Identification and characterisation of LGR4/beta-catenin signalling in acute myeloid leukaemic stem cells

Abstract

Acute myeloid leukaemia (AML) is a deadly form of leukaemia resulting in the highest number of leukaemia-associated deaths. The high mortality rate is due to frequent relapse caused by the persistence of drug-resistant leukaemic stem cells (LSCs). We have previously demonstrated an essential role for beta-catenin signalling in regulating LSCs in AML. Leucine-rich repeat containing G protein-coupled receptor 4 (Lgr4) has recently been identified as the receptor for R-spondin (Rspo) proteins to activate beta-catenin. This study showed that Rspo2/Rspo3 cooperating with Wnt3a potently potentiated beta-catenin activation in haematopoietic stem cell (HSC)-derived pre-LSCs. Overexpression of Lgr4 augmented activation of Wnt/beta-catenin signalling and promoted leukaemogenesis in vivo. Inhibition of Lgr4 reduced beta-catenin activity, completely abolished Rspo3/Wnt3a/beta-catenin signalling and prevented leukaemia development. A microarray experiment of 104 AML patient samples showed that high Lgr4 expression was associated with poor outcomes of AML patients. Altogether, these findings provide strong evidence demonstrating Lgr4 to be a critical regulator of beta-catenin signalling in AML. Gene expression profiling identified Rgs1 (regulator of G protein signalling 1) to be a major component of Lgr4 signalling. Rgs1 has been shown to bind directly to G protein subunits Gaq and Gai (Moratz et al., J Immunol, 2000), and treatment of pre-LSCs with Gaq and Gai inhibitors indicated Gaq to be a key downstream effector of Lgr4/Rgs1 signalling. Further functional studies showed that Gaq knockdown reduced beta-catenin expression, attenuated the effect of Wnt3a/Rspo3-potentiated beta-catenin activation and impaired LSC self-renewal, recapitulating the role of Lgr4 in LSC regulation. These data support the view that Gaq is an integral component of Lgr4 signalling in LSCs. Gene expression analysis also showed that Lgr4 knockdown increased expression of Gadd45a (growth arrest and DNA damage-inducible gene) and repressed several mitochondrial associated genes. Functional studies showed that Gadd45a deletion significantly increased in vivo LSC self-renewal and enhanced AML progression. Blockade of Lgr4 signalling inhibited mitochondrial energy metabolism, on which LSCs rely for survival. Collectively, this study has identified a novel Wnt3a/Rspo2/Rspo3-Lgr4-Gaq-beta-catenin signalling pathway governing LSCs and interference with components of this pathway may represent a promising therapeutic approach for eradicating LSCs in AML.