{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T20:20:03Z","timestamp":1787948403020,"version":"build-2784847793"},"posted":{"date-parts":[[2025,1,14]]},"group-title":"Cancer Biology","reference-count":58,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,1,14]],"date-time":"2025-01-14T00:00:00Z","timestamp":1736812800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"Department of Defence, USA","award":["#HT9425 24-1-0357 (OC230398)"],"award-info":[{"award-number":["#HT9425 24-1-0357 (OC230398)"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,26]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Despite treatment advances, ovarian cancer (OC) remains the deadliest gynecological cancer, with a \u223c70% mortality rate. OCs initially respond to platinum chemotherapy but often develop resistance. While PARP inhibitors improve relapse-free survival in responsive cases, they do not extend overall survival. This underscores the need for effective combination therapies beyond BRCA status. The lysosomal protein cathepsin L (CTSL) can traffic to the nucleus, where it can regulate DNA repair and serve as a chromatin modifier. Here, we tested the hypothesis that nucleoside analog clofarabine (CLF) induced nuclear CTSL (nCTSL) serves as a biomarker of sensitivity to DNA-damaging agents in ovarian cancer.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>Role of nuclear CTSL in DNA damage, synergy with PARPis and CRM1 inhibitor, tumor regression was analyzed by 3D-spheroid culture, colony forming assay, subcellular fractions, comet assay, and IFC\/ western-blotting using PARP inhibitor-sensitive and-resistant OC cell lines, ex vivo cultures of patient-derived OC ascites cells (OVA), primary OCs, and patient-derived xenografts (PDXs).<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>In OC cell lines, treatment with CLF induced nCTSL in a subset of models-designated CLF-responsive (CLF-r)-and sensitized them to the PARP inhibitors olaparib and rucaparib. In CLF-non-responsive (CLF-nr) models, nCTSL trafficking and drug synergy were achieved with the CLF+olaparib combination. Synergy was observed in 47% of CLF-r and 24% of CLF-nr OVA samples. Mechanistically, CLF monotherapy induced nuclear import of CTSL via KPNB1 in CLF-r cells, while combination treatment with CLF+olaparib was required in CLF-nr cells. CLF also downregulated the nuclear export protein CRM1 in both CLF-r and CLF-nr models. CTSL knockdown conferred resistance to CLF+olaparib in both cohorts, supporting a functional role for nCTSL in therapeutic response. Notably, in a subset of OVAs (29%) classified as CLF-resistant (CLF-Res), CLF failed to downregulate CRM1; however, pharmacological inhibition of CRM1 with KPT8602 restored sensitivity to CLF+PARP inhibitor treatment. In vivo, CLF+olaparib treatment in CLF-r and CLF-nr PDX models led to enhanced DNA damage, reduced tumor burden, and prolonged survival.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion<\/jats:title>\n                  <jats:p>Collectively, these findings identify nCTSL as a predictive marker of response to DNA-damaging therapies and support the CLF+olaparib combination as a promising strategy for overcoming drug resistance in OC via CTSL-mediated DNA damage.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.01.09.632284","type":"posted-content","created":{"date-parts":[[2025,1,14]],"date-time":"2025-01-14T13:55:16Z","timestamp":1736862916000},"source":"Crossref","is-referenced-by-count":1,"title":["Drug-induced nuclear cathepsin L (nCTSL) defines a targetable DNA damage response axis and PARP inhibitor sensitivity in ovarian cancer"],"prefix":"10.64898","author":[{"given":"Prabhu","family":"Thirusangu","sequence":"first","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ling","family":"Jin","sequence":"additional","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shruti","family":"Rao","sequence":"additional","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Upasana","family":"Ray","sequence":"additional","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"},{"name":"Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anya","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julie","family":"Staub","sequence":"additional","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xinyan","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"},{"name":"Department of Oncology, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaonan","family":"Hou","sequence":"additional","affiliation":[{"name":"Department of Oncology, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jamison L","family":"VanBlaricom","sequence":"additional","affiliation":[{"name":"Department of Oncology, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Syed Mohamed Musheer","family":"Aalam","sequence":"additional","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sun-Hee","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Oncology, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann L","family":"Oberg","sequence":"additional","affiliation":[{"name":"Department of Health Science Research, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8825-2178","authenticated-orcid":false,"given":"Nagarajan","family":"Kannan","sequence":"additional","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Weroha","sequence":"additional","affiliation":[{"name":"Department of Oncology, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeremy","family":"Chien","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Medicine, University of California Davis Medical Center, Sacramento, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scott H","family":"Kaufmann","sequence":"additional","affiliation":[{"name":"Department of Oncology, Mayo Clinic, Rochester, MN, USA"},{"name":"Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, 55905, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jamie N","family":"Bakkum-Gamez","sequence":"additional","affiliation":[{"name":"Department of Obstetrics and Gynecology, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viji","family":"Shridhar","sequence":"additional","affiliation":[{"name":"Department of Experimental Pathology and Medicine, Mayo Clinic, Rochester, MN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026082812551355000_2025.01.09.632284v2.1","doi-asserted-by":"crossref","unstructured":"Andrei Havasi SSC , Ana Teodora Havasi , and Calin Cainap . Ovarian Cancer\u2014Insights into Platinum Resistance and Overcoming It. Medicina (Kaunas) (Review) 2023.","DOI":"10.3390\/medicina59030544"},{"key":"2026082812551355000_2025.01.09.632284v2.2","doi-asserted-by":"publisher","DOI":"10.1136\/ijgc-2022-004149"},{"key":"2026082812551355000_2025.01.09.632284v2.3","doi-asserted-by":"publisher","DOI":"10.1158\/1078-0432.CCR-05-2664"},{"key":"2026082812551355000_2025.01.09.632284v2.4","doi-asserted-by":"publisher","DOI":"10.1200\/JCO.2005.03.8554"},{"key":"2026082812551355000_2025.01.09.632284v2.5","doi-asserted-by":"publisher","DOI":"10.1517\/14656560902997990"},{"key":"2026082812551355000_2025.01.09.632284v2.6","doi-asserted-by":"publisher","DOI":"10.1002\/cncr.21005"},{"key":"2026082812551355000_2025.01.09.632284v2.7","doi-asserted-by":"crossref","first-page":"471","DOI":"10.15171\/apb.2015.064","article-title":"Clofarabine Has Apoptotic Effect on T47D Breast Cancer Cell Line via P53R2 Gene Expression","volume":"5","year":"2015","journal-title":"Adv Pharm Bull"},{"key":"2026082812551355000_2025.01.09.632284v2.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejphar.2013.11.021"},{"key":"2026082812551355000_2025.01.09.632284v2.9","doi-asserted-by":"publisher","DOI":"10.1158\/1535-7163.MCT-08-0427"},{"key":"2026082812551355000_2025.01.09.632284v2.10","doi-asserted-by":"publisher","DOI":"10.1080\/15257770.2018.1453075"},{"key":"2026082812551355000_2025.01.09.632284v2.11","doi-asserted-by":"crossref","unstructured":"Khalafi S , Zhu S , Khurana R , Lohse I , Giordano S , Corso S et al. A novel strategy for combination of clofarabine and pictilisib is synergistic in gastric cancer. Transl Oncol 2022; 15: 101260.","DOI":"10.1016\/j.tranon.2021.101260"},{"key":"2026082812551355000_2025.01.09.632284v2.12","doi-asserted-by":"publisher","DOI":"10.5483\/BMBRep.2015.48.3.105"},{"key":"2026082812551355000_2025.01.09.632284v2.13","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1158\/0008-5472.CAN-21-1987","article-title":"Targeting Ribonucleotide Reductase Induces Synthetic Lethality in PP2A-Deficient Uterine Serous Carcinoma","volume":"82","year":"2022","journal-title":"Cancer Res"},{"key":"2026082812551355000_2025.01.09.632284v2.14","doi-asserted-by":"publisher","DOI":"10.1200\/jco.2005.23.16_suppl.7109"},{"key":"2026082812551355000_2025.01.09.632284v2.15","doi-asserted-by":"publisher","DOI":"10.1016\/j.bcp.2009.06.094"},{"key":"2026082812551355000_2025.01.09.632284v2.16","doi-asserted-by":"publisher","DOI":"10.1016\/S1097-2765(04)00209-6"},{"key":"2026082812551355000_2025.01.09.632284v2.17","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.201204053"},{"key":"2026082812551355000_2025.01.09.632284v2.18","doi-asserted-by":"publisher","DOI":"10.1016\/S1534-5807(04)00141-8"},{"key":"2026082812551355000_2025.01.09.632284v2.19","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.00135-06"},{"key":"2026082812551355000_2025.01.09.632284v2.20","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2016.01.102"},{"key":"2026082812551355000_2025.01.09.632284v2.21","doi-asserted-by":"publisher","DOI":"10.1038\/onc.2009.466"},{"key":"2026082812551355000_2025.01.09.632284v2.22","doi-asserted-by":"publisher","DOI":"10.1038\/s41401-019-0286-9"},{"key":"2026082812551355000_2025.01.09.632284v2.23","doi-asserted-by":"crossref","unstructured":"Holzen L , Parigiani MA , Reinheckel T . Tumor cell-and microenvironment-specific roles of cysteine cathepsins in mouse models of human cancers. Biochim Biophys Acta Proteins Proteom 2020; 1868: 140423.","DOI":"10.1016\/j.bbapap.2020.140423"},{"key":"2026082812551355000_2025.01.09.632284v2.24","doi-asserted-by":"publisher","DOI":"10.1038\/s41388-020-01621-4"},{"key":"2026082812551355000_2025.01.09.632284v2.25","doi-asserted-by":"crossref","unstructured":"Jung D , Khurana A , Roy D , Kalogera E , Bakkum-Gamez J , Chien J et al. Quinacrine upregulates p21\/p27 independent of p53 through autophagy-mediated downregulation of p62-Skp2 axis in ovarian cancer. Sci Rep 2018; 8: 2487.","DOI":"10.1038\/s41598-018-20531-w"},{"key":"2026082812551355000_2025.01.09.632284v2.26","doi-asserted-by":"crossref","unstructured":"Thirusangu P , Pathoulas CL , Ray U , Xiao Y , Staub J , Jin L et al. Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal\/Mitochondrial Membrane Permeabilization and Cell Death. Cancers (Basel ) 2021; 13.","DOI":"10.3390\/cancers13092004"},{"key":"2026082812551355000_2025.01.09.632284v2.27","doi-asserted-by":"crossref","first-page":"2100","DOI":"10.1038\/nprot.2014.138","article-title":"The cellular thermal shift assay for evaluating drug target interactions in cells","volume":"9","year":"2014","journal-title":"Nature Protocols"},{"key":"2026082812551355000_2025.01.09.632284v2.28","doi-asserted-by":"crossref","unstructured":"Sevim H , \u00c7elik H , D\u00fcs\u00fcnceli L , Ceyhan CS , Molotkova A , Nakazawa K et al. Clofarabine induces ERK\/MSK\/CREB activation through inhibiting CD99 on Ewing sarcoma cells. Plos One 2021; 16.","DOI":"10.1371\/journal.pone.0253170"},{"key":"2026082812551355000_2025.01.09.632284v2.29","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2006.5"},{"key":"2026082812551355000_2025.01.09.632284v2.30","doi-asserted-by":"crossref","unstructured":"Kanakkanthara A , Huntoon CJ , Hou X , Zhang M , Heinzen EP , O\u2019Brien DR et al. ZC3H18 specifically binds and activates the BRCA1 promoter to facilitate homologous recombination in ovarian cancer. Nat Commun 2019; 10: 4632.","DOI":"10.1038\/s41467-019-12610-x"},{"key":"2026082812551355000_2025.01.09.632284v2.31","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-10-3059"},{"key":"2026082812551355000_2025.01.09.632284v2.32","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-09-1947"},{"key":"2026082812551355000_2025.01.09.632284v2.33","doi-asserted-by":"publisher","DOI":"10.1158\/2159-8290.CD-20-0818"},{"key":"2026082812551355000_2025.01.09.632284v2.34","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-06-1410"},{"key":"2026082812551355000_2025.01.09.632284v2.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2017.07.039"},{"key":"2026082812551355000_2025.01.09.632284v2.36","doi-asserted-by":"publisher","DOI":"10.1021\/cb2000296"},{"key":"2026082812551355000_2025.01.09.632284v2.37","doi-asserted-by":"publisher","DOI":"10.1038\/s41388-023-02785-5"},{"key":"2026082812551355000_2025.01.09.632284v2.38","doi-asserted-by":"publisher","DOI":"10.1254\/jphs.FP0050980"},{"key":"2026082812551355000_2025.01.09.632284v2.39","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-08-3065"},{"key":"2026082812551355000_2025.01.09.632284v2.40","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2017.07.039"},{"key":"2026082812551355000_2025.01.09.632284v2.41","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1017300108"},{"key":"2026082812551355000_2025.01.09.632284v2.42","doi-asserted-by":"publisher","DOI":"10.1016\/j.cellsig.2018.01.012"},{"key":"2026082812551355000_2025.01.09.632284v2.43","doi-asserted-by":"publisher","DOI":"10.1016\/j.pharmthera.2015.08.007"},{"key":"2026082812551355000_2025.01.09.632284v2.44","doi-asserted-by":"publisher","DOI":"10.1038\/onc.2008.310"},{"key":"2026082812551355000_2025.01.09.632284v2.45","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M008944200"},{"key":"2026082812551355000_2025.01.09.632284v2.46","doi-asserted-by":"publisher","DOI":"10.1158\/1541-7786.MCR-07-0160"},{"key":"2026082812551355000_2025.01.09.632284v2.47","doi-asserted-by":"publisher","DOI":"10.1038\/emboj.2011.225"},{"key":"2026082812551355000_2025.01.09.632284v2.48","doi-asserted-by":"publisher","DOI":"10.1002\/ijc.24275"},{"key":"2026082812551355000_2025.01.09.632284v2.49","doi-asserted-by":"crossref","first-page":"1490","DOI":"10.1016\/j.chembiol.2016.10.011","article-title":"Proteome-wide Profiling of Clinical PARP Inhibitors Reveals Compound-Specific Secondary Targets","volume":"23","year":"2016","journal-title":"Cell Chem Biol"},{"key":"2026082812551355000_2025.01.09.632284v2.50","doi-asserted-by":"crossref","first-page":"1442","DOI":"10.1016\/j.chembiol.2016.12.003","article-title":"PARP Inhibitors: Staying on Target?","volume":"23","year":"2016","journal-title":"Cell Chem Biol"},{"key":"2026082812551355000_2025.01.09.632284v2.51","doi-asserted-by":"crossref","unstructured":"Zampieri LX , Sboarina M , Cacace A , Grasso D , Thabault L , Hamelin L et al. Olaparib Is a Mitochondrial Complex I Inhibitor That Kills Temozolomide-Resistant Human Glioblastoma Cells. International Journal of Molecular Sciences 2021; 22: 11938.","DOI":"10.3390\/ijms222111938"},{"key":"2026082812551355000_2025.01.09.632284v2.52","doi-asserted-by":"crossref","first-page":"1296","DOI":"10.1021\/acschembio.5b01018","article-title":"Conformation-Selective Analogues of Dasatinib Reveal Insight into Kinase Inhibitor Binding and Selectivity","volume":"11","year":"2016","journal-title":"Acs Chemical Biology"},{"key":"2026082812551355000_2025.01.09.632284v2.53","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.C000345200"},{"key":"2026082812551355000_2025.01.09.632284v2.54","doi-asserted-by":"publisher","DOI":"10.1016\/j.exphem.2014.11.001"},{"key":"2026082812551355000_2025.01.09.632284v2.55","unstructured":"C RM, Restuadi R , Zhang G , Marks D , Ortega-Prieto P , Doherty E et al. Multimodal imaging reveals a lysosomal drug reservoir that drives heterogeneous distribution of PARP inhibitors. Nat Commun 2026."},{"key":"2026082812551355000_2025.01.09.632284v2.56","doi-asserted-by":"crossref","first-page":"688","DOI":"10.1038\/nsmb.1831","article-title":"53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers","volume":"17","year":"2010","journal-title":"Nature Structural & Molecular Biology"},{"key":"2026082812551355000_2025.01.09.632284v2.57","doi-asserted-by":"publisher","DOI":"10.4161\/cc.26582"},{"key":"2026082812551355000_2025.01.09.632284v2.58","doi-asserted-by":"crossref","unstructured":"Tholen M , Hillebrand LE , Tholen S , Sedelmeier O , Arnold SJ , Reinheckel T . Out-of-frame start codons prevent translation of truncated nucleo-cytosolic cathepsin L in vivo. Nat Commun 2014; 5: 4931.","DOI":"10.1038\/ncomms5931"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.01.09.632284","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T19:55:25Z","timestamp":1787946925000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.01.09.632284"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,14]]},"references-count":58,"URL":"https:\/\/doi.org\/10.1101\/2025.01.09.632284","relation":{},"subject":[],"published":{"date-parts":[[2025,1,14]]},"subtype":"preprint"}}