Abstract
Lung cancer is the leading cause of cancer mortality worldwide, accounting for approximately 1.6 million deaths per annum. The most common form of lung cancer is non-small cell lung cancer (NSCLC), of which anaplastic lymphoma kinase (ALK) fusions occur with echinoderm microtubule-associated protein like-4 (EML4) in approximately 6% of cases, known as ALK+ NSCLC. The ALK tyrosine kinase inhibitor (TKI), alectinib, prolongs patient survival; however, resistance develops within a few years of treatment commencement. Co-targeting secondary oncogenic drivers such as Src homology region 2-containing protein tyrosine phosphatase (SHP2) is a potential strategy for improving efficacy and preventing emerging resistance. Given that the expression of SHP2 is ubiquitous, and ALK is restricted to cancer cells, the combination of ALK and SHP2 inhibitors may provide a way to limit synergistic cytotoxicity to cancer cells and minimise SHP2 toxicity. Therefore, it was proposed that adding SHP099, a SHP2 inhibitor, to alectinib, will increase the efficacy of alectinib and decrease the toxicity of SHP099 in ALK+ NSCLC cells.
The first section of this thesis examined the synergy of the combination of alectinib and SHP099 and the underlying mechanism of action in variant 1 and 3 ALK+ NSCLC monolayer cell lines. The results demonstrated that at low concentrations, the combination exhibited synergistic suppression of cell growth in both cell lines according to the Bliss combination index (CI) as a measure of drug interaction. The mechanism behind this synergy is likely due to the strong suppression of phosphorylated mitogen-activated protein kinase 1 (pERK), which induces a G1 phase cell cycle arrest from a decrease of cyclin D1. Furthermore, the variant 1 H3122 cells had an associated decrease of cyclin B1 and an increase of phosphorylated cyclin-dependent kinase 1 (pCDK1). In both variants, there was an increase in p27 expression and induction of intrinsic apoptosis, evidenced by increased Bcl-2 interacting protein (Bim) and cleaved caspase-3 expression.
The second section of this thesis focused on developing an in vitro semi-synthetic, high throughput 3D spheroid to allow testing of the combination in a higher model. The H3122 spheroid model underwent direct validation against an excised xenograft over time. A comparable percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive cells were observed in an inner core at each time point, suggesting apoptosis or necrosis; however, further investigations are required to determine the nature of these TUNEL-positive cells. This central core is an essential component to mimic the oxygen and nutrient gradients for the xenografts, as the presence of a hypoxic core decreases drug sensitivity. Investigation of the combination of alectinib and SHP099 in the spheroid model demonstrated synergistic suppression at low concentrations, with the decrease in cell viability being greater than the Bliss CI predicted.
The last section of this thesis investigated the development of an alectinib-resistant cell line and trialling the combination of alectinib and SHP099. At the lowest concentration, synergistic suppression occurred, greater than that predicted by the Bliss CI, indicating that this combination could benefit patients who have already developed alectinib resistance.
This thesis is consistent with the hypothesis that combining alectinib and SHP099 produces a synergistic suppression of variants 1, 3 and alectinib-resistant ALK+ NSCLC cells at low concentrations and, thus, can be a potential therapeutic strategy. Furthermore, this study identified differing responses regarding the mechanism of cell suppression between the two EML4-ALK variants; hence, it could influence potential treatment options for patients. Nevertheless, future investigations will further validate the spheroid model and delve into toxicity testing of this combination, in addition to combination testing with CDK1 or cyclin B1 inhibitors for improving the response of variant 3 EML4-ALK.