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Expression of the AR splice variant, androgen receptor variant 7 (AR-V7), in prostate cancer is correlated with poor patient survival and resistance to AR targeted therapies and taxanes. Currently, there is no specific inhibitor of AR-V7, while the molecular mechanisms regulating its biological function are not well elucidated. Here, we report that AR-V7 has unique biological features that functionally differentiate it from canonical AR-fl or from the second most prevalent variant, AR-v567. First, AR-V7 exhibits fast nuclear import kinetics via a pathway distinct from the nuclear localization signal dependent importin-α/β pathway used by AR-fl and AR-v567. We also show that the dimerization box domain, known to mediate AR dimerization and transactivation, is required for AR-V7 nuclear import but not for AR-fl. Once in the nucleus, AR-V7 is transcriptionally active, yet exhibits unusually high intranuclear mobility and transient chromatin interactions, unlike the stable chromatin association of liganded AR-fl. The high intranuclear mobility of AR-V7 together with its high transcriptional output, suggest a Hit-and-Run mode of transcription. Our findings reveal unique mechanisms regulating AR-V7 activity, offering the opportunity to develop selective therapeutic interventions.

Current treatments for retinoblastoma, the most common intraocular malignancy, rely on a limited number of drugs repurposed from other pediatric cancer therapies. Drug toxicity and relapse of the disease necessitate new therapeutic strategies. The implementation of drug candidates in the clinic is usually followed by in vitro validation, which requires accurate and clinically relevant in vitro models. We report here a robust 3D tumoroid-based platform, for testing various clinical protocols, e.g. therapeutic agents following a single or repeated exposure, alone or in combination with focal therapy (thermotherapy). The platform, generated from the Y79 retinoblastoma cell line, consists of matrix-embedded tumoroids, which retain the key features of the disease and allow prolonged culture time. Following the optimization of growth conditions, the effect of single and repeated exposure to chemotherapeutic agents has been measured by high-throughput automated fluorescence imaging followed by quantification. The clinical conditions of chemothermotherapy have been recapitulated with a setup consisting of: an infrared diode laser (810 nm, 0.3 W) that heats the tumoroids in the presence of indocyanine green (50 µg/mL); a thermal camera to enable real-time temperature measurement; and an environment-controlled inverted microscope to ensure physiological conditions and precise focusing of the laser. Melphalan and carboplatin, two drugs currently used to treat retinoblastoma in clinical protocols of chemotherapy and chemothermotherapy respectively, have been applied to validate the system.Tumoroid viability and size progressively decreased over 4 weeks of repeated drug exposure with melphalan, reflecting the response of retinoblastoma in advanced clinical cases. Carboplatin treatment showed an activity enhancement in the tumoroid model similar to that observed in the clinic, when combined with focal therapy, demonstrating that the platform system closely recapitulates the clinical conditions. Inversely, the thermotherapy experimental protocol designed for 2D in vitro cell culture and commonly used to date, i.e. an incubation temperature increased to 42 °C for 1 h, did not show an improvement of the carboplatin cytotoxicity, confirming that our laser-based thermotherapy protocol provides a more accurate and clinically relevant drug response. Such an approach, combining physiologically relevant in vitro cancer models and investigation of drugs in well-established treatment modalities, can significantly reduce the number of new drug candidates that give false-positives or overestimated efficacies when evaluated in relevant in vivo models. It is expected that the platform will contribute to the implementation of new therapeutic strategies to treat retinoblastoma, and could also be adapted to other diseases for which repeated drug exposure and/or chemothermotherapy is relevant. Citation Format: Irina Sinenko, Fabien Kuttler, Valentin Simeonov, Alexandre Moulin, Christina Stathopoulos, Gerardo Turcatti, Adeline Berger, Francis Munier, Paul Dyson. Tumoroid-based screening platform to test focal, chemo- and combination therapy for retinoblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3541.

Background: Lamb coccidiosis, caused by intestinal parasites of the Eimeria genus, has pronounced health and economic impacts throughout the world. Eimeria ovinoidalis and Eimeria crandallis are the most pathogenic species in sheep. Control of these parasites requires the use of anticoccidial drugs such as sulfonamides, diclazuril, and toltrazuril. In this study, resistance to anticoccidial drugs was suspected in two farms as clinical signs (diarrhea) persisted after treatment. Method: On each farm, 4.5-month-old rams were divided into three groups so that they were either (i) left untreated (Control group), (ii) treated with diclazuril (1 mg/kg body weight), or (iii) treated with toltrazuril (20 mg/kg body weight). Animals were treated at day 0 (D0) and fecal samples were collected at D0 and eight days later (D8) to evaluate the reduction in intensity of Eimeria oocyst excretion. Oocyst morphology and morphometry were used to identify Eimeria species at both sampling dates. Results: Reduction of oocyst shedding was incomplete in both farms (92.44% and 93.58%) after diclazuril treatment. More specifically, the efficacy was reduced in both farms for E. ovinoidalis/Eimeria marsica (92.59% and 91.87%) and E. crandallis/Eimeria weybridgensis (75.34% and 80.10%). The general efficacy of toltrazuril was high in both farms (97.6% and 97.96%). However, a slightly reduced efficacy was noted in farm 1 for E. crandallis/E. weybridgensis (93.26%) while this efficacy was high in farm 2 (98.88%). Conclusions: We suggest a simple protocol to investigate the efficacy of anticoccidial treatments in sheep and to rapidly identify potentially resistant species. In these two farms, treating animals with diclazuril will select pathogenic species, and toltrazuril could favor resistant E. crandallis/E. weybridgensis in one of them.