Lee, Sun Hee

Abstract Introduction: 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. We found that nucleoside analog clofarabine (CLF) induces CTSL nuclear trafficking in OC cells. Here we tested the hypothesis that accumulation nuclear CTSL (nCTSL) is a biomarker of sensitivity to DNA damaging therapy in OCs for improved therapeutic options. Methods: To determine the role of CLF induced nuclear CTSL in DNA damage and its synergy with PARP and CRM1 inhibitors in PARP inhibitor–sensitive and –resistant ovarian cancer (OC) cell lines, ex vivo cultures of ascites-derived patient OC cells (OVA-3D spheroids), primary ovarian tumors, and patient-derived xenograft (PDX) models and validated by colony formation assays. Nuclear CTSL localization was determined in subcellular fractionated lysates by western blots and immunofluorescence cytometry (IFC). Tumor regression was determined in vivo using ex vivo cultures of PDX derived cells. Results: In ovarian cancer (OC) cell lines, CLF monotherapy induced nuclear CTSL (nCTSL) in a subset of models classified as CLF-responsive (CLF-r), sensitizing them to the PARP inhibitors olaparib and rucaparib. In CLF-non-responsive (CLF-nr) models, nCTSL induction and therapeutic synergy were observed only with the CLF+olaparib combination. Synergy was detected in 47% of CLF-r and 24% of CLF-nr patient-derived ascites samples (OVA). Mechanistically, CLF induced nuclear import of CTSL via KPNB1 in CLF-r cells, while this import was triggered by the CLF+olaparib combination in CLF-nr cells. CLF also downregulated the nuclear export protein CRM1 (XPO1) in both groups. Knockdown of CTSL in either CLF-r or CLF-nr cells abrogated sensitivity to the combination. Notably, in 29% of OVA samples classified as CLF-resistant (CLF-Res), CLF failed to suppress CRM1 expression. In these cases, CRM1 inhibition with KPT8602 restored CLF+PARP inhibitor synergy. In vivo, CLF-r and CLF-nr PDX models treated with the combination exhibited increased DNA damage, reduced tumor burden, and extended survival. Conclusion: These findings identify nuclear CTSL (nCTSL) induction as a potential biomarker of response to DNA-damaging therapies. The combination of CLF and olaparib represents a promising strategy to overcome drug resistance in ovarian cancer by promoting CTSL nuclear localization and enhancing DNA damage. Citation Format: Prabhu Thirusangu, Ling Jin, Upasana Ray, Anya Zhao, Julie Staub, Xinyan Wu, Xiaonan Hou, Jamison L. VanBlaricom, Syed Mohamed Musheer Aalam, Sun-Hee Lee, Ann Oberg, Nagarajan Kannan, John Weroha, Jeremy Chien, Scott H. Kaufmann, Jamie N. Bakkum-Gamez, Viji Shridhar. Role of drug induced nuclear CTSL (nCTSL) in DNA damage response in cancer- therapeutic implications [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Ovarian Cancer Research; 2025 Sep 19-21; Denver, CO. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl):Abstract nr A050.

Abstract Although inhibitors of the kinases CHK1, ATR, and WEE1 are undergoing clinical testing, it remains unclear how these three classes of agents kill susceptible cells and whether they utilize the same cytotoxic mechanism. Here we observed that CHK1 inhibition induces apoptosis in a subset of acute leukemia cell lines in vitro, including TP53-null acute myeloid leukemia (AML) and BCR/ABL–positive acute lymphoid leukemia (ALL), and inhibits leukemic colony formation in clinical AML samples ex vivo. In further studies, downregulation or inhibition of CHK1 triggered signaling in sensitive human acute leukemia cell lines that involved CDK2 activation followed by AP1-dependent TNF transactivation, TNFα production, and engagement of a TNFR1- and BID-dependent apoptotic pathway. AML lines that were intrinsically resistant to CHK1 inhibition exhibited high CHK1 expression and were sensitized by CHK1 downregulation. Signaling through this same CDK2–AP1–TNF cytotoxic pathway was also initiated by ATR or WEE1 inhibitors in vitro and during CHK1 inhibitor treatment of AML xenografts in vivo. Collectively, these observations not only identify new contributors to the antileukemic cell action of CHK1, ATR, and WEE1 inhibitors, but also delineate a previously undescribed pathway leading from aberrant CDK2 activation to death ligand–induced killing that can potentially be exploited for acute leukemia treatment. Significance: This study demonstrates that replication checkpoint inhibitors can kill AML cells through a pathway involving AP1-mediated TNF gene activation and subsequent TP53-independent, TNFα-induced apoptosis, which can potentially be exploited clinically.

Abstract Purpose: Clear cell ovarian carcinoma (CCOC) is an aggressive disease that often demonstrates resistance to standard chemotherapies. Approximately 25% of patients with CCOC show a strong APOBEC mutation signature. Here, we determine which APOBEC3 enzymes are expressed in CCOC, establish clinical correlates, and identify a new biomarker for detection and intervention. Experimental Designs: APOBEC3 expression was analyzed by IHC and qRT-PCR in a pilot set of CCOC specimens (n = 9 tumors). The IHC analysis of APOBEC3B was extended to a larger cohort to identify clinical correlates (n = 48). Dose-response experiments with platinum-based drugs in CCOC cell lines and carboplatin treatment of patient-derived xenografts (PDXs) were done to address mechanistic linkages. Results: One DNA deaminase, APOBEC3B, is overexpressed in a formidable subset of CCOC tumors and is low or absent in normal ovarian and fallopian tube epithelial tissues. High APOBEC3B expression associates with improved progression-free survival (P = 0.026) and moderately with overall survival (P = 0.057). Cell-based studies link APOBEC3B activity and subsequent uracil processing to sensitivity to cisplatin and carboplatin. PDX studies extend this mechanistic relationship to CCOC tissues. Conclusions: These studies demonstrate that APOBEC3B is overexpressed in a subset of CCOC and, contrary to initial expectations, associated with improved (not worse) clinical outcomes. A likely molecular explanation is that APOBEC3B-induced DNA damage sensitizes cells to additional genotoxic stress by cisplatin. Thus, APOBEC3B is a molecular determinant and a candidate predictive biomarker of the therapeutic response to platinum-based chemotherapy. These findings may have broader translational relevance, as APOBEC3B is overexpressed in many different cancer types.

Abstract Bcl-2 is phosphorylated on Ser70 after treatment of cells with spindle poisons. On the basis of effects observed in cells overexpressing Bcl-2 S70E or S70A mutants, various studies have concluded that Ser70 phosphorylation either enhances or diminishes Bcl-2 function. In the present study, the ability of phosphorylated Bcl-2, as well as the S70E and S70A mutants, to bind and neutralize proapoptotic Bcl-2 family members under cell-free conditions and in intact cells was examined in an attempt to resolve this controversy. Surface plasmon resonance indicated that phosphorylated Bcl-2, Bcl-2 S70E, and Bcl-2 S70A exhibit enhanced binding to Bim and Bak compared with unmodified Bcl-2. This enhanced binding reflected a readily detectable conformation change in the loop domain of Bcl-2. Furthermore, Bcl-2 S70E and S70A bound more Bak and Bim than wild-type Bcl-2 in pull-downs and afforded greater protection against several chemotherapeutic agents. Importantly, binding of endogenous Bcl-2 to Bim also increased during mitosis, when Bcl-2 is endogenously phosphorylated, and disruption of this mitotic Bcl-2/Bim binding with navitoclax or ABT-199, like Bcl-2 downregulation, enhanced the cytotoxicity of paclitaxel. Collectively, these results provide not only a mechanistic basis for the enhanced antiapoptotic activity of phosphorylated Bcl-2, but also an explanation for the ability of BH3 mimetics to enhance taxane sensitivity. Cancer Res; 73(23); 6998–7008. ©2013 AACR.