G-protein coupled receptors (GPCRs) are the most successful class of drug targets. PAR1 is a GPCR that is significantly elevated in aggressive leukaemia, however, its function in AML remains unknown. Here, we show that shRNA-mediated knockdown of PAR1 markedly decreased active β-catenin in MLL-AF9 mediated leukaemia stem cells (LSCs). PAR1 deficiency decreased LSC colony formation (p=0.0002) and induced apoptosis (12-fold). Microarray expression analysis revealed that PAR1 inhibition significantly reduced the expression of several genes responsible for maintenance of mitochondrial integrity and energy metabolism (mtND4L (p=0.0013), mtND2 (p<0.0001) and mtCytB (p<0.0001)). We detected a significant increase in ROS production (p=0.0003) following PAR1 inhibition indicative of mitochondrial destabilisation. Furthermore, PAR1 deficient LSCs had significantly reduced oxygen consumption (p=0.0066) and lower basal ATP levels (p=0.012) compared to control LSCs. PAR1 inhibition, therefore, induces substantial oxidative stress which triggers apoptosis.
To assess the therapeutic value of PAR1 inhibition, we used a selective non-peptide PAR1 inhibitor. Cell viability assays showed that PAR1 inhibition was potent against LSCs and had no cytotoxic effects on lineage-negative normal mouse bone marrow cells, identifying a therapeutic window to eliminate LSCs while preserving normal blood cells. PAR1 inhibitor treatment induced a 2-fold reduction in colony formation, 3.8-fold enhanced ROS production and inhibited β-catenin activity. BrdU labelling revealed a significant reduction in in vivo short-term proliferation of LSCs that were pre-treated with PAR1 inhibitor prior to transplantation into recipient mice (p=0.0008).
Western blot analysis showed activation of JNK in response to inhibition of PAR1. Sustained JNK activation has been reported to promote survival signals during leukaemogenesis. By treating primary human patient samples we have shown that co-inhibition of PAR1 and JNK induced a potent anti-LSC effect, significantly enhancing apoptosis compared to single treatment. Our data suggest a novel LSC-eliminating treatment strategy targeting PAR1/β-catenin/JNK signalling for aggressive AML.