Investigating the role of driver mutations in mesothelioma using proteogenomics

Abstract

Pleural mesothelioma (PM) is a cancer of the mesothelial cells lining the pleural cavity of the lungs. Although rare, it is an aggressive cancer, and currently one of the relatively most deadly cancers after diagnosis, with a low median survival of 8-14 months following diagnosis. Current treatment strategies are therefore essentially palliative care, as there is a lack of effective therapeutics for PM. Though there has been some recent progress in treating certain histological subtypes of PM, there are no curative therapies. This highlights a need for a deeper understanding of the disease, in order to inform future therapeutic developments. PM is mainly linked to exposure to asbestos – a group of naturally occurring mineral fibres found to be fire retardant and insulating, and consequently with a wide range of applications. Chronic inflammation caused due to frustrated phagocytosis in immune cells and prolonged cellular stress owing to the bio-persistence and structure of the asbestos fibres leads to malignant transformation of mesothelial cells, albeit usually after a long latency period of 20-40 years. Additionally, nano fibres currently in use in a wide range of applications have been linked to pathogenesis of mesothelioma like disease in mice models, emphasising the need to better understand the molecular mechanism leading to PM for developing improved therapeutics for patients.

Prior genomic studies investigating predominant asbestos induced genetic alterations in PM have identified BAP1 and NF2 as tumour suppressor genes with loss of function mutations in a large proportion of PM patients. Currently, there is a lack of understanding on the deeper molecular effects of the loss of function of NF2 and BAP1, highlighting the need to investigate the downstream effects of their loss of function in mesothelioma. BAP1 is a deubiquitylase enzyme while NF2 is an upstream regulator of the hippo pathway. The hippo pathway is a signalling pathway that controls differentiation, proliferation, and migration of cells, and where dysregulation is commonly linked to abnormal cell growth and cancer. The lack of effective therapeutics for PM, and prominent mutations of two distinct tumour suppressor genes in patients presents an opportunity to potentially stratify therapeutics based on the mutational status of patients.

This project aimed to investigate the role of loss of NF2 and BAP1 in PM, by using an established and clinically relevant human mesothelial cellular model with NF2 or BAP1 knockout (KO). The investigation was done employing a novel -omics approach called proteogenomics, integrating proteomic and mRNA level expression data to gain insights on molecular effects of NF2 and BAP1 loss.

The investigation revealed proteomic dysregulation linked to NF2 and BAP1 loss in the cellular model, with insights on pathways involved obtained and qualification of dysregulated proteins for further investigation as potential therapeutic targets. Additionally, on treatment with inhibitors for the YAP/TAZ (Hippo pathway effectors) activated TEAD transcription factors, reduction in anchorage independent growth was also seen in both NF2 and BAP KO cellular models.

Further, effects of different culture conditions were tested, where to create conditions similar to the tissue microenvironment, cells were grown in 3D (as spheroids). Proteomics on 3D cultured cellular models revealed mostly conserved effects of NF2 and/or BAP1 loss, while proteome level changes linked to specific molecular pathways were observed on 3D cell culture. Additionally, a new cellular model with double KO (dKO) of NF2 and BAP1 was also generated to represent a patient subgroup where both NF2 and BAP1 incur loss of function mutations in patients.

The thesis lays out a basis for future work, where identified candidate targets from proteomics can be investigated, along with additional assays studying interactions with immune cells such as macrophages.