Abstract P140: Chromomycin A5, a marine-derived antibiotic, targets the oncogenic TBX2 in breast cancer

Abstract

Abstract Breast cancer (BC) continues to pose a major health burden. In 2020 alone, over 2 million people were diagnosed with BC globally and of these 684 996 people succumbed to the disease. Cancer cell drug resistance and the notion of transcription factors as undruggable targets has slowed progress for novel BC therapies. The transcription factor, TBX2, is commonly over-expressed in breast cancer with various hormone receptor statuses where it functions as a powerful pro-proliferative factor by directly repressing p21, PTEN, CST-6, and NDGR1 expression. Furthermore, TBX2 mediates Cisplatin drug resistance by inducing an S-phase cell cycle arrest allowing for cisplatin-induced DNA damage repair and breast cancer cell survival. Targeting TBX2 with DNA-damaging chemotherapies therefore emerges as a promising anti-cancer strategy to treat BC. To this end, we performed a reverse affinity screen to fish for marine-derived compounds with DNA-damaging- and TBX2 binding- activity and identified the antibiotic Chromomycin A5 (CMA5) as a promising agent. This project confirms the binding affinity of CMA5 for TBX2 by thermophoresis and fully characterizes the in vitro anticancer activity of CMA5 in TBX2-driven BC cells. Short-term and long-term selective cytotoxicity of CMA5 were investigated in two TBX2-driven BC cell lines (MCF-7 and T47D) and a non-malignant breast epithelial cell line (MCF-12A) using MTT and clonogenic assays. Our results show that CMA5 exhibited potent (IC50 ≤6.5 nM) and selective (SI ~ 3.3 ) cytotoxicity against BC cells. Importantly, CMA5 inhibited TBX2 protein levels and co-treatment of the BC cells with CMA5 and the proteasome 26S inhibitor MG132 rescued TBX2 levels. These results show that CMA5 targets TBX2 for protein degradation. Furthermore, whereas knocking down TBX2 using shRNA blocked the cytotoxicity of CMA5 (IC50 increased by 5-fold), ectopically over-expressing TBX2 increased the cytotoxic activity of CMA5 (IC50 decreased by 1.2-fold). These results suggest that CMA5 cytotoxicity is dependent, in part, on the presence of TBX2. To further explore the significance of TBX2 inhibition by CMA5 we investigated the impact of CMA5 on expression levels of the TBX2 target genes p21, PTEN, CST-6 and NDGR1 by quantitative real-time PCR analyses. The results revealed that all four genes were upregulated in the presence of CMA5 treatment which suggested that CMA5 inhibits BC cell viability and survival by targeting TBX2 and thus releasing its ability to repress these tumor suppressors. To elucidate the mechanism(s) underpinning the cytotoxic activity of CMA5 we investigated its effects on several markers of DNA damage (yH2AX expression) and apoptosis (cleaved caspase 3, 7, 8, 9 and PARP) by western blotting and immunocytochemistry. Our results show that CMA5 induced DNA damage and killed cells, in part, by both the intrinsic and extrinsic apoptotic programmed cell death pathways. Overall, this study identifies CMA5 as a promising anti-breast cancer agent because of its capability to induce DNA damage and to inhibit the oncogenic TBX2. Citation Format: Claire Bellis, Suparna Chakraborty, Mihlali Mlaza, Bianca Del Bianco Sahm, Paula Rezende Teixeira, Leticia Costa-Lotufo, Sharon Prince. Chromomycin A5, a marine-derived antibiotic, targets the oncogenic TBX2 in breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P140.