ETV6-NCOA2 is a transcriptional regulator that induces a gene expression pattern similar to that in ETV6-NCOA2 pediatric leukemia. Human cord blood CD34⁺ progenitors were transduced with MSCV-MIGR1-IRES-GFP retroviruses expressing EN2 or empty vector. The cells were cultured in vitro (SCF, FLT3L, and thrombopoietin (TPO) for 5 days and sorted for GFP⁺. In vivo EN2 engrafted cells (n = 3) were sorted for CD45⁺NGFR⁺, EN2 + NOTCH1-L1601PdP leukemia cells were sorted for CD45⁺GFP⁺NGFR⁺ (n = 3), and EN2 PDX cells were sorted for CD45⁺ (n = 3) from BM of transplanted sick NSG mice. The sorted cells were sent for bulk RNA-seq. (A) Unsupervised clustering of the samples based on the 500 top differentially expressed genes in the RNA-seq experiments. (B) GSEA analysis of EN2 + NOTCH1-L1601PdP (left) and EN2 (right) compared with the 250 top-ranked upregulated genes of human EN2 PDXs. The differentially expressed genes of the EN2 and EN2 + NOTCH1-L1601PdP samples were ranked according to their log₁₀ (P value) and compared with the patient’s gene set. (C) Venn diagram of significantly upregulated genes in the human in vivo EN2 engrafted cells, in vivo EN2 + NOTCH1-L1601PdP leukemia, and EN2 PDX samples. (D) GSEA pre-ranked analysis of in vivo EN2–engrafted cells, in vivo EN2 + NOTCH1-L1601PdP leukemia, and EN2 PDX samples compared with the T-ALL gene set (top) or B-cell gene set (bottom).