Fig. 7: MYC targeting is beneficial in Enzalutamide-resistant conditions.

a Drug sensitivity curves of Enzalutamide-naïve, or Enzalutamide-resistant (EnzaRes) C42B cells treated with MYCi975. Data are presented as mean values ± SEM from n = 3 biologically independent experiments. b Colony formation assay using Enzalutamide-resistant (EnzaRes) C42B cells in Intact (i.e., treated with DMSO), treated with Enzalutamide (10 µM), MYCi975 (2 µM), or a combination of Enzalutamide+MYCi975 (10 µM + 2 µM). Cells were grown in the presence of respective drugs. Representative images are shown, and data are presented as mean values ± SEM from n = 6 biologically independent experiments. P value was estimated using a one-tailed Welch t-test. *p value < 0.05, ***p value ≤ 0.001. c Boyden chamber-based in vitro migration assay using Enzalutamide-resistant (EnzaRes) C42B cells in Intact (i.e., treated with DMSO), treated with Enzalutamide (10 µM), MYCi975 (2 µM), or a combination of Enzalutamide+MYCi975 (10 µM + 2 µM). Representative images are shown, data are presented as mean values ± SEM from n = 5 biologically independent experiments, indicating the quantification of Crystal Violet trapped by migrated cells. Scale bars, 100 μm. P value was estimated using a one-tailed Welch t-test. *p value < 0.05, **p value ≤ 0.01. d Expression of NME2 in Intact and Enzalutamide-resistant (EnzaRes) C42B cells, using qRT-PCR, data are presented as mean values ± SEM from n = 6 biologically independent experiments. P value was estimated using the one-tailed Welch t-test. ***p value ≤ 0.001. e Two different siRNAs targeting NME2 were used to downregulate NME2 (left panel) and its effect on MYC expression using qRT-PCR is shown (right panel), data are presented as mean values ± SEM from n = 6 biologically independent experiments. P value was estimated using one-tailed Welch t-test. * p value < 0.05. f Boyden chamber-based in vitro migration assay using Enzalutamide-resistant (EnzaRes) C42B cells treated with DMSO or MYCi975 (2 μM) with or without knockdown of NME2. Representative images are shown, data are presented as mean values ± SEM from n = 4 biologically independent experiments, indicating the cell count quantification of Crystal Violet trapped by migrated cells, normalized to control (DMSO with siScramble). Scale bars, 100 μm. P value was estimated using 2-way ANOVA, *p value < 0.05, **p value < 0.01, ***p value < 0.001, ****p value < 0.0001. g Comparison of anti-proliferative effects of Enzalutamide on C42B EnzaRes cells that have NME2 knocked down via CRISPR (sgNME2_V1 and sgNME2_V2) versus C42B EnzaRes which have received non-targeting sgRNA control (sgNT). Data are presented as mean values ± SEM from n = 3 independent biological replicates for each cell line. h Western blot of NME2, MYC and GAPDH protein levels in C42B shNME2 EnzaRes cells treated with Doxycycline for 96 h at the indicated concentrations. (n = 3 biologically independent experiments, representative blot shown). i Schematic representation and tumor volumes for mice bearing established C42B EnzaRes shNME2 xenografts treated with vehicle (n = 7 mice), Enzalutamide (10 mg/kg QD i.p) and/or Doxycycline (2 mg/mL in drinking water) (n = 6 mice for Dox, Enza, Enza + Dox arms) for 24 days. Statistical significance was performed via two-way ANOVA. *p value < 0.05, ***p value ≤ 0.001, ****p value ≤ 0.0001. Source data for all the panels in this figure are provided as a Source Data file.