Combination of ALK and MEK inhibitors for the treatment of ALK-positive non-small cell lung cancer
Anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer (NSCLC) most commonly arises through Echinoderm microtubule-associated protein like 4 (EML4)-ALK chromosomal fusion and is associated with younger aged patients with non-smoking or light smoking history. Patients harbouring EML4-ALK fusion oncoprotein are highly sensitive to ALK-tyrosine kinase inhibitors (TKIs). Crizotinib, a first generation ALK-TKI, was approved by the Food and Drug Administration (FDA) for the treatment of ALK-positive NSCLC patients. Crizotinib has demonstrated superior efficacy compared to standard platinum-based chemotherapy with longer progression-free survival and a higher objective response rate. However, the clinical benefit of crizotinib (and other ALK inhibitors) is limited due to the development of acquired resistance typically within a year of therapy. Mechanism of resistance includes copy number gain, secondary mutations in ALK, or activation of bypass signalling pathways Thus, this study investigated the possibility of improving the efficacy of ALK inhibition, and overcoming resistance, by employing the combined effect of ALK inhibitor, crizotinib and MEK inhibitor, selumetinib in both crizotinib naïve (H3122) and crizotinib resistant (CR-H3122) ALK-positive non-small cell lung cancer cells. The combination of crizotinib and selumetinib significantly decreased the cell viability of both H3122 and CR-H3122 cells. The mechanism behind this synergistic effect of combination treatment was due to the marked suppression of RAS/MAPK signalling pathway that led to a decrease in cell proliferation and increase in apoptosis. Cell cycle analysis showed that the combination treatment significantly increased the percentage of cells in the G1 phase by ≥ 8 % compared to either single drug treatment. And this was mediated by an increase in expression of p27 (≥ 1.2-fold) and a decrease in cyclin D1 expression (≥ 80%) by the combination treatment compared to either of single drug treatment. Similarly, there was a significant increase in the percentage of apoptotic cells (≥ 17%) with combination treatment compared to either of single drug treatment. The drug combination elicited greater than 3-fold increase in expression of Bim compared to crizotinib alone that in turn upregulated the expression of cleaved caspase and cleaved PARP. Furthermore, the efficacy of combination treatment in xenograft model of ALK-positive NSCLC was investigated. Results showed that crizotinib (25 mg/kg) and selumetinib (25 mg/kg) decreased the tumour volume by 52 % and 59 %, respectively compared to vehicle control. Interestingly, their combination further markedly reduced tumour volume compared to both crizotinib and selumetinib monotherapy. Moreover, the preclinical toxicity study on Balb/c mice demonstrated that both the single and combined treatment of crizotinib and selumetinib were non-toxic at the dose of 25 mg/kg with values of alanine transaminase (ALT) (< 80 U/L) and creatinine ( < 2 mg/dL) within the normal range. Also, the combination treatment did not modulate the activity of the two drugs major metabolizing enzyme CYP3A. In summary, the finding in this thesis supports the hypothesis that marked inhibition of tumour growth can be obtained by the combination of ALK inhibitor with MEK inhibitor and thus can be a potential strategy in the treatment of ALK-positive NSCLC. This study further raises the possibility of gaining the benefit of combination treatment by the use of a sufficient dose of MEK inhibitor alone in crizotinib resistant NSCLC. In addition to this, the study identifies Bim, PARP and cyclin dependent kinase (CDK) as the druggable target for possible triple drug therapy.
Advisor: Ashton, John; Rosengren, Rhonda
Degree Name: Doctor of Philosophy
Degree Discipline: Department of Pharmacology and Toxicology
Publisher: University of Otago
Keywords: Lung cancer; ALK-positive NSCLC; ALK inhibitor; MEK inhibitor; Crizotinib; Selumetinib; Combination treatment
Research Type: Thesis