Fig. 5: HDAC-mediated suppression of EVI1 modulates Myc signaling.

A Heatmap showing ssGSEA enrichment of MSigDB of gene signature in MOLM1 and UCSD/AML1 HDACi-treated cells. Hot or cold colors indicate correlation or anticorrelation of the top enriched gene sets from each functional group (P ≤ 0.05 in bold, calcolated according to³⁰) among cell lines and treatments (bottom). B Uniform manifold approximation and projection (UMAP) plot of clustering results of PR#002 bone marrow cells before and after therapy. Colors indicate the cell populations on the basis of the reference mapping approach. C UMAP plot of clustering results of PR#002 bone marrow cells before (left) and after (right) therapy, colored according to the UCell score of leukemic markers. D GSEA running score plot of the top enriched MYC targets V1 pathway in HSC, LMPP, and LP (Adj.P = 0.0011) cell populations of PR#002. Each graph indicates the running enrichment score (ES) of the pathway (top), the location of single genes of the gene set in the ranking (central), and the distribution of the ranking metric (bottom). E MYC expression (brownish) in bone marrow leukemia cells of PR#002 at diagnosis and following two cycles of azacitidine and entinostat (left). Scale bar: 100 µm. Right, the scatter dot blot indicates the mean ± SD of the percentage of the MYC-positive cells in FFPE tissue sections (n = 10 fields per condition). F EVI1, ∆EVI1, and MYC expression in EVI1^High AML cell lines, 6 days after shRNA transduction. NT = non-targeting, sh#87 = shRNA directed against EVI1 (n = 3 biological replicates). G In vivo antileukemic effect of entinostat and azacitidine. Mice (PDLX_PR#008) were treated with vehicle (DMSO), azacitidine (1 mg/kg) for 5 days, entinostat (10 mg/kg) (5 days/week) or the combination of both for 3 weeks. Histograms show the percentage hCD45⁺ in bone marrow (top) and the percentage of EVI1 mRNA relative to the control gene RPL13A (ΔΔCT) in CD45+ cells (bottom) at the end of treatment. H UMAP plot of clustering results of PDLX_PR#008 bone marrow LP before (i) and after entinostat (ii), azacytidine (iii) or the combination of both (iv), colored according to the UCell score of leukemic markers. I GSEA running score plot of the top enriched MYC targets V1 pathway in PDLX_PR#008 LP after the treatment with azacitidine/entinostat (Adj.P = 3.40 × 10^-8) or entinostat as a single agent (Adj.P = 2.17 × 10^-12). Each graph indicates the running enrichment score (ES) of the pathway (top), the location of single genes of the gene set in the ranking (central), and the distribution of the ranking metric (bottom). J Dot plot of GSEA results illustrating Molecular Signatures Database (MsigDB) biological processes associated with the indicated treatments compared to the vehicle in PDLX_PR#008 LP. Set size refers to the number of genes associated with each (MsigDB) biological process. Dot color indicates the range of Adj.P for each pathway. K EVI1, MYC, and Ki67 expression (brownish) in bone marrow leukemia cells (PDLX_PR#008) following three weeks treatment of azacitidine and entinostat as described in panel G. FFPE tissue were stained with anti-EVI1, anti-MYC and anti-Ki67 antibodies revealed by immunoperoxidase. Scale bar: 100 μm. L Histograms display the mean ± SD of the percentage of the EVI1, MYC or Ki67-positive cells in FFPE tissue sections. Statistical significance was determined by a two-tailed non-parametric t-test (Mann-Whitney) (E), and one-way ANOVA with Tukey’s correction for multiple comparison testing (G, L). GSEA enrichment score significance was based on a weighted Kolmogorov Smirnov (WKS) test corrected for multiple hypotheses testing: Benjamini & Hochberg (BH or FDR) (D, I, J). Data are presented as mean ± SD in E (n = 10 fields per condition), G (vehicle n = 7, treated n = 5 per group; n = 9 for the bottom panel), K (vehicle n = 7, treated n = 5 per group), L (n = 10 fields per condition). Source data are provided as a Source Data file. See also Supplementary Fig. 7-8 and Supplementary Data 3.