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CBFA2T3-GLIS2 oncogenic fusion is sufficient for leukemic transformation

ABSTRACT: Purpose: To investigate whether human cord blood CD34+ hematopoietic stem and progenitor cells (CB HSPCs) transduced with CBFA2T3-GLIS2 (C/G) expression construct recapitulates primary C/G acute myeloid leukemia (AML). Methods: CB HSPCs were transduced with either C/G fusion/GFP or GFP control contruct (C/G-CB or GFP-CB cells, respectively) and cultured in endothelial cell (EC) co-culture or myeloid promoting culture (MC) for 12 weeks. RNA from transduced cells were isolated at week 1, 6 and 12 in either culture condition. Whole transcriptome RNA-sequencing was conducted using 75bp strand-specific paired-end mRNA libraries and sequenced on the Illumina HiSeq 2000/2500. Results: Unsurpervised clustering analysis of RNA-sequencing of C/G-CB cells cultured with ECs or in MC demonstrated that the C/G-CB cells from weeks 6 and 12 in EC co-culture clustered with primary C/G-positive patient samples, but not C/G-CB cells cultured in MC nor GFP controls. In addition, EC C/G-CB cells had signficant enrichemnt of WNT, HEDGEHOG and TGF-beta pathways. However, both EC and MC cultures revealed up-regulation of ERG and BMP2, genes compared to GFP control, which are genes previously shown to be strongly upregulated in C/G AML, and concomitant down-regulation of the erythroid-megakaryocyte differentiation gene GATA1. Conclusions: We find that lentiviral transduction of C/G fusion is sufficient to induce malignant transformation of human CB HSPCs that recapitulates the transcriptome of primary C/G AML.

ORGANISM(S): Homo sapiens

PROVIDER: GSE181726 | GEO | 2022/09/21

REPOSITORIES: GEO

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Project description:The occurrence of clonal perturbations and leukemia in patients transplanted with retrovirally-transduced autologous hematopoietic stem and progenitor cells (HSPCs) has stimulated extensive investigation, demonstrating that proviral insertions perturb adjacent proto-oncogene expression. Although enhancer-deleted lentiviruses are less likely to result in insertional oncogenesis, there is evidence that they may perturb transcript splicing, and one patient with a benign clonal expansion of lentivirally-transduced HPSC has been reported. The rhesus macaque model provides an opportunity for informative long-term analysis to ask whether transduction impacts on long-term HSPC properties. We utilized two techniques to examine whether lentivirally-transduced HSPCs from eight rhesus macaques transplanted 1-13.5 years previously are perturbed at a population level, comparing telomere length as a measure of replicative history and gene expression profile of vector positive versus vector negative cells. There were no differences in telomere lengths between sorted GFP+ and GFP- blood cells, suggesting that lentiviral transduction did not globally disrupt replicative patterns. Bone marrow GFP+ and GFP- CD34+ cells showed no differences in gene expression using unsupervised and principal component analysis. These studies did not uncover any global long-term perturbation of proliferation, differentiation, or other important functional parameters of transduced HSPCs in the rhesus macaque model. CD34+ hematopoietic stem and progenitor cells were purified from bone marrow aspirates obtained from 4 rhesus macaques 3-9 years following transplantation with lentivirally-transduced autologous hematopoietic stem and progenitor cells. All lentiviral vectors contained the GFP marker gene. The CD34+ cells from each animal were sorted via flow cytometry into GFP+ and GFP- fractions, and RNA from these cells was used for Affymetrix gene expression analysis as detailed below. There were two samples, GFP+ and GFP-, from each of 4 animals.

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CBFA2T3-GLIS2 oncogenic fusion is sufficient for leukemic transformation

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