From Mechanisms of Leukaemogenic Dysregulation to New Candidate Therapeutics
The maintenance of a continuous flow of mature blood cells from a reservoir of specialised haematopoietic stem cells requires regulation from an intricate network of regulatory programmes. Gene regulatory networks are well evolved to control multi-lineage commitment and differentiation while maintaining self-renewal capacity in haematopoietic stem and progenitor cells. In leukaemia, mutations commonly hijack these regulatory programs causing both a block in normal differentiation and the misplacement of self-renewal capabilities. Rearrangements involving the *MLL1* gene (*Kmt2a*) produce powerful oncogenic proteins occurring predominantly in paediatric and infant acute myeloid and lymphoid leukaemias. Despite having more than 80 fusion partners, MLL-AF9, MLL-AF4 and MLL-ENL make up more than 60% of cases involving *MLL1* translocations. Conventional retroviral transduction models commonly use lineage depleted or c-Kit enriched mouse bone marrow haematopoietic stem and progenitor cells (HSPCs) that display distinct heterogeneity when profiled at the single-cell level which could explain inconsistencies across previously reported models. Conversely, our model, referred to as ME-Transformed cells, implements a conditionally blocked multipotent haematopoietic progenitor murine cell line (HoxB8-FL) allowing for the reproducible tracking of early leukaemic transformation and transcriptional changes due to MLL-ENL expression (Basillico., *et al* 2020). Using ME-Transformed cells, we knocked out 64 genes via CRISPR-Cas9 followed by small-bulk RNA-Sequencing. The 64 targets include transcription factors (TFs) and key transcriptional regulators implicated in the aetiology of MLL rearranged leukaemia. Bioinformatic analysis revealed a regulatory network connecting 19,437 links across 7,548 genes. Previous work in the Göttgens lab used a similar approach to establish the gene regulatory function of 39 TFs in the wild-type parental HoxB8-FL cells (Kucinski *et al*. 2020). The first aim of this thesis was to use ME-Transformed cells to establish a gene regulatory network underpinning the connection of 64 key TFs and transcriptional regulators in MLL rearranged leukaemogenesis. Thereby enabling the use of our newly established leukaemia transcription factor network to comparatively identify differences between the leukaemic and normal counterparts as a direct result of MLL-ENL expression. The second aim of this thesis was to investigate the molecular response to new candidate therapeutics. Using conventional cellular assays and RNA-Sequencing, this research project identified potential therapeutic vulnerabilities within several promising therapeutic avenues that need to be explored beyond the limits of this thesis. This experimental strategy makes the most of the nature of experimental system due to the presence of a known and accessible cell of origin (HoxB8-FL) allowing us to pinpoint candidates based on their sensitivity and specificity for the leukaemic model. The third aim was to ascertain whether prolonged exposure to AML therapeutics induces consistent molecular changes characteristic of resistant cells. Intratumoural heterogeneity underpins resistance to cancer therapies through both genetic and non-genetic means. Long term exposure of ME-Transformed cells to single small molecule inhibitory agents targeting the kinase activity of ATM recapitulated drug resistant mechanisms seen in patients. The fourth and final aim of this thesis was to determine potential combinations of small molecule therapeutics that could be combined to increase both the specificity and sensitivity of treatment while preventing the emerge of drug resistance populations. Subsequent single-cell RNA-Sequencing of ATM inhibitor resistant cells was able to identify crucial transcriptional changes between resistant and drug naive cells highlighting a new therapeutic vulnerability and a potential target for the eradication of drug resistant cells. In summary, the integration of experimental work and novel computational methods has allowed us to begin to decipher whether the perturbation of specific genes results in the identification of potential candidate therapeutic vulnerabilities as well offering a reference for which to project future perturbations, be it genetic or pharmacological.
leukaemia, MLL-ENL, network, therapeutics, transcription factor
Networked Digital Library of Theses and Dissertations