TET2 and WT1 mutations accelerate CEBPA P30 leukemia development

Bi-allelic (bi) mutations in the myeloid transcription factor CEBPA are found in about 5% of acute myeloid leukemia (AML) patients and frequently co-occur with mutations in TET2, WT1 and GATA2. Co-occurrence with TET2 and WT1 mutations give a worse overall survival whereas co-occurrence with GATA2 mutations improve overall survival. To study the underlying molecular mechanisms, we modeled these mutations in primary human bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) using CRISPR/Cas9 gene editing.. Targeting the N-terminal part of CEBPA using Cas9 ribonucleoproteins resulted in loss of the full-length CEBPA protein (p42) and marked upregulation of the shorter p30 isoform as seen in patients. CEBPAbi mutations resulted in loss of monocytic colonies and increased serial replating capacity in CFU assays. CEBPAbi combined with TET2 or WT1 loss-of-function mutations resulted in leukemic transformation and uncontrolled cell proliferation in a mouse stromal-5 co-culture assay. Transplantation of CEBPAbi-mutated HSPCs into immunodeficient mice expressing human cytokines (NSGS mice) revealed a myeloid skewing, and when combined with TET2 or WT1 mutations, leukemic transformation. Interestingly, while CEBPAbi- and co-mutated HSPCs could engraft in the BM of NSG mice, we did not observe dissemination to the peripheral blood and no leukemic transformation. Subsequent cytokine dropout experiments revealed a dependency on human stem cell factor in CEBPAbi leukemia development.. Proteome analysis showed that protein differences were mainly driven by CEBPAbi mutations and strengthened by loss of TET2 or WT1. These data suggest that an altered chromatin state due to TET2 or WT1 loss further enhances CEBPA p30-mediated transcription. We are currently performing scRNAseq and ATACseq to further substantiate these findings and decipher the underlying mechanisms..