The interaction between RUNX2 and core binding factor beta as a potential therapeutic target in canine osteosarcoma.

Osteosarcoma remains the most common primary bone tumour in dogs with half of affected dogs unable to survive 1 year beyond diagnosis. New therapeutic options are needed to improve outcomes for this disease. Recent investigations into potential therapeutic targets have focused on cell surface molecules with little clear therapeutic benefit. Transcription factors and protein interactions represent underdeveloped areas of therapeutic drug development. We have utilized allosteric inhibitors of the core binding factor transcriptional complex, comprised of core binding factor beta (CBFß) and RUNX2, in four canine osteosarcoma cell lines Active inhibitor compounds demonstrate anti-tumour activities with concentrations demonstrated to be achievable in vivo while an inactive, structural analogue has no activity. We show that CBFß inhibitors are capable of inducing apoptosis, inhibiting clonogenic cell growth, altering cell cycle progression and impeding migration and invasion in a cell line-dependent manner. These effects coincide with a reduced interaction between RUNX2 and CBFß and alterations in expression of RUNX2 target genes. We also show that addition of CBFß inhibitors to the commonly used cytotoxic chemotherapeutic drugs doxorubicin and carboplatin leads to additive and/or synergistic anti-proliferative effects in canine osteosarcoma cell lines. Taken together, we have identified the interaction between components of the core binding factor transcriptional complex, RUNX2 and CBFß, as a potential novel therapeutic target in canine osteosarcoma and provide justification for further investigations into the anti-tumour activities we describe here.

T-cell derived acetylcholine aids host defenses during enteric bacterial infection with Citrobacter rodentium.

The regulation of mucosal immune function is critical to host protection from enteric pathogens but is incompletely understood. The nervous system and the neurotransmitter acetylcholine play an integral part in host defense against enteric bacterial pathogens. Here we report that acetylcholine producing-T-cells, as a non-neuronal source of ACh, were recruited to the colon during infection with the mouse pathogen Citrobacter rodentium. These ChAT+ T-cells did not exclusively belong to one Th subset and were able to produce IFNγ, IL-17A and IL-22. To interrogate the possible protective effect of acetylcholine released from these cells during enteric infection, T-cells were rendered deficient in their ability to produce acetylcholine through a conditional gene knockout approach. Significantly increased C. rodentium burden was observed in the colon from conditional KO (cKO) compared to WT mice at 10 days post-infection. This increased bacterial burden in cKO mice was associated with increased expression of the cytokines IL-1ß, IL-6, and TNFα, but without significant changes in T-cell and ILC associated IL-17A, IL-22, and IFNγ, or epithelial expression of antimicrobial peptides, compared to WT mice. Despite the increased expression of pro-inflammatory cytokines during C. rodentium infection, inducible nitric oxide synthase (Nos2) expression was significantly reduced in intestinal epithelial cells of ChAT T-cell cKO mice 10 days post-infection. Additionally, a cholinergic agonist enhanced IFNγ-induced Nos2 expression in intestinal epithelial cell in vitro. These findings demonstrated that acetylcholine, produced by specialized T-cells that are recruited during C. rodentium infection, are a key mediator in host-microbe interactions and mucosal defenses.