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, and within this, ALK mutations occur in approximately 6% of cases worldwide. The first-generation ALK inhibitor crizotinib had great success in prolonging ALK-positive lung cancer patient survival. However, resistance to crizotinib typically develops in within a year of treatment. One mechanism by which resistance can occur is through activation of ‘bypass’ kinase signalling pathways. It has been previously determined that phosphorylated Src, a downstream mediator of ALK, is upregulated in a crizotinib resistant ALK-positive cell line. Therefore, this study aimed to investigate the effect of combining crizotinib with the Src inhibitor dasatinib in crizotinib-naïve (H3122) and crizotinib-resistant (CR-H3122) ALK-positive non-small cell lung cancer cells as a treatment strategy to overcome acquired crizotinib resistance.
In cell culture, it was first confirmed that CR-H3122 cells were significantly less sensitive to crizotinib than H3122 cells using cell viability assays (p < 0.01). By contrast, the sensitivity of CR-H3122 cells to dasatinib was mildly increased (p < 0.05); therefore, no cross-resistance to dasatinib had occurred. Combination drug application significantly inhibited the growth of CR-H3122 (p < 0.0001) cells with there being synergy at lower concentrations and additivity at higher concentrations. However, the combination in the H3122 cells had additivity at lower concentrations but was only more effective than crizotinib alone at decreasing the cell viability (p < 0.001).
Western blot with densitometry analysis determined that there was significant suppression of phosphorylated ALK/ALK levels by crizotinib alone and in combination with dasatinib in H3122 cells (p < 0.02) but not with dasatinib alone (p > 0.05). Also, phosphorylated ALK/ALK levels in CR-H3122 cells were significantly decreased by crizotinib alone and the drugs in combination (p < 0.01) but not from dasatinib alone (p > 0.05), despite being crizotinib-resistant. The opposite was the case for phosphorylated Src/Src, with levels being significantly reduced by dasatinib alone and the drugs in combination in both the CR-H3122 (p < 0.001) and H3122 (p < 0.05) but not by crizotinib alone (p > 0.05). Therefore, it can be concluded that crizotinib and dasatinib are acting on independent molecular targets.
These results are consistent with the hypothesis that the combination of ALK and Src inhibition can be more effective than the individual drugs at decreasing the viability of crizotinib resistant ALK-positive lung cancer cells, through acting on different molecular targets and therefore may aid in overcoming crizotinib resistance, both as an upfront treatment option and as a possible treatment following progression on ALK inhibitors alone.