The role of canonical WNT signalling in normal haematopoiesis and in acute myeloid leukaemia

Leckenby, Adam M. 2022. The role of canonical WNT signalling in normal haematopoiesis and in acute myeloid leukaemia. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Acute myeloid leukaemia (AML) is a heterogeneous haematological malignancy which presents with the clonal expansion of immature myeloid lineage cells that are incapable of terminal differentiation. AML cells accumulate, resulting in a dysfunctional bone marrow. Induction chemotherapy is often applied as a treatment, but relapse of AML is frequent, possibly due to a quiescent population of leukaemic stem cells. WNT/-catenin signalling is critical to normal developmental processes in adults. Without an external WNT agonist, the pathway remains in a state of active suppression through constitutive degradation of the central mediator, -catenin (CTNNB1). When stimulated, the destruction complex becomes disrupted and stabilised -catenin translocates to the nucleus. Here, -catenin binds transcription factors of the TCF family which form a transcription complex promoting the expression of target genes involved in cell proliferation and other roles. WNT/-catenin signalling is active in haematopoiesis, with WNT genes influencing proliferation and differentiation. Self-renewal is a fundamental characteristic of haematopoietic stem cells and leukaemic stem cells. WNT/-catenin signalling represents an important pathway for roles in disease onset and progression as it can conferself-renewal, proliferative, and survival effects. -catenin is found to be overexpressed in approximately 60% of AML patients, regardless of genotype. Further, it has been shown to be a good prognostic marker for poor prognosis patients. In vitro studies in cell lines suggest that -catenin enhances proliferation whilst decreasing apoptosis and differentiation, though in vitro studies in primary haematopoietic cells have failed to reproduce this. A close homolog, -catenin, is also overexpressed in AML. Directly, -catenin has the capacity to induce TCF-dependent transcription in some contexts. -catenin may also indirectly promote TCF-dependent transcription though -catenin stabilisation as a consequence of destruction complex saturation. If -catenin is critical to AML maintenance or leukaemic stem cell self-renewal, it would represent an attractive induction or maintenance therapeutic target. By utilising CRISPR-Cas9 to derive complete knockout clones, this study has revealed that -catenin is disposable for normal haematopoietic functions. Deletion of -catenin and - catenin had no consequence for AML maintenance, sensitivity to standard chemotherapeutics, or differentiation status; however, knockout of -catenin was incompatible with clonal expansion in AML cell lines with active WNT/-catenin signalling and in primary AML samples. -catenin had no compensative capacity for -catenin loss and was not required for the translocation of -catenin into the nuclear compartment. Transcriptomic analysis by RNA sequencing of WNT/-catenin signalling active and knockout clones revealed that WNT/- catenin signalling activated the RAP1 signalling pathway in AML. Re-activation of RAP1 signalling was able to rescue clonal expansion capacity in -catenin knockout cells. Further investigation using CRISPR-mediated activation revealed that the WNT/-catenin target gene and activator of RAP1, RAPGEF4, was able to restore clonal growth significantly, whilst CRISPR-mediated inhibition of SIPA1L1, an inhibitor of RAP1 signalling increased clonogenicity modestly. Applied together, activation of RAPGEF4 and inhibition of SIPA1L1 elevated clonogenicity above scrambled controls. Whilst these studies, alongside recent in vivo literature, revealed WNT/-catenin signalling is disposable for AML, further investigation of the unexplored downstream RAP1 signalling pathway represents a promising opportunity with potential clinical benefits.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Medicine
Date of First Compliant Deposit:	28 February 2023
Last Modified:	27 Feb 2024 02:30
URI:	https://orca.cardiff.ac.uk/id/eprint/157375

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