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. 2017 Sep;102(9):1558-1566.
doi: 10.3324/haematol.2017.164046. Epub 2017 Jun 1.

CRISPR-Cas9-induced t(11;19)/MLL-ENL Translocations Initiate Leukemia in Human Hematopoietic Progenitor Cells in vivo

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Free PMC article

CRISPR-Cas9-induced t(11;19)/MLL-ENL Translocations Initiate Leukemia in Human Hematopoietic Progenitor Cells in vivo

Jana Reimer et al. Haematologica. .
Free PMC article

Abstract

Chromosomal translocations that generate oncogenic fusion proteins are causative for most pediatric leukemias and frequently affect the MLL/KMT2A gene. In vivo modeling of bona fide chromosomal translocations in human hematopoietic stem and progenitor cells is challenging but essential to determine their actual leukemogenic potential. We therefore developed an advanced lentiviral CRISPR-Cas9 vector that efficiently transduced human CD34+ hematopoietic stem and progenitor cells and induced the t(11;19)/MLL-ENL translocation. Leveraging this system, we could demonstrate that hematopoietic stem and progenitor cells harboring the translocation showed only a transient clonal growth advantage in vitro In contrast, t(11;19)/MLL-ENL-harboring CD34+ hematopoietic stem and progenitor cells not only showed long-term engraftment in primary immunodeficient recipients, but t(11;19)/MLL-ENL also served as a first hit to initiate a monocytic leukemia-like disease. Interestingly, secondary recipients developed acute lymphoblastic leukemia with incomplete penetrance. These findings indicate that environmental cues not only contribute to the disease phenotype, but also to t(11;19)/MLL-ENL-mediated oncogenic transformation itself. Thus, by investigating the true chromosomal t(11;19) rearrangement in its natural genomic context, our study emphasizes the importance of environmental cues for the pathogenesis of pediatric leukemias, opening an avenue for novel treatment options.

Figures

Figure 1.
Figure 1.
An improved lentiviral vector system for generation of CRISPR-Cas9-induced chromosomal rearrangements. (A) Schematic presentation of lentiviral vector architecture including genomic RNA-generating promoter assembly: published architecture (L-CRISPR) (i), improved architecture with cytomegalovirus enhancer (CMV) and simian virus 40 enhancer (SV40) and exchangeability of the hU6 promoter for a H1 promoter (L40C) (ii), and lentiviral vector for dual sgRNA delivery (L-CRISPR-CTN (iii). (B) Analysis of viral titers in three independent cell lines with two different sgRNAs and three replicates each. (C) Knock-out efficacies (fluorescence reporter assay) of sgRNAs targeting intronic sequences of ENL and MLL. Selected sgRNAs are marked. (D) Knock-out efficacies of selected sgRNAs expressed from a human U6 or H1 promoter, as indicated. Knock-out efficacies of selected sgRNAs in L-CRISPR-CTN dual sgRNA configuration (DV) (neg ctrl= anti-luciferase (Luc) sgRNA, pos ctrl= Tet2 sgRNA). (E) T7-endonuclease-I assay for on-target sites and the top five predicted off-target sites in HEL cells (OT-1-OT-5). Indel frequencies at endogenous loci are indicated below. Analysis in cells transduced with targeting (+) and Luc (-) sgRNAs: MLL-I9-#1(i) and ENL-I1-#4 (ii).
Figure 2.
Figure 2.
CRISPR-Cas9-induced MLL-ENL rearrangements cause clonal expansion of human CD34+ hematopoietic stem and progenitor cells. (A) Schematic depiction of CRISPR-Cas9-induced chromosomal rearrangements at the MLL and ENL loci. (B) Reverse transcriptase polymerase chain reaction-based detection of MLL-ENL transcript in K562 cells. Ctrl = MLL-I9-#1 + Luc sgRNAs. (C) RT-PCR-based detection of reciprocal ENL-MLL transcript in K562 cells. Ctrl = MLL-I9-#1 + Luc sgRNA. (D) Serial plating of CD34+ HSPC after transduction with L-CRISPR-CTN. (E) Detection of MLL-ENL expression in CD34+ HSPCs at fifth plating. Control (MLL-I9-#1 + Luc sgRNAs) template from third plating. (F) Detection of the genomic MLL-ENL breakpoint in CD34+ HSPCs at fifth plating (control (MLL-I9-#1 + Luc sgRNAs) template from second plating) compared to the first plating. (G) Analysis of MLL target genes in MLL-ENL-expressing cells (fifth plating) compared to controls (third plating). Differential regulation marked above. Ctrl = MLL-I9-#1 + Luc sgRNAs.
Figure 3.
Figure 3.
CRISPR-Cas9-induced MLL-ENL rearrangements are leukemogenic in a CD34+ hematopoietic stem and progenitor cell xenotransplantation model. (A) Schematic depiction of the xenotransplantation model. (B) Survival of mice transplanted with L-CRISPR-CTN-transduced CD34+ HSPC. Mice that succumbed to non-hematopoietic disease were censored and are indicated (ticked). (C) Flow cytometric analysis of one mouse with hematopoietic disease compared to a control, with markers as indicated (ctrl = MLL-I9-#1 + Luc sgRNAs). (D) Bone marrow (BM) cytospin analysis of a diseased mouse (MGG, 1000X). (E) Detection of an MLL translocation with fluorescence in situ hybridization on interphase nuclei (Vysis LSI MLL probe; Abbott Laboratories) in BM cells from one diseased mouse. (F) Histopathological analysis of liver tissue from a healthy control mouse (ctrl = MLL-I9-#1 + Luc sgRNA) (top) and a mouse with monocytic leukemia-like disease transplanted with L-CRISPR-CTN(11;19)-containing CD34+ HSPC (bottom) (HE, 100x). (G) Alignment of Sanger sequencing-derived genomic t(11;19)/MLL-ENL breakpoints of mice with a detectable MLL-ENL breakpoint. (H) MLL-ENL and ENL-MLL genomic breakpoints detected in the BM of two mice with a monocytic leukemia-like disease. (I) Expression of the MLL-ENL (left) and reciprocal ENL-MLL (right) fusion genes, measured by quantitative polymerase chian reaction from the BM of mice with a detectable MLL-ENL breakpoint compared to control mice (MLL-I9-#1 + Luc sgRNAs).
Figure 4.
Figure 4.
In vivo environment affects the oncogenic transformation of primary human hematopoietic stem and progenitor cells by t(11;19) (A) Survival of serially transplanted mice with human cell engraftment. Donor: primary recipients with [L-CRISPR-CTN(11;19)] a monocytic leukemia-like disease (Mono #1/#2), a healthy mouse with a detectable MLL-ENL breakpoint but no disease in the primary recipient, and two control donors (MLL-I9-#1 + Luc sgRNAs) (n = 3 per donor). (B) Analysis of bone marrow (BM) cell morphology (left: MGG, 1000x) and liver histopathology (right: HE, 100X) showing severe infiltration of a secondary recipient transplanted with monocytic leukemia-like disease cells. (C) Flow cytometry analysis of monocytic leukemia cells for monocytic and progenitor cell surface marker expression. (D) Flow cytometry analysis of B-ALL cells. (E) Analysis of BM cell morphology (MGG, 1000x) of a secondary recipient with B-ALL. (F) Detection of an MLL translocation with fluorescence in situ hybridization on interphase nuclei of B-ALL cells (Vysis LSI MLL probe; Abbott Laboratories). (G) Alignment of Sanger sequencing-derived genomic t(11;19)/MLL-ENL breakpoints of mice with B-ALL (Secondary) compared to the healthy primary (Primary) mouse.

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