3jc48-3

The proto-oncogene cellular myelocytomatosis (c-Myc) is a transcription factor that is upregulated in several human cancers. Therapeutic targeting of c-Myc remains a challenge because of a disordered protein tertiary structure. The basic helical structure and zipper protein of c-Myc forms an obligate heterodimer with its partner MYC-associated factor X (MAX) to function as a transcription factor. An attractive strategy is to inhibit MYC/MAX dimerization to decrease c-Myc transcriptional function. Several methods have been described to inhibit MYC/MAX dimerization including small molecular inhibitors and proteomimetics. We studied the effect of a second-generation small molecular inhibitor 3JC48-3 on prostate cancer growth and viability. In our experimental studies, we found 3JC48-3 decreases prostate cancer cells' growth and viability in a dose-dependent fashion in vitro. We confirmed inhibition of MYC/MAX dimerization by 3JC48-3 using immunoprecipitation experiments. We have previously shown that the MYC/MAX heterodimer is a transcriptional repressor of a novel kinase protein kinase D1 (PrKD1). Treatment with 3JC48-3 upregulated PrKD1 expression and phosphorylation of known PrKD1 substrates: the threonine 120 (Thr-120) residue in beta-catenin and the serine 216 (Ser-216) in Cell Division Cycle 25 (CDC25C). The mining of gene expression in human metastatic prostate cancer samples demonstrated an inverse correlation between PrKD1 and c-Myc expression. Normal mice and mice with patient-derived prostate cancer xenografts (PDX) tolerated intraperitoneal injections of 3JC48-3 up to 100 mg/kg body weight without dose-limiting toxicity. Preliminary results in these PDX mouse models suggest that 3JC48-3 may be effective in decreasing the rate of tumor growth. In conclusion, our study demonstrates that 3JC48-3 is a potent MYC/MAX heterodimerization inhibitor that decreases prostate cancer growth and viability associated with upregulation of PrKD1 expression and kinase activity.

c‐Myc is a basic helix‐loop‐helix‐leucine zipper (bHLH‐ZIP) transcription factor that is responsible for the transcription of a wide range of target genes involved in many cancer‐related cellular processes. Over‐expression of c‐Myc has been observed in, and directly contributes to, a variety of human cancers including those of the hematopoietic system, lung, prostate and colon. To become transcriptionally active, c‐Myc must first dimerize with Myc‐associated factor X (Max) via its own bHLH‐ZIP domain. A proven strategy towards the inhibition of c‐Myc oncogenic activity is to interfere with the structural integrity of the c‐Myc–Max heterodimer. The small molecule 10074‐G5 is an inhibitor of c‐Myc–Max dimerization (IC50=146 μM) that operates by binding and stabilizing c‐Myc in its monomeric form. We have identified a congener of 10074‐G5, termed 3jc48‐3 (methyl 4′‐methyl‐5‐(7‐nitrobenzo[c][1,2,5]oxadiazol‐4‐yl)‐[1,1′‐biphenyl]‐3‐carboxylate), that is about five times as potent (IC50=34 μM) at inhibiting c‐Myc–Max dimerization as the parent compound. 3jc48‐3 exhibited an approximate twofold selectivity for c‐Myc–Max heterodimers over Max–Max homodimers, suggesting that its mode of action is through binding c‐Myc. 3jc48‐3 inhibited the proliferation of c‐Myc‐over‐expressing HL60 and Daudi cells with single‐digit micromolar IC50 values by causing growth arrest at the G0/G1 phase. Co‐immunoprecipitation studies indicated that 3jc48‐3 inhibits c‐Myc–Max dimerization in cells, which was further substantiated by the specific silencing of a c‐Myc‐driven luciferase reporter gene. Finally, 3jc48‐3′s intracellular half‐life was >17 h. Collectively, these data demonstrate 3jc48‐3 to be one of the most potent, cellularly active and stable c‐Myc inhibitors reported to date.