American Society of Hematology

Figure 5.

ETV6-NCOA2 causes de-repression of ETV6 targets. (A) Transcription factor binding site analysis (i-Cis target37). The analysis was performed separately for the significantly upregulated and downregulated differentially expressed genes of the bulk RNA-seq of CD34+ cells transduced with EN2 or empty vector, and for the in vivo EN2 engrafted cells, in vivo EN2 + NOTCH1-L1601PdP leukemia, and EN2 PDX samples. (B) GSEA analysis of EN2 in vitro and EN2 PDX sample pre-ranked RNA-seq results compared with the 500 top upregulated genes in the LOUCY cell line treated with short interfering RNA (siRNA) against ETV6 (siETV6).14 (C) ChIP-seq for H3K27Ac was performed on CD34+ cells transduced with EN2 or empty vector in vitro, and EN2 PDX. H3K27 differential acetylation of HES1 and DLL4 was examined by integrative genomics viewer63 analysis. (D) Left: Co-IP EN2/ETV6: HEK-293T cells were transfected with empty vector, EN2-Flag, ETV6-HA, or a combination of both. The proteins were immunoprecipitated with either Flag antibody (EN2) or HA antibody (ETV6). Right: Co-IP EN2/p300: EN2-Flag or ETV6-Flag stable DND41 cells were immunoprecipitated with either Flag antibody (top) or endogenous p300 antibody (bottom). (E) Left: 293T cells stably expressing ETV6 luciferase reporter vector were transfected with 200 ng ETV6 alone, increasing concentrations of EN2 (200, 300, and 400 ng) or a combination of both (ANOVA test P < .0001) (n = 6). Right: ETV6-luciferase reporter stable transfected 293T cells were transfected with 200 ng ETV6 alone or with 200 ng ETV6 with 1 of the following: EN2 200 ng, EN2 + 1 200 ng, or 5 μM A485 (a p300 inhibitor)51 or EN2 EN2 200 ng in addition to 1 or 5 μM A485 or A486 (inactive A485 analog) (ANOVA test P < .0001). (F) A scheme for proposed EN2 mechanism of leukemogenesis. Upper panel: ETV6-HDAC repression of genes in wt hematopoietic progenitors. Lower panel: EN2-ETV6-p300 complex in EN2 hematopoietic progenitors. This figure was created with BioRender.com. **P < .008; ***P < .0006; ****P < .0001. Chr, chromosome; HDAC, histone deacetylase.

ETV6-NCOA2 causes de-repression of ETV6 targets. (A) Transcription factor binding site analysis (i-Cis target³⁷). The analysis was performed separately for the significantly upregulated and downregulated differentially expressed genes of the bulk RNA-seq of CD34⁺ cells transduced with EN2 or empty vector, and for the in vivo EN2 engrafted cells, in vivo EN2 + NOTCH1-L1601PdP leukemia, and EN2 PDX samples. (B) GSEA analysis of EN2 in vitro and EN2 PDX sample pre-ranked RNA-seq results compared with the 500 top upregulated genes in the LOUCY cell line treated with short interfering RNA (siRNA) against ETV6 (siETV6).¹⁴ (C) ChIP-seq for H3K27Ac was performed on CD34⁺ cells transduced with EN2 or empty vector in vitro, and EN2 PDX. H3K27 differential acetylation of HES1 and DLL4 was examined by integrative genomics viewer⁶³ analysis. (D) Left: Co-IP EN2/ETV6: HEK-293T cells were transfected with empty vector, EN2-Flag, ETV6-HA, or a combination of both. The proteins were immunoprecipitated with either Flag antibody (EN2) or HA antibody (ETV6). Right: Co-IP EN2/p300: EN2-Flag or ETV6-Flag stable DND41 cells were immunoprecipitated with either Flag antibody (top) or endogenous p300 antibody (bottom). (E) Left: 293T cells stably expressing ETV6 luciferase reporter vector were transfected with 200 ng ETV6 alone, increasing concentrations of EN2 (200, 300, and 400 ng) or a combination of both (ANOVA test P < .0001) (n = 6). Right: ETV6-luciferase reporter stable transfected 293T cells were transfected with 200 ng ETV6 alone or with 200 ng ETV6 with 1 of the following: EN2 200 ng, EN2 + 1 200 ng, or 5 μM A485 (a p300 inhibitor)⁵¹ or EN2 EN2 200 ng in addition to 1 or 5 μM A485 or A486 (inactive A485 analog) (ANOVA test P < .0001). (F) A scheme for proposed EN2 mechanism of leukemogenesis. Upper panel: ETV6-HDAC repression of genes in wt hematopoietic progenitors. Lower panel: EN2-ETV6-p300 complex in EN2 hematopoietic progenitors. This figure was created with BioRender.com. **P < .008; ***P < .0006; ****P < .0001. Chr, chromosome; HDAC, histone deacetylase.

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