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Title: Novel therapeutic targets in NSCLC resistance to Erlotinib
Author: Chater, Emily
ISNI:       0000 0004 6423 7269
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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Approximately 10% of non-small cell lung cancers (NSCLCs) have an activating mutation within the kinase domain of epidermal growth factor receptor (EGFR). Competitive tyrosine kinase inhibitors (TKIs), such as Erlotinib and Gefitinib, have been developed to therapeutically exploit this. These compounds have shown excellent clinical responses in approximately 77% of patients with EGFR-mutant driven lung cancer. However, despite positive initial response, patients develop resistance, and this is often attributed (50% of cases) to the T790M secondary mutation in the ATP binding pocket of EGFR. This mutation increases the receptors affinity for ATP which competes out reversible first-generation inhibitors such as erlotinib and gefitinib. This thesis aims to better understand the underlying mechanisms involved in the resistance to this compound. Changes in metabolism have frequently been associated with tumour progression and drug resistance in many cancer types. Here, to identify novel resistance mechanisms, 1H-NMR metabolic profiling was performed on EGFR-driven NSCLC cells with and without, the additional T790M-EGFR resistance mutation. This study identified both glutathione (GSH) and phosphatidylcholine metabolism as two key pathways perturbed in erlotinib-resistant cells. Interestingly, 1H-NMR revealed lower levels of both oxidised and reduced forms of GSH in erlotinib-resistant cell lines and this was verified by colorimetric assays. This change correlated with reduced mRNA expression of GSH synthesizing enzymes in resistant cell lines. Increasing cellular GSH levels by siRNA-mediated targeting of GSH degrading enzymes sensitised erlotinib-resistant cells to erlotinib treatment. In addition, the use of GSH synthesizing enzyme inhibitors such as Buthionine Sulfoximine (BSO), which targets the gamma-glutamylcysteine synthetase (GCS, GSH synthesising enzyme) and degrading enzyme inhibitors such as Ethacrynic acid (EA) (inhibits glutathione S-transferase) increased and decreased resistance to erlotinib, respectively. EA also showed promising results in vivo as BALB/c nude mice, injected subcutaneously with PC9ER cells, showed increased sensitivity to erlotinib treatment when co-administered with EA. Furthermore, we demonstrated that mutant EGFR signalling can modulate the transcription of GSH enzymes likely via changes in the regulation of the transcription factor, Nuclear Factor (Erythroid-Derived 2)-Like 2 (NRF2). Targeting NRF2 using siRNAs decreased GSH abundance and increased erlotinib resistance. However, at present we do not understand how raised GSH levels might cause resistance to erlotinib in lung cancer cells expressing activated T790M-EGFR. To discover other differences between erlotinib-resistant and sensitive cells, we employed reverse phase protein array (RPPA) and, following DAVID bio-informatics analysis, 4 identified changes in both beta-oxidation and ataxia telangiectasia mutated kinase (ATM)/Ataxia Telangiectasia and Rad3-related kinase (ATR) cell cycle checkpoint proteins. Changes in these signalling molecules were confirmed by Western blotting. Yet, only targeting the ATR pathway affected the resistance to erlotinib in PC9ER cell lines. Indeed, inhibition of WEE1 with the compound MK1775 may hold promise as a treatment for patients with erlotinib-resistant NSCLC. In short, this work has identified GSH degrading enzymes and WEE1 as novel targets for reversing erlotinib resistance in EGFR-driven NSCLC. However, further investigation is needed to understand the mechanisms involved.
Supervisor: Pardo, Olivier Sponsor: Association for International Cancer Research (AICR)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral