Establishment of a patient-specific iPSC model of stepwise leukemogenesis in severe congenital neutropenia

Abstract

Severe congenital neutropenia (CN) is a pre-leukemia bone marrow failure syndrome with profoundly diminished terminal granulocytic differentiation of hematopoietic stem cells (HSCs). Life-long treatment with granulocyte-colony stimulating factor (G-CSF) increases the number of neutrophils. About 20 % of CN patients develop AML or MDS. The mechanism of leukemia transformation in CN patients is mostly unclear. Limited numbers of available primary HSC of pediatric CN patients and a lack of animal models hinder the research attempts on the CN and CN/AML pathophysiology. In my PhD Thesis, I aimed to overcome these obstacles by implementing advanced techniques, including patient-derived induced pluripotent cells (iPSCs) in combination with CRISPR/Cas9 gene-editing, for the establishment of the in vitro model of congenital neutropenia and of step-wise leukemia development. Using this model, I was able to (1) reproduce “maturation arrest” of granulopoiesis seen in the bone marrow of CN patients; (2) to generate sufficient numbers of hematopoietic stem and progenitor cells (HSPCs) for the analysis of myeloid differentiation, multi-omics analysis and functional validation of identified targets. The ultimate aim was to identify novel molecules that may be further explored for their therapeutic potential for CN/AML. Our group recently reported an extremely high frequency of cooperating acquired CSF3R (colony-stimulating factor 3 receptor) and RUNX1 (runt-related transcription factor 1) mutations in CN patients who developed acute myeloid leukemia (CN/AML). I established an in vitro model of stepwise leukemogenesis in CN/AML through CRISPR/Cas9 mediated gene-editing of iPSCs from two CN patients with acquired CSF3R and RUNX1 mutations and overt AML. Using this model, I identified BAALC (brain and acute leukemia, cytoplasmic) upregulation as a key leukemogenic event in CN. Importantly, CRISPR/Cas9-mediated knockout of BAALC in CN/AML-iPSCs derived hematopoietic cells of CN/AML patients restored defective myeloid differentiation to the levels observed in healthy donor hematopoietic stem and progenitor cells (HSPCs). Using transcriptomics data analysis, I found that CMPD1, an inhibitor of p38-mediated MK2a phosphorylation, re-establishes gene expression signature similar to that of BAALC knockout. Intriguingly, in vitro treatment of primary blasts of CN/AML patients with CMPD1 resulted in a marked reduction of cell proliferation without affecting differentiation of healthy donor HSPCs. In summary, these observations suggest that targeting of BAALC in hematopoietic cells of CN patients may prevent leukemogenic transformation or eliminate AML blasts in CN/AML individuals.