FIGURE 1 from Dabrafenib Alters MDSC Differentiation and Function by Activation of GCN2

Dabrafenib (DAB) impairs MDSC proliferation and differentiation. A, Representative flow cytometry histograms showing antigen-specific proliferation of CD8⁺ T cells from OTI mice modulated by bone marrow–derived MDSC. T cells were cocultured with OVA_257–264 peptide-pulsed DCs and MDSC at ratio DC: T cell: MDSC 1:10:30, where 1 = 10,000 cells, in 96-well plates for 60 hours. Experiment repeated three times with similar results. DAB = dabrafenib. B, Frequency of OTI CD8⁺ T undergoing one (D1), two (D2), three (D3), four (D4), or more than five (D5) cycles of division from plot A. NP = nonproliferative cells. Data are expressed as mean value. Experiment repeated three times with similar results. C, Mean Arg1, Nos2, and Eif2ak4 expression relative to 18s RNA, assessed by qPCR (n = 4). MDSCs were generated in presence ± 1.5 µmol/L DAB for 4 days. P value was determined by two-tailed unpaired Student t test. Significance considered P < 0.05. Experiment repeated three times with similar results. D, Representative flow cytometry histograms showing antigen-specific proliferation of CD8⁺ T cells from OTI mice modulated by mature bone marrow–derived MDSC. T cells were cocultured with OVA_257–264 peptide-pulsed DCs and MDSC at ratio DC: T cell: MDSC 1:10:30, where 1 = 10,000 cells, in 96-well plates for 60 hours in presence of ± 1.5 µmol/L DAB. Experiment repeated three times with similar results. DAB = dabrafenib. E, Frequency of OTI CD8⁺ T undergoing one (D1), two (D2), three (D3), four (D4), or more than five (D5) cycles of division from D. NP = nonproliferative cells. Data are expressed as mean value. Experiment repeated three times with similar results. F, Number of MDSCs generated in vitro in the presence of ± 1.5 µmol/L DAB after 4 days in culture (n = 4). P value was determined by two-tailed unpaired Student t test. Significance considered P < 0.05. Experiment repeated three times with similar results. G, Dose–response for inhibition of proliferation after 4 days in culture. DNA fluorescence was measured for cultures with the indicated concentration of dabrafenib. IC₅₀ value was calculated at 1.59 µmol/L. Lines represent the mean ± SEM of four independent experiments. H, Dose–response for viability after 4 days in culture in presence of 1.5 µmol/L DAB. Each datapoint indicates the percentage of total live cells assessed by flow cytometry, normalized to control samples. Lines represent the mean ± SEM of four independent experiments. I, Percentage of live cells in MDSC in vitro cultures ± 1.5 µmol/L DAB assessed by flow cytometry. Error bars indicate ± SD (n = 4). P value was determined by two-tailed unpaired Student t test. Significance considered P < 0.05. Experiment repeated four times with similar results. J, Mean frequency and total cell number of different subsets of immune cells in fresh bone marrow (BM) and MDSC. A total of 1 × 10⁶ BM cells were seeded at the start of the experiment and differentiated for 4 days ± 1.5 µmol/L DAB (n = 4). Color coding represents each subtype. Experiment repeated two times with similar results. K, Representative flow cytometry contour plots of MDSC surface phenotype when expanded for 4 days with ± 1.5 µmol/L DAB compared with the ones found in fresh bone marrow. Gates show mean frequency (n = 4). Experiment repeated two times with similar results. L and M, Mean frequency of the PMN population CD11b⁺MHCII^negLy6C⁺Ly6G⁺ in MDSC ± 1.5 µmol/L DAB (J) or increasing doses of DAB (K) (n = 4). Experiment repeated three times with similar results. In J, P value was determined by two-tailed unpaired Student t test. Significance considered P < 0.05. N, Representative flow cytometry histograms showing antigen-specific proliferation of CD8⁺ T cells from OTI mice modulated by FACS sorted m-MDSC generated in presence of ± 1.5 µmol/L DAB. T cells were cocultured with OVA_257–264 peptide-pulsed DCs and MDSC at ratio DC: T cell: MDSC 1:10:20, where 1 = 5,000 cells, in 96-well plates for 60 hours. Experiment repeated three times with similar results. DAB = dabrafenib.