Deciphering pre-leukaemic dysregulation caused by mutant C/EBPα at the level of the single cell

Abstract Deciphering pre-leukaemic dysregulation caused by mutant C/EBPα at the level of the single cell Moosa Rashid Qureshi C/EBPα is an ideal candidate to explore pre-leukaemic dysregulation of transcriptional networks because it is a significant player in normal myelopoiesis, it interacts with other transcription factors (TFs) in lineage specification, and the CEBPA gene has been identified as an early mutation in acute myeloid leukaemia (AML). N321D is a mutation affecting the leucine zipper domain of C/EBPα which has previously generated an aggressive AML phenotype in a mouse model. The first objective of my thesis was to construct a novel in vitro cellular model to recapitulate pre-leukaemic changes associated with the CEBPA N321D mutation. The conditionally immortalised murine Hoxb8-FL cell line can differentiate into relatively homogenous populations of functional myeloid, lymphoid and dendritic cells in appropriate cell culture conditions. These cells were transduced with retroviral vectors which expressed either no transgene or “Empty Vector” (EV), the CEBPA wildtype (WT) or N321D mutation. CEBPA N321D-transduced cells had immature morphological appearance in Flt3L differentiation media, proliferated indefinitely, and were characterised by a plasmacytoid dendritic cell (pDC)-like immunophenotype. These cells were capable of long-term engraftment in a mouse model but did not recapitulate leukaemia for several months after transplantation. The second objective was to characterise the transcriptional deregulation associated with the phenotypes demonstrated by EV, CEBPA WT and N321D-expressing cells in the presence of Flt3L. Single cell RNA-seq (scRNA-seq) confirmed phenotypic observations that CEBPA N321D-transduced cells had a pDC precursor-like gene expression signature when placed in Flt3L conditions of differentiation. Single cell analysis also identified transcriptional heterogeneity in the CEBPA N321D compartment at single cell resolution. The third objective was to use ChIP-seq to investigate the effect of the N321D mutation on C/EBPα’s TF binding profile, and correlate this with scRNA-seq data to identify a set of candidate genes (NOTCH2, JAK2, SIRPA, FOS, and others) where gene expression changes were associated with direct binding events by the mutant TF. In addition to characterising the transcriptional effects of the CEBPA mutation, a fourth objective was to update and expand the Göttgens group’s CODEX database of TF-binding and histone modification data for human blood cells, increasing annotation of the genome to 14.83%. This online database now provides significant coverage of the non-coding portion of the genome, and is therefore potentially of interest to the wider haematology research community.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2020

Author dc:creator

• Qureshi, Moosa

Advisors dc:contributor.advisor

• Gottgens, Berthold
• Hodson, daniel

• C/EBPα
• CEBPA
• AML
• scRNA-seq
• dendritic cell
• single cell

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/24fc51bc-26ac-47af-8b34-271ca7befb11/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.53215

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/306136