Targeting the Cytoskeleton: Design, Mechanisms, and Therapeutic Applications of Synthetic Actin-Disrupting Toxins in Cancer Metastasis

Abstract

Despite advances in cancer therapies overall, there are no proven therapies targeting the metastatic spread of tumors, which is the leading cause of cancer deaths. Our objective is to develop a new therapeutic strategy that disrupts actin dynamics used by cancer cells to invade surrounding tissues and metastasize. Building on our discovery that the marine toxin Mycalolide B (MycB) blocks metastasis in mice by severing filamentous actin (F-actin), we synthesized a series of analogues to improve drug suitability. Among these, one lead analogue (2a) emerged as most effective in suppression of cancer cell invasion and F-actin severing in multiple cancer cell models, albeit at approximately 100-fold reduced potency compared to MycB. Analogue 2a synthesis was scalable and accommodated additional moieties, such as a linker for antibody conjugation, making it a promising candidate for therapeutic development. We investigated the effects of both MycB and 2a treatments on cell division in A549 cells, showing that these treatments caused aborted cytokinesis and the accumulation of aneuploid cells. This correlated with defects in F-actin accumulation within the contractile ring, despite normal septin ring localization and mitotic microtubules with actin toxin treatments. Cytotoxicity and removal of aneuploid cells were increased by combining actin toxins with Nutlin-3 to activate the p53 pathway in A549 cells, with synergistic effects observed. Since most cancers acquire mutations in p53, we extended testing to PANC-1 pancreatic cancer cells treated with the p53-reactivating drug PRIMA-1, which was synergistic with MycB in cytotoxicity assays. These results indicate that the cytostatic and cytotoxic outcomes of actin-targeting toxins are modulated by p53 status and provide a rationale for combination therapy testing in cancer models. Finally, we engineered antibody-drug conjugates (ADCs) targeting Human Epidermal Growth Factor Receptor-2 (HER2), using 2a as a payload connected via a maleimide-cleavable linker. These ADCs retained HER2 binding properties and specificity towards HER2-positive cancer cells in severing F-actin and suppressing cell invasion. Together, these findings establish a new framework for developing actin-targeting ADCs that selectively impair cancer cell growth and invasion for further optimization and testing in metastatic cancer models.