The Transplant Bellwether: Endothelial Cells in Antibody-Mediated Rejection.

Ab-mediated rejection of organ transplants remains a stubborn, frequent problem affecting patient quality of life, graft function, and grant survival, and for which few efficacious therapies currently exist. Although the field has gained considerable knowledge over the last two decades on how anti-HLA Abs cause acute tissue injury and promote inflammation, there has been a gap in linking these effects with the chronic inflammation, vascular remodeling, and persistent alloimmunity that leads to deterioration of graft function over the long term. This review will discuss new data emerging over the last 5 y that provide clues into how ongoing Ab-endothelial cell interactions may shape vascular fate and propagate alloimmunity in organ transplants.

PG545 sensitizes ovarian cancer cells to PARP inhibitors through modulation of RAD51-DEK interaction.

PG545 (Pixatimod) is a highly sulfated small molecule known for its ability to inhibit heparanase and disrupt signaling mediated by heparan-binding-growth factors (HB-GF). Previous studies indicated that PG545 inhibits growth factor-mediated signaling in ovarian cancer (OC) to enhance response to chemotherapy. Here we investigated the previously unidentified mechanisms by which PG545 induces DNA damage in OC cells and found that PG545 induces DNA single- and double-strand breaks, reduces RAD51 expression in an autophagy-dependent manner and inhibits homologous recombination repair (HRR). These changes accompanied the ability of PG545 to inhibit endocytosis of the heparan-sulfate proteoglycan interacting DNA repair protein, DEK, leading to DEK sequestration in the tumor microenvironment (TME) and loss of nuclear DEK needed for HRR. As a result, PG545 synergized with poly (ADP-ribose) polymerase inhibitors (PARPis) in OC cell lines in vitro and in 55% of primary cultures of patient-derived ascites samples ex vivo. Moreover, PG545/PARPi synergy was observed in OC cells exhibiting either de novo or acquired resistance to PARPi monotherapy. PG545 in combination with rucaparib also generated increased DNA damage, increased antitumor effects and increased survival of mice bearing HRR proficient OVCAR5 xenografts compared to monotherapy treatment in vivo. Synergistic antitumor activity of the PG545/rucaparib combination was likewise observed in an immunocompetent syngeneic ID8F3 OC model. Collectively, these results suggest that targeting DEK-HSPG interactions in the TME through the use of PG545 may be a novel method of inhibiting DNA repair and sensitizing cells to PARPis.

Exploiting LRRC15 as a Novel Therapeutic Target in Cancer.

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.

Quinacrine Induces Nucleolar Stress in Treatment-Refractory Ovarian Cancer Cell Lines.

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.

Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal/Mitochondrial Membrane Permeabilization and Cell Death.

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.

Repurposing quinacrine for treatment-refractory cancer.

Quinacrine, also known as mepacrine, has originally been used as an antimalarial drug for close to a century, but was recently rediscovered as an anticancer agent. The mechanisms of anticancer effects of quinacrine are not well understood. The anticancer potential of quinacrine was discovered in a screen for small molecule activators of p53, and was specifically shown to inhibit NFκB suppression of p53. However, quinacrine can cause cell death in cells that lack p53 or have p53 mutations, which is a common occurrence in many malignant tumors including high grade serous ovarian cancer. Recent reports suggest quinacrine may inhibit cancer cell growth through multiple mechanisms including regulating autophagy, FACT (facilitates chromatin transcription) chromatin trapping, and the DNA repair process. Additional reports also suggest quinacrine is effective against chemoresistant gynecologic cancer. In this review, we discuss anticancer effects of quinacrine and potential mechanisms of action with a specific focus on gynecologic and breast cancer where treatment-refractory tumors are associated with increased mortality rates. Repurposing quinacrine as an anticancer agent appears to be a promising strategy based on its ability to target multiple pathways, its selectivity against cancer cells, and the synergistic cytotoxicity when combined with other anticancer agents with limited side effects and good tolerability profile.