Epithelial-to-mesenchymal transition supports ovarian carcinosarcoma tumorigenesis and confers sensitivity to microtubule-targeting with eribulin

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumour type with limited treatment options. OCS is hypothesised to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analysed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumours. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts (PDX). Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a down-regulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate EMT plays a key role in OCS tumourigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes

Degron features of the regulatory domain of squalene monooxygenase - a rate limiting enzyme in cholesterol synthesis

Cholesterol is an essential lipid associated with many important biological functions. At both the cellular and physiological levels, cholesterol is acquired through two main sources. One source is uptake, while the other source is de novo cholesterol synthesis. Squalene monooxygenase (SM) is a rate-limiting enzyme of cholesterol synthesis. A few studies have suggested SM could be a promising treatment target to lower cholesterol levels in the blood and as another metabolic target in certain cancers. Thus, there is an increasing need to understand the regulation of this enzyme. One critical mode of regulation is the cholesterol-accelerated degradation of SM. This process requires the first 100 amino acids of SM (termed SM N100). The SM N100 regulatory domain represents a degron region (a degradation signal), which allows SM to be regulated by cholesterol. However, insights into cholesterol sensing by SM N100 and the mechanisms by which SM N100 confers instability were unknown. To investigate this degron, we utilised SM N100 fused to green fluorescent protein, a fusion protein which recapitulates the cholesterol-accelerated degradation of SM. Here, we have performed a series of point mutations, truncations and domain swaps based on our understanding of known degron features. We identified that an amphipathic helix (residues Gln62–Leu73) in SM N100 is required for cholesterol-accelerated degradation. We also present evidence that the cholesterol-driven disorder of the amphipathic helix lengthens the disordered region surrounding the helix and exposes a hydrophobic patch which accelerates SM N100 degradation. Attempts to identify ubiquitination sites revealed SM N100 undergoes non-canonical ubiquitination at serine residues to signal SM N100 for degradation. Finally, we identified valosin-containing protein (VCP) as a key protein which mediates the removal of the SM N100 degron from the endoplasmic reticulum into the cytosol for degradation. In summary, we have increased our understanding of the SM N100 degron architecture, furthering insights into how cholesterol sensing in the endoplasmic reticulum is coupled to protein quality control

Development of NanoLuc-targeting protein degraders and a universal reporter system to benchmark tag-targeted degradation platforms

Modulation of protein abundance using tag-Targeted Protein Degrader (tTPD) systems targeting FKBP12(F36V) (dTAGs) or HaloTag7 (HaloPROTACs) are powerful approaches for preclinical target validation. Interchanging tags and tag-targeting degraders is important to achieve efficient substrate degradation, yet limited degrader/tag pairs are available and side-by-side comparisons have not been performed. To expand the tTPD repertoire we developed catalytic NanoLuc-targeting PROTACs (NanoTACs) to hijack the CRL4(CRBN) complex and degrade NanoLuc tagged substrates, enabling rapid luminescence-based degradation screening. To benchmark NanoTACs against existing tTPD systems we use an interchangeable reporter system to comparatively test optimal degrader/tag pairs. Overall, we find the dTAG system exhibits superior degradation. To align tag-induced degradation with physiology we demonstrate that NanoTACs limit MLKL-driven necroptosis. In this work we extend the tTPD platform to include NanoTACs adding flexibility to tTPD studies, and benchmark each tTPD system to highlight the importance of comparing each system against each substrate