Role of drug induced nuclear CTSL (nCTSL) in DNA damage response in cancer- therapeutic implications

Thirusangu, P.; Jin, L.; Ray, U.; Zhao, A.; Wu, X.; Hou, X.; VanBlaricom, J. L.; Aalam, S. M. M.; Oberg, A. L.; Kannan, N.; Weroha, J.; Chien, J.; Kaufmann, S. H.; Bakkum-Gamez, J. N.; Shridhar, V.

doi:10.1101/2025.01.09.632284

bioRxiv · 10.1101/2025.01.09.632284

Role of drug induced nuclear CTSL (nCTSL) in DNA damage response in cancer- therapeutic implications

Thirusangu, P.·Jin, L.·Ray, U.·Zhao, A.·Wu, X.·Hou, X.·VanBlaricom, J. L.·Aalam, S. M. M.·Oberg, A. L.·Kannan, N.·Weroha, J.·Chien, J.·Kaufmann, S. H.·Bakkum-Gamez, J. N.·Shridhar, V.

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Abstract

ABSTRACT Background: Despite treatment advances, ovarian cancer (OC) remains the deadliest gynecological cancer, with a ~70% 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. Methods: 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). Results: 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. Conclusion: 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.

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Thirusangu, P., Jin, L., Ray, U., Zhao, A., Wu, X., Hou, X., VanBlaricom, J. L., Aalam, S. M. M., Oberg, A. L., Kannan, N., Weroha, J., Chien, J., Kaufmann, S. H., Bakkum-Gamez, J. N., Shridhar, V.. 2025-01-14. Role of drug induced nuclear CTSL (nCTSL) in DNA damage response in cancer- therapeutic implications. https://doi.org/10.1101/2025.01.09.632284

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