270 In vitro modelling and high-throughput drug screening for sporadic arteriovenous malformations

Sporadic arteriovenous malformations (AVMs) are congenital disorders of blood vessels which present with strokes, life threating haemorrhages, pain and/or disfigurement. Current management options limited to high-risk, invasive procedures often ineffective. Recent genetic studies have identified post-zygotic variants in three genes the MAPK/ERK pathway as causative a third cutaneous AVM patients. The drivers many cancers, and pharmacologically targetable repurposing licensed inhibitors. Although some earlier work using MAPKi inhibitors showed promise vitro vivo experiments, anecdotal case reports results variable. We hypothesised that an unbiased drug screen could provide invaluable approach for identification novel therapies or combinations. A CRISPR protocol was developed optimised knock-in point mutations immortalised human endothelial cell line HUVEC/TERT2, generating KRAS c.35G>T (p.G12V) be compared its wildtype (WT) control. Characterisation intracellular signaling activation, global gene expression profiles, well cellular characteristics undertaken, revealing increased MAPK activation proliferation. Extensive optimisation tissue culture conditions, time points measurement, allowed determination most robust readouts FDA-approved high throughput drug-screen, namely proliferation primary endpoint. further readout created by lines expressing genetically encoded FRET-based ERK activity biosensor, EKAREV, used secondary screening round. This model KRAS-mutant is important step study this disease.