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The Preclinical Development of Phyllanthusmins for the Treatment of High Grade Serous Ovarian Cancer
thesisposted on 01.05.2021, 00:00 by Alexandria Young
High grade serous ovarian cancer (HGSOC) is a lethal gynecological malignancy with an unmet need for new therapeutics. Many of the most widely used chemotherapeutic drugs are derived from natural products or their semi-synthetic derivatives. We have developed potent synthetic analogues of a class of compounds known as phyllanthusmins, inspired by natural products isolated from the tropical plant, Phyllanthus poilanei Beille. The most potent analogue, PHY34, had the highest potency in HGSOC cell lines in vitro and displayed cytotoxic activity through activation of apoptosis. PHY34 exerts its cytotoxic effects by inhibiting autophagy at a late stage in the pathway, involving the disruption of lysosomal function. The autophagy activator, rapamycin, combined with PHY34 eliminated apoptosis, suggesting that autophagy inhibition may be required for apoptosis. PHY34 was readily bioavailable through intraperitoneal administration in vivo where it significantly inhibited the growth of cancer cell lines in hollow fibers, as well as reduced ovarian tumor burden in a xenograft model. In order to identify its molecular target/s, we undertook an unbiased approach utilizing photo affinity labeling, pulldown, and mass spectrometry. Protein targets from the nucleocytoplasmic transport pathway were identified, with the cellular apoptosis susceptibility (CAS) protein, also known as CSE1L, representing the top hit. No inhibitors currently exist targeting CAS/CSE1L, an essential protein highly expressed in cancer cells. When compared to known inhibitors of other nucleocytoplasmic transport proteins, such as KPNB1 and XPO1, PHY34 activity was distinct, suggesting a mechanism apart from the activity of these previously developed compounds. PHY34 treatment precipitated various cellular changes consistent with CAS/CSE1L inhibition, including the cytoplasmic shift of nuclear localization sequence (NLS)-mCherry and cell cycle arrest. Analysis of patient datasets demonstrate increased CAS/CSE1L expression in high grade serous ovarian cancer, correlating with severity of disease. In vivo toxicity studies and in vitro assays utilizing non-tumorigenic cell lines suggest PHY34 treatment may be well tolerated by normal cells. Inhibition of CAS/CSE1L by PHY34 is a viable therapeutic strategy for the treatment of high grade serous ovarian cancer. We demonstrate that PHY34 acts as a late-stage autophagy inhibitor with nanomolar potency and significant antitumor efficacy as a single agent against HGSOC in vivo, possibly through direct interaction with CAS/CSE1L. This class of compounds holds promise as a potential, novel chemotherapeutic and demonstrates the effectiveness of targeting the autophagic and nucleocytoplasmic pathways as viable strategies for combating ovarian cancer.