Targeting ß-catenin interactions in acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a clonal disorder of haematopoietic stem cells (HSCs), characterised by the expansion of abnormal myeloblasts. AML arises from leukaemia stem cells (LSCs) which clonally evolve to evade chemotherapy and mediate relapse. Survival rates have improved over 50 years; however, the prognosis remains dismal for the elderly or those harbouring adverse cytogenetic events. There’s an urgent unmet clinical need for targeted efficacious drugs in AML which reduce side-effects and induce robust remissions. One potential molecular drug target is ß-catenin, the central mediator of Wnt signalling which is frequently dysregulated in myeloid leukaemia. ?-Catenin is overexpressed, mislocalised, and overactive in AML, where it confers inferior patient survival and drives the emergence, maintenance, and drug resistance of LSC. The level, localisation and activity of ß-catenin is governed heavily through protein interactions, and we recently characterised the first ?- catenin interactome study in myeloid leukaemia cells. One putative novel interactor of ß- catenin identified was Wilms Tumour 1 (WT1) protein which is frequently mutated and overexpressed in AML. This study explored the physical and functional interaction between ?-catenin and WT1 in myeloid leukaemia cells. This thesis identified a non-direct, RNA independent association between ß-catenin and WT1 in a variety of myeloid cell lines, and primary AML blasts. Functionally, WT1 knockdown significantly decreased ß-catenin nuclear expression and Wnt signalling in the KG-1 cell line. Furthermore, induction of WT1 mutations (exons 8 and 9) increased ß-catenin expression and augmented Wnt signalling output. Reciprocally, we showed that ß-catenin knockdown repressed WT1 expression and signalling which was at least party transcriptionally driven. Following on from interactome analyses we validated ?-catenin interaction with two RNA binding proteins (RBP; MSI2 and LIN28B), and isolated ß-catenin from initiation complexes in polysome profiling suggesting ?-catenin might influence post-transcriptional gene expression. To assess which RNAs are associated with ?- catenin we performed RBP immunoprecipitation (RIP) for ?-catenin coupled to RNA sequencing (RIP-seq) and transcripts related to critical processes such as myeloid differentiation, IL-18 signalling and the canonical Wnt signalling pathway itself. Overall, this study reports the first physical and functional interaction between ß-catenin and WT1 in AML and reveals a potential novel role for ?-catenin in the regulation of post-transcriptional gene control, both of which could inform novel ?-catenin targeting strategies in AML.