MLL leukemia induction by t(9;11) chromosomal translocation in human hematopoietic stem cells using genome editing

Genome editing provides a potential approach to model de novo leukemogenesis in primary human hematopoietic stem and progenitor cells (HSPCs) through induction of chromosomal translocations by targeted DNA double-strand breaks. However, very low efficiency of translocations and lack of markers for translocated cells serve as barriers to their characterization and model development. Here we utilized TALENs to generate t(9;11) chromosomal translocations encoding MLL-AF9 and reciprocal AF9-MLL fusion products in CD34+ human cord blood cells. Selected cytokine combinations enabled monoclonal outgrowth and immortalization of initially rare translocated cells, which were distinguished by elevated MLL target gene expression, high surface CD9 expression, and increased colony forming ability. Subsequent transplantation into immune-compromised mice induced myeloid leukemias within 48 weeks, whose pathologic and molecular features extensively overlap with de novo patient MLL-rearranged leukemias. No evidence of secondary pathogenic mutations was revealed by targeted exome sequencing and whole genome RNA-seq analyses, suggesting the genetic sufficiency of t(9;11) translocation for leukemia development from human HSPCs. Thus, genome editing enables modeling of human acute MLL-rearranged leukemia in vivo reflecting the genetic simplicity of this disease and provides an experimental platform for biological and disease modeling applications. For RNA-sequencing, RNA was purified from xenografted leukemic mouse bone marrow and cultured cells using Trizol (Invitrogen) followed by RNeasy kit (Qiagen) as manufacturer’s instructions, and sample quality was evaluated using Agilent RNA 6000 nano chip and Agilent 2100 Bioanalyzer (Agilent, St. Laurent, QC, Canada). More than 2mg of total RNA per sample was submitted for RNA-sequening. For targeted exome sequencing, genomic DNA was extracted from cultured cells and mouse bone marrow cells using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) and analyzed using the TruSight myeloid sequencing panel targeting 54 frequently mutated genes in blood cancers (Illumina, CA, USA). Paired-end sequencing runs were performed on a MiSeq (Illumina) with reagent kit v3 according to manufacturer’s instructions. Paired sequences obtained from each sample were mapped to human genome reference GRCh37/hg19. Variant calls were annotated by Variant Effect Predictor (VEP).