Genomic profiling of a multi-lineage and multi-passage patient-derived xenograft biobank reflects heterogeneity of ovarian cancer.

Ovarian cancer (OC) manifests as a complex disease characterized by inter- and intra-patient heterogeneity. Despite enhanced biological and genetic insights, OC remains a recalcitrant malignancy with minimal survival improvement. Based on multi-site sampling and a multi-lineage patient-derived xenograft (PDX) establishment strategy, we present herein the establishment of a comprehensive PDX biobank from histologically and molecularly heterogeneous OC patients. Comprehensive profiling of matched PDX and patient samples demonstrates that PDXs closely recapitulate parental tumors. By leveraging multi-lineage models, we reveal that the previously reported genomic disparities of PDX could be mainly attributed to intra-patient spatial heterogeneity instead of substantial model-independent genomic evolution. Moreover, DNA damage response pathway inhibitor (DDRi) screening uncovers heterogeneous responses across models. Prolonged iterative drug exposure recapitulates acquired drug resistance in initially sensitive models. Meanwhile, interrogation of induced drug-resistant (IDR) models reveals that suppressed interferon (IFN) response and activated Wnt/ß-catenin signaling contribute to acquired DDRi drug resistance.

BRD4 inhibition impairs DNA mismatch repair, induces mismatch repair mutation signatures and creates therapeutic vulnerability to immune checkpoint blockade in MMR-proficient tumors.

BACKGROUND: Mismatch repair deficiency (dMMR) is a well-recognized biomarker for response to immune checkpoint blockade (ICB). Strategies to convert MMR-proficient (pMMR) to dMMR phenotype with the goal of sensitizing tumors to ICB are highly sought. The combination of bromodomain containing 4 (BRD4) inhibition and ICB provides a promising antitumor effect. However, the mechanisms underlying remain unknown. Here, we identify that BRD4 inhibition induces a persistent dMMR phenotype in cancers. METHODS: We confirmed the correlation between BRD4 and mismatch repair (MMR) by the bioinformatic analysis on The Cancer Genome Atlas and Clinical Proteomic Tumor Analysis Consortium data, and the statistical analysis on immunohistochemistry (IHC) scores of ovarian cancer specimens. The MMR genes (MLH1,MSH2,MSH6,PMS2) were measured by quantitative reverse transcription PCR, western blot, and IHC. The MMR status was confirmed by whole exome sequencing, RNA sequencing, MMR assay and hypoxanthine-guanine phosphoribosyl transferase gene mutation assay. The BRD4i AZD5153 resistant models were induced both in vitro and in vivo. The transcriptional effects of BRD4 on MMR genes were investigated by chromatin immunoprecipitation among cell lines and data from the Cistrome Data Browser. The therapeutic response to ICB was testified in vivo. The tumor immune microenvironment markers, such as CD4, CD8, TIM-3, FOXP3, were measured by flow cytometry. RESULTS: We identified the positive correlation between BRD4 and MMR genes in transcriptional and translational aspects. Also, the inhibition of BRD4 transcriptionally reduced MMR genes expression, resulting in dMMR status and elevated mutation loads. Furthermore, prolonged exposure to AZD5153 promoted a persistent dMMR signature both in vitro and in vivo, enhancing tumor immunogenicity, and increased sensitivity to α-programmed death ligand-1 therapy despite the acquired drug resistance. CONCLUSIONS: We demonstrated that BRD4 inhibition suppressed expression of genes critical to MMR, dampened MMR, and increased dMMR mutation signatures both in vitro and in vivo, sensitizing pMMR tumors to ICB. Importantly, even in BRD4 inhibitors (BRD4i)-resistant tumor models, the effects of BRD4i on MMR function were maintained rendering tumors sensitive to ICB. Together, these data identified a strategy to induce dMMR in pMMR tumors and further, indicated that BRD4i sensitive and resistant tumors could benefit from immunotherapy.

Inhibiting the IRE1α Axis of the Unfolded Protein Response Enhances the Antitumor Effect of AZD1775 in TP53 Mutant Ovarian Cancer.

Targeting the G2/M checkpoint mediator WEE1 has been explored as a novel treatment strategy in ovarian cancer, but mechanisms underlying its efficacy and resistance remains to be understood. Here, it is demonstrated that the WEE1 inhibitor AZD1775 induces endoplasmic reticulum stress and activates the protein kinase RNA-like ER kinase (PERK) and inositol-required enzyme 1α (IRE1α) branches of the unfolded protein response (UPR) in TP53 mutant (mtTP53) ovarian cancer models. This is facilitated through NF-κB mediated senescence-associated secretory phenotype. Upon AZD1775 treatment, activated PERK promotes apoptotic signaling via C/EBP-homologous protein (CHOP), while IRE1α-induced splicing of XBP1 (XBP1s) maintains cell survival by repressing apoptosis. This leads to an encouraging synergistic antitumor effect of combining AZD1775 and an IRE1α inhibitor MKC8866 in multiple cell lines and preclinical models of ovarian cancers. Taken together, the data reveal an important dual role of the UPR signaling network in mtTP53 ovarian cancer models in response to AZD1775 and suggest that inhibition of the IRE1α-XBP1s pathway may enhance the efficacy of AZD1775 in the clinics.

AZD5153 reverses palbociclib resistance in ovarian cancer by inhibiting cell cycle-related proteins and the MAPK/PI3K-AKT pathway.

The CDK4/6 inhibitor, palbociclib has recently entered clinic-trial stage for breast cancer treatment. However, translating its efficacy to other solid tumors has been challenging, especially for aggressive solid tumors. We found that the effect of palbociclib as a single agent was limited due to primary and acquired resistance in multiple ovarian cancer (OC) models. Among these, patient-derived organoid and xenograft models are two most representative models of drug responsiveness in patients with OC. In preclinical models, this study demonstrated that activated MAPK/PI3K-AKT pathway and cell cycle-related proteins induced the resistance to palbociclib, which was overcome by the addition of the bromodomain protein 4 (BRD4) inhibitor AZD5153. Moreover, this study revealed that AZD5153 and palbociclib had a synergistic lethal effect on inducing the cell cycle arrest and increasing apoptosis, even in RB-deficient cell lines. Based on these results, it is anticipated that this class of drugs, including AZD5153, which inhibit the cell cycle-related protein and MAPK/PI3K-AKT pathway, will exhibit synergistic effects with palbociclib in OC.

Dragon's Blood Regulates Rac1-WAVE2-Arp2/3 Signaling Pathway to Protect Rat Intestinal Epithelial Barrier Dysfunction Induced by Simulated Microgravity.

Dragon's Blood is a red resin from Dracaena cochinchinensis (Lour.) S.C. Chen (Yunnan, China). As a traditional Chinese medicinal herb, it has shown protective effects on intestinal disorders. Microgravity could alter intestinal homeostasis. However, the potential herbal drugs for preventing intestine epithelial barrier (IEB) dysfunction under microgravity are not available. This study aimed to investigate the effects of Dragon's Blood (DB) on microgravity-induced IEB injury and explore its underlying mechanism. A rat tail-suspension model was used to simulate microgravity (SMG). Histomorphology, ultrastructure, permeability, and expression of junction proteins in jejunum, ileum, and colon of SMG rats were determined. Proteomic analysis was used to identify differentially expressed proteins (DEPs) in rat ileum mucosa altered by DB. The potential mechanism of DB to protect IEB dysfunction was validated by western blotting. The effects of several components in DB were evaluated in SMG-treated Caco-2 cells. DB protected against IEB disruption by repairing microvilli and crypts, inhibiting inflammatory factors, lowering the permeability and upregulating the expression of tight and adherens junction proteins in the ileum of SMG rats. Proteomic analysis showed that DB regulated 1080 DEPs in rat ileum mucosa. DEPs were significantly annotated in cell-cell adhesion, focal adhesion, and cytoskeleton regulation. DB increased the expression of Rac1-WAVE2-Arp2/3 pathway proteins and F-actin to G-actin ratio, which promoted the formation of focal adhesions. Loureirin C in DB showed a protective effect on epithelial barrier injury in SMG-treated Caco-2 cells. DB could protect against IEB dysfunction induced by SMG, and its mechanism is associated with the formation of focal adhesions mediated by the Rac1-WAVE2-Arp2/3 pathway, which benefits intestinal epithelial cell migration and barrier repair.

The Immunological Role of CDK4/6 and Potential Mechanism Exploration in Ovarian Cancer.

Background: Ovarian cancer (OC) is one of the most lethal gynecologic cancers. Growing evidence has proven that CDK4/6 plays a key role in tumor immunity and the prognosis of many cancers. However, the expression and function of CDK4/6 in OC remain unclear. Therefore, we aimed to explore the influence of CDK4/6 in OC, especially on immunity. Methods: We analyzed CDK4/6 expression and prognosis using The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and Genotype Tissue Expression (GTEx) data. Subsequently, we used the cytoHubba plug-in of Cytoscape software and starBase to identify the noncoding RNAs (ncRNAs) regulating CDK4/6. Finally, we verified the effect of CDK4/6 on immunity in OC cell lines and animal models. Results: CDK4/6 expression was higher in OC tissues than in normal ovarian tissues, and the high expression levels of CDK4/6 contributed to the immunosuppressive state of OC and were thus related to the poor prognosis of OC patients. This was also in general agreement with the results of OC cell line and animal experiments. Mechanistically, the CDK4/6 inhibitor palbociclib increased the secretion of interferon (IFN)-γ and the interferon-stimulated gene (ISG) response, thereby upregulating the expression of antigen-presenting molecules; this effect was partly dependent on the STING pathway and thus activated immunity in OC. Additionally, according to public data, the LRRC75A-AS1-hsa-miR-330-5p axis could inhibit the immune response of OC patients by upregulating CDK4/6, leading to a poor prognosis. Conclusion: CDK4/6 affects the immune microenvironment of OC and correlates with the prognosis of OC patients.

Electrophysiological actions of cyclosporin A and tacrolimus on rat hippocampal CA1 pyramidal neurons.

AIM: The aim of the present study was to investigate the electrophysiological actions of cyclosporin A (CsA) and tacrolimus (FK506) on neurons in the brain, and to elucidate the relevant mechanisms. METHODS: Whole-cell current-clamp recording was made in CA1 pyramidal neurons in rat hippocampal slices; whole-cell voltage-clamp recording was made in dissociated hippocampal CA1 pyramidal neurons of rats. RESULTS: CsA (100 micromol/L) and FK506 (50 micromol/L) did not significantly alter the passive electrical properties of hippocampal CA1 pyramidal neurons, but slowed down the repolarizing phase of the action potential. CsA (10-100 micromol/L) selectively inhibited the delayed rectifier K+ current (IK) in a concentration-dependent manner. CsA did not affect the kinetic properties of IK. Intracellular dialysis of CsA (100 micromol/L) had no effect on IK. The inhibition of IK by CsA (100 micromol/L) persisted under the low Ca2+ conditions that blocked the basal activity of calcineurin. CONCLUSION: CsA exerted calcineurin-independent inhibition on the IK in rat hippocampal pyramidal neurons. Taken together with our previous finding with FK506, it is conceivable that the spike broadening caused by the immunosuppressant drugs is due to direct inhibition on the IK.

Calcineurin-independent inhibition of the delayed rectifier K+ current by the immunosuppressant FK506 in rat hippocampal neurons.

The immunosuppressant drug FK506 was found to be a potent neuroprotective agent in animal models of brain ischemia. However, the mechanisms underlying the action remain to be elucidated. The delayed rectifier K(+) channel has been implicated in ischemic injury and neuronal death in the brain. The aim of the present study is to investigate whether the neuroprotective action of FK506 results from blocking the K(+) channel. In acutely dissociated CA1 pyramidal neurons of rat hippocampus, superfusion of FK506 (0.01-100 microM) selectively inhibited the delayed rectifier K(+) current (I(K)) with an IC(50) value of 13.2+/-4.9 microM. The inhibition of I(K) by FK506 (10 microM) had a rapid onset, and then gradually reached a steady-state level. The inhibition was voltage-dependent, became more potent when the currents were elicited by strong depolarization. Moreover, FK506 (10 microM) caused marked negative shifts of the steady-state activation and inactivation curves of I(K), and accelerated its recovery from inactivation. Intracellular dialysis of FK506 (30 microM) was ineffective. The inhibition of I(K) by FK506 (10 microM) persisted under the low-Ca(2+) conditions that blocked the basal activity of protein phosphatase 2B (calcineurin). Rapamycin did not antagonize FK506 but mimicked it. Cyclosporin A inhibited I(K) only at 30 and 100 microM. Taken together, the results suggest that FK506 exert a direct inhibition on the delayed rectifier K(+) channel without involvement of calcineurin.