Fig 2: MYC-bound promoter-distal and MYCi975-sensitive chromatin accessibility sites exhibit significant CTCF and FOX factor motif enrichment.(A) MYC consensus peaks were separated into promoter proximal (±2 kb from TSS) or promoter distal (>±2 kb from TSS). The mean MYC ChIP-seq read coverage is plotted at both promoter-proximal and promoter-distal MYC-bound sites in vehicle control and 48-hour MYCi975-treated 22Rv1 cells. (B) Motif enrichment analysis of MYC-bound promoter-proximal peaks, listing the top five enriched motifs, in addition to CTCF (#19) and FOXA1 (#55) for contrast (%TWM, percentage of targets with motif; %BWM, percentage of background with motif). (C) Motif enrichment analysis of MYC-bound promoter-distal peaks, listing the top five and including c-MYC (#17). (D) Heatmap representation of all MYC-bound sites separated into promoter-proximal and promoter-distal sites, showing MYC ChIP-seq coverage and 22Rv1 ATAC-seq coverage in DMSO- versus 48-hour MYCi975-treated 22Rv1 cells. (E) Venn diagram demonstrating overlap of the differential MYCI975 types 1 to 3 with differential ATAC-seq peaks in DMSO- versus 48-hour MYCi975-treated 22Rv1 cells. (F) Motif enrichment analysis of the differentially lost ATAC-seq peaks (left) in MYCi975-treated 22Rv1 cells. Motif enrichment analysis was run on differential ATAC-seq peaks outside of the MYC cistrome, showing enrichment for both CTCF and FOX factors (right).

Fig 3: MYCi975 enhances the efficacy of antihormone therapy.(A) Immunoblot showing loss of FOXM1, FOXA1, AR, and AR variant protein levels in sonicated nuclear fractions of MYCi975-treated 22Rv1 cells. Replicates (1 and 2) represent biological replicates of cells treated with 10 µM MYCi975 for 48 hours. (B) Genome browser tracks of MYC (green) ChIP-seq data at the FOXA1, FOXM1, and AR gene loci in MYCi975-treated 22Rv1 cells. TSS of each gene is indicated by an arrow. (C) Representative 4 × 5 dose-response matrices showing percentage viability (left) and Bliss index (right) analysis of predicted versus observed cell viability of prostate cancer cells (LNCaP, C4-2B, and 22RV1) treated with MYCi975 and ENZ or the breast cancer line MCF-7 treated with MYCi975 and 4-OHT (n = 3). Bliss scores > 0 indicate synergy, scores close to zero indicate additivity, and scores < 0 denote antagonism. (D) Fold change tumor volumes of 22Rv1 xenografts in nude mice treated with MYCi975 (100 mg/kg BID p.o.) or vehicle, 5 days a week for 3 weeks. n = 4 to 7 grafts per group (from three to four mice). (E) Fold change tumor volumes of 22Rv1 xenografts in nude mice treated with MYCi975 (100 mg/kg QD p.o.), ENZ (25 mg/kg QD i.p.), combination of MYCi975/ENZ, or vehicle for 18 days. n = 7 to 8 grafts per group (from four to five mice). Error bars represent means ± SEM and analyzed by two-way analysis of variance (ANOVA) in GraphPad Prism for (D) and (E) (*P < 0.05; ****P < 0.0001).