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. 2021 Oct 12;139(3):399–412. doi: 10.1182/blood.2020010405

Figure 1.

Figure 1

ETV6-NCOA2 is a T-cell oncogene that induces immature T-cell arrest in murine BM progenitors. (A) Schematic representation of ETV6-NCOA2 (EN2) fusion. bHLH-PAS, basic helix-loop-helix Per-ARNT-SIM domain; NID, nuclear receptor interaction domain. (B) Murine BM progenitors treated with fluorouracil were transduced with MSCV-MIGR1-IRES-GFP retroviruses expressing ETV6-NCOA2, empty vector, or KAT6A-NCOA2 (KN2) and plated in methylcellulose (IL-3, IL-6, and SCF). Colonies were counted and re-plated every 7 to 10 days. EN2 induces self-renewal of transduced cells on methylcellulose culture compared with the empty vector–transduced cells in the second and third replate (Mann-Whitney U test P = .014) (n = 3). (C) Lineage-negative cells (lin–) were enriched from wt-C57BL/6 mice; the cells were transduced with EN2, KN2, or empty vector, incubated in liquid culture (IL-3, IL-6, and SCF) for 5 days, and sorted for GFP+. The RNA of the GFP+ sorted cells was sent for bulk RNA-seq. Gene set enrichment analysis (GSEA) of the EN2 vs empty vector–transduced cells demonstrated enrichment of Notch1 signature (NES, 1.67; FDR, 0.011), ETP (NES, 1.98; FDR, 0.006), and early thymic signature (NES, 2.65; FDR, 0.0). (D) Murine BM HSPCs treated with fluorouracil were transduced with either EN2 or empty vector and plated on OP9-DL4 stroma (IL-7, Flt3L) for 3 weeks and then immunophenotyped by flow cytometry (n = 4). Left panel: average immunophenotype results (Mann-Whitney U test for EN2 vs empty vector P = .02 in DN1, DN2, and DN3). Right panel: representative example of CD44 and CD25 flow cytometry results. AF700, Alexa Fluor 700; HSC, hematopoietic stem cell; SS, side scatter.