Jin, Ling

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.

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.

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.