GAS6-AXL Inhibition by AVB-500 Overcomes Resistance to Paclitaxel in Endometrial Cancer by Decreasing Tumor Cell Glycolysis.

Chemotherapy is often ineffective in advanced-stage and aggressive histologic subtypes of endometrial cancer. Overexpression of the receptor tyrosine kinase AXL has been found to be associated with therapeutic resistance, metastasis, and poor prognosis. However, the mechanism of how inhibition of AXL improves response to chemotherapy is still largely unknown. Thus, we aimed to determine whether treatment with AVB-500, a selective inhibitor of GAS6-AXL, improves endometrial cancer cell sensitivity to chemotherapy particularly through metabolic changes. We found that both GAS6 and AXL expression were higher by immunohistochemistry in patient tumors with a poor response to chemotherapy compared with tumors with a good response to chemotherapy. We showed that chemotherapy-resistant endometrial cancer cells (ARK1, uterine serous carcinoma and PUC198, grade 3 endometrioid adenocarcinoma) had improved sensitivity and synergy with paclitaxel and carboplatin when treated in combination with AVB-500. We also found that in vivo intraperitoneal models with ARK1 and PUC198 cells had decreased tumor burden when treated with AVB-500 + paclitaxel compared with paclitaxel alone. Treatment with AVB-500 + paclitaxel decreased AKT signaling, which resulted in a decrease in basal glycolysis. Finally, multiple glycolytic metabolites were lower in the tumors treated with AVB-500 + paclitaxel than in tumors treated with paclitaxel alone. Our study provides strong preclinical rationale for combining AVB-500 with paclitaxel in aggressive endometrial cancer models.

GAS6/AXL Inhibition Enhances Ovarian Cancer Sensitivity to Chemotherapy and PARP Inhibition through Increased DNA Damage and Enhanced Replication Stress.

Over 80% of women with high-grade serous ovarian cancer (HGSOC) develop tumor resistance to chemotherapy and die of their disease. There are currently no FDA-approved agents to improve sensitivity to first-line platinum- and taxane-based chemotherapy or to PARP inhibitors. Here, we tested the hypothesis that expression of growth arrest-specific 6 (GAS6), the ligand of receptor tyrosine kinase AXL, is associated with chemotherapy response and that sequestration of GAS6 with AVB-S6-500 (AVB-500) could improve tumor response to chemotherapy and PARP inhibitors. We found that GAS6 levels in patient tumor and serum samples collected before chemotherapy correlated with ovarian cancer chemoresponse and patient survival. Compared with chemotherapy alone, AVB-500 plus carboplatin and/or paclitaxel led to decreased ovarian cancer-cell survival in vitro and tumor burden in vivo. Cells treated with AVB-500 plus carboplatin had more DNA damage, slower DNA replication fork progression, and fewer RAD51 foci than cells treated with carboplatin alone, indicating AVB-500 impaired homologous recombination (HR). Finally, treatment with the PARP inhibitor olaparib plus AVB-500 led to decreased ovarian cancer-cell survival in vitro and less tumor burden in vivo. Importantly, this effect was seen in HR-proficient and HR-deficient ovarian cancer cells. Collectively, our findings suggest that GAS6 levels could be used to predict response to carboplatin and AVB-500 could be used to treat platinum-resistant, HR-proficient HGSOC. IMPLICATIONS: GAS6/AXL is a novel target to sensitize ovarian cancers to carboplatin and olaparib. Additionally, GAS6 levels can be associated with response to carboplatin treatment.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

BACKGROUND: Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin (IL)-1ß. IL-1ß plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders. OBJECTIVE: The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and IL-1ß processing. METHOD: Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, IL-1ß enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays. RESULTS: TETBr and TMTOH induced inflammasome aggregate formation and IL-1ß release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or LPS+ATP challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways. CONCLUSION: Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314.

Homologous recombination deficiency real-time clinical assays, ready or not?

Cancers with deficiencies in homologous recombination-mediated DNA repair (HRR) demonstrate improved clinical outcomes and increased survival. Approximately 50% of high-grade serous ovarian cancers (HGSOC) exhibit homologous recombination deficiency (HRD). HRD can be caused by germline or somatic mutations of genes involved in the HR pathway. Given platinum-based chemotherapy and poly (ADP-ribose) polymerase inhibitors (PARPis) are used in HGSOC, double-strand breaks (DSBs) are common. Unrepaired DSBs are toxic to cells as genomic instability ensues and cells eventually die. Thus, tumor cells with DSBs utilize the high-fidelity HRR as one of the central pathways for repair. In tumors that have HRD, an alternate pathway such as non-homologous end-joining (NHEJ) is used and leads to error-prone repair. To date, methods for clinical detection of homologous recombination deficiency (HRD) are limited to genomic changes of HRR genes and genomic mutation patterns resulting from HRD genes involved in HR-mediated DNA repair. However, these tests detect genomic scars that might not always correlate well with PARP inhibitor or platinum sensitivity in the current state. Therefore, a functional HRD assay should be able to more accurately predict tumor response in real-time. RAD51 foci formation has been used as a functional assay to define HRD and closely correlates with chemotherapy and PARPi sensitivity. The inability to form RAD51 foci is a common feature of HRD. DNA damage can also cause transient slowing or stalling of replication forks defined as replication stress. Replication fork stalling can lead to fork degradation and decreased cell viability if forks do not resume DNA synthesis. Fork degradation has been found to lead to chemosensitivity in BRCA-deficient tumors. To determine this fork degradation phenotype, replication fork/DNA fiber assays are utilized. This review will highlight functional assays for HRD in the context of translating these to real-time clinical assays.

Subjective seizure counts by epilepsy clinical drug trial participants are not reliable.

PURPOSE: Self-reported seizure counts of patients with epilepsy guide individual treatment decisions and often represent the primary outcome measure of pharmacological trial in epileptology. The validity of these data has recently been challenged and the question is whether this applies to participants of clinical studies as well. Here we compared self-estimated seizure counting and documentation accuracy in participants and nonparticipants of former epilepsy clinical drug trials. METHODS: Adult participants (N=100) from a total of twenty-two phase II, III or IV clinical drug studies performed at our unit (2002-2015) underwent a structured telephone interview on self-estimated seizure awareness and seizure documentation accuracy. Data were compared to data from a recent study in adult epilepsy patients (N=132) who never participated in clinical trials and who answered the same questions (Blachut et al., Seizure 2015; 29:97-103). RESULTS: Reported seizure frequencies, self-estimated seizure documentation accuracy (at best 46-53%), and the motivation for seizure-documentation were almost identical in both groups and no group effect was found except for higher self-reported awareness for nocturnal seizures in former study participants. CONCLUSION: Epilepsy patients having participated in clinical drug trials report comparable erroneous seizure counts as do epilepsy patients in general. These data further corroborate the notion that most clinical trials in epileptology are based on inaccurate measures. Implications and possible solutions for patients, physicians, and research are discussed.

Novel techniques for automated seizure registration: Patients' wants and needs.

OBJECTIVES: Patient-reported seizure frequency is essential for therapy management and clinical research but lacks validity mainly due to seizure-induced seizure unawareness. Automated seizure detection by mobile monitoring devices promises to settle this serious methodological issue. Here, we explored attitudes and preferences towards future devices for seizure detection in adult patients with therapy-refractory epilepsies. METHODS: A total of 102 inpatients enrolled and underwent a newly constructed semistructured 30-minute interview on automated seizure registration. RESULTS: Most patients would generally apply and permanently use seizure registration devices. Removable devices were preferred (e.g., wristband sensors), but also patch electrodes at invisible body sites appeared acceptable. Only a minority of patients would accept implantations for seizure registration (especially of depth electrodes). Also, permanent optical or acoustical surveillance were accepted by a few patients only. Most patients were ready to care for the device (e.g., charging battery), to have doctor's appointments for device control, and even to pay for the device. Seizure prediction was evaluated as an essential additional function. Only half of the patients wanted emergency calls in case of a seizure. SIGNIFICANCE: Patients would accept automated seizure registration if the device had as little as possible negative effect on daily living. High acceptance might, therefore, be expected for hardware equipment as it is nowadays used by many healthy subjects for physiological self-monitoring and life-logging. The proper medical engineering task of the future, therefore, is to optimize sensors in those highly feasible devices and to establish reliable biomarkers and outcome measures for a diversity of diseases (including epilepsy) from data obtained by this generic hardware.

Counting seizures: The primary outcome measure in epileptology from the patients' perspective.

PURPOSE: Patient-reported seizure counts represent a key outcome measure for individual treatments and clinical studies in epileptology. Video-EEG based research, however, demonstrated lack of validity due to underreporting. Here we examined the practice of keeping seizure diaries and the patients' attitudes toward seizure counting. METHODS: Anticipating a low return rate, a comprehensive survey was mailed to 1100 adult outpatients. Besides methods and reasons to document or not to document seizures, the questionnaire addressed clinical, personality and sociodemographic characteristics as well as the subjective experience of seizures. RESULTS: Questionnaires from 170 patients (15%) could be included in our analysis. Patients estimated to be aware of 5.3 out of 10 daytime seizures (nocturnal seizures: 2.6) while they supposed that relatives/colleagues noticed 7.1 (nocturnal: 4.6). Almost two-thirds of the patients reported to keep a seizure diary with a self-estimated documentation rate of 8.7 out of 10 noticed daytime seizures (nocturnal: 7.7). Documenters and non-documenters showed only marginal group differences with regard to clinical, personality and sociodemographic characteristics. Importantly, patients were more committed to keep a seizure diary when they judged it to be relevant for clinical treatment decisions. CONCLUSION: Patients appear to know that they underreport seizures. According to their view, seizure unawareness as induced by seizures themselves seems to be a more important factor than omitting documentation of noticed seizures. Thus, the potential to improve the validity of seizure diaries of electronic devices which facilitate documenting noticed seizures appears limited.

An efficient synthesis of fluorinated azaheterocycles by aminocyclization of alkenes.

A general and efficient approach to important fluorinated azaheterocycles has been developed by incorporating nucleophilic fluorination into alkene difunctionalization. This intramolecular aminofluorination transformation of alkenes has been achieved via the aminocyclization of reactive unsaturated N-iodoamines, which can be generated in situ from either unsaturated N-chloramines or their amine precursors in a one-pot protocol.