Identification and characterisation of genetic lesions that predispose to and gene expression patterns that contribute to Myeloid malignancies

Abstract

Acute Myeloid Leukaemia (AML) is a heterogeneous disease caused by multiple genetic lesions. Our laboratory focuses on understanding the genetics of both inherited and acquired haematopoietic malignancies. In this thesis, I have investigated both inherited and acquired genetic changes that contribute to myeloid malignancies. One of the key factors regulating haematopoiesis is GATA2, a zinc finger transcription factor. Germline mutations in GATA2 have been associated with several clinical phenotypes such as myelodysplastic syndrome (MDS)/AML, immunodeficiency disorders (MonoMAC syndrome, DCML deficiency, congenital neutropenia, NK cell deficiency, aplastic anaemia) and Emberger syndrome. Moreover, several somatic mutations in GATA2 have been reported in MDS/AML. Intriguingly, missense somatic and germline mutations reported to date are mutually exclusive, and several clinical phenotypes are associated with specific mutations. We generated a zinc finger 2 (ZF2) mutant allelic series representing a range of clinical phenotypes to investigate how each mutation effects transactivation, DNA binding, protein structure, protein partner interactions and in vitro differentiation. Specific GATA2 mutations perturb the interactions and functions in distinct ways that are beginning to explain differences in observed clinical phenotypes. We performed gene expression analysis of 91 selected MDS/AML genes, including GATA2, on 166 well annotated primary AML samples, bone marrow mononuclear cells (BMMNC) and CD34 controls. Correlation analyses of GATA2 expression levels with expression of other genes and other mutational and clinical data, was performed to help identify genetic aberrations that cooperate with abnormal levels of GATA2 in AML. Statistical correlations of expression levels of various other genes with outcome and mutation status were also identified. One such correlation was reduced GATA2 expression with oncogenic RAS mutations. A pilot study was carried out to evaluate and optimise an NRAS G12D-induced leukaemia model. All mice transplanted with mutant NRAS G12D rapidly developed haematopoietic disease post-transplantation whereas the control group did not. Based on these pilot studies, we have initiated transplantation experiments in a conditional GATA2 knockout model to investigate the requirement of GATA2 in NRAS G12D induced myeloid disease. Recipient mice continue to be monitored, but are yet to develop disease. We also identified gene expression patterns of prognostic significance in AML and narrowed down a combination of three genes that are highly predictive of outcome. We devised a strategy integrating these genes into currently used risk stratification strategies and significantly improved risk stratification of AML patients at diagnosis. Among syndromes that predispose to MDS/AML, is Diamond Blackfan Anaemia (DBA), a congenital disorder characterised by red blood cell deficiency. The underlying genetic cause of DBA in a child was identified using whole genome sequencing (WGS), targeted massively parallel sequencing (MPS) and high density SNP array. A complex scenario of germline and somatic aberrations were identified in two genetic loci that helped to explain the clinical features seen in the patient and the progression of this disease. These have led to the discovery of a mechanism by which spontaneous remissions occur in DBA patients. Together, these studies have given us valuable insights into malignant myeloid disease biology and offer potential applications in improving therapeutic approaches in AML patients.