Identification of targetable components of beta-catenin signalling in Acute Myeloid Leukaemic Stem Cells

Abstract

Acute myeloid leukaemia (AML) remains the leading cause of leukaemia-related death with a relative five-year survival rate of only 24% in Australia. This poor prognosis is mainly due to disease relapse, which is thought to be attributable to the persistence of leukaemic stem cells (LSC) after chemotherapy. Previous studies have shown that β-catenin is required for the establishment of LSC in mixed lineage leukaemia (MLL)-rearranged AML. Targeted inhibition of β-catenin signalling has been hampered by the lack of pathway components amenable to pharmacoinhibition. In this study we have identified a novel β-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in AML LSC compared to normal HSC (P=0.0114). ShRNA-mediated inhibition of GPR84 impaired cell proliferation (P=0.0009) and AML reconstitution in vivo (P<0.0001). The GPR84- deficient phenotype could be rescued by expression of constitutively active β-catenin in vivo (P=0.0039). Furthermore, GPR84 overexpression accelerated leukaemogenesis in vivo (P=0.0039). These data suggest that inhibiting GPR84/β-catenin signalling may provide a promising novel therapeutic strategy in AML. However, since a GPR84-specific inhibitor has not yet been developed, we aimed to further delineate the GPR84/β- catenin axis in order to pharmacologically inhibit other components of this pathway. Preliminary data from our lab has implicated the G protein GNA13 as a potential downstream target of GPR84. GNA13 overexpression rescued GPR84 deficiency (P=0.0012) through augmentation of cell proliferation in vivo (P<0.0001). In contrast, shRNA-mediated ablation of GNA13 impeded cell proliferation (P=0.0066) and delayed disease onset in vivo (P<0.0001). However, GNA13 may not integrate into a therapeutic regimen since GNA13-deficiency did not sensitize LSC to bortezomib as implicated by previous reports. Hence we conducted microarray studies to identify novel downstream target genes of GPR84 potentially amenable to therapeutic intervention. Bcl11a, a proto-oncogene previously shown to be involved in leukaemogenesis, was highly upregulated in cells overexpressing GPR84 compared to control (P=0.0032). Interestingly, GPR84-deficiency substantially increased the efficacy of the Bcl11a inhibitor simvastatin. Hence simvastatin may be a candidate inhibitor in combination with GPR84-directed chemotherapy in AML.