Ray, Upasana

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 In our efforts to enhance sensitivity to PARP inhibitors, we identified clofarabine (CLF) as a potential therapy for drug-resistant ovarian cancer and nuclear trafficking of Cathepsin L (CTSL) as a treatment- responsive biomarker. Using PARP inhibitor-sensitive and -resistant OC cell lines, ex vivo cultures of patient-derived ovarian ascites (OVA), primary ovarian tumors, and xenografts (PDX), we found that CLF monotherapy induces nuclear CTSL (nCTSL) in CLF-responsive cells (CLF-r) and sensitizes them to PARP inhibitors olaparib and rucaparib. In CLF non-responsive cells (CLF-nr), a combination of CLF with olaparib is necessary for nCTSL trafficking and synergy. CLF+olaparib synergy was observed in 47% of CLF-r and 24% of CLF-nr OVA samples. Drug-induced nCTSL is crucial for DNA damage response, including cell cycle arrest and apoptosis. Knockdown of CTSL in both CLF-r and CLF-nr cells conferred resistance to the CLF+olaparib combination, emphasizing nCTSL’s role in the DNA damage response pathway (DDR). Mechanistically, CLF facilitates CTSL nuclear import via KPNB1 in CLF-r cells. In CLF-nr cells, both olaparib and CLF are needed to facilitate CTSL nuclear import. Additionally, CLF downregulates the nuclear export protein CRM1 (XPO1) in both cohorts. Interestingly, CLF does not downregulate CRM1 in a subset of OVAs (29%), and they were classified as CLF-resistant (CLF- Res). In these samples, inhibiting CRM1 with KPT8602 restored synergy between CLF and PARP inhibitors. In vivo, CLF-r and CLF-nr PDX models exhibited enhanced DDR, reduced tumor burden, and prolonged survival with the CLF+olaparib combination. These findings suggest the CLF+olaparib combination is a promising therapeutic strategy for drug-resistant OC by inducing DDR through CTSL nuclear localization.

Abstract A problematic feature of many human cancers is a lack of understanding of mechanisms controlling organ-specific patterns of metastasis, despite recent progress in identifying many mutations and transcriptional programs shown to confer this potential. To address this gap, we developed a methodology that enables different aspects of the metastatic process to be comprehensively characterized at a clonal resolution. Our approach exploits the application of a computational pipeline to analyze and visualize clonal data obtained from transplant experiments in which a cellular DNA barcoding strategy is used to distinguish the separate clonal contributions of two or more competing cell populations. To illustrate the power of this methodology, we demonstrate its ability to discriminate the metastatic behavior in immunodeficient mice of a well-established human metastatic cancer cell line and its co-transplanted LRRC15 knockdown derivative. We also show how the use of machine learning to quantify clone-initiating cell (CIC) numbers and their subsequent metastatic progeny generated in different sites can reveal previously unknown relationships between different cellular genotypes and their initial sites of implantation with their subsequent respective dissemination patterns. These findings underscore the potential of such combined genomic and computational methodologies to identify new clonally-relevant drivers of site-specific patterns of metastasis.

Ovarian cancer ranks fifth in terms of cancer mortality in women due to lack of early diagnosis and poor clinical management. Characteristics like high cellular proliferation, EMT and metabolic alterations contribute to oncogenicity. Cancer, being a “metabolic disorder,” is governed by various key regulatory factors like metabolic enzymes, oncogenes, and tumor suppressors. Sirtuins (SIRT1‐SIRT7) belong to the group of NAD + deacetylase and ADP‐ribosylation enzymes that function as NAD + sensors and metabolic regulators. Among sirtuin orthologs, SIRT6 emerges as an important oncogenic player, although its possible mechanistic involvement in ovarian cancer advancement is still elusive. Our data indicated a higher expression of SIRT6 in ovarian cancer tissues compared with the non‐malignant ovarian tissue. Further, we observed that overexpression of SIRT6 enhances glycolysis and oxidative phosphorylation in ovarian cancer cells. The energy derived from these processes facilitates migration and invasion through invadopodia formation by reorganization of actin fibers. Mechanistically, SIRT6 has been shown to promote ERK1/2‐driven activatory phosphorylation of DRP1 at serine‐616, which has an obligatory role in inducing mitochondrial fission. These fragmented mitochondria facilitate cell movement important for metastases. siRNA‐mediated downregulation of SIRT6 was found to decrease cellular invasion through compromised mitochondrial fragmentation and subsequent reduction in stress fiber formation in ovarian cancer cells. Thus, the present report establishes the impact of SIRT6 in the regulation of morphological and functional aspects of mitochondria that modulates invasion in ovarian cancer cells.

Abstract Abundant fibrotic stroma is a typical feature of most solid tumors, and stromal activation promotes oncogenesis, therapy resistance, and metastatic dissemination of cancer cells. Therefore, targeting the tumor stroma in combination with standard-of-care therapies has become a promising therapeutic strategy in recent years. The leucine-rich repeat-containing protein 15 (LRRC15) is involved in cell–cell and cell–matrix interactions and came into focus as a promising anticancer target owing to its overexpression in mesenchymal-derived tumors such as sarcoma, glioblastoma, and melanoma and in cancer-associated fibroblasts in the microenvironment of breast, head and neck, lung, and pancreatic tumors. Effective targeting of LRRC15 using specific antibody–drug conjugates (ADC) has the potential to improve the outcome of patients with LRRC15-positive (LRRC15+) cancers of mesenchymal origin or stromal desmoplasia. Moreover, LRRC15 expression may serve as a predictive biomarker that could be utilized in the preclinical assessment of cancer patients to support personalized clinical outcomes. This review focuses on the role of LRRC15 in cancer, including clinical trials involving LRRC15-targeted therapies, such as the ABBV-085 ADC for patients with LRRC15+ tumors. This review spans perceived knowledge gaps and highlights the clinical avenues that need to be explored to provide better therapeutic outcomes in patients.

Abstract Dissemination of ovarian cancer cells can lead to inoperable metastatic lesions in the bowel and omentum that cause patient death. Here we show that LRRC15, a type-I 15-leucine–rich repeat-containing membrane protein, highly overexpressed in ovarian cancer bowel metastases compared with matched primary tumors and acts as a potent promoter of omental metastasis. Complementary models of ovarian cancer demonstrated that LRRC15 expression leads to inhibition of anoikis-induced cell death and promotes adhesion and invasion through matrices that mimic omentum. Mechanistically, LRRC15 interacted with β1-integrin to stimulate activation of focal adhesion kinase (FAK) signaling. As a therapeutic proof of concept, targeting LRRC15 with the specific antibody–drug conjugate ABBV-085 in both early and late metastatic ovarian cancer cell line xenograft models prevented metastatic dissemination, and these results were corroborated in metastatic patient-derived ovarian cancer xenograft models. Furthermore, treatment of 3D-spheroid cultures of LRRC15-positive patient-derived ascites with ABBV-085 reduced cell viability. Overall, these data uncover a role for LRRC15 in promoting ovarian cancer metastasis and suggest a novel and promising therapy to target ovarian cancer metastases. Significance: This study identifies that LRRC15 activates β1-integrin/FAK signaling to promote ovarian cancer metastasis and shows that the LRRC15-targeted antibody–drug conjugate ABBV-085 suppresses ovarian cancer metastasis in preclinical models.

A considerable subset of gynecologic cancer patients experience disease recurrence or acquired resistance, which contributes to high mortality rates in ovarian cancer (OC). Our prior studies showed that quinacrine (QC), an antimalarial drug, enhanced chemotherapy sensitivity in treatment-refractory OC cells, including artificially generated chemoresistant and high-grade serous OC cells. In this study, we investigated QC-induced transcriptomic changes to uncover its cytotoxic mechanisms of action. Isogenic pairs of OC cells generated to be chemoresistant and their chemosensitive counterparts were treated with QC followed by RNA-seq analysis. Validation of selected expression results and database comparison analyses indicated the ribosomal biogenesis (RBG) pathway is inhibited by QC. RBG is commonly upregulated in cancer cells and is emerging as a drug target. We found that QC attenuates the in vitro and in vivo expression of nucleostemin (NS/GNL3), a nucleolar RBG and DNA repair protein, and the RPA194 catalytic subunit of Pol I that results in RBG inhibition and nucleolar stress. QC promotes the redistribution of fibrillarin in the form of extranuclear foci and nucleolar caps, an indicator of nucleolar stress conditions. In addition, we found that QC-induced downregulation of NS disrupted homologous recombination repair both by reducing NS protein levels and PARylation resulting in reduced RAD51 recruitment to DNA damage. Our data suggest that QC inhibits RBG and this inhibition promotes DNA damage by directly downregulating the NS–RAD51 interaction. Additionally, QC showed strong synergy with PARP inhibitors in OC cells. Overall, we found that QC downregulates the RBG pathway, induces nucleolar stress, supports the increase of DNA damage, and sensitizes cells to PARP inhibition, which supports new therapeutic stratagems for treatment-refractory OC. Our work offers support for targeting RBG in OC and determines NS to be a novel target for QC.

Abstract Objective: Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Increasing evidence suggests that Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, a recently recognized critical regulator of lipid metabolism is associated with cancer metastasis. Though recent studies reveal that high expression of PLA2G3 significantly correlates with poor prognosis, however, there is lack of a single paradigm that determines the contribution of secreted PLAs in ovarian cancer (OC) progression that may open new therapeutic opportunities. This study aimed to investigate the role and molecular regulatory mechanism of PLA2G3 in OC. Methods: CRISPR and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal microscopy, cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Metabolic inhibitor PFK-158 induced autophagy in combination with platinum drugs was assessed by immunofluorescence assay, immunoblot and MTT cell viability assay. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and in combination with chemotherapy was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. Results: CRISPR and shRNA mediated downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. Accumulating evidence also shows that increased lipogenic activity in cancer cells is associated with loss of primary cilia (PC), which coordinates cellular signaling homeostasis, and is thus associated with tumorigenesis. We found that both PFK158-mediated downregulation of PLA2G3 and genetic KD of PLA2G3 resulted in increased number of percent ciliated cells and inhibited oncogenesis. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore PC in OC cells. Of clinical relevance, PFK158 induced an increase in percent ciliated cells in patient-derived primary ascites culture through autophagic degradation of PLA2G3 to reduce cell viability and sensitized cells to chemotherapy. Conclusion: Taken together, this will be the first comprehensive study that emphasizes the in depth crucial role of PLA2G3 in regulating the OC metastasis and further suggests the therapeutic potential of targeting phospholipases and/or restoration of primary cilia in cancer cells for future treatment. Thus, the elucidation of pathways and molecular inhibitors towards ciliogenesis in cancer cells will be of interest to forge forward to be tested in a novel clinical setting. Citation Format: Upasana Ray, Debarshi Roy, Ling Jin, Prabhu Thirusangu, Julie Staub, Yinan Xiao, Viji Shridhar. Autophagy-dependent therapeutic targeting of group III phospholipase A2 attenuates ovarian cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1936.

We previously reported that the antimalarial compound quinacrine (QC) induces autophagy in ovarian cancer cells. In the current study, we uncovered that QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels, implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. Using a Magic Red® cathepsin L activity assay and LysoTracker red, we discerned that QC-induced CTSL activation promotes lysosomal membrane permeability (LMP) resulting in the release of active CTSL into the cytosol to promote apoptotic cell death. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, mitochondrial outer membrane permeabilization (MOMP), and mitochondrial cytochrome-c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas induced overexpression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL, suggesting that CTSL promotes QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockout cells or with the anti-malarial autophagy-inhibiting drug chloroquine. Importantly, we showed that upregulation of CTSL in QC-treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome-c, tBid, cleaved PARP, and caspase3. Taken together, the data suggest that QC-induced autophagy and CTSL upregulation promote a positive feedback loop leading to excessive autophagic flux, LMP, and MOMP to promote QC-induced cell death in ovarian cancer cells.

Abstract Background Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, is recognized as a regulator of lipid metabolism associated with oncogenesis. Though recent studies reveal that high PLA2G3 expression significantly correlates with poor prognosis in several cancers, however, role of PLA2G3 in ovarian cancer (OC) pathogenesis is still undetermined. Methods CRISPR-Cas9 and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal and Transmission electron microscopy analysis, and the cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Regulation of primary ciliation by PLA2G3 downregulation both genetically and by metabolic inhibitor PFK-158 induced autophagy was assessed by immunofluorescence-based confocal analysis and immunoblot. Transient transfection with GFP-RFP-LC3B and confocal analysis was used to assess the autophagic flux in OC cells. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and with platinum drugs was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. Results Downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. PLA2G3 knockdown in HeyA8MDR-resistant cells showed sensitivity to carboplatin treatment. We found that both PFK158 inhibitor-mediated and genetic downregulation of PLA2G3 resulted in increased number of percent ciliated cells and inhibited oncogenesis. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore primary cilia in OC cells. Of clinical relevance, PFK158 also induces percent ciliated cells in human-derived primary ascites cells and reduces cell viability with sensitization to chemotherapy. Conclusions Taken together, our study for the first time emphasizes the role of PLA2G3 in regulating the OC metastasis. This study further suggests the therapeutic potential of targeting phospholipases and/or restoration of PC for future OC treatment and the critical role of PLA2G3 in regulating ciliary function by coordinating interface between lipogenesis and metastasis.