Figure 4. Evaluation of the role of MYC in mTORi/HDACi combination response and cooperative signature expression.

A) Heatmap of the fold enrichment of cMyc ChIP-seq (ChIP signal versus input signal) in the promoter region for each gene in MM1s cells (see Figure S5A for other gene regions). B) Representative genomic view of MYC binding on E2f2 in MM1s cells, C) ChIP-qPCR analysis of seven genomic proximal promoters of human MYC target genes in L363 (mean ± SE, *p<0.05, **p<0.001). D) Western blot for MYC in P493-6 tet-repressible cell line. Cells treated with 0.1mg/ml doxycycline for 72 hours for full repression of exogenous MYC expression (timepoint 0), before washing and changing media to tet (dox)-free. E) Cooperative signature heatmap of log₂ fold change versus P493-6 (1-48hrs) after removal of tet treatment. F) Cell viability relative to untreated control for MYC-ON and -OFF P493 cells after 48 hours of single agent or combination treatment (n = 2 experiments with 3 technical replicates in each). The interaction of combination treatment and MYC effect was significant by ANOVA (p<0.0001) (See also Figure S6). G) Combined mTOR and HDAC inhibition affects MYC protein stability. MYC stability following cycloheximide treatment. Representative western blot pseudo-images (Simple Western automated capillary immunoassay) for MYC in L363 cells after 18 hours of single agent rapamycin (10nM) or MS-275 (500nM) or combination treatment. Cells were treated with cycloheximide to block protein translation to determine drug effects on MYC half-life. This experiment was repeated 3×. Graph showing percent MYC protein relative to no cycloheximide/untreated control over an 80 minute time course (time 0 is 5 minutes after addition of cycloheximide); MYC protein half-life is estimated from the slope of lines (see Fig. S7 for equation and half-life estimations immediately and 4 hours after initiating mTORi/HDACi treatment), H) Western blots of MYC phospho-threonine 58 residue (known to destabilize the protein) and MYC phospho-serine 62 residue (protein stabilizing). Cells were pre-treated with cycloheximide to inhibit translation and MG-132 to prevent immediate degradation of the phosphorylated MYC species.