The tumor-maintaining function of UTX/KDM6A in DNA replication and the PARP1-dependent repair pathway

The tumor-maintaining function of UTX/KDM6A in DNA replication and the PARP1-dependent repair pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The tumor-maintaining function of UTX/KDM6A in DNA replication and the PARP1-dependent repair pathway Shu-Ping Wang, Lin-Wen Yeh, Je-Wei Chen, Jia-Yun Yeh, Mei-Han Kao, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4620473/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Histone H3K27 demethylase UTX (aka KDM6A) is mutated in many human cancers, suggesting its tumor suppressive role during cancer development. However, most tumors still express wild-type UTX/KDM6A and its function is not always linked to tumor suppression. This suggests that UTX could be a pleiotropic factor in tumorigenesis; its deficiency promotes tumor initiation, while its presence is required for tumor maintenance. Here, we present evidence of UTX/KDM6A's role in sustaining tumor growth, revealing its function in tumor maintenance. We find that UTX/KDM6A sustains tumor cell cycling and survival via regulating DNA replication-associated transcriptional programs in a demethylase-independent manner. UTX/KDM6A can also interact with PARP1 and facilitate its recruitment to DNA lesions. Therefore, UTX/KDM6A depletion disrupts DNA replication and repair pathways, activating ATM–CHK2 and ATR–CHK1 signaling pathways and triggering S and G2/M checkpoints, leading to a pronounced defect in tumor growth. Analysis of human cancer xenograft models further demonstrates that knockdown of UTX/KDM6A by RNA-interference, rather than inhibition of its enzymatic activity via GSK-J4, shows potent anticancer effects. Dual inhibition of UTX/KDM6A and ATR further demonstrates synergistic anticancer activities. Our work highlights UTX/KDM6A as a potential therapeutic target for cancer treatment, especially when combined with ATR inhibition. Biological sciences/Molecular biology/Epigenetics Biological sciences/Molecular biology/DNA damage and repair Biological sciences/Molecular biology/DNA replication Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations There is NO conflict of interest to disclose. Supplementary Files WangLinWenOncogeneSupplementaryinformation.pdf Cite Share Download PDF Status: Posted Version 1 posted 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-4620473","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":319343330,"identity":"850d0dce-8576-49f7-b0ad-0b80df9dbaf5","order_by":0,"name":"Shu-Ping Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYPACGx4DMG3AwAOmeQhrScPUIkFAy2EGAxQ+Pi3ys3sMPxf8Oi9jLnb8AeOPgjsy/O0HGB+8bWOoMziAXYvBnTPG0jP7bvNYzs4xYOYxeMYjcSaB2XBuG4METi0SuRukeXtu8xjczmFgZjA4DPRUAps0L1CLGQ4t8jNyN//m7TkH1JIOdBhIC/8D9t/4tDDcyN0mzfPjAFBLAjC4QFokEtiY8WkxuHP+mzVvQzLYL4dBWiRuPGyWnHNOQnI/LofNbku+zfPHzt5cOv3hwx9/Dtvz9ycf/PCmzIZfsgGHw0DBz9gGYUONZWxgwBuTYKk/uOVHwSgYBaNgFDAAAIKJVr2FL+3OAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-5895-1269","institution":"Academia Sinica","correspondingAuthor":true,"prefix":"","firstName":"Shu-Ping","middleName":"","lastName":"Wang","suffix":""},{"id":319343331,"identity":"4458c2ba-4434-4796-9687-648be739fa83","order_by":1,"name":"Lin-Wen Yeh","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Lin-Wen","middleName":"","lastName":"Yeh","suffix":""},{"id":319343332,"identity":"4841c0e2-50f3-439e-b09b-9f7dcbaf58c7","order_by":2,"name":"Je-Wei Chen","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Je-Wei","middleName":"","lastName":"Chen","suffix":""},{"id":319343333,"identity":"e5f90965-5827-4485-84e7-3f17384a5eb2","order_by":3,"name":"Jia-Yun Yeh","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Jia-Yun","middleName":"","lastName":"Yeh","suffix":""},{"id":319343334,"identity":"a01f8edd-53e6-4e8a-956d-a3be53a94861","order_by":4,"name":"Mei-Han Kao","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Mei-Han","middleName":"","lastName":"Kao","suffix":""},{"id":319343335,"identity":"58c8af45-6340-4879-9119-423790706455","order_by":5,"name":"Hsiao-Chin Hong","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Hsiao-Chin","middleName":"","lastName":"Hong","suffix":""},{"id":319343336,"identity":"06a0f9e4-a474-4c1c-9d12-73f46a233a7a","order_by":6,"name":"Sean Wu","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Sean","middleName":"","lastName":"Wu","suffix":""},{"id":319343337,"identity":"1b231441-b82e-4d09-a800-7abf40abe24c","order_by":7,"name":"Wai-Mui Cheung","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Wai-Mui","middleName":"","lastName":"Cheung","suffix":""},{"id":319343338,"identity":"003f8db2-4d9a-41f5-a07e-b56426b863b4","order_by":8,"name":"Ta-Yu Liu","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Ta-Yu","middleName":"","lastName":"Liu","suffix":""},{"id":319343339,"identity":"eed6850d-a6b7-41a8-b0f0-fbca992c1979","order_by":9,"name":"Marvin Aberin","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Marvin","middleName":"","lastName":"Aberin","suffix":""},{"id":319343340,"identity":"aab9792b-6ed7-492d-9214-af3c59bf7b94","order_by":10,"name":"Ernesto Paas-Oliveros","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ernesto","middleName":"","lastName":"Paas-Oliveros","suffix":""},{"id":319343341,"identity":"666b486e-54d3-47b4-bb3a-4e3628b2433a","order_by":11,"name":"Arian Escajeda","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Arian","middleName":"","lastName":"Escajeda","suffix":""},{"id":319343342,"identity":"3b3a07ad-5ece-4614-bbde-8ce47cff7587","order_by":12,"name":"Edward Shih","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Edward","middleName":"","lastName":"Shih","suffix":""},{"id":319343343,"identity":"88bad836-2ac0-44e1-aad1-6f6251a7aa7b","order_by":13,"name":"Woan-Yuh Tarn","email":"","orcid":"","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Woan-Yuh","middleName":"","lastName":"Tarn","suffix":""},{"id":319343344,"identity":"01606187-dae0-455b-a4b5-64cf6b7d442c","order_by":14,"name":"Yao-Ming Chang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yao-Ming","middleName":"","lastName":"Chang","suffix":""},{"id":319343345,"identity":"f33d6430-6979-4a8f-b536-28e26cb1bbb7","order_by":15,"name":"Lan-Hsin Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Lan-Hsin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-06-22 06:55:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4620473/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4620473/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60547215,"identity":"8ef13625-28dc-4cf5-8fdb-48daddedfe36","added_by":"auto","created_at":"2024-07-18 04:09:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1696304,"visible":true,"origin":"","legend":"\u003cp\u003eUnveiling a potential tumor-maintaining role of UTX in supporting tumor cell growth. (A) Western blot analysis was conducted with the specified antibodies on lysates from control (shCtrl) and UTX/KDM6A-shRNAs (shUTX-7762 or shUTX-7763)-transduced cancer cell lines. (B) Stable UTX/KDM6A-knockdown or control cells were subjected to colony formation assays, with representative plate images shown. (C) The colony formation ability of stable cell clones in (B) was assessed by crystal violet assays. Quantification is presented as mean ± SEM for three independent experiments. (D) Top: A schematic illustrating the procedure for Doxinduced UTX/KDM6A-knockdown. Stably Dox-inducible (Tet-on) shUTX-expressing HeLa or control (shCtrl) cells were generated through lentiviral transduction. shRNA-mediated knockdown of UTX/KDM6A was induced by differential doses of Dox treatment as indicated. Lysates were collected at 4-, 5-, or 6-days post-Dox induction and subjected to Western blot analysis using the indicated antibodies. (E) Western blot analysis was performed using the specified antibodies on lysates obtained from Dox-induced UTX/KDM6A-knockdown NCI-H1975 cells. Lysates were collected at 3 days after Dox induction. (F) Cell viability was measured in Dox-induced UTX/KDM6A-knockdown HeLa or control cells treated with or without Dox. Cells were exposed to Dox treatment for 3 days and subjected to MTT assays as indicated time points. Data are presented as mean ± SEM for three independent experiments. (G and H) Left: Colony formation assays were conducted for the indicated cells, HeLa for (G) and NCI-H1975 for (H), treated with or without Dox as the indicated doses. Representative images of the plates are shown. Right: The colony formation ability was assessed by crystal violet assays. Quantification is presented as mean ± SEM for three independent experiments. **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 (Ordinary one-way ANOVA). Ctrl, control. Dox, doxycycline.\u003c/p\u003e","description":"","filename":"WangLinWenOncogeneMainFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-4620473/v1/315df948c3da53e7bf204396.png"},{"id":60547842,"identity":"dce522e7-918b-4989-86e2-67fe223c771b","added_by":"auto","created_at":"2024-07-18 04:17:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":712446,"visible":true,"origin":"","legend":"\u003cp\u003eUTX/KDM6A orchestrates transcriptional programs associated with DNA replication and repair pathways. (A) Pathway enrichment analysis was conducted on genes (n = 3,349) downregulated by UTX/KDM6A depletion in HeLa cells, utilizing KEGG pathways. (B) A schematic representation delineates the components of the replisome converging on the replication fork. (C) Heatmaps illustrates genes associated with DNA replication and mismatch repair that exhibited downregulation in UTX/KDM6A shRNA-expressing HeLa cells compared to control cells. (D and E) RT-qPCR analysis was performed to assess mRNA levels of DNA replication-associated genes in HeLa (D) and NCI-H1975 (E) cells transduced with control (shCtrl) or UTX/KDM6A-shRNAs (shUTX-7762 or shUTX-7763). RPL37A served as an endogenous control. Data are represented as mean ± SEM from three independent experiments. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 (Two-way ANOVA). (F–H) Enrichments of enhancer histone marks (H3K4me1, H3K27ac, and H3K27me3) were analyzed at three representative gene enhancers in control (shCtrl) and UTX/KDM6A-shRNAs (shUTX-7762 or shUTX-7763)- transduced NCI-H1975 cells. The gene enhancers examined include PNCA (F), LIG1 (G), and MCM3 (H). Fold enrichments were normalized to H3, and relative fold enrichment was further normalized to the control cells (n = 2 biological replicates from 2 independent experiments). *p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001, ns, no significant (Ordinary one-way ANOVA).\u003c/p\u003e","description":"","filename":"WangLinWenOncogeneMainFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-4620473/v1/e13cd349f5856ae594f0ef12.png"},{"id":60547213,"identity":"28b1528b-a48d-4c78-aed7-4ad5d0cb7c1c","added_by":"auto","created_at":"2024-07-18 04:09:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1774835,"visible":true,"origin":"","legend":"\u003cp\u003eUTX/KDM6A deficiency disrupts S-phase and G2/M-phase cell cycle progression. (A) Flow cytometric analysis of the cell cycle was performed for Dox-inducible (Tet-on) shUTXexpressing HeLa or control (shCtrl) cells with or without Dox treatment. The analysis quantified sub-G1, G1, S, and G2/M phases from the PI-labeled histograms. Cells were treated with 50 ng/mL Dox for 72 h. (B) Quantification of cells in each phase is presented as mean ± SEM for three independent experiments. (C) Flow cytometric analysis of the cell cycle was conducted for Dox-inducible (Tet-on) shUTX-expressing NCI-H1975 or control (shCtrl) cells with or without Dox treatment. Cell cycle analysis calculated sub-G1, G1, S, and G2/M phases from the PI-labeled histograms following serum starvation for 16 h. Cells were treated with 50 ng/ml Dox for 72 h. (D) Quantification of cells in each phase is shown as mean ± SEM for three independent experiments. ****p \u0026lt; 0.0001 (Two-way ANOVA). (E) Immunostaining for histone H3 Ser10 phosphorylation (H3S10P) in Dox-inducible (Tet-on) shUTX-expressing HeLa or control (shCtrl) cells with or without Dox treatment (50 ng/mL). (F) Quantification of H3S10P signal intensity is presented as mean ± SEM for three independent experiments. ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 (Two-way ANOVA). (G) Monitoring of defective S-phase progression in UTX/KDM6A-deficient cells. Dox-inducible (Tet-on) shUTX-expressing HeLa or control (shCtrl) cells were treated with 50 ng/mL Dox. Three days later, cells were pulse-labeled with BrdU and harvested at the indicated time intervals. Cells were stained with anti-BrdU monoclonal antibody and DAPI and analyzed by two-parameter FACS to measure DNA synthesis (BrdU-APC, y axis) and DNA content (DAPI, x axis). (H) Quantification of cells in early, middle, and late S phase is shown as mean ± SEM for three independent experiments. (I) Western blot analysis was performed using the specified antibodies on lysates obtained from control (shCtrl) and UTX/KDM6A-shRNA (shUTX-7763)-transduced HeLa (left) or NCI-H1975 (right) cells. Numbers below the immunoblots panels indicate the densitometric values normalized to the respective GAPDH value. * denotes a non-specific band. (J) Cytotoxic effects of Dox-induced knockdown of UTX/KDM6A on NCI-H1975 cells were asseseed by flow cytometry with Annexin V-PI staining. Annexin V-positive and PI-negative populations represent cells in early apoptosis, while Annexin V and PI double-positive staining indicates cells in late apoptosis. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001 (Two-way ANOVA). Ctrl, control. Dox, doxycycline.\u003c/p\u003e","description":"","filename":"WangLinWenOncogeneMainFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-4620473/v1/7ac87b1149d8ac276f819341.png"},{"id":60547216,"identity":"10fd08fb-6866-4283-bee7-e7f953909808","added_by":"auto","created_at":"2024-07-18 04:09:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2085074,"visible":true,"origin":"","legend":"\u003cp\u003eUTX/KDM6A deficiency impairs DDR and DSB repair. (A) Dox-inducible (Tet-on) shUTX-expressing HeLa cells treated with or without Dox (50 ng/mL) and 0 or 2mM of hydroxyurea (HU) for 72 h were stained for γH2AX. (B) Quantification of γH2AX signal intensity is presented as mean ± SEM for three independent experiments. (C) Dox-inducible (Tet-on) shUTX-expressing HeLa or control (shCtrl) cells treated with or without Dox (50 ng/mL) and 0 or 2 Gy γ-radiation, followed by recovery for indicated time points, were stained for γH2AX. (D) Quantification of γH2AX signal intensity is presented as mean ± SEM for three independent experiments. *p \u0026lt; 0.05, **p \u0026lt; 0.01 (Two-way ANOVA). (E) The schematic diagram illustrates a unique DSB-repair monitoring system. Site-specific DSBs were introduced via CRISPR/Cas9- mediated genomic DNA cutting, followed by integration of the unique marker sequence (incorporated in ssODN or dsODN) via HR or NHEJ pathways. PCR analysis was then employed to quantitatively evaluate the marker sequence in the genomic DNA. ODN, oligodeoxynucleotide. (F) Genomic DNA was collected 24 h after CRISPR/Cas9-mediated site-specific DSBs. The activity of HR or NHEJ was determined by PCR analysis. PCR products amplified with different primer sets (i.e., FR, FM, RM) were analyzed by electrophoresis in a 2% agarose gel. The arrow indicates the FM PCR product, and the arrowhead indicates the RM PCR product. (G) HR activity was quantified by qPCR analysis. Quantification of HR activity is presented as mean ± SEM for three independent experiments. *p \u0026lt; 0.05, ***p \u0026lt; 0.001, (Ordinary one-way ANOVA). Ctrl, control. Dox, doxycycline.\u003c/p\u003e","description":"","filename":"WangLinWenOncogeneMainFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-4620473/v1/36cbd90dc110a47732d4fb77.png"},{"id":60547218,"identity":"a5e46d0e-1d25-4ff1-887a-a7f027f3a058","added_by":"auto","created_at":"2024-07-18 04:09:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1546835,"visible":true,"origin":"","legend":"\u003cp\u003eUTX/KDM6A facilitates PARP-1 recruitment to DNA damages sites during DNA damage response. (A) Two independent U2OS subclones expressing GFP-PARP-1 (U2OS/GFP-PARP-1 #2 and #4) with stable transduction of shCtrl, shUTX-7762, or shUTX-7763 were subjected to laser micro-irradiation and monitored using time-lapse microscopy. Representative images at 0, 7, 12, 27, and 97 seconds are shown after laser micro-irradiation. (B and C) The accumulation of GFP-PARP1 on the laser damage tracks in U2OS/GFP-PARP-1 #2 (B, n = 22) or #4 (C, n = 25) cells was quantified and plotted. Increased fluorescence on the damage tracks is plotted over time. (D and E) Direct binding of purified Flag-tagged PARP-1 (FPARP- 1) to full-length (D) or deletion fragments (E) of HA-tagged UTX (HA-UTX) immobilized on anti-HA beads (monitored by antibodies as indicated). (F) Schematic representation and summary of the interaction between PARP-1 and various deletion fragments of UTX/KDM6A. Ctrl, control. (G) PARP-1 interacts with UTX/KDM6A in vivo. HEK293T cells were co-transfected with PARP44 1 and 3XHA–UTX (or an empty vector) for 36 h. Subsequently, cell lysates were immunoprecipitated (IP) with anti-HA or mouse IgG and subjected to immunoblotting using the specified antibodies. (H) Interactions among PARP-1, UTX/KDM6A, and MLL4/KMT2D in cells. HEK293T cells were co-transfected with PARP-1, 3XHA–UTX, Flag-MLL4-C (C-terminal region, amino acids 4507-5537), or the corresponding empty vectors for 36 h. Following transfection, cell lysates were immunoprecipitated (IP) with anti-HA or mouse IgG and subjected to Western blot analysis using the specified antibodies.\u003c/p\u003e","description":"","filename":"WangLinWenOncogeneMainFigures5.png","url":"https://assets-eu.researchsquare.com/files/rs-4620473/v1/42c1d45f881a3abaed4eeac0.png"},{"id":60547212,"identity":"4629b709-669c-40c6-bed7-8a431a0ec244","added_by":"auto","created_at":"2024-07-18 04:09:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":697162,"visible":true,"origin":"","legend":"\u003cp\u003eThe tumor-maintaining function of UTX/KDM6A supports tumor growth in vivo. (A) Tumor growth rates of Dox-inducible (Tet-on) shUTX-expressing NCI-H1975 or control (shCtrl) cell lines upon Dox treatment as indicated in xenograft assays (shCtrl, n=8; shUTX, n=9). Data are represented as means ± SEM. *p \u0026lt; 0.05, ****p \u0026lt; 0.0001 (Two-way ANOVA followed by Bonferroni correction). (B) Gross pathological photographs at necropsy of the NCI-H1975 tumors that arose in mice treated with Dox-induced shCtrl or shUTX (shCtrl, n=8; shUTX, n=9). #, Two mice in the shCtrl group were euthanized prior to the scheduled endpoint (Day 17 post-treatment) due to their tumor sizes surpassing 2000 mm³. (C) The weight of each tumor in the indicated tumor groups. **p \u0026lt; 0.01 (Unpaired t test with Welch’s correction). Ctrl, control. Dox, doxycycline. (D) Proposed model illustrating the tumor-maintaining functions of UTX/KDM6A in the regulation of DNA replication and repair pathways to support tumor cell growth. In UTX/KDM6A-proficient tumor cells, UTX/KDM6A, independently of its H3K27 demethylase activity, plays a key role in activating DNA replication-associated gene expression, probably by physically bridging the MLL3/4 (KMT2C/D) complex to target gene enhancers and promoters. UTX/KDM6A may also facilitate the interaction between PARP-1 and the MLL3/4 (KMT2C/D) complex (and probably the MRN complex) to DNA damage sites in response to intrinsic and extrinsic genomic stresses. In UTX/KDM6A-deficient tumor cells, loss of UTX/KDM6A results in defective DNA replisome function, which may slow down the S-phase progression, induce replication stress, and lead to DNA damage accumulation. Due to the critical roles of UTX/KDM6A in DDR and DNA repair (e.g., HR-repair), UTX/KDM6A-deficient tumor cells fail to repair the replication stress-induced DNA damage. Consequently, loss of UTX/KDM6A results in the accumulation of substantial DNA damage, triggering DNA replication and DSB checkpoint signaling pathways, provoking cellular apoptosis, and ultimately leading to severe tumor cell death. Created with BioRender.com.\u003c/p\u003e","description":"","filename":"WangLinWenOncogeneMainFigures6.png","url":"https://assets-eu.researchsquare.com/files/rs-4620473/v1/e6a664604d74a94916ce840a.png"},{"id":105564484,"identity":"21b0d30d-2834-4df5-8c1b-71f2111db305","added_by":"auto","created_at":"2026-03-27 12:49:44","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3025176,"visible":true,"origin":"","legend":"","description":"","filename":"LinWenOncogeneMaintext.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4620473/v1_covered_a02077da-e99f-4c14-8c95-fd64f7c822a4.pdf"},{"id":60547217,"identity":"389b7db7-d2cb-4125-a8b3-c66e7bb2bae0","added_by":"auto","created_at":"2024-07-18 04:09:57","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15144740,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"WangLinWenOncogeneSupplementaryinformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4620473/v1/4162412c6f65e16f51909faa.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"The tumor-maintaining function of UTX/KDM6A in DNA replication and the PARP1-dependent repair pathway","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4620473/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4620473/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Histone H3K27 demethylase UTX (aka KDM6A) is mutated in many human cancers, suggesting its tumor suppressive role during cancer development. However, most tumors still express wild-type UTX/KDM6A and its function is not always linked to tumor suppression. This suggests that UTX could be a pleiotropic factor in tumorigenesis; its deficiency promotes tumor initiation, while its presence is required for tumor maintenance. Here, we present evidence of UTX/KDM6A's role in sustaining tumor growth, revealing its function in tumor maintenance. We find that UTX/KDM6A sustains tumor cell cycling and survival via regulating DNA replication-associated transcriptional programs in a demethylase-independent manner. UTX/KDM6A can also interact with PARP1 and facilitate its recruitment to DNA lesions. Therefore, UTX/KDM6A depletion disrupts DNA replication and repair pathways, activating ATM–CHK2 and ATR–CHK1 signaling pathways and triggering S and G2/M checkpoints, leading to a pronounced defect in tumor growth. Analysis of human cancer xenograft models further demonstrates that knockdown of UTX/KDM6A by RNA-interference, rather than inhibition of its enzymatic activity via GSK-J4, shows potent anticancer effects. Dual inhibition of UTX/KDM6A and ATR further demonstrates synergistic anticancer activities. Our work highlights UTX/KDM6A as a potential therapeutic target for cancer treatment, especially when combined with ATR inhibition.","manuscriptTitle":"The tumor-maintaining function of UTX/KDM6A in DNA replication and the PARP1-dependent repair pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 04:09:52","doi":"10.21203/rs.3.rs-4620473/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10f6b1fd-6d3e-4074-8a78-9cb7a57b9d0a","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33765301,"name":"Biological sciences/Molecular biology/Epigenetics"},{"id":33765302,"name":"Biological sciences/Molecular biology/DNA damage and repair"},{"id":33765303,"name":"Biological sciences/Molecular biology/DNA replication"}],"tags":[],"updatedAt":"2026-03-23T16:16:53+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 04:09:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4620473","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4620473","identity":"rs-4620473","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}