FIGURE 4 from Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2

<p>Dabrafenib-induced GCN2 activation alters proliferation and differentiation of MDSCs. <b>A,</b> Total cell counts in MDSC <i>in vitro</i> cultures from wild-type (B6) and GCN2-deficient (B6.GCN2^−/−) mice differentiated in presence of ± 1.5 µmol/L DAB. <i>P</i> values were determined by two-way ANOVA with Šidák correction post-test. Significance considered <i>P</i> < 0.05. <b>B,</b> Representative flow cytometry contour plot of Ly6C and Ly6G expression in wild-type and GCN2^−/− MDSCs differentiated in presence of ± 1.5 µmol/L DAB. <b>C,</b> Frequency of CD11b⁺MHCII^negLy6C⁺Ly6G⁺ MDSCs in wild-type and GCN2^−/− MDSCs differentiated in presence of ± 1.5 µmol/L DAB. <i>P</i> values were determined by two-way ANOVA with Šidák correction post-test. Significance considered <i>P</i> < 0.05. <b>D,</b> qPCR of <i>Asns</i> and <i>Atf4</i> in MDSCs lysates in wild-type and GCN2^−/− MDSCs differentiated in presence of ± 1.5 µmol/L DAB, relative to <i>18s</i> mRNA. <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. Western blot analysis (<b>E)</b> and densitometry quantification (<b>F</b>) of ERK1/2, eIF2α, p38, and 4EBP1 phosphorylation relative to total protein in whole culture MDSC lysates. Quantification is shown as fold change in the intensity signal of samples treated with 1.5 µmol/L DAB compared with Ctrl sample for each genotype (wild-type or GCN2^−/−). <i>P</i> value was determined by two-tailed unpaired Student <i>t</i> test. Significance considered <i>P</i> < 0.05. <b>G–I,</b> Ribosomal profiling analyzed in wild-type and GCN2^−/− MDSC lysates. <b>G,</b> Optical density (O.D.) at 254 nm. The area designated as “polysomes” represents the fraction of RNA-forming complexes of two or more ribosomes. <b>H,</b> AUC of the region marked as polysomes in G. Bars indicate mean ± SD (<i>n</i> = 4). <i>P</i> values were determined by two-way ANOVA with Šidák correction post-test. Significance considered <i>P</i> < 0.05. <b>I,</b> Relative quantification (%) of the AUC in polysomes versus monosomes (40s, 60s, and 80s region). Data are expressed as mean ± SD (<i>n</i> = 4). <i>P</i> values were determined by two-way ANOVA with Šidák correction post-test. Significance considered <i>P</i> < 0.05. For all panels, experiments were repeated three times with similar results. DAB = dabrafenib.</p>