Abstract P3113: RAF 'Paradox Breaker' Inhibitor, PLX8394, Enhances Inflammation And Apoptosis In Murine Hypertension In Vivo

Abstract

Introduction: The ERK1/2 cascade, activated by RAF kinases, is a key pathway for cardiac remodelling and cytoprotection. Due to activating oncogenic mutations in BRAF, small molecule RAF inhibitors have been developed. However, owing to inhibitor resistance or paradoxical ERK1/2 pathway activation, a new generation of ‘paradox breaking’ drugs are currently in clinical trials. Our previous work characterised RAF targeting in hypertension, where it is instrumental in driving cardiac remodelling. Here we explored whether use of the RAF paradox breakers (e.g. PLX8394) are viable modalities for targeting RAF in hypertension. Hypothesis: We hypothesize that RAF ‘paradox breakers’ will protect cardiac function by inhibiting maladaptive hypertensive remodelling. Methods & Results: To assess the role of RAF inhibition in cardiac adaptation to hypertension in vivo , C57Bl/6J mice were treated with angiotensin-II (AngII; 0.8mg/kg/d, 7d n=8;) without/with 5mg/kg/d PLX8394 (n=11). PLX8394 alone had no detrimental effect on cardiac function or remodelling. AngII promoted cardiac hypertrophy, by increasing left ventricular (LV) wall thickness (WT) and decreasing LV internal diameter (ID; assessed by echocardiography). PLX8394 had no impact on AngII-induced hypertrophy (WT:ID, p=0.45), or abundance of AngII-induced hypertrophic Nppa (p=0.77) or Nppb (p=0.49) mRNAs. Additionally, PLX8394 did not affect AngII-induced perivascular fibrosis (p=0.69; picrosirius red histology) or moderate Col1a1 (p=0.22) and Col3a1 (p=0.82) mRNAs. However, PLX8394 use in AngII-hypertension was pro-inflammatory, with increased pro-inflammatory cytokines ( Il1b , p=0.02; Il6 , p=0.008) and the inflammatory cell marker CD45 (p=0.001) mRNAs; a response echoed in hearts labelled by immunohistochemistry for CD45 (p=0.02). Moreover, western blotting revealed elevated PARP cleavage (p=0.005), indicating increased apoptosis with PLX8394 use. Conclusion: Despite no adverse effect on cardiac function or global hypertensive remodelling at 7 days, RAF inhibition by PLX8394 appears to be pro-inflammatory and pro-apoptotic. Thus, long-term use of RAF ‘paradox breakers’ in hypertensive individuals may be cardiotoxic and promote adverse cardiac events.