Fig. 8: Targeting AR overcomes BRAFi resistance.

a RT-qPCR analysis of EGFR and SERPINE1 expression normalized to RPLP0, in two melanoma cells (M121224BR and WM983ABR) propagated in the presence of Dabrafenib and treated with the AR inhibitors AZD3514 and ARCC4 or DMSO for 48 h. Mean ± SD n(biological replicates)=3, one-way ANOVA, M121224BR *p = 0.0114; WM983ABR *p = 0.0123. b IF with anti-PAI-1 antibodies of melanoma cells (M121224BR) treated with the AR inhibitors AZD3514 or ARCC4 or DMSO control for 48 h as in the previous panel. Representative images and quantification of the PAI-1signal per individual cells (arbitrary units). n(cells per sample)≥100, unpaired, two-tailed t-test, **** p < 0.0001. Color scale: gray, DAPI; cyan, PAI-1. Scale bar: 40 μm. AR IF in parallel cultures is in Supplementary Fig. 6. c Live-cell imaging proliferation assays (IncuCyte) of the indicated BRAFi-resistant cells treated with AR inhibitors or DMSO, as in a, b. n(biological replicates) = 3, (four images per well every 4 h for 150 h). Mean ± SD; Pearson r correlation test, ****p < 0.0001. d Cell death as detected by live-cell staining (IncuCyte, Cytotox Red) of cells as in the previous panel at 72 h of treatment with the AR inhibitors versus DMSO control. Four images per cell culture, n (biological replicates)=3, median, box (25-75%) and whiskers (5-95%), unpaired t-test, *, p < 0.05; **p < 0.01; ****p < 0.0001. e Clonogenicity assays of three drug-naive melanoma cell lines treated with Dabrafenib (0.5 µM) individually or in combination with AZD3514 (10 µM) or ARCC4 (1 µM). Quantification of crystal violet stained colonies. n(dishes)=3, Mean ± SD, unpaired, two-tailed t-test, (M14) ***p = 0.0001; (SKMEL28) **p = 0.0018; WM983A) *p = 0.0130. f Tumor volume quantification 14 days after intradermal injection into immunodeficient mice (NOD.CB17-Prkdcscid/J) of BRAFi-resistant A375 cells pretreated with AZD3514 (10 μΜ) or DMSO vehicle 24 h prior to injection. n(tumors)=5, Paired, two-tailed t-test, *p < 0.05. Scale bar: 1 mm. g IF of tumors as in f using anti-Ki-67- and anti-MelanA antibodies. Representative images and quantification of Ki67⁺ cells and MelanA⁺ area in arbitrary units. n(tumors)=5, Paired, two-tailed t-test, *p < 0.05, **p = 0.0063. Color scale: cyan, DAPI; magenta, Ki-67. Scale bar: 40 µm.