S119: CEBPA AND TET2 MUTATIONS COOPERATE TO INDUCE AGGRESSIVE AML VIA GATA-2 DOWNREGULATION

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Background: The transcription factor CCAAT-enhancer-binding protein alpha (C/EBPα) is a master regulator of granulopoiesis and is mutated in 10-15 % of Acute Myeloid Leukemia (AML) patients. N-terminal frameshifts represent the predominant type of lesions and ablate the expression of the full-length protein p42, leading to overexpression of the shorter isoform p30. Mutations in the C-terminal basic-region leucine zipper (bZip) can disrupt the DNA-binding ability of C/EBPα. AML patients harbor mono- or biallelic CEBPA mutations (CEBPAmo or CEBPAbi). The most commonly co-occurring mutations are loss-of-function mutations in the methylcytosine dioxygenase TET2, resulting in adverse overall survival. We hypothesized that combinatorial effects of CEBPA mutations together with TET2 loss specifically rewire transcriptional and epigenetic circuitries in AML cells, thereby strongly influencing disease outcome. Aims: We aimed to elucidate the molecular mechanisms behind cooperative effects of CEBPA and TET2 mutations through state-of-the-art transcriptomic and epigenomic analyses of relevant in vitro and in vivo models as well as data from AML patients. Methods: We used the CRISPR/Cas9 technology to introduce Tet2 mutations in murine cell lines expressing only p30 (Cebpap30/p30) or mimicking biallelic CEBPA mutations (Cebpap30/C-mut.) to study the functional cooperation of these mutations in vitro. A Cebpa-/p30Tet2-/- mouse model was used to study effects of Tet2 loss in CEBPA mutated AML. We performed RNA-, C/EBPα-ChIP-, ATAC-, Bisulfite-, CUT&RUN and CRISPR/Cas9-mediated enhancer screens to generate a comprehensive dataset for in-depth comparative analysis and correlation with relevant patient data from the beatAML collection. Results: Integration of transcriptomic and epigenomic data from in vitro and in vivo models of CEBPA-TET2 co-mutated AML, in combination with gene expression analyses in AML patients, identified the transcription factor GATA2 as a conserved target of the CEBPA-TET2 axis. p30 and TET2 were strongly bound to the -77 kb enhancer of the Gata2 gene, and CRISPR/Cas9-induced enhancer deletions diminished Gata2 expression. Furthermore, TET2 loss reduced chromatin accessibility and increased DNA methylation of the Gata2 promoter, resulting in decreased Gata2 mRNA levels. RNAi-mediated silencing revealed a dose-dependent effect of Gata2 expression on leukemia cell fitness in vivo. Reduction of Gata2 levels by 25-50 % provided a strong competitive advantage to Cebpap30/p30 cells while near-complete downregulation of Gata2 expression (>75 %) dramatically reduced cellular fitness. Finally, treatment with the demethylating agent 5-azacytidine restored Gata2 expression in an AML model with Cebpa and Tet2 mutations and caused a significant survival benefit. Summary/Conclusion: The datasets generated from these models enable deeper insights into the epigenetic and transcriptomic changes that depend on CEBPA and TET2 mutations in a physiologically relevant mutational context. Our results reveal that mutational disruption of CEBPA and TET2 results in down-regulated GATA2 expression, causing aggressive AML. We propose a mechanism in which C/EBPα p30 mediates recruitment of TET2 to regulatory regions in the GATA2 gene to maintain its expression. Conversely, loss of TET2 leads to reduced GATA2 levels, which is restored by 5-azacytidine treatment. Thus, interference with the C/EBPα-TET2 axis may provide entry points for the development of rational targeted therapies in AML patients with these mutations.