Crispr modeling of acute myeloid leukemia mutations in primary hematopoietic stem cells to study (pre-) leukemia development
Experimental Hematology(2022)
Abstract
The development of acute myeloid leukemia (AML) is a stepwise process with early founder mutations that give rise to hematopoietic stem cells (HSCs) with a clonal advantage, followed by subsequent driver mutations that result in transformation and full-blown leukemia. To study the development and progression of AML through time requires consecutive samples of (pre-)leukemic cells, however, this also comes with many practical challenges. Therefore, we implemented a recently published CRISPR methodology that allows modeling of AML mutations including DNMT3A, TET2, ASXL1, RUNX1, GATA2, and CEBPA p30 in primary HSCs. In short, bone marrow (BM) derived CD34+ cells are expanded, electroporated with ribonucleoprotein complexes, and in case of homologous directed repair, directly followed by HDR-template delivery using adeno-associated viruses. The HDR template consists of a SFFV-GFP, which is integrated in an early exon resulting in a loss of function (LOF) that can be traced through GFP expression. In general, we reach editing efficiencies ranging from 60-90% and HDR efficiencies of around 30-50%. Modeling CEBPA p30 in BM CD34+ cells resulted in a block in differentiation and increased colony forming potential in multiple replates, and uncontrolled cell growth. Proteome analysis revealed both known and unknown CEBPA p30 targets being differentially expressed. Injecting CEBPA p30 cells in NSGS mice resulted in a (pre-)leukemic phenotype with increased myeloid output, aberrant plasma membrane marker expression, and an immature phenotype. Currently, we are further evaluating the additional effect of recurrently co-mutated genes TET2 and GATA2. In addition, we also study the consequences of LOF mutations in DNMT3A, ASXL1, TET2, and RUNX1 in NSG and NSGS mice potentially followed by secondary hits, stresses and treatments in order to study the leukemic evolution through time. The development of acute myeloid leukemia (AML) is a stepwise process with early founder mutations that give rise to hematopoietic stem cells (HSCs) with a clonal advantage, followed by subsequent driver mutations that result in transformation and full-blown leukemia. To study the development and progression of AML through time requires consecutive samples of (pre-)leukemic cells, however, this also comes with many practical challenges. Therefore, we implemented a recently published CRISPR methodology that allows modeling of AML mutations including DNMT3A, TET2, ASXL1, RUNX1, GATA2, and CEBPA p30 in primary HSCs. In short, bone marrow (BM) derived CD34+ cells are expanded, electroporated with ribonucleoprotein complexes, and in case of homologous directed repair, directly followed by HDR-template delivery using adeno-associated viruses. The HDR template consists of a SFFV-GFP, which is integrated in an early exon resulting in a loss of function (LOF) that can be traced through GFP expression. In general, we reach editing efficiencies ranging from 60-90% and HDR efficiencies of around 30-50%. Modeling CEBPA p30 in BM CD34+ cells resulted in a block in differentiation and increased colony forming potential in multiple replates, and uncontrolled cell growth. Proteome analysis revealed both known and unknown CEBPA p30 targets being differentially expressed. Injecting CEBPA p30 cells in NSGS mice resulted in a (pre-)leukemic phenotype with increased myeloid output, aberrant plasma membrane marker expression, and an immature phenotype. Currently, we are further evaluating the additional effect of recurrently co-mutated genes TET2 and GATA2. In addition, we also study the consequences of LOF mutations in DNMT3A, ASXL1, TET2, and RUNX1 in NSG and NSGS mice potentially followed by secondary hits, stresses and treatments in order to study the leukemic evolution through time.
MoreTranslated text
Key words
leukemia mutations,crispr modeling,acute myeloid leukemia,myeloid leukemia
AI Read Science
Must-Reading Tree
Example
![](https://originalfileserver.aminer.cn/sys/aminer/pubs/mrt_preview.jpeg)
Generate MRT to find the research sequence of this paper
Chat Paper
Summary is being generated by the instructions you defined