Development and optimisation of high throughput screening systems in Mammalian cells

Abstract

Synthetic lethality is based on the recognition that the inactivation of both genes (either by mutation or deletion) in a specific gene pair can lead to cell death. This approach has become a promising cancer treatment strategy since it allows the selective elimination of cancer cells whilst sparing the healthy ones. Over the years a wide range of drugs utilising synthetic lethality have been developed and applied in clinical practice. For example, the PARP inhibitors which target only BRCA1/2 mutated cancer cells. To identify synthetically lethal compounds, SICLOPPS (Split Intein mediated Cyclisation Of Peptides and Proteins) was used. SICLOPPS describes a genetically encoded method allowing cyclic peptide libraries to be produced and subsequently screened within cells. Cyclic peptides are attractive therapeutic targets and SICLOPPS libraries can be generated using standard molecular biology techniques. In this thesis an SX5 SICLOPPS library containing 3.2 million cyclic peptides is screened in an isogenic cell line containing an MTAP deletion. MTAP is ametabolic enzyme which is deleted in 15% of all cancer types, rising to >40%of glioblastoma multiforme. MTAP deletion cancers (MTAP-/-) have a worse prognosis, according to abundant clinical studies. MTAP is expressed ubiquitously in healthy cells, whilst MTAP deleted cells have a wide range of metabolic vulnerabilities. Hence compounds targeting cells with MTAP deletions may present a novel cancer therapeutic strategy. A high throughput dropout screen was carried out to identify cyclic peptides that are synthetically lethal to MTAP-/- cells. Using Next Generation sequencing and data analysis, motifs were identified in the dropout screen for further testing. By generating a focused library and subsequent bio-panning, the 3.2 million members library was narrowed down to a small list of 29 cyclic peptides. Three cyclic peptides, CP25, CP18 and CP14 displayed a significant decrease of viability of MTAP-/-cells while not affecting the normal HCT116 cells. Conditional synthetic lethality considers the impacts of the tumour microenvironment on cells as well as genetic alterations. The core of a cancer tumour is often hypoxic, due to the large diffusion barrier that oxygen must cross to reach the core of the tumour. In response to hypoxia, the HIF-1α and HIF-1β subunits dimerise in the nucleus, forming the HIF-1 transcription factor. HIF-1 can trigger the expression of several hypoxic genes, including those implicated in angiogenesis and metastasis which are observed in aggressive tumours. The thesis presents the outcome of extensive hypoxia screening, which has identified six cyclic peptides consequently tested in viability experiments. A subset of these candidates has demonstrated a significant decrease in cell viability of hypoxicTRex293 cells which is an encouraging result for further investigation. 

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