Abstract P6-10-17: Pharmacological Strategies to Target Circadian Clock Genes in TNBC

Abstract

Abstract Pharmacological Strategies to Target Circadian Clock Genes in TNBC Yuanzhong Pan, Priya Jayachandran, Evanthia T. Roussos Torres, Steve A. Kay Background: Triple-negative breast cancer (TNBC) remains the most aggressive form of breast cancer and more targeted treatment options remain challenging. Our recent analysis of clinical data showed that circadian clock genes (including positive regulators BMAL1 and CLOCK, and negative regulators CRYs, PERs, and REV-ERBs) play important roles in breast cancer, and most prominently in TNBC. We have also shown in other cancer types that cells with a mesenchymal signature and stem-cell like state rely on BMAL1/CLOCK activity to support proliferation. BMAL1 and CLOCK are transcription factors that have remained “undruggable” to date. Alternatively, our lab developed small molecule compounds that target the negative regulators CRY, REV-ERB and CK2. We are therefore prompted to study if the clock genes can serve as a therapeutic target in TNBC using our small molecules that target clock regulators in TNBC. In addition, BMAL1 and CLOCK require ubiquitin E3 ligases and proteasomes for their transcriptional function, thus we hypothesize proteosome inhibitors may provide synergy to improve response to our novel clock-based therapeutics. Methods: We first used shRNA-mediated gene knockdown (KD) to disrupt the core circadian clock regulator BMAL1 and CLOCK in a panel of TNBC cell lines across different molecular subtypes (MDA-MB-231, MDA-MB-157 and MDA-MB-436, MDA-MB-453, HCC70, HCC1143, BT549, Hs578T). After KD, cell proliferation was quantified using CellTiter-Glo. We then tested our small molecule compounds SHP656—that stabilizes CRY, SR29065 and derivatives—that agonizes REV-ERB, and GO289—an inhibitor of CK2 that stabilizes PER. Based on our knowledge of the clock regulators, we tested drug synergy between SHP1705 and proteasome inhibitors MG132 and Carfilzomib across a range of concentrations. RNA-seq of cells treated with either single drug or drug combination were performed to study the mechanism of the synergistic effect. Results: A subset of the tested TNBC cell lines showed significantly impaired proliferation after BMAL1 or CLOCK KD. All cells in the mesenchymal-like molecular subtype responded to BMAL1/CLOCK KD, which is consistent with our previous data in other cancer types. It also confirmed BMAL1 and CLOCK have the potential to serve as therapeutic targets for mesenchymal-like TNBC. We then tested the clock compounds in our TNBC panel. We found SR29065 and GO289 both inhibit cancer cell proliferation at a clinically relevant EC50. Combination of our novel small molecule SHP1705 with the proteosome inhibitor MG132 and carfilzomib, demonstrated significant synergistic effects in vitro. In order to understand the mechanism of the synergistic effect between clock compounds and proteasome inhibitors, we performed RNA-seq on single drug and combo-treated cells. Differential expression analysis revealed that over two-thousand genes are specifically changed in the combo group. GO analysis showed that these genes are enriched in MYC target genes. Because MYC is also, like BMAL1 and CLOCK, a E-box binding transcription factor, this result implies that the mechanism driving synergy may be due to a disruption of E-box-dependent transcription. Conclusions: Here we showed that the core circadian clock regulator BMAL1 and CLOCK are potential therapeutic targets in mesenchymal-like TNBCs. Using REV-ERB agonists and CK2 inhibitors to target BMAL1/CLOCK transcription activity, we can achieve compound EC50s in single-micromolar range. In vivo experiments in murine models of TNBC are underway to determine the efficacy of single agent and combination therapy with proteosome inhibitors. Given the recently established safety of CRY stabilizers there is great potential for translation of these findings to clinical trials in patients with TNBC. Citation Format: Yuanzhong Pan, Priya Jayachandran, Evanthia T. Roussos Torres, Steve A. Kay. Pharmacological Strategies to Target Circadian Clock Genes in TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-17.