Fig. 4: Effect of HDACis in human 3q26 EVI1^High AML.

A H&E histological sections of PR#003 bone marrow cells grown in collagen type I scaffolds with vehicle (DMSO) or HDACis (AR-42, belinostat, and entinostat) for 72 hr. Scale bar: 40 µm. B Percentage of cell viability of primary 3q26 AML grown in collagen type I scaffolds and treated with HDACis. Scale bar: 100 µm. Drug concentration (IC₅₀) used in (A) and (B) were established following a dose-response titration assay presented in Supplementary Fig. 6A. C AUC effect of HDACis and chemotherapy agents in EVI1^High (samples PR#002-008, and PR#023-024) and EVI1^Low (samples PR#010-022, and PR#025-039) AML blasts. D EVI1, ∆EVI1, and cleaved caspase 3 protein expression of primary 3q26 AML samples after 24 h of treatment with the indicated concentrations of HDACis. E Representative images of IF staining of primary 3q26 EVI1^High AML cell showing EVI1 nuclear content (in red) following 24 hr of treatment with DMSO or HDACis at the indicated concentrations (n = 3 biological replicates). Nuclei in blue (DAPI), scale bar: 100 µm. F Quantitative IF analysis of nuclear EVI1 content in 3q26 EVI1^High primary AML cells after 24 h of treatment with indicated compounds. G Effect of entinostat (left) or ara-C (right) on EVI1 nuclear localization (in red) following 6 hr of treatment in PR#003 or PR#004, respectively. The nuclei were stained with DAPI (blue). Scale bar: 100 µm. IF quantification of EVI1 nuclear content is presented at the bottom. H EVI1 expression (brownish) in bone marrow leukemia cells at diagnosis and following two cycles of azacitidine and entinostat (PR#002, left) or three cycles of ara-C and daunorubicin (3 + 7) (PR#004, right). Top, formalin-fixed, paraffin-embedded (FFPE) tissue sections were stained with anti-EVI1 antibody revealed by immunoperoxidase. Scale bar: 100 µm. Statistical significance was determined by a two-tailed non-parametric t-test (Mann-Whitney) (C, G, H) or one-way ANOVA with Tukey’s correction for multiple comparison testing (B, F). Data are presented as mean ± SD in B (PR#003 n = 3, PR#004 n = 2), C (EVI1^High n = 9, EVI1^Low n = 28), F (PR#002 n = 318, PR#003 n = 573), G (PR#003 n = 235, PR#004 n = 214), and H (n = 10 fields per condition). Source data are provided as a Source Data file. See also Supplementary Fig. 5-6 and Supplementary Data 2.