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Title: Novel therapeutic strategies for targeting EGFR mutated non-small cell lung cancer
Author: Drobnitzky, Neele
ISNI:       0000 0004 7232 0740
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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Background: NSCLC patients with EGFR M+ tumours have marked clinical responses to EGFR-TKIs. Most patients experience tumour regrowth, as drug resistance emerges through on-target secondary EGFR-mutations or other activating mutations that reactivate the downstream signalling. However, a significant number of patients become drug resistant through unknown mechanisms of resistance and these currently represent an area of high unmet clinical need. In this work, we aim to understand biological responses to sustained EGFR inhibition in EGFR mutated NSCLC cells, to generate EGFR-TKI resistant cell lines, and to utilise these resistant cell lines to identify novel treatment strategies to overcome or prevent EGFR-TKI resistance. Methods: We used human NSCLC cell lines with activating EGFR mutations commonly found in the clinic and treated them with both 1st and 3rd generation EGFR-TKIs (gefitinib and AZD9291, respectively). We also utilised 3D spheroids and in vivo tumour xenograft growth in both subcutaneous and orthotopic settings. Cell lines were analysed by western blotting, RPPA and RNAseq and a bioactive small molecule library was used to identify combination targets. Key findings: Our data demonstrate that EGFR mutant cells respond to EGFR-TKI treatment by acute changes in intracellular signalling, reduction in cell migration and proliferation, and the induction of apoptosis. Thereafter, surviving cells re-engage signalling and a resistant cell population resumes growth even in the presence of high concentrations of the drug. Broadly equivalent responses are seen in 2D, 3D and in in vivo models. Resistant cell lines included those both with (PC9GS and PC99291S) and without (HCC827GS and H19759291S) known resistance-conferring mutations (EGFR T790M, NRAS Q61R). Our screening approach using a bioactive small molecule library identified IGF-1R as a promising target for preventing and overcoming resistance to EGFR-TKIs in EGFR-TKI sensitive H1975 and EGFR-TKI resistant H19759291S cell lines, respectively. The effects on prevention of emergence of resistance were apparent in 2D and 3D, though these findings need to be confirmed in in vivo models. Of particular interest was the identification of AURK via our screen for targets for overcoming resistance and inhibitors of AURK selectively inhibited the growth of EGFR-TKI resistant cells in the presence but also absence of EGFR-TKIs. The underlying mechanisms for these newly arisen sensitivities remain to be determined. Conclusions: Sustained exposure of EGFR M+ NSCLC cells in vitro to EGFR-TKIs identifies three phases of drug response; sensitive, tolerant and resistant, associated with distinct phenotypic responses, culminating in the emergence of a highly resistant population of cells. Combination treatment of EGFRTKIs with IGF-1R inhibitors appears to be an attractive approach for overcoming and preventing resistance if suitable biomarkers were available for patient selection. AURK-inhibition is a novel and potentially more broadly applicable approach to overcoming and potentially preventing EGFR-TKI resistance. Further validation work is required to demonstrate the in vivo activity and therapeutic potential of these combinations.
Supervisor: Ryan, Anderson Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available