The AML-associated K313 mutation enhances C/EBP\u03b1 activity by leading to C/EBP\u03b1 overexpression

Ian Edward Gentle,Mohamed Tarek Badr,Isabel Moelter,Michael Lübbert,Konstanze Döhner,Georg Häcker

doi:10.1038/s41419-021-03948-6

Abstract

Mutations in the transcription factor C/EBPα are found in ~10% of all acute myeloid leukaemia (AML) cases but the contribution of these mutations to leukemogenesis is incompletely understood. We here use a mouse model of granulocyte progenitors expressing conditionally active HoxB8 to assess the cell biological and molecular activity of C/EBPα-mutations associated with human AML. Both N-terminal truncation and C-terminal AML-associated mutations of C/EBPα substantially altered differentiation of progenitors into mature neutrophils in cell culture. Closer analysis of the C/EBPα-K313-duplication showed expansion and prolonged survival of mutant C/EBPα-expressing granulocytes following adoptive transfer into mice. C/EBPα-protein containing the K313-mutation further showed strongly enhanced transcriptional activity compared with the wild-type protein at certain promoters. Analysis of differentially regulated genes in cells overexpressing C/EBPα-K313 indicates a strong correlation with genes regulated by C/EBPα. Analysis of transcription factor enrichment in the differentially regulated genes indicated a strong reliance of SPI1/PU.1, suggesting that despite reduced DNA binding, C/EBPα-K313 is active in regulating target gene expression and acts largely through a network of other transcription factors. Strikingly, the K313 mutation caused strongly elevated expression of C/EBPα-protein, which could also be seen in primary K313 mutated AML blasts, explaining the enhanced C/EBPα activity in K313-expressing cells.

Highlights

Acute myeloid leukaemia (AML) is characterised by the accumulation of granulocyte or monocyte precursors in the bone marrow and peripheral blood
Analysis of transcription factor enrichment in the differentially regulated genes indicated a strong reliance of SPI1/PU.1, suggesting that despite reduced DNA binding, C/EBPα-K313 is active in regulating target gene expression and acts largely through a network of other transcription factors
Cell culture Hoxb8 neutrophil progenitors were cultured in progenitor outgrowth medium (Optimem Glutamax supplemented with 10% foetal bovine serum (FCS), 1% Pen/Strep, 30 μM β-mercaptoethanol, 1% stem cell factor (SCF) supernatant, 1 μM β-estradiol)

Summary

Introduction

Acute myeloid leukaemia (AML) is characterised by the accumulation of granulocyte or monocyte precursors in the bone marrow and peripheral blood. Genomic alterations in AML effect numerous transcription factor genes [1, 2]. In ~10% of AML cases, mutations in the gene encoding the transcription factor C/EBPα are found [3, 4]. The most notable haematopoietic defect of C/ EBPα-deficient mice is the lack of mature granulocytes [5]. C/EBPα is required for commitment to the granulocytic lineage and in early granulopoiesis, but probably not at later stages [6]. Overexpression of C/EBPα forces the differentiation of bi-potential progenitor cells along the granulocytic lineage [7]. C/EBPα−/− mice do not develop myeloid leukaemia even upon expression of Bcr-abl [8], and C/EBPα is required for the development of leukaemia in other models (MLLfusion or Hoxa9/Meis1-induced leukaemia) [9, 10]

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Results

Conclusion