HSP90 inhibition disrupts telomere maintenance and promotes chromosomal instability (CIN) in cancer cells

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Telomere dysfunction triggers chromosomal instability (CIN). To quantify telomere-directed activity and separate it from general mitotic effects, we utilized a human artificial chromosome (HAC) assay with isogenic lines carrying a linear, telomere-containing EGFP HAC or a circular, telomere-lacking EGFP HAC. Methods Four HSP90 inhibitors (TAS-116, XL-888, SNX-2112, 17-AAG) were tested at each compound’s cell-specific LC50 in HT1080 (linear and circular HACs) and HEK293 (linear HAC) cells, with GRN163L as a positive control. HAC loss was quantified by flow cytometry. Telomere length was measured by Southern blot and qPCR in parental HT1080 and HEK293 cells, and telomere signal intensity by FISH in HEK293. Telomere dysfunction (TIFs; γH2AX/TRF2) and micronuclei (MNi) were also scored. Results TAS-116 showed the strongest telomere-specific activity among HSP90 inhibitors, significantly increasing linear HAC loss in both HT1080 and HEK293 while circular HACs showed only minimal instability. TAS-116 shortened telomeres in both lines (qPCR: ~1.8-fold in HT1080; ~2-fold in HEK293) and, in HEK293, reduced FISH telomere signal to 59.66% of control. TAS-116 also increased telomere-associated damage (~ 1.8 TIFs per TIF-positive nucleus), DNA double-strand breaks, and micronuclei relative to the other HSP90 inhibitors. Conclusions TAS-116 consistently disrupts telomere maintenance—driving linear HAC loss, telomere shortening, reduced FISH signal, elevated TIFs, and increased MNi—thereby validating the HAC-based framework for discriminating telomere-directed activity. These findings support the therapeutic promise of HSP90 inhibition against telomere maintenance in cancer. HSP90 inhibitors chromosomal instability CIN telomere dysfunction human artificial chromosome HAC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Telomeres, repetitive TTAGGG sequences at chromosome ends, preserve genomic integrity by preventing DNA damage responses and aberrant repair events such as chromosome fusions [ 1 ]. In most somatic cells, telomeres progressively shorten with each division due to the end-replication problem. In contrast, 85–90% of cancers reactivate telomerase, a ribonucleoprotein complex comprising the catalytic subunit hTERT and the RNA template hTR, to sustain telomere length and enable unlimited proliferation [ 2 ]. Because telomerase activity is nearly absent in normal somatic cells yet crucial for tumor immortality, it remains a prime therapeutic target. Telomerase inhibitors such as GRN163L (Imetelstat) induce telomere shortening and growth arrest in a variety of cancer models [ 3 , 4 ]. However, resistance and inconsistent efficacy have also been reported, for example in pancreatic cancer cell lines where GRN163L-treated cells maintained very short but stable telomeres for extended periods before entering crisis [ 5 ]. These observations highlight the need for alternative approaches to disrupt telomere maintenance. One such approach is the use of G-quadruplex (G4) stabilizing ligands, which target guanine-rich telomeric DNA to block telomerase access and trigger DNA damage responses [ 6 – 8 ]. G4s are noncanonical secondary DNA structures formed in guanine-rich regions, including telomeres [ 9 ]. Stabilization of G4s with small molecules induces telomere dysfunction, providing a therapeutic avenue [ 10 ]. Our previous work established a quantitative assay for telomere-specific CIN using isogenic cell lines: one carrying a linear, telomere-containing EGFP-expressing HAC [ 11 ] and another with a circular, telomere-lacking HAC [ 12 , 13 ]. This system distinguishes telomere-specific activity from off-target mitotic effects and validated G4 ligands as effective telomere-targeting agents [ 11 , 14 – 17 ]. In addition to telomeric DNA, telomerase itself can be targeted at the protein level. HSP90 is a key chaperone required for hTERT folding, stability, and nuclear localization [ 18 , 19 ]. Prior studies demonstrated that HSP90 inhibitors such as geldanamycin and 17-AAG destabilize hTERT, reducing telomerase activity [ 20 – 23 ]. Building on this, we tested a panel of HSP90 inhibitors (TAS-116, XL-888, SNX-2112, 17-AAG) for their ability to impair telomere maintenance. We further included GRN163L as a positive control. Here we show that these compounds disrupt telomerase function, induce telomere shortening, increase TIFs, and trigger CIN in human cancer cells, with TAS-116 exhibiting the strongest effects. These results underscore the therapeutic potential of HSP90 inhibitors, particularly TAS-116, as telomere-targeting anticancer agents. Methods Cell lines and culture HT1080 cells carrying either a circular alphoidtetO-HAC-EGFP (lacking telomeres)[ 26 ] or a linear 21DqHAC-EGFP (containing telomeres) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Clontech Laboratories, Inc.) and maintained under blasticidin S selection (4 µg/ml for circular HAC and 8 µg/ml for linear HAC). HEK293 cells carrying a GFP-21HAC2 were grown in the same medium supplemented with 10% FBS and maintained with 200µg/ml hygromycin B selection [ 25 ]. All cells were incubated at 37°C in a humidified 5% CO₂ atmosphere. After initiation of drug treatment, cells were cultured in non-selective medium to allow detection of HAC loss. For experiments assessing mitotic abnormalities, parental HT1080 and HEK293 cells without HACs were cultured in DMEM supplemented with 10% FBS under non-selective conditions. Four HSP90 inhibitors—TAS-116, XL-888, SNX-2112, and 17-AAG—were tested. For CIN assays, drug concentrations were adjusted to the LC₅₀ value of each compound in the respective cell line (Supplementary Table S1). For assays examining mitotic abnormalities, the LC₅₀ value concentrations were used, as confirmed experimentally for HT1080 and HEK293 cells. Calculation of HAC loss and Flow cytometry The rate of spontaneous HAC loss and HAC loss after HSP90 inhibitor treatment was calculated as previously described [ 12 ]. EGFP fluorescence expressions from HACs were measured by flow cytometry using a FACSCalibur instrument (BD Biosciences) with CellQuest software. Data was analyzed with FlowJo software. Cells were harvested by trypsinization, and at least 4 × 10⁴ cells were analyzed for each sample. Southern blot Southern blot analysis of telomere length was performed using a dual 5′ biotin-labeled, telomere-specific probe. Genomic DNA (5 µg) was digested with HphI and MnlI restriction enzymes and separated on a 1% agarose gel by electrophoresis at 12 V for 20–24 h. DNA was transferred to a nylon membrane (Hybond-N+, GE Healthcare Life Sciences), denatured with an alkaline solution, and crosslinked by UV irradiation. The membrane was hybridized with ~ 200 pmol of a 20-bp telomere-specific probe (5′-CCCTAACCCTAACCCTAACC-3′; 5′ dual biotin-labeled) in ULTRAhyb® ultrasensitive hybridization buffer (Invitrogen, AM8670) according to the manufacturer’s instructions. Detection was carried out using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific, 89880). Quantitative PCR (qPCR) telomere length analysis Telomere length was measured using the Absolute Human Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. Genomic DNA was extracted from drug-treated cells, and qPCR reactions were performed following the manufacturer’s protocol. Data analysis and calculation of telomere length were carried out using the standard curve method provided by the manufacturer. Immunofluorescence and micronuclei analysis The number of γH2AX foci and the percentage of γH2AX foci colocalizing with telomeric sequences (identified by TRF2 staining) were quantified as previously described [ 11 ]. For each compound, approximately 20–30 nuclei were analyzed (Fig. 4 and Supplementary Table S5). After drug treatment, cells were fixed, permeabilized, and blocked as described in [ 11 ]. Samples were mounted with Vectashield Vibrance mounting medium containing DAPI. Images were acquired on a DeltaVision Core system (Applied Precision). Z-series were collected at 0.25 µm intervals, and image stacks were deconvolved using SoftWorx software. Micronuclei (MNi) formation was assessed under the same experimental conditions. The presence of micronuclei was identified by DAPI staining and confirmed by fluorescence microscopy. The frequency of MNi formation was calculated as the percentage of nuclei containing at least one micronucleus. Telomere fluorescence in situ hybridization (Telo-FISH) To obtain metaphase chromosome spreads, drug-treated cells were incubated overnight in growth medium containing 0.1 mg/ml Colcemid [ 11 , 12 , 35 ]. Metaphase cells were harvested, prepared, and hybridized with a TelC-PNA telomere probe (Cy3-OO-TAACCCTAACCCTAACCC; PNA Bio) [ 35 ]. Drug concentrations were the same as those used for Southern blot and qPCR assays, and GRN163L was included as a positive control (Supplementary Table S3). Slides were analyzed by fluorescence microscopy using the CRC/LRBGE Fluorescence Imaging Facility at NIH. Results HAC-based assay reveals telomere-specific effects of HSP90 inhibitors To identify compounds that specifically target telomeres or telomerase, we used three cell lines each carrying a distinct HAC (Fig. 1 ). Two HT1080-derived lines carried either a linear, telomere-containing EGFP-expressing HAC (21DqHAC) [ 24 , 25 ] or a circular, telomere-lacking alphoid tetO -HAC [ 26 – 28 ]. A third line, derived from HEK293, carried the same linear HAC. Linear HACs (~ 5 Mb) [ 11 , 25 , 29 ] contained functional kinetochores and segregated stably, while the circular HAC lacked telomeres but maintained mitotic stability [ 26 – 28 ]. EGFP expression enabled detection of HAC loss by flow cytometry (Fig. 1 ). Four HSP90 inhibitors were evaluated in this study: TAS-116, XL-888, SNX-2112, 17-AAG. (Fig. 2 ). LC50 values were determined for each compound in each cell line (Supplementary Table S1). HAC loss was measured after 1- and 4-day treatments (Fig. 2 B and 2 C). After 4 days, all HSP90 inhibitors increased linear HAC loss, whereas only mild effects were observed in circular HACs. As shown in Fig. 2 C, fold increases in HAC loss relative to circular HACs were 6.8 (TAS-116), 8.3 (XL-888), 7.0 (SNX-2112), and 5.3 (17-AAG). One-day treatment did not produce significant effects. Comparable HAC loss was observed in both HT1080 and HEK293 cells carrying linear HACs, though HT1080 showed slightly higher sensitivity (Fig. 2 and Supplementary Figure S1). 17-AAG induced marked HAC loss in HT1080 cells but had weaker effects in HEK293, whereas TAS-116, XL-888, and SNX-2112 showed more consistent activity. GRN163L produced robust effects in both cell lines. (Supplementary Figure S1). The circular HAC remained stable under all treatments. These results validate the HAC-based assay as a tool for detecting telomere-targeting activity and identify TAS-116 as a potent inducer of telomere-specific CIN [ 30 – 32 ]. HSP90 inhibitors induce telomere shortening in genomic chromosomes We next measured genomic telomere length in HT1080 and HEK293 cells by Southern blot and qPCR after 20 days of treatment with HSP90 inhibitors (TAS-116, XL-888, SNX-2112, 17-AAG) or GRN163L as positive control (Fig. 3 ). Concentrations were optimized for each line (Supplementary Table S3). TAS-116 induced the strongest shortening in both cell types (Fig. 3 A, 3 B). qPCR confirmed that TAS-116 reduced telomere length 1.8-fold in HT1080 and 2-fold in HEK293. GRN163L produced a 1.5-fold reduction in HT1080 and a 3-fold reduction in HEK293. Southern blot analysis supported these patterns, with statistical values provided in Supplementary Table S3B (HT1080) and S3C (HEK293). Collectively, TAS-116 emerged as the most effective HSP90 inhibitor for telomere shortening, with effects comparable to the positive control GRN163L. FISH confirms TAS-116–induced telomere dysfunction in HEK293 cells To validate these findings, telomere signal intensity was quantified in HEK293 cells after 20 days of treatment with TAS-116, XL-888, or GRN163L (positive control), with untreated cells serving as a negative control (Supplementary Figure S2). Using FISH, positive control GRN163L reduced signal intensity to 55.15% of control (p < 0.0001). TAS-116 reduced signal intensity to 59.66% of control (p < 0.0001), while XL-888 reduced it to 70.78% (p < 0.0001) (Supplementary Figure S2B and Supplementary Table S4). These results corroborate the Southern blot and qPCR findings and confirm that TAS-116 is the most potent HSP90 inhibitor for inducing telomere dysfunction under these conditions. TAS-116 induces telomere-associated DNA damage and micronuclei formation To investigate the mechanisms of telomere dysfunction, we quantified DNA double-strand breaks (DSBs), telomere dysfunction–induced foci (TIFs), and micronuclei (MNi) in HEK293 cells treated with HSP90 inhibitors, with GRN163L included as a positive control (Fig. 4 ). γH2AX immunostaining revealed that GRN163L induced the highest number of DSBs (28 foci/cell, p < 0.0001), followed by TAS-116 (9 foci/cell, p < 0.0001). XL-888 (2.8 foci/cell), SNX-2112 (1.8 foci/cell), and 17-AAG (1.8 foci/cell) had weaker effects (Fig. 4 A and Supplementary Table S5A). TIFs, defined as colocalization of γH2AX with the telomeric protein TRF2, showed that TAS-116 induced on average 1.8 TIFs per TIF-positive nucleus, whereas GRN163L induced 3.8, and no TIFs were detected with the other inhibitors (Fig. 4 A and Supplementary Table S5A). MNi formation, a marker of CIN, was highest with GRN163L (26.62%, p < 0.0001) and TAS-116 (22.98%, p < 0.0001). Lower rates were observed with XL-888 (5.61%), SNX-2112 (4.31%), and 17-AAG (5.66%) compared to 1.79% in controls (Fig. 4 B and Supplementary Table S5B). Representative images are shown in Fig. 4 B. These findings, consistent with prior assays, confirm that TAS-116 and GRN163L induce strong telomere-specific DNA damage and CIN, with TAS-116 emerging as the most potent HSP90 inhibitor in this context. Discussion The rigorous evaluation of telomere-targeting agents is critical for advancing cancer therapeutics, yet few assays can reliably distinguish telomere-specific activity from general cytotoxicity. By employing an HAC-based system with linear and circular HACs, combined with direct analyses of telomere length and function, we established a versatile framework for identifying compounds that disrupt telomere integrity. Our findings demonstrate that TAS-116, an HSP90 inhibitor currently in clinical development, exhibits robust telomere-targeting effects across multiple assays. TAS-116 induced linear HAC loss, genomic telomere shortening, reduced telomere signal intensity, elevated TIFs, and increased micronuclei formation. These effects were comparable in magnitude to those of the positive control GRN163L, a direct telomerase inhibitor, confirming the validity of our assay. All HSP90 inhibitors tested promoted telomere dysfunction to varying degrees, underscoring that HSP90 inhibition broadly impacts telomere integrity, with TAS-116 showing the strongest and most reproducible effects across assays. Mechanistically, TAS-116 binds to the ATP-binding pocket of HSP90 and arrests its chaperone cycle, thereby impairing proper folding, stabilization, and assembly of hTERT (Fig. 5 ). Consequently, telomerase is rendered functionally inactive, consistent with defective holoenzyme assembly and impaired telomere elongation. This explains how TAS-116 indirectly suppresses telomere maintenance while simultaneously destabilizing other oncogenic client proteins of HSP90. Importantly, GRN163L was included as a positive control because our previous work had established its strong activity as a telomerase inhibitor capable of inducing telomere dysfunction when tested in the same HAC-based system. Its reproducible activity across both linear and circular HAC assays provides a reliable benchmark against which novel compounds can be evaluated. As expected, GRN163L consistently triggered robust telomere shortening, DNA damage, and CIN, thereby validating the sensitivity of our experimental framework. Beyond these results, our study contributes to the broader understanding of telomere-directed therapies. Cancer cells rely heavily on telomere maintenance for survival, and strategies such as telomerase inhibition, G-quadruplex stabilization, and Shelterin disruption have all been pursued with varying success. HSP90 inhibition offers an alternative route, indirectly destabilizing telomerase through impaired hTERT folding and assembly while simultaneously impairing multiple oncogenic pathways. The dual functionality of TAS-116—chaperone inhibition and telomere destabilization—may enhance its therapeutic potential by exploiting a fundamental vulnerability of cancer cells. In line with this, TAS-116 has undergone evaluation in a first-in-human phase I clinical trial for solid tumors, supporting the translational relevance of its telomere-targeting activity [ 33 , 34 ]. This work also underscores the methodological value of HAC-based assay. By directly comparing linear and circular HACs, this approach distinguishes telomere-dependent from off-target effects, providing clarity that traditional assays cannot achieve. Such precision makes this system a powerful tool for preclinical evaluation of telomere-targeting agents. Limitations of this study include its reliance on in vitro models and a relatively short treatment window, which may not fully capture long-term telomere erosion or the complexity of in vivo tumor biology. Nevertheless, these limitations point to clear next steps: extending analyses to animal models and expanding compound screening. Importantly, they do not diminish the strong and consistent evidence supporting TAS-116 as a telomere-targeting agent. In conclusion, our results establish TAS-116 as a potent telomere-targeting compound. They also validate the HAC-based assay as an effective framework for evaluating telomere-directed therapeutics. As illustrated in Fig. 5 , HSP90 normally supports telomerase activity by stabilizing and folding hTERT in an ATP-dependent manner, enabling assembly of an active holoenzyme. TAS-116 blocks this cycle by occupying the ATP-binding pocket of HSP90, thereby rendering telomerase functionally inactive. Together, these findings highlight the clinical promise of TAS-116 while underscoring the broader utility of HAC-based and telomere assays in guiding the development of novel cancer therapies. Conclusions TAS-116 consistently disrupts telomere maintenance—inducing linear HAC loss, telomere shortening, reduced telomere FISH signal, elevated TIF formation, and increased micronuclei—thereby validating the HAC-based system as a robust platform for detecting telomere-directed activity. Collectively, these findings highlight the therapeutic potential of HSP90 inhibition as an effective strategy to target telomere maintenance in cancer cells. Abbreviations CIN Chromosome Instability HSP90 Heat Shock Protein 90 HAC Human Artificial Chromosome FISH Fluorescence in situ Hybridization TIF Telomere dysfunction-Induced Foci MNi Micronuclei EGFP Enhanced Green Fluorescent Protein TRF2 Telomeric Repeat-binding Factor 2 Declarations Competing interests The authors declare they have no competing interests. Authors’ contributions HSL, JHK, and LM performed the experiments and analyzed the data. VL, HSL, and JHK contributed to the study design and drafting of the manuscript. LN provided support for the HSP90 inhibitors used in this study. VL, HSL, JHK, and NK were involved in data interpretation and discussion. All authors have read and approved the final manuscript. Data availability All data used and/or analyzed during this study are available from the corresponding author on reasonable request. Ethics approval and consent to participate This study used only established and de-identified human cell lines (HEK293, HT1080, and HAC-engineered derivatives) and did not involve human participants, identifiable human data, or animal subjects. According to NIH policy and federal regulations (45 CFR 46), research using established de-identified cell lines does not require Institutional Review Board (IRB) review or ethics approval. Consent for publication Not applicable Acknowledgements This work was supported by the intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, USA. The authors are grateful to Dr. Shay from University of Texas, Southwestern Medical Center for providing GRN163L and the late Dr. Len Neckers from Urologic Oncology Branch, CRC, National Cancer Institute for providing us HSP 90 inhibitors, TAS-116, XL-888, SNX-2112 and 17-AAG. The authors also would like to thank Dr. Yasuhiro Kazuki from Tottori University for providing HEK293GFP linear HAC cells. Author details 1 Developmental Therapeutics Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA 2 Urologic Oncology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA References de Lange T: Shelterin: the protein complex that shapes and safeguards human telomeres . Genes Dev 2005, 19 (18):2100-2110. Shay JW, Wright WE: Role of telomeres and telomerase in cancer . Semin Cancer Biol 2011, 21 (6):349-353. Dikmen ZG, Gellert GC, Jackson S, Gryaznov S, Tressler R, Dogan P, Wright WE, Shay JW: In vivo inhibition of lung cancer by GRN163L: a novel human telomerase inhibitor . Cancer Res 2005, 65 (17):7866-7873. Tefferi A, Lasho TL, Begna KH, Patnaik MM, Zblewski DL, Finke CM, Laborde RR, Wassie E, Schimek L, Hanson CA et al : A Pilot Study of the Telomerase Inhibitor Imetelstat for Myelofibrosis . N Engl J Med 2015, 373 (10):908-919. 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Whitesell L, Lindquist SL: HSP90 and the chaperoning of cancer . Nat Rev Cancer 2005, 5 (10):761-772. Shimomura A, Yamamoto N, Kondo S, Fujiwara Y, Suzuki S, Yanagitani N, Horiike A, Kitazono S, Ohyanagi F, Doi T et al : First-in-Human Phase I Study of an Oral HSP90 Inhibitor, TAS-116, in Patients with Advanced Solid Tumors . Mol Cancer Ther 2019, 18 (3):531-540. Rastogi S, Joshi A, Sato N, Lee S, Lee MJ, Trepel JB, Neckers L: An update on the status of HSP90 inhibitors in cancer clinical trials . Cell Stress Chaperones 2024, 29 (4):519-539. Liskovykh M, Petrov NS, Noskov VN, Masumoto H, Earnshaw WC, Schlessinger D, Shabalina SA, Larionov V, Kouprina N: Actively transcribed rDNA and distal junction (DJ) sequence are involved in association of NORs with nucleoli . Cell Mol Life Sci 2023, 80 (5):121. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFiles.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 27 Feb, 2026 Reviewers agreed at journal 18 Feb, 2026 Reviewers invited by journal 09 Feb, 2026 Editor invited by journal 19 Dec, 2025 Editor assigned by journal 16 Dec, 2025 Submission checks completed at journal 16 Dec, 2025 First submitted to journal 15 Dec, 2025 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8369668","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588585605,"identity":"ac1cab2e-7960-4987-b550-dd34e991f472","order_by":0,"name":"Hee-Sheung Lee","email":"","orcid":"","institution":"National Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Hee-Sheung","middleName":"","lastName":"Lee","suffix":""},{"id":588585608,"identity":"a1bfbbd1-9f0b-4978-ba76-89ae12ba9d40","order_by":1,"name":"Jung-Hyun Kim","email":"","orcid":"","institution":"National Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Jung-Hyun","middleName":"","lastName":"Kim","suffix":""},{"id":588585610,"identity":"6a150cd2-9f37-49ae-be8b-9ee6e931540d","order_by":2,"name":"Mikhail Liskovykh","email":"","orcid":"","institution":"National Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Mikhail","middleName":"","lastName":"Liskovykh","suffix":""},{"id":588585611,"identity":"1c0104a8-2ca6-46db-9783-af900f24e526","order_by":3,"name":"Neckers Len","email":"","orcid":"","institution":"National Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Neckers","middleName":"","lastName":"Len","suffix":""},{"id":588585612,"identity":"f4ba112f-6bea-491a-91e3-c1cbda823f1b","order_by":4,"name":"Natalay Kouprina","email":"","orcid":"","institution":"National Cancer Institute","correspondingAuthor":false,"prefix":"","firstName":"Natalay","middleName":"","lastName":"Kouprina","suffix":""},{"id":588585613,"identity":"a5a6298e-da61-486e-a4dc-e23dcfac264c","order_by":5,"name":"Vladimir Larionov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIie3RMQrCMBTG8UDBLk+ztiie4UFBEQpeJS7p4iC4ODrVKyg9hiCOlYAuOri41KFF6BxvYKK4KKS6ieQPgW/58YYQYrP9YA31Uj2oHjkhgFWk9iT+VA32KbmHGjI9Kol7KNLrOhwsT6JQV86tLnGPFzARiHCz2PPBKuOoSAm9KYwDIyGciHosgk7GNBGAKXB/biK0fJAgieSHxHtcaWNz+Lzibj1pJCXZLGLe9rLhKGWoiAAHTYRS7shrHAJNoqWUE9HH3azImYG8pD/Fqf7Nt9z8a2Kz2Wx/3Q1HcU0lbAhjLAAAAABJRU5ErkJggg==","orcid":"","institution":"National Cancer Institute","correspondingAuthor":true,"prefix":"","firstName":"Vladimir","middleName":"","lastName":"Larionov","suffix":""}],"badges":[],"createdAt":"2025-12-15 20:08:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8369668/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8369668/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102446835,"identity":"8d6856fa-90d0-4bb9-8fb8-073a14b5799f","added_by":"auto","created_at":"2026-02-11 17:49:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":124438,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design of the HAC-based assay for measuring chromosomal instability (CIN) induced by telomere dysfunction.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Human HT1080 cells carried either the circular alphoid\u003csup\u003etetO\u003c/sup\u003e-HAC (lacking telomeres) or the linear 21DqHAC (containing telomeres), while HEK293 cells carried the linear 21DqHAC. Both HACs stably segregate during normal cell division. Each HAC contains an EGFP transgene, allowing cells that inherit the HAC to display enhanced green fluorescence, whereas cells that lose the HAC lack fluorescence. In untreated control populations, cells maintain stable HACs and show uniform green fluorescence. In contrast, treatment with telomerase inhibitors leads to a greater loss of linear HACs compared to circular HACs, producing a mixed population of EGFP-positive and EGFP-negative cells. (B) The percentage of HAC-containing cells was quantified by flow cytometry (FACS). Compounds that promote HAC loss are thereby identified as inducers of chromosomal mis-segregation and CIN. Importantly, comparison of linear versus circular HACs allows telomere-specific effects of compounds to be distinguished from general cytotoxicity or mitotic defects.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8369668/v1/ff17714e40dd7edf9e9dcd72.png"},{"id":102745940,"identity":"f2cbd489-f01b-4d4b-a982-273785e192cb","added_by":"auto","created_at":"2026-02-16 08:54:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of HSP90 inhibitors on the stability of circular versus linear HACs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Chemical structures of the four HSP90 inhibitors used in this study. (B) Rates of HAC loss one day after treatment in HT1080 cells carrying either a circular or a linear HAC. (C) Rates of HAC loss four days after treatment under the same conditions. HT1080 cells containing either the circular alphoid\u003csup\u003etetO\u003c/sup\u003e-HAC or the linear 21DqHAC were treated with HSP90 inhibitors at the concentrations described in the Supplementary Table S2. The rate of HAC loss was calculated based on the percentage of EGFP-positive versus EGFP-negative cells, as determined by flow cytometry. Untreated controls correspond to the frequency of spontaneous HAC loss. Compared with controls, all HSP90 inhibitors increased linear HAC loss, with TAS-116, XL-888, and SNX-2112 showing the strongest effects after 4 days of treatment, whereas the circular HAC remained largely stable.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8369668/v1/9fa29ee26f0ac3123978a99e.png"},{"id":102446840,"identity":"174fd52f-359d-406f-845d-8dc5dc33164c","added_by":"auto","created_at":"2026-02-11 17:49:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":137630,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of HSP90 inhibitors on telomere length in HT1080 and HEK293 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Telomere length of HT1080 cells was assessed by Southern blot analysis and quantitative PCR after 20 days of treatment with HSP90 inhibitors. (B) Telomere length of HEK293 cells was evaluated under the same conditions. Different concentrations of each inhibitor were applied depending on the cell line, as described in the Supplementary Table S3. GRN163L, a telomerase inhibitor, was included as a positive control.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8369668/v1/a1bc2ab02bd79657d44226f9.png"},{"id":102745808,"identity":"5903ffd2-64b9-4f4f-8e75-887b9f272a95","added_by":"auto","created_at":"2026-02-16 08:54:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":322121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTAS-116 induces telomere-associated DNA damage and micronuclei formation in HEK293 cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Colocalization of γH2AX foci (green) with the telomeric protein TRF2 (red) in HEK293 cells treated for 20 days with TAS-116, XL-888, SNX-2112, 17-AAG, or the telomerase inhibitor GRN163L. Representative immunofluorescence images are shown (DNA counterstained with DAPI, blue). Colocalization of γH2AX and TRF2 signals indicates telomere dysfunction–induced foci (TIFs). Quantification of γH2AX foci and TIFs is presented in the accompanying graphs; at least 100 nuclei were analyzed per condition. (B) Micronuclei (MNi) formation in HEK293 cells after treatment with TAS-116, XL-888, SNX-2112, 17-AAG, or GRN163L. Representative images (arrows) illustrate MNi, and quantification is shown in the graph. TAS-116 and GRN163L induced significantly higher rates of MNi formation compared with other inhibitors and untreated controls. Statistical significance was determined by t-test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8369668/v1/eaf4ef82d33a35a519469d0d.png"},{"id":102746096,"identity":"ab9acc1b-21be-472b-a7dd-5922a9a9d4ef","added_by":"auto","created_at":"2026-02-16 08:55:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of telomerase regulation by HSP90 and its inhibition by TAS-116.\u003c/strong\u003e\u003cbr\u003e\nSchematic representation of the role of HSP90 in telomerase folding and activation, and how TAS-116 disrupts this process. Under normal conditions (right panels), hTERT is associated with HSP90 and co-chaperones such as p23 in an ATP-dependent manner. ATP binding and hydrolysis drive the HSP90 chaperone cycle, allowing proper folding, stabilization, and assembly of hTERT into an active telomerase holoenzyme with hTR and associated factors. This results in a functionally active telomerase complex capable of elongating telomeres and maintaining chromosomal stability. In the presence of TAS-116 (left panel), the inhibitor occupies the ATP-binding pocket of HSP90, blocking its ATPase activity and halting the chaperone cycle. Consequently, hTERT fails to achieve correct folding and assembly, leaving telomerase functionally inactive. This inhibition of telomerase activity contributes to telomere dysfunction and chromosomal instability (CIN), providing a mechanistic basis for TAS-116’s anticancer effects.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8369668/v1/8f975ea8d1309256c12454aa.png"},{"id":104397526,"identity":"f4f52a1f-0a23-476b-b3c9-8599bda7d195","added_by":"auto","created_at":"2026-03-11 11:50:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2942357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8369668/v1/c84a60b6-7fd0-46eb-9403-535dd4e9b0d3.pdf"},{"id":103049261,"identity":"f44fc1b2-e281-4d94-9a5d-ad6d0f3f1224","added_by":"auto","created_at":"2026-02-20 07:39:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3480767,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-8369668/v1/2c57cfb403145926cb0ba49a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"HSP90 inhibition disrupts telomere maintenance and promotes chromosomal instability (CIN) in cancer cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTelomeres, repetitive TTAGGG sequences at chromosome ends, preserve genomic integrity by preventing DNA damage responses and aberrant repair events such as chromosome fusions [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In most somatic cells, telomeres progressively shorten with each division due to the end-replication problem. In contrast, 85\u0026ndash;90% of cancers reactivate telomerase, a ribonucleoprotein complex comprising the catalytic subunit hTERT and the RNA template hTR, to sustain telomere length and enable unlimited proliferation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Because telomerase activity is nearly absent in normal somatic cells yet crucial for tumor immortality, it remains a prime therapeutic target.\u003c/p\u003e \u003cp\u003eTelomerase inhibitors such as GRN163L (Imetelstat) induce telomere shortening and growth arrest in a variety of cancer models [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, resistance and inconsistent efficacy have also been reported, for example in pancreatic cancer cell lines where GRN163L-treated cells maintained very short but stable telomeres for extended periods before entering crisis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. These observations highlight the need for alternative approaches to disrupt telomere maintenance. One such approach is the use of G-quadruplex (G4) stabilizing ligands, which target guanine-rich telomeric DNA to block telomerase access and trigger DNA damage responses [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. G4s are noncanonical secondary DNA structures formed in guanine-rich regions, including telomeres [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Stabilization of G4s with small molecules induces telomere dysfunction, providing a therapeutic avenue [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Our previous work established a quantitative assay for telomere-specific CIN using isogenic cell lines: one carrying a linear, telomere-containing EGFP-expressing HAC [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and another with a circular, telomere-lacking HAC [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This system distinguishes telomere-specific activity from off-target mitotic effects and validated G4 ligands as effective telomere-targeting agents [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to telomeric DNA, telomerase itself can be targeted at the protein level. HSP90 is a key chaperone required for hTERT folding, stability, and nuclear localization [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Prior studies demonstrated that HSP90 inhibitors such as geldanamycin and 17-AAG destabilize hTERT, reducing telomerase activity [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Building on this, we tested a panel of HSP90 inhibitors (TAS-116, XL-888, SNX-2112, 17-AAG) for their ability to impair telomere maintenance. We further included GRN163L as a positive control. Here we show that these compounds disrupt telomerase function, induce telomere shortening, increase TIFs, and trigger CIN in human cancer cells, with TAS-116 exhibiting the strongest effects. These results underscore the therapeutic potential of HSP90 inhibitors, particularly TAS-116, as telomere-targeting anticancer agents.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and culture\u003c/h2\u003e \u003cp\u003eHT1080 cells carrying either a circular alphoidtetO-HAC-EGFP (lacking telomeres)[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] or a linear 21DqHAC-EGFP (containing telomeres) were cultured in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Clontech Laboratories, Inc.) and maintained under blasticidin S selection (4 \u0026micro;g/ml for circular HAC and 8 \u0026micro;g/ml for linear HAC). HEK293 cells carrying a GFP-21HAC2 were grown in the same medium supplemented with 10% FBS and maintained with 200\u0026micro;g/ml hygromycin B selection [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. All cells were incubated at 37\u0026deg;C in a humidified 5% CO₂ atmosphere. After initiation of drug treatment, cells were cultured in non-selective medium to allow detection of HAC loss. For experiments assessing mitotic abnormalities, parental HT1080 and HEK293 cells without HACs were cultured in DMEM supplemented with 10% FBS under non-selective conditions. Four HSP90 inhibitors\u0026mdash;TAS-116, XL-888, SNX-2112, and 17-AAG\u0026mdash;were tested. For CIN assays, drug concentrations were adjusted to the LC₅₀ value of each compound in the respective cell line (Supplementary Table S1). For assays examining mitotic abnormalities, the LC₅₀ value concentrations were used, as confirmed experimentally for HT1080 and HEK293 cells.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCalculation of HAC loss and Flow cytometry\u003c/h3\u003e\n\u003cp\u003eThe rate of spontaneous HAC loss and HAC loss after HSP90 inhibitor treatment was calculated as previously described [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. EGFP fluorescence expressions from HACs were measured by flow cytometry using a FACSCalibur instrument (BD Biosciences) with CellQuest software. Data was analyzed with FlowJo software. Cells were harvested by trypsinization, and at least 4 \u0026times; 10⁴ cells were analyzed for each sample.\u003c/p\u003e\n\u003ch3\u003eSouthern blot\u003c/h3\u003e\n\u003cp\u003eSouthern blot analysis of telomere length was performed using a dual 5\u0026prime; biotin-labeled, telomere-specific probe. Genomic DNA (5 \u0026micro;g) was digested with HphI and MnlI restriction enzymes and separated on a 1% agarose gel by electrophoresis at 12 V for 20\u0026ndash;24 h. DNA was transferred to a nylon membrane (Hybond-N+, GE Healthcare Life Sciences), denatured with an alkaline solution, and crosslinked by UV irradiation. The membrane was hybridized with ~\u0026thinsp;200 pmol of a 20-bp telomere-specific probe (5\u0026prime;-CCCTAACCCTAACCCTAACC-3\u0026prime;; 5\u0026prime; dual biotin-labeled) in ULTRAhyb\u0026reg; ultrasensitive hybridization buffer (Invitrogen, AM8670) according to the manufacturer\u0026rsquo;s instructions. Detection was carried out using the Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific, 89880).\u003c/p\u003e\n\u003ch3\u003eQuantitative PCR (qPCR) telomere length analysis\u003c/h3\u003e\n\u003cp\u003eTelomere length was measured using the Absolute Human Telomere Length Quantification qPCR Assay Kit (ScienCell Research Laboratories, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Genomic DNA was extracted from drug-treated cells, and qPCR reactions were performed following the manufacturer\u0026rsquo;s protocol. Data analysis and calculation of telomere length were carried out using the standard curve method provided by the manufacturer.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence and micronuclei analysis\u003c/h3\u003e\n\u003cp\u003eThe number of γH2AX foci and the percentage of γH2AX foci colocalizing with telomeric sequences (identified by TRF2 staining) were quantified as previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. For each compound, approximately 20\u0026ndash;30 nuclei were analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Supplementary Table S5). After drug treatment, cells were fixed, permeabilized, and blocked as described in [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Samples were mounted with Vectashield Vibrance mounting medium containing DAPI. Images were acquired on a DeltaVision Core system (Applied Precision). Z-series were collected at 0.25 \u0026micro;m intervals, and image stacks were deconvolved using SoftWorx software.\u003c/p\u003e \u003cp\u003eMicronuclei (MNi) formation was assessed under the same experimental conditions. The presence of micronuclei was identified by DAPI staining and confirmed by fluorescence microscopy. The frequency of MNi formation was calculated as the percentage of nuclei containing at least one micronucleus.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTelomere fluorescence in situ hybridization (Telo-FISH)\u003c/h2\u003e \u003cp\u003eTo obtain metaphase chromosome spreads, drug-treated cells were incubated overnight in growth medium containing 0.1 mg/ml Colcemid [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Metaphase cells were harvested, prepared, and hybridized with a TelC-PNA telomere probe (Cy3-OO-TAACCCTAACCCTAACCC; PNA Bio) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Drug concentrations were the same as those used for Southern blot and qPCR assays, and GRN163L was included as a positive control (Supplementary Table S3). Slides were analyzed by fluorescence microscopy using the CRC/LRBGE Fluorescence Imaging Facility at NIH.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHAC-based assay reveals telomere-specific effects of HSP90 inhibitors\u003c/h2\u003e \u003cp\u003eTo identify compounds that specifically target telomeres or telomerase, we used three cell lines each carrying a distinct HAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two HT1080-derived lines carried either a linear, telomere-containing EGFP-expressing HAC (21DqHAC) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] or a circular, telomere-lacking alphoid\u003csup\u003etetO\u003c/sup\u003e-HAC [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A third line, derived from HEK293, carried the same linear HAC. Linear HACs (~\u0026thinsp;5 Mb) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] contained functional kinetochores and segregated stably, while the circular HAC lacked telomeres but maintained mitotic stability [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. EGFP expression enabled detection of HAC loss by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFour HSP90 inhibitors were evaluated in this study: TAS-116, XL-888, SNX-2112, 17-AAG. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). LC50 values were determined for each compound in each cell line (Supplementary Table S1). HAC loss was measured after 1- and 4-day treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). After 4 days, all HSP90 inhibitors increased linear HAC loss, whereas only mild effects were observed in circular HACs. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, fold increases in HAC loss relative to circular HACs were 6.8 (TAS-116), 8.3 (XL-888), 7.0 (SNX-2112), and 5.3 (17-AAG). One-day treatment did not produce significant effects. Comparable HAC loss was observed in both HT1080 and HEK293 cells carrying linear HACs, though HT1080 showed slightly higher sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Figure S1). 17-AAG induced marked HAC loss in HT1080 cells but had weaker effects in HEK293, whereas TAS-116, XL-888, and SNX-2112 showed more consistent activity. GRN163L produced robust effects in both cell lines. (Supplementary Figure S1). The circular HAC remained stable under all treatments. These results validate the HAC-based assay as a tool for detecting telomere-targeting activity and identify TAS-116 as a potent inducer of telomere-specific CIN [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHSP90 inhibitors induce telomere shortening in genomic chromosomes\u003c/h2\u003e \u003cp\u003eWe next measured genomic telomere length in HT1080 and HEK293 cells by Southern blot and qPCR after 20 days of treatment with HSP90 inhibitors (TAS-116, XL-888, SNX-2112, 17-AAG) or GRN163L as positive control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Concentrations were optimized for each line (Supplementary Table S3). TAS-116 induced the strongest shortening in both cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). qPCR confirmed that TAS-116 reduced telomere length 1.8-fold in HT1080 and 2-fold in HEK293. GRN163L produced a 1.5-fold reduction in HT1080 and a 3-fold reduction in HEK293. Southern blot analysis supported these patterns, with statistical values provided in Supplementary Table S3B (HT1080) and S3C (HEK293). Collectively, TAS-116 emerged as the most effective HSP90 inhibitor for telomere shortening, with effects comparable to the positive control GRN163L.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFISH confirms TAS-116\u0026ndash;induced telomere dysfunction in HEK293 cells\u003c/h2\u003e \u003cp\u003eTo validate these findings, telomere signal intensity was quantified in HEK293 cells after 20 days of treatment with TAS-116, XL-888, or GRN163L (positive control), with untreated cells serving as a negative control (Supplementary Figure S2). Using FISH, positive control GRN163L reduced signal intensity to 55.15% of control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). TAS-116 reduced signal intensity to 59.66% of control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), while XL-888 reduced it to 70.78% (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Supplementary Figure S2B and Supplementary Table S4). These results corroborate the Southern blot and qPCR findings and confirm that TAS-116 is the most potent HSP90 inhibitor for inducing telomere dysfunction under these conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eTAS-116 induces telomere-associated DNA damage and micronuclei formation\u003c/h2\u003e \u003cp\u003eTo investigate the mechanisms of telomere dysfunction, we quantified DNA double-strand breaks (DSBs), telomere dysfunction\u0026ndash;induced foci (TIFs), and micronuclei (MNi) in HEK293 cells treated with HSP90 inhibitors, with GRN163L included as a positive control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). γH2AX immunostaining revealed that GRN163L induced the highest number of DSBs (28 foci/cell, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), followed by TAS-116 (9 foci/cell, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). XL-888 (2.8 foci/cell), SNX-2112 (1.8 foci/cell), and 17-AAG (1.8 foci/cell) had weaker effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Supplementary Table S5A).\u003c/p\u003e \u003cp\u003eTIFs, defined as colocalization of γH2AX with the telomeric protein TRF2, showed that TAS-116 induced on average 1.8 TIFs per TIF-positive nucleus, whereas GRN163L induced 3.8, and no TIFs were detected with the other inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Supplementary Table S5A).\u003c/p\u003e \u003cp\u003eMNi formation, a marker of CIN, was highest with GRN163L (26.62%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and TAS-116 (22.98%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Lower rates were observed with XL-888 (5.61%), SNX-2112 (4.31%), and 17-AAG (5.66%) compared to 1.79% in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Supplementary Table S5B). Representative images are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB.\u003c/p\u003e \u003cp\u003eThese findings, consistent with prior assays, confirm that TAS-116 and GRN163L induce strong telomere-specific DNA damage and CIN, with TAS-116 emerging as the most potent HSP90 inhibitor in this context.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe rigorous evaluation of telomere-targeting agents is critical for advancing cancer therapeutics, yet few assays can reliably distinguish telomere-specific activity from general cytotoxicity. By employing an HAC-based system with linear and circular HACs, combined with direct analyses of telomere length and function, we established a versatile framework for identifying compounds that disrupt telomere integrity.\u003c/p\u003e \u003cp\u003eOur findings demonstrate that TAS-116, an HSP90 inhibitor currently in clinical development, exhibits robust telomere-targeting effects across multiple assays. TAS-116 induced linear HAC loss, genomic telomere shortening, reduced telomere signal intensity, elevated TIFs, and increased micronuclei formation. These effects were comparable in magnitude to those of the positive control GRN163L, a direct telomerase inhibitor, confirming the validity of our assay. All HSP90 inhibitors tested promoted telomere dysfunction to varying degrees, underscoring that HSP90 inhibition broadly impacts telomere integrity, with TAS-116 showing the strongest and most reproducible effects across assays.\u003c/p\u003e \u003cp\u003eMechanistically, TAS-116 binds to the ATP-binding pocket of HSP90 and arrests its chaperone cycle, thereby impairing proper folding, stabilization, and assembly of hTERT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Consequently, telomerase is rendered functionally inactive, consistent with defective holoenzyme assembly and impaired telomere elongation. This explains how TAS-116 indirectly suppresses telomere maintenance while simultaneously destabilizing other oncogenic client proteins of HSP90.\u003c/p\u003e \u003cp\u003eImportantly, GRN163L was included as a positive control because our previous work had established its strong activity as a telomerase inhibitor capable of inducing telomere dysfunction when tested in the same HAC-based system. Its reproducible activity across both linear and circular HAC assays provides a reliable benchmark against which novel compounds can be evaluated. As expected, GRN163L consistently triggered robust telomere shortening, DNA damage, and CIN, thereby validating the sensitivity of our experimental framework.\u003c/p\u003e \u003cp\u003eBeyond these results, our study contributes to the broader understanding of telomere-directed therapies. Cancer cells rely heavily on telomere maintenance for survival, and strategies such as telomerase inhibition, G-quadruplex stabilization, and Shelterin disruption have all been pursued with varying success. HSP90 inhibition offers an alternative route, indirectly destabilizing telomerase through impaired hTERT folding and assembly while simultaneously impairing multiple oncogenic pathways. The dual functionality of TAS-116\u0026mdash;chaperone inhibition and telomere destabilization\u0026mdash;may enhance its therapeutic potential by exploiting a fundamental vulnerability of cancer cells. In line with this, TAS-116 has undergone evaluation in a first-in-human phase I clinical trial for solid tumors, supporting the translational relevance of its telomere-targeting activity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis work also underscores the methodological value of HAC-based assay. By directly comparing linear and circular HACs, this approach distinguishes telomere-dependent from off-target effects, providing clarity that traditional assays cannot achieve. Such precision makes this system a powerful tool for preclinical evaluation of telomere-targeting agents.\u003c/p\u003e \u003cp\u003eLimitations of this study include its reliance on in vitro models and a relatively short treatment window, which may not fully capture long-term telomere erosion or the complexity of \u003cem\u003ein vivo\u003c/em\u003e tumor biology. Nevertheless, these limitations point to clear next steps: extending analyses to animal models and expanding compound screening. Importantly, they do not diminish the strong and consistent evidence supporting TAS-116 as a telomere-targeting agent.\u003c/p\u003e \u003cp\u003eIn conclusion, our results establish TAS-116 as a potent telomere-targeting compound. They also validate the HAC-based assay as an effective framework for evaluating telomere-directed therapeutics. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003e, HSP90 normally supports telomerase activity by stabilizing and folding hTERT in an ATP-dependent manner, enabling assembly of an active holoenzyme. TAS-116 blocks this cycle by occupying the ATP-binding pocket of HSP90, thereby rendering telomerase functionally inactive. Together, these findings highlight the clinical promise of TAS-116 while underscoring the broader utility of HAC-based and telomere assays in guiding the development of novel cancer therapies.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTAS-116 consistently disrupts telomere maintenance\u0026mdash;inducing linear HAC loss, telomere shortening, reduced telomere FISH signal, elevated TIF formation, and increased micronuclei\u0026mdash;thereby validating the HAC-based system as a robust platform for detecting telomere-directed activity. Collectively, these findings highlight the therapeutic potential of HSP90 inhibition as an effective strategy to target telomere maintenance in cancer cells.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCIN\u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Chromosome Instability\u003c/p\u003e\n\u003cp\u003eHSP90\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Heat Shock Protein 90\u003c/p\u003e\n\u003cp\u003eHAC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Human Artificial Chromosome\u003c/p\u003e\n\u003cp\u003eFISH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fluorescence in situ Hybridization\u003c/p\u003e\n\u003cp\u003eTIF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Telomere dysfunction-Induced Foci\u003c/p\u003e\n\u003cp\u003eMNi\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Micronuclei\u003c/p\u003e\n\u003cp\u003eEGFP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Enhanced Green Fluorescent Protein\u003c/p\u003e\n\u003cp\u003eTRF2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Telomeric Repeat-binding Factor 2\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHSL, JHK, and LM performed the experiments and analyzed the data. VL, HSL, and JHK contributed to the study design and drafting of the manuscript. LN provided support for the HSP90 inhibitors used in this study. VL, HSL, JHK, and NK were involved in data interpretation and discussion. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data used and/or analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study used only established and de-identified human cell lines (HEK293, HT1080, and HAC-engineered derivatives) and did not involve human participants, identifiable human data, or animal subjects. According to NIH policy and federal regulations (45 CFR 46), research using established de-identified cell lines does not require Institutional Review Board (IRB) review or ethics approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research, USA. The authors are grateful to Dr. Shay from University of Texas, Southwestern Medical Center for providing GRN163L and the late Dr. Len Neckers from Urologic Oncology Branch, CRC, National Cancer Institute for providing us HSP 90 inhibitors, TAS-116, XL-888, SNX-2112 and 17-AAG. The authors also would like to thank Dr. Yasuhiro Kazuki from Tottori University for providing HEK293GFP linear HAC cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDevelopmental Therapeutics Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA \u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eUrologic Oncology Branch, National Cancer Institute, NIH, Bethesda, MD 20892, USA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ede Lange T: \u003cstrong\u003eShelterin: the protein complex that shapes and safeguards human telomeres\u003c/strong\u003e. \u003cem\u003eGenes Dev \u003c/em\u003e2005, \u003cstrong\u003e19\u003c/strong\u003e(18):2100-2110.\u003c/li\u003e\n\u003cli\u003eShay JW, Wright WE: \u003cstrong\u003eRole of telomeres and telomerase in cancer\u003c/strong\u003e. \u003cem\u003eSemin Cancer Biol \u003c/em\u003e2011, 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\u003cstrong\u003e80\u003c/strong\u003e(5):121.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"HSP90 inhibitors, chromosomal instability, CIN, telomere dysfunction, human artificial chromosome, HAC","lastPublishedDoi":"10.21203/rs.3.rs-8369668/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8369668/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHeat shock protein 90 (HSP90) stabilizes numerous oncogenic client proteins, including factors required for telomere maintenance. Telomere dysfunction triggers chromosomal instability (CIN). To quantify telomere-directed activity and separate it from general mitotic effects, we utilized a human artificial chromosome (HAC) assay with isogenic lines carrying a linear, telomere-containing EGFP HAC or a circular, telomere-lacking EGFP HAC.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFour HSP90 inhibitors (TAS-116, XL-888, SNX-2112, 17-AAG) were tested at each compound\u0026rsquo;s cell-specific LC50 in HT1080 (linear and circular HACs) and HEK293 (linear HAC) cells, with GRN163L as a positive control. HAC loss was quantified by flow cytometry. Telomere length was measured by Southern blot and qPCR in parental HT1080 and HEK293 cells, and telomere signal intensity by FISH in HEK293. Telomere dysfunction (TIFs; γH2AX/TRF2) and micronuclei (MNi) were also scored.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTAS-116 showed the strongest telomere-specific activity among HSP90 inhibitors, significantly increasing linear HAC loss in both HT1080 and HEK293 while circular HACs showed only minimal instability. TAS-116 shortened telomeres in both lines (qPCR: ~1.8-fold in HT1080; ~2-fold in HEK293) and, in HEK293, reduced FISH telomere signal to 59.66% of control. TAS-116 also increased telomere-associated damage (~\u0026thinsp;1.8 TIFs per TIF-positive nucleus), DNA double-strand breaks, and micronuclei relative to the other HSP90 inhibitors.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTAS-116 consistently disrupts telomere maintenance\u0026mdash;driving linear HAC loss, telomere shortening, reduced FISH signal, elevated TIFs, and increased MNi\u0026mdash;thereby validating the HAC-based framework for discriminating telomere-directed activity. These findings support the therapeutic promise of HSP90 inhibition against telomere maintenance in cancer.\u003c/p\u003e","manuscriptTitle":"HSP90 inhibition disrupts telomere maintenance and promotes chromosomal instability (CIN) in cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 17:49:24","doi":"10.21203/rs.3.rs-8369668/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-02-27T14:42:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158640411459342670076510146828135742657","date":"2026-02-18T14:40:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-09T09:32:20+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-19T10:10:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-17T04:04:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-17T04:02:47+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-12-15T19:59:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3093a53d-6e2a-44d1-9c79-03e507059e89","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-11T17:49:24+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 17:49:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8369668","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8369668","identity":"rs-8369668","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}