Developing an iPSC model of infant B-cell acute lymphoblastic leukaemia

Abstract

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common cancer in childhood. Despite overall survival rates of over 85%, high-risk subtypes still correlate with poor clinical outcome. Infant BCP-ALL presents the highest frequency of rearrangements involving the MLL/11q23 gene, a negative prognostic factor found in approximately 70% of cases. The infant form harbouring the t(4;11) MLL-AF4 translocation is characterised by a particularly dismal prognosis and a unique disease biology, as the main leukaemogenic event is thought to be largely completed during prenatal life. Further understanding of t(4;11) biology has been hampered by the limitations of currently available in vivo models in recapitulating the human disease, warranting the development of novel human-based bona-fide models. Induced pluripotent stem cell (iPSC) modelling offers the unique opportunity of recapitulating the developmental progression of malignancies, and could enable the modelling of in utero leukaemogenesis of infant BCP-ALL. To this end, I have derived iPSCs from BCP-ALL cell lines and primary patient samples using integrating and non-integrating reprogramming methods. Lentiviral reprogramming generated two iPS-like colonies from the non-MLL-rearranged NALM6 cell line with varying degrees of partial pluripotency and leukaemic gene expression. Sendai virus-OSKM delivery in CD19- enriched primary MLL-AF4+ and BCR-ABL+ patient samples gave rise to translocation-negative, fully reprogrammed iPSCs that could differentiate to lineages of the three germ layers in vitro and in vivo. Strikingly, episomal reprogramming of MLL-AF4+ SEM cells resulted in a partially-reprogrammed cell line that harboured the leukaemic translocation, albeit in abnormal FISH variant patterns compared to parental SEM cells. SEM iPS-like cells, however, showed an impaired differentiation potential and could not be directed towards the haematopoietic fate in vitro for disease modelling studies. Nonetheless, partially-reprogrammed iPSCs could provide valuable insight into the mechanisms of failed BCP-ALL reprogramming. Therefore, global transcriptome analysis of iPS-like cell lines was performed to determine the transcriptional basis of partial reprogramming in BCP-ALL. Taken together, the data presented in this thesis work support an overall refractoriness of BCP-ALL reprogramming to pluripotency, as recently reported in the literature.

Date of Award	31 Dec 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Valerie Kouskoff (Supervisor) & Georges Lacaud (Supervisor)