Fig. 3
SAMHD1 triphosphohydrolase activity controls DAC response. a, b IC50 values for (a) DAC or (b) AZA in AML cell lines THP-1 and OCI-AML3 with experimentally modulated SAMHD1 levels. SAMHD1 was depleted by either CRISPR/Cas9-mediated SAMHD1 knockout in THP-1 cells (THP-1 KO), by shRNA-mediated silencing of SAMHD1 in OCI-AML3 cells, or by transduction with VLPs carrying SAMHD1-degrading Vpx protein (Vpx-VLPs). Unedited THP-1 cells (THP-1 Ctr.), cells transduced with control-shRNAs (shCTRL), or cells treated with Vpr-carrying VLPs (Vpr-VLPs) served as controls. Each point represents an average of three technical replicates, and in each case, as shown, three independent experiments were performed. Horizontal lines and error bars represent means ± s.d. The numbers above the data points indicate the factor of decrease of IC50 values in SAMHD1-depleted cells relative to control cells. Representative SAMHD1 immunoblots for the different experimental conditions are shown. β-Actin served as a loading control. c, d Representative LC–MS/MS analyses of (c) DAC-TP levels or (d) AZA-TP levels in THP-1 Ctr. or THP-1 KO cells. e Representative immunoblots of proteins involved in DNA damage response and DNA methylation in THP-1 Ctr. or THP-1 KO cells following treatment with increasing concentrations (0, 0.08, 0.4, 2, and 10 µM) of either AZA (left panels) or DAC (right panels) for 72 h. f, g IC50 values of (f) DAC, AZA and daunorubicin or (g) DAC, AZA and lomustine without or with the RAD51 inhibitor B02 in parental HEL cells and HEL cells overexpressing either SAMHD1-WT or the dNTPase-defective SAMHD1 mutants SAMHD1-D311A or SAMHD1-D207N. Each closed circle represents a technical replicate (n = 3) of one representative experiment out of three. Horizontal lines and error bars represent mean ± s.d. Numbers indicate the factor of decrease of IC50 values in SAMHD1-depleted relative to control cells. Statistical analyses were performed using unpaired two-tailed Students’ t-test. **p < 0.01; ***p < 0.001
