Abstract 3715: Pharmacological targeting of circadian clock genes reveals regulatory mechanisms of E-box regulated genes in cancer

Abstract

Abstract Transcription factors (TFs) are key regulators of homeostasis and cancer. Recent advances in pharmacological targeting of TFs makes them valuable targets in various diseases, but systems-level understanding of the function and regulation of TFs from different families in cancers remains lacking. Our lab and collaborators have been developing new small molecules to modulate the functions of key regulators of the circadian clock BMAL1 and CLOCK, which are bHLH family TFs that activate gene expression by binding to a DNA motif called E-box, with a consensus sequence of CANNTG. E-boxes are ubiquitous in the genome and broadly regulate essential physiological processes of the cell, but their molecular biology in cancer is largely unknown. In the current work we tested the ability of small molecules to regulate the expression of BMAL1 and CLOCK target genes. Across different cell lines of various cancer types, we found that stabilization of one of the negative regulators of CLOCK, CRY2, by our novel compound called SHP1705, is the most consistent to suppress canonical BMAL1 target genes. GO and GSEA analysis of RNA-seq data shows that SHP1705 also significantly changed most core regulators of circadian rhythms as well as other E-box-binding proteins. However, SHP1705 has IC50 values above 10µM in most tested cells, creating a challenge for efficacy in a clinical setting. Because BMAL1 requires the proteasome to sustain the turnover of a transcription burst, we hypothesized proteasome inhibitors (PI) MG132 and carfilzomib may synergize with SHP1705. Consequently, we found strong synergy with both PIs. Analysis of TF-target genes in combination-treated cells reveals significant changes in expression profiles, suggesting that these genes are regulated by E-box, and other related/alternative promoter elements: CCAAT-box, CG-rich motifs, and ATGGC. Co-enrichment of these elements revealed the components of E-box containing promoters and showed potential properties of E-box-regulated gene expression. Co-existence of these motifs in the same proximal promoter also potentially allows RNAPII multiple choices of alternative promoters and could promote expression of variants in cancer cells. Our results support the necessity of inhibiting the whole promoter and provide an example of how to approach designer pharmacological strategies to achieve this purpose. Citation Format: Yuanzhong Pan, Heinz-Josef Lenz, Evanthia Roussos Torres, Steve A. Kay. Pharmacological targeting of circadian clock genes reveals regulatory mechanisms of E-box regulated genes in cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3715.