Fig. 2: ZM enforces aberrant activation of a pro-leukemic stemness gene-expression program including Hoxa, Meis1, Sox4, Myc and Myb.

a Heatmap of 259 genes showing higher expression in both normal LSK (Lin-/Sca1+/c-Kit+) and ZM-immortalized murine AML cells, relative to mature blood cell types. Expression values represent mean-centered log2(TPM). Genes are sorted in a descending order based on their expression values in ZM+ AML cells. A9M and MLL-AF9 represent the murine AML lines established by the coexpressed Hoxa9 plus Meis1a (A9M) and MLL-AF9, respectively. b GSEA shows enrichment of the HSC-related and mutant-NPM1-upregulated genes in ZM+ relative to A9M+ cells, as well as enrichment of genes upregulated upon myeloid differentiation or the indicated TF-related transcripts in A9M+ relative to ZM+ cells. The p value (n = 15,282 genes per group) was calculated by an empirical phenotype-based permutation test. The FDR is adjusted for gene set size and multiple hypotheses testing while the p value is not. c Volcano plot displays differentially expressed genes (DEGs) identified in ZM+ versus A9M+ AML cells. The up- and down-regulated DEGs, defined with the indicated cutoff, are represented by red and blue dots, respectively, with example stemness-related genes highlighted. d DAVID functional annotation of DEGs identified in ZM+ versus A9M+ AML cells, with the enriched terms for upregulated (n = 1200 genes) and downregulated (n = 300 genes) DEGs indicated by red bars and blue bars, respectively. The p value was calculated by Fisher’s Exact test. e Venn diagram showing overlaps among ZM-upregulated genes (versus A9M), MLL-AF9-upregulated genes (relative to A9M), and LSK stemness genes. f RT-PCR of Hoxa7, Hoxa9 and Meis1 in ZM- and MLL-AF9-transformed AML cells. qPCR signals from three independent experiments were normalized to those of 18S RNA and presented as mean ± SD. g RT-PCR of AML-related proto-oncogenes in murine HSPCs post-transduction of EV or the indicated ZM. qPCR signals from three independent experiments were normalized to those of 18S RNA and then WT ZM and presented as mean ± SD.