3259 – C-TERMINAL CEBPA MUTATIONS INDUCE DOSE-DEPENDENT MYELOID DIFFERENTIATION BLOCK IN ISOGENIC IPSC MODELS

Abstract

Background

CCAAT/enhancer-binding protein α (CEBPA) encodes a leucine zipper transcription factor required for cell cycle arrest and commitment of hematopoietic stem and progenitor cells (HSPCs) to the granulocytic lineage. C-terminal in-frame mutations affecting the basic leucine zipper (bZIP) domain impair dimerization and DNA binding, contributing to the differentiation block typical of acute myeloid leukemia (AML). Since monoallelic and biallelic mutations are associated with distinct clinical outcomes, genotype-specific mechanisms remain to be clarified.

CCAAT/enhancer-binding protein α (CEBPA) encodes a leucine zipper transcription factor required for cell cycle arrest and commitment of hematopoietic stem and progenitor cells (HSPCs) to the granulocytic lineage. C-terminal in-frame mutations affecting the basic leucine zipper (bZIP) domain impair dimerization and DNA binding, contributing to the differentiation block typical of acute myeloid leukemia (AML). Since monoallelic and biallelic mutations are associated with distinct clinical outcomes, genotype-specific mechanisms remain to be clarified.

Aims

To generate isogenic induced pluripotent stem cell (iPSC) lines carrying C-terminal CEBPA mutations and evaluate the impact of monoallelic and biallelic genotypes on HSPC maturation and myeloid differentiation.

To generate isogenic induced pluripotent stem cell (iPSC) lines carrying C-terminal CEBPA mutations and evaluate the impact of monoallelic and biallelic genotypes on HSPC maturation and myeloid differentiation.

Methods

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was used to introduce C-terminal deletions in two hiPSC lines, generating wild-type (WT), monoallelic, and biallelic clones. Cells were differentiated into CD34+ HSPCs and subsequently induced toward granulocytic and monocytic lineages. Differentiation was assessed by flow cytometry and cytomorphology. CD34+ cells were analyzed using single-cell RNA sequencing (scRNA-seq) and colony-forming assays.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was used to introduce C-terminal deletions in two hiPSC lines, generating wild-type (WT), monoallelic, and biallelic clones. Cells were differentiated into CD34+ HSPCs and subsequently induced toward granulocytic and monocytic lineages. Differentiation was assessed by flow cytometry and cytomorphology. CD34+ cells were analyzed using single-cell RNA sequencing (scRNA-seq) and colony-forming assays.

Results

All genotypes efficiently generated CD34+ HSPCs, indicating preserved early hematopoietic potential. WT clones produced mature granulocytes and monocytes, whereas monoallelic mutants showed reduced myeloid output and biallelic mutants displayed a marked differentiation block. scRNA-seq revealed preserved lymphoid potential but a genotype-dependent loss of granulocytic signatures in mutant cells. Colony assays showed enrichment of immature colony-forming unit (CFU)-granulocyte, erythrocyte, monocyte, and megakaryocyte (GEMM) and CFU-granulocyte/macrophage (GM) progenitors with reduced CFU-G and CFU-M frequencies.

All genotypes efficiently generated CD34+ HSPCs, indicating preserved early hematopoietic potential. WT clones produced mature granulocytes and monocytes, whereas monoallelic mutants showed reduced myeloid output and biallelic mutants displayed a marked differentiation block. scRNA-seq revealed preserved lymphoid potential but a genotype-dependent loss of granulocytic signatures in mutant cells. Colony assays showed enrichment of immature colony-forming unit (CFU)-granulocyte, erythrocyte, monocyte, and megakaryocyte (GEMM) and CFU-granulocyte/macrophage (GM) progenitors with reduced CFU-G and CFU-M frequencies.

Conclusions

CEBPA plays a central role in granulopoiesis, and C-terminal mutations induce genotype-dependent differentiation defects. This iPSC-based model provides a platform for functional characterization of CEBPA alterations in AML.

CEBPA plays a central role in granulopoiesis, and C-terminal mutations induce genotype-dependent differentiation defects. This iPSC-based model provides a platform for functional characterization of CEBPA alterations in AML.