Discerning the spatio-temporal disease patterns of surgically induced OA mouse models

Osteoarthritis (OA) is the most common cause of disability in ageing societies, with no effective therapies available to date. Two preclinical models are widely used to validate novel OA interventions (MCL-MM and DMM). Our aim is to discern disease dynamics in these models to provide a clear timeline in which various pathological changes occur. OA was surgically induced in mice by destabilisation of the medial meniscus. Analysis of OA progression revealed that the intensity and duration of chondrocyte loss and cartilage lesion formation were significantly different in MCL-MM vs DMM. Firstly, apoptosis was seen prior to week two and was narrowly restricted to the weight bearing area. Four weeks post injury the magnitude of apoptosis led to a 40–60% reduction of chondrocytes in the non-calcified zone. Secondly, the progression of cell loss preceded the structural changes of the cartilage spatio-temporally. Lastly, while proteoglycan loss was similar in both models, collagen type II degradation only occurred more prominently in MCL-MM. Dynamics of chondrocyte loss and lesion formation in preclinical models has important implications for validating new therapeutic strategies. Our work could be helpful in assessing the feasibility and expected response of the DMM- and the MCL-MM models to chondrocyte mediated therapies

In vivo E2F reporting reveals efficacious schedules of MEK1/2–CDK4/6 targeting and mTOR–s6 resistance mechanisms

Targeting cyclin-dependent kinases 4/6 (CDK4/6) represents a therapeutic option in combination with BRAF inhibitor and/or MEK inhibitor (MEKi) in melanoma; however, continuous dosing elicits toxicities in patients. Using quantitative and temporal in vivo reporting, we show that continuous MEKi with intermittent CDK4/6 inhibitor (CDK4/6i) led to more complete tumor responses versus other combination schedules. Nevertheless, some tumors acquired resistance that was associated with enhanced phosphorylation of ribosomal S6 protein. These data were supported by phospho-S6 staining of melanoma biopsies from patients treated with CDK4/6i plus targeted inhibitors. Enhanced phospho-S6 in resistant tumors provided a therapeutic window for the mTORC1/2 inhibitor AZD2014. Mechanistically, upregulation or mutation of NRAS was associated with resistance in in vivo models and patient samples, respectively, and mutant NRAS was sufficient to enhance resistance. This study utilizes an in vivo reporter model to optimize schedules and supports targeting mTORC1/2 to overcome MEKi plus CDK4/6i resistance. SIGnIFICAnCE: Mutant BRAF and NRAS melanomas acquire resistance to combined MEK and CDK4/6 inhibition via upregulation of mTOR pathway signaling. This resistance mechanism provides the preclinical basis to utilize mTORC1/2 inhibitors to improve MEKi plus CDK4/6i drug regimens

Liganded Androgen Receptor Interaction with β-Catenin: NUCLEAR CO-LOCALIZATION AND MODULATION OF TRANSCRIPTIONAL ACTIVITY IN NEURONAL CELLS

A yeast two-hybrid assay was employed to identify androgen receptor (AR) protein partners in gonadotropin-releasing hormone neuronal cells. By using an AR deletion construct (AR-(Delta371-485)) as a bait, beta-catenin was identified as an AR-interacting protein from a gonadotropin-releasing hormone neuronal cell library. Immunolocalization of co-transfected AR and FLAG-beta-catenin demonstrated that FLAG-beta-catenin was predominantly cytoplasmic in the absence of androgen. In the presence of 5alpha-dihydrotestosterone, FLAG-beta-catenin completely co-localized to the nucleus with AR. This effect was specific to AR because liganded progesterone, glucocorticoid, or estrogen alpha receptors did not translocate FLAG-beta-catenin to the nucleus. Agonist-bound AR was required because the AR antagonists casodex and hydroxyflutamide failed to translocate beta-catenin. Time course experiments demonstrated that co-translocation occurred with similar kinetics. Nuclear co-localization was independent of the glycogen synthase kinase-3beta, p42/44 ERK mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways because inhibitors of these pathways had no effect. Transcription assays demonstrated that liganded AR repressed beta-catenin/T cell factor-responsive reporter gene activity. Conversely, co-expression of beta-catenin/T cell factor repressed AR stimulation of AR-responsive reporter gene activity. Our data suggest that liganded AR shuttles beta-catenin to the nucleus and that nuclear interaction of AR with beta-catenin may modulate transcriptional activity in androgen target tissues