Investigating the anti-cancer activity of novel osmiumarene compounds using CRISPR screening technology

Acquired resistance to chemotherapeutic agents is a common problem in the treatment of cancer. Whilst combination therapy of cisplatin and taxol is the first line treatment for ovarian cancer, around 15% of patients do not respond to this regimen and 80% of those that do relapse within 18-24 months with cisplatin-resistant disease. Thus, novel drugs are required with new mechanisms of action that can be used to treat cisplatinresistant ovarian and other cancers. This thesis is focussed on the pre-clinical evaluation of two structurally similar osmium-arene complexes as candidates for use as anti-cancer agents. The half-sandwich ‘piano-stool’ iodido complex FY12 was able to overcome platinum resistance in ovarian carcinoma cell lines as compared to the chlorido complex FY05, even though there is only a single halide change between the two complexes. FY12 was also active in a wide range of cancer cell lines with lower acute toxicity than FY05 in a model organism, zebrafish. In order to progress to the clinic, more detailed analysis of the mechanism of action for these complexes is required. Interestingly, FY12-induced cell death is dependent on autophagy and the intrinsic apoptosis pathway, working in synergy with one another. Furthermore, glycolysis and oxidative phosphorylation were also shown to play a role in the mechanism of FY12-induced cytotoxicity, while DNA damage did not appear to be involved. This demonstrated a novel mechanism of action of FY12
as compared to cisplatin. The single halide change in FY05 affected the mechanism of complex-induced cell death, as this complex did not induce intrinsic apoptosis and autophagy, but was dependent on p21 and p53.
Genome-wide CRISPR screens were utilized to identify genes whose loss could confer resistance to FY12, or to cisplatin as a control, thereby providing insights into the mechanism of action of this complex. CRISPR datasets demonstrated that FY12 targets many different genes and their pathways. RPRML and PINLYP were validated as genes involved in the mechanism of FY12-induced cell death in IGROV1 ovarian cancer cells and four other genes, ARRDC4, SYDE2, RFNG and EIF4EBP1, were also found to contribute. The high levels of mutation in DNA damage genes in IGROV1 cells compromised the ability to identify genes associated with cisplatin resistance. However, the CRISPR screen implicated C1S, C14orf10S, EGFR, MERTK, ZNF8 and RARB as genes whose loss could confer resistance to cisplatin.
Due to the hypermutator phenotype of IGROV1 cells, a subsequent genome-wide
CRISPR screen was performed in HEK293T human embryonic kidney 293 cells
containing the SV40 T-antigen. In this screen HS6ST1, RAMP2, ASIC2 and OTOP2
loss was found to give rise to FY12 resistance, whilst C20orf196, AQP11 and
STARD9 contributed to cisplatin resistance. Out of the four unique gene targets
identified for FY12 in HEK293T cells, three were cation channels, which is intriguing given that FY12 is a small positively-charged molecule.
These studies have characterised the pathways by which FY12 and FY05 complexes induce cell death, and initiated studies to unravel the specific mechanisms responsible for the development of acquired resistance. Whilst further work is required to further probe the contribution of the genes identified in this project, this study provides important pre-clinical data to support the progress of the FY12 complex towards clinical trials for the treatment of cisplatin-resistant ovarian cancer.