Multiplex CRISPR/Cas9-Based Genome Editing in Human Hematopoietic Stem Cells Models Clonal Hematopoiesis and Myeloid Neoplasia

Abstract

Hematologic malignancies are driven by combinations of genetic lesions that have been difficult to model in human cells. We used CRISPR/Cas9 genome engineering of primary adult and umbilical cord blood CD34⁺ human hematopoietic stem and progenitor cells (HSPCs), the cells of origin for myeloid pre-malignant and malignant diseases, followed by transplantation into immunodeficient mice to generate genetic models of clonal hematopoiesis and neoplasia. Human hematopoietic cells bearing mutations in combinations of genes, including cohesin complex genes, observed in myeloid malignancies generated immunophenotypically defined neoplastic clones capable of long-term, multi-lineage reconstitution and serial transplantation. Employing these models to investigate therapeutic efficacy, we found that TET2 and cohesin-mutated hematopoietic cells were sensitive to azacitidine treatment. These findings demonstrate the potential for generating genetically defined models of human myeloid diseases, and they are suitable for examining the biological consequences of somatic mutations and the testing of therapeutic agents.

Keywords: CHIP; SMC3; azacitidine; clonal hematopoiesis; cohesin; gene editing; genome engineering; human CD34(+) cells; human HSPC; myeloid neoplasm.

Figure 1. Genetic editing of a single and multiple leukemia drivers in CD34+ cells in vitro and in vivo

A: Multiplex targeting of UCB CD34+ cells leads to efficient indel formation across all genes targeted without evidence of major selection for a dominant clone during 5 days of in vitro culture (p=1.0, Shannon entropy). B: Analysis of single cell-derived colonies demonstrates multiplex gene targeting in a single cell and suggests distinct patterns of zygosity (SMC3, p=0.02; ASXL1, p=0.04; RUNX1, p=0.04; Barnard’s exact test). C: Indel type analysis for SMC3 gene pre-injection (AF=0.05) and 5 months post-transplantation in mouse ZM178 (AF=0.02, recipient #1 for adult CD34 donor ADT1 in Table S1B) shows stable representation of major LOF clones with dropout of predicted non-LOF clones. Corresponding colors denote the same indel type. D=deletion, I=insertion, followed by size and hg19 genomic coordinate in brackets. n=3; mean ± SD indel VAF amongst edited alleles for “D1: 112341796”, “D2: 112341794”, and “D8: 112341797” are 0.379 ± 0.032, 0.165 ± 0.046 and 0.091 ± 0.046, respectively, with pre-injection indel VAF amongst edited alleles of 0.363, 0.193, and 0.042, respectively. D: Multiplex targeting of UCB CD34+ cells with LOF mutations in 11 leukemia drivers followed by in vivo expansion shows evidence of mutated clone expansion in 10/17 transplanted mice. Incidence of CRISPR/Cas9 induced indels in experimental genes is statistically significantly different from control genes SRSF2 and U2AF1 (p=0.01, Fisher’s exact test, two-tailed). Clones with AF>0.01 at 5 months in the sorted bone marrow CD45+ cells are shown. Pre-injection AF: SMC3(0.17), STAG2(0.28), U2AF1(0.05), TET2(0.16), NF1(0.14), DNMT3A(0.10), EZH2(0.22), SRSF2(0.04), TP53(0.60), RUNX1(0.20). See also Figure S1, S2, and Table S1, S3, S4.

Figure 2. Functional and morphologic characterization of in vivo models generated by targeting CD34+ cells

A: IHC shows expansion of immature CD45+ MPO+ myeloid cells in the bone marrow of mouse 1783 characterized by a major SMC3/FLT3-ITD mutated clone. Comparison is made to the control mouse 1764 transplanted with mock infected CD34+ cells and mouse 1782 characterized by a FLT3-ITD only mutated clone. B: Expansion of CD45+ CD163+ histiocytic/macrophage cells in the bone marrow mouse 1785 characterized by LOF mutations in 7 genes and NPM1 mutation as demonstrated by IHC. Comparison is made to the control mouse 1764 and mouse 1787 characterized by an NPM1 only mutated clone. All images are 40X magnification (scale bar = 0.125 mm) with H&E insets at 100X magnification (scale bar = 0.025mm). C–D: Human engraftment over time in peripheral blood (C) and bone marrow at 5 months (D) of mouse 1783, wildtype (WT) control mice (1763 and 1764) and mouse 1782 with FLT3-ITD only mutant clone as determined by flow cytometry using hCD45 staining. Mean %+/− SD of peripheral blood (PB) and bone marrow (BM) engraftment is shown for control WT mice 1763 and 1764. See also Figure S2 and Table S3.

Figure 3. Clonal dynamics, multi-lineage reconstitution and serial transplantability of mutant clones

A: Emergence of a single dominant clone in mouse 1785 at 5 months post-transplantation as assessed by NGS and indel type analysis. D=deletion, I=insertion: followed by size of indel and (Hg19 genomic coordinate). Indel fractions observed at the time of pre-injection and at 5 months: TP53(0.06; 0.43), NF1(0.14; 0.32), RUNX1(0.20; 0.32), TET2(0.17; 0.23), EZH2(0.22; 0.24), ASXL1(0.12; 0.24), and U2AF1(0.05; 0.13). B: Sorting of B-(CD45+CD19+CD33-CD3-), T-(CD45+CD3+CD19-CD33-), myeloid-(CD45+CD33+CD19-CD3-) and immature (CD45+CD34+CD19-CD3-CD33-) cells followed by NGS supports presence of the mutant clone in all lineages of mouse 1783. Representative flow analysis panels are shown; gating as outlined in each panel, % viable CD45+ cells shown. C: Patterns of genetic lesions among primary and secondary transplant recipients of 3 independent donor mice, 1783, 1785 and 1786. Clones with AF>0.01 in the sorted CD45+ bone marrow are shown. Mutant clones identified in the primary transplant are more likely to be detected in the secondary transplant recipients than mutant clones not identified in the primary transplant (p<0.0001, Fisher’s exact test). See also Figure S3 and Table S4.

Figure 4. Genotype-specific responses to treatment with azacitidine in vitro and in vivo

A: In vitro 72-hour treatment of TET2 and SMC3 mutant clones shows increased sensitivity to hypomethylating agent azacitidine (250nM). Mean of 3 technical replicates with SD is plotted for each condition. % viability is normalized to DMSO treated controls for each genetic condition. * p<0.05 (two–tailed unpaired t-test) B: Schedule of azacitidine dosing in vivo. After engraftment of TET2 and ASXL1 mutant clones, 2.5mg/kg azacitidine or PBS (vehicle) was administered intraperitoneally once daily on Days 1–5 of a 14 day cycle. Mice were treated for a total of 12 weeks (6 cycles). C: Bone marrow analysis of TET2 and ASXL1 indel fraction by NGS demonstrates a genotype specific response of TET2 mutant clones (n=3–4 per group). * p<0.05 (two–tailed unpaired t-test) D: Analysis of TET2 indel fraction in sorted populations from azacitidine and vehicle treated TET2 mice (n=3 per group). * p<0.05 (two–tailed unpaired t-test) See also Figure S4.