PHF8/IFN-I response protein signature is frequent in patients with AML and provides novel opportunities for future targeted therapies. (A) Unsupervised hierarchical clustering of samples from patients with AML (n = 204 samples) based on proteins participating in GO term IFN-I–mediated signaling pathway. The clustering identifies 2 major sample groups (group-1 and group-2) as highlighted in the heat map. Annotations at the top of the heat map show patient disease status and PHF8 protein expression. (B) Box plot showing the differential expression of PHF8 in AML samples based on IFN protein expression cluster (group-1 = 102 patient samples vs group-2 = 102 patient samples). Two sample t test, permutation-based FDR <0.05 represented as significant. (C) Dot plot for PHF8 expression vs PC2 scores of 204 samples from patients with AML (primary, n = 177; relapsed AML, n = 27) based on the IFN-I signature subproteome. (D) Immunoblot analysis of total PHF8 levels of 9 samples (P1-P9) from patients with AML. Actin was used as loading control. (E) Immunoprecipitated PHF8 from cells from patients with primary AML expressing high PHF8 levels, untreated (control) or under combinatorial treatment with OKA/ATRA (O/A), and immunoblotted for phospho-Ser (upper panel) as well as for total PHF8 protein after membrane stripping (lower panel). (F) Violin plot depicting total numbers of colonies of 5 samples from patients with primary AML with high expression of PHF8 (n = 5). Significance was tested using the Tukey multiple comparison test (2-way ANOVA, ∗P < .02; ∗∗P < .008; ∗∗∗P < .0002). (G) Samples from patients with AML with low expression of PHF8 (n = 4), treated with the indicated concentrations of ATRA and OKA (OKA 20 minutes incubation). Tukey multiple comparison test, ns. (H-I) Colony morphology of primary human AML cells expressing high (H) or low (I) PHF8 levels. Cells were treated with ATRA (10^–8 M) or/and OKA (500 nM, 20 minutes incubation). IP, Immunoprecipitation; WB, western blot.