lead to therapeutic failure. Conventionally, the resistance to TMZ has been closely associated with the expression MGMT enzyme. This enzyme is known to repair the DNA damage inflicted by TMZ, thereby reducing the effectiveness of the drug. Yet, recent research has unveiled a more complex landscape. Interestingly, the potential involvement of the mismatch repair (MMR) complex is associated with TMZ-acquired resistance. Furthermore, emerging evidence suggests that proteins responsible for repairing double-strand breaks (DSBs) in DNA may also be a major contributor in TMZ resistance. The augmented activity of these DSB repair proteins could potentially enhance the ability of the cancer cells to overcome the damage inflicted by TMZ, rendering the treatment less effective.\u003c/p\u003e \u003cp\u003eTLK1, a DNA repair enzyme, was identified as one of the highly up-regulated kinase in glioblastoma using genetic screening. The overexpression of TLK1 in TMZ-resistant cells is likely to trigger the DNA damage response (DDR) cascade, leading to an augmented DNA repair process. In our study, we found that the introduction of TLK1 inhibitor, J54, hampers the DDR pathway, leading to the accumulation of DNA damage and double-strand breaks (DSBs), ultimately culminating in cell death in TMZ resistance-acquired cells. The findings from this study suggest that J54 holds immense promise as a potential therapeutic agent for GBM, as it targets TLK1, an important kinase involved in DNA damage response and repair.\u003c/p\u003e \u003cp\u003eFurther investigation into the precise mechanisms underlying the role of TLK1 in drug resistance and the potential of J54 as a sensitizing agent is warranted for the development of effective therapeutic strategies. The development of targeted therapies that interfere with the TLK1 signaling pathway may offer new avenues for the treatment of TMZ-acquired resistance GBM cells.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of Interest:\u003c/h2\u003e \u003cp\u003eThe authors disclose no conflicts.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe project was conceptualized by BP and SK, with BP conducting the investigation. BP performed all the experiments. Data analysis was carried out by SK and BP.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors extend their sincere gratitude to Prof. Benedetti for generously providing the pNek1 antibody. We acknowledge the help from Dimple Chhabria, Janhvi Dhongdi in some experiments. SK sincerely thanks the support from the Kankuben Bakshirambhai Gelot Chair.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStupp, R. et al. Radiotherapy plus Concomitant and Adjuvant Temozolomide for Glioblastoma. \u003cem\u003eN Engl. J. Med.\u003c/em\u003e \u003cb\u003e352\u003c/b\u003e (10), 987\u0026ndash;996 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohammed, S., Dinesan, M. \u0026amp; Ajayakumar, T. 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A dynamic in vivo model of epithelial-to-mesenchymal transitions in circulating tumor cells and metastases of breast cancer. \u003cem\u003eOncogene 2012 3133\u003c/em\u003e. \u003cb\u003e31\u003c/b\u003e (33), 3741\u0026ndash;3753 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalil, M. I., Singh, V., King, J. \u0026amp; De Benedetti, A. TLK1-mediated MK5-S354 phosphorylation drives prostate cancer cell motility and may signify distinct pathologies. \u003cem\u003eMol. Oncol.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (13), 2537\u0026ndash;2557 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalil, M. I. \u0026amp; De Benedetti, A. The TLK1\u0026ndash;MK5 Axis Regulates Motility, Invasion, and Metastasis of Prostate Cancer Cells. \u003cem\u003eCancers (Basel)\u003c/em\u003e ;\u003cb\u003e14\u003c/b\u003e(23). (2022).\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":"Blood-Brain-Barrier, glioblastoma, temozolomide, phenothiazine, tousled-like kinase-1, DNA damage response.","lastPublishedDoi":"10.21203/rs.3.rs-5229526/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5229526/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe acquired resistance to existing therapies poses a grave concern in achieving successful therapeutic outcomes. Temozolomide (TMZ), a widely used alkylating chemotherapeutic in Glioblastoma therapy, often encounters resistance, necessitating the investigation of the underlying mechanisms of TMZ-acquired resistance. To study TMZ resistance, a cell-based model system was generated by intermittently exposing glioblastoma cells to increasing concentrations and time of TMZ over six months. The survival response of cells at higher concentrations confirmed TMZ-resistant cells, which exhibited a phenotypic shift toward a mesenchymal-like state, with decreased epithelial traits, indicating mesenchymal-epithelial transition (MET). This transition likely facilitates the stabilization and clonal growth of TMZ-resistant cells.\u003c/p\u003e \u003cp\u003eSubsequent analysis revealed elevated expression of TLK1, a DNA repair protein, thus reinforcing its potential involvement in mechanisms associated with acquired resistance. To explore the therapeutic aspect of TLK1 inhibition, we utilized an in-house developed TLK1 inhibitor, J54. The inhibition of TLK1 in TMZ-resistant cells enhanced cytotoxicity, indicating TLK1 as a potential target to combat TMZ resistance. Moreover, TLK1 inhibition reduced cell migration and invasion, implying its role in promoting metastasis. In conclusion, our study sheds light on the role of TLK1 in the context of TMZ resistance, highlighting its potential as a valuable target for therapeutic intervention.\u003c/p\u003e","manuscriptTitle":"TLK1 as a Druggable Target in TMZ-Resistant Glioblastoma: Evaluating Small Molecule as a Promising Drug Candidate","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-20 09:20:05","doi":"10.21203/rs.3.rs-5229526/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-04T07:44:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-01T19:58:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235912144350353831443435848588215660634","date":"2024-10-18T21:52:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326939495097110095869028119751537244193","date":"2024-10-16T14:44:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-16T14:35:07+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-16T14:31:58+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-10-14T04:27:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-10T09:12:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-09T05:36:19+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":"807bedd7-a594-4a2b-acbd-332149e4b46c","owner":[],"postedDate":"November 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39769141,"name":"Biological sciences/Chemical biology/Kinases"},{"id":39769142,"name":"Biological sciences/Chemical biology/Small molecules"},{"id":39769143,"name":"Biological sciences/Chemical biology/Target validation"},{"id":39769144,"name":"Biological sciences/Cancer"}],"tags":[],"updatedAt":"2025-04-28T16:00:36+00:00","versionOfRecord":{"articleIdentity":"rs-5229526","link":"https://doi.org/10.1038/s41598-025-86599-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-26 15:57:16","publishedOnDateReadable":"April 26th, 2025"},"versionCreatedAt":"2024-11-20 09:20:05","video":"","vorDoi":"10.1038/s41598-025-86599-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-86599-3","workflowStages":[]},"version":"v1","identity":"rs-5229526","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5229526","identity":"rs-5229526","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}