Definition and interrogation of the gene regulatory network of CEBPA double-mutated AML

Adamo, Assunta (2022). Definition and interrogation of the gene regulatory network of CEBPA double-mutated AML. University of Birmingham. Ph.D.

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Abstract

Mutations in C/EBPα and RUNX1 transcription factors are oncogenic drivers in acute myeloid leukaemia (AML). The CEBPA gene is usually hit by two different biallelic mutations. This genetic change results in the expression of two distinct proteins: one shows impaired DNA binding or dimerization abilities, the other lacks a transactivation domain. To this day the molecular mechanisms that promote leukaemogenesis in affected patients, referred to as CEBPA double-mutated (CEBPAN/C), are still not completely understood. The RUNX1 gene is frequently affected by the t(8;21) translocation, generating the fusion product RUNX1-ETO. Previous work from our lab has demonstrated that CEBPAN/C and t(8;21) AMLs are epigenetically related, and that a similar gene regulatory network is corrupted in both forms of leukaemia. In the study presented here we confirmed this observation with a larger dataset, including 10 CEBPAN/C and 7 t(8;21) AML samples. To address the question of the molecular mechanisms governing the maintenance of CEBPA double-mutated leukaemia, we employed multi-omics approaches to comprehensively characterize CEBPAN/C AMLs using purified primary patient-derived cells and a CEBPAN/C AML cell line. We studied the gene expression at both the bulk and single cell level in blasts and leukaemic stem cells, determined open chromatin regions, mapped mutant C/EBPα binding sites using chromatin immunoprecipitation assays followed by sequencing (ChIP-Seq) and linked these regions to their rightful genes using promoter capture HiC. We used ChIP-Seq to show that a large number of C/EBPα binding sites overlap with RUNX1 and c-FOS binding sites. We found that C/EBP binding is associated with a composite C/EBP:AP-1 binding motif capable to bind a heterodimer of both proteins, a feature specific for CEBPAN/C AML. Based on this data, we determined the core gene regulatory network (GRN) of CEBPAN/C AML and compared it to normal cells. Finally, we interrogated this GRN and identified AP-1 and the C/EBP family itself as important factors maintaining CEBPAN/C AML. Our work has enhanced our insight into CEBPAN/C AML pathogenesis and represents an important resource for identifying novel targets for therapeutic intervention.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):