Investigating the mechanisms of acquired resistance to ALK inhibitors in EML4-ALK-driven lung cancer

Abstract

Targeted therapies have had a great impact on the management of oncogene-driven non-small cell lung cancer, where a subset of lung adenocarcinomas is driven by the EML4-ALK translocation. While ALK inhibitors such as crizotinib or alectinib lead to excellent initial responses in the clinic, the majority of patients show disease progression due to the development of acquired resistance. Mutations in the ALK kinase domain occur in a significant percentage of patients and lead to poor ALK inhibition and therefore, drug resistance. Parallel pathway alterations also significantly contribute to the development of acquired resistance. In this thesis, the aim was to gather new insight into the mechanisms of development of resistance, as well as evaluate the role of non-coding RNAs in differential drug response. EML4-ALKmut cell lines treated long-term with ALK inhibitors were used as models of acquired resistance. Data from RNA sequencing & subsequent protein analysis evidenced an upregulation of CDK1, CDK6, cyclin B1, and cyclin E1. Treatment of cells with the pan-CDK inhibitors alvocidib and dinaciclib, as well as the CDK7/12 inhibitor THZ1 led to decreased cell proliferation as well as induction of apoptosis. Furthermore, there was specificity of these compounds for EML4-ALK cells compared with normal epithelial cells or compared with other lung cancer cell lines of different genetic background. Mechanistically, apoptosis was evidenced through the downregulation of the anti-apoptotic proteins Survivin and MCL-1, initiated by transcriptional inhibition. These data were translated in vivo, where treatment of a mouse xenograft model of crizotinib & alectinib resistance with alvocidib led to delayed tumour growth. In addition, the role of the non-coding genome was examined. Through transcriptomic analysis, it was found that crizotinib-resistant cells exhibit upregulation of the oncogenic miR-25 and miR-30c and the tumour suppressing miR-149 and miR-103. It was further shown that CDK6 is an indirect target of miR-149 and a direct target of miR-103. The upregulation of miR-25 and miR-30c was clinically relevant since some patients who developed resistance to ALK inhibitors in the clinic had increased levels of circulating miR-25 and miR-30c in their plasma. Taken together, these results suggest a multifactorial development of resistance to ALK inhibitors, which can be targeted by exploiting a vulnerability of EML4-ALKmut cells to transcriptional inhibition. Through these findings, clinical testing of CDKi compounds could lead to the addition of new therapeutics to the armamentarium intended for a cohort of patients with very limited treatment options.

Date of Award	31 Dec 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Angeliki Malliri (Supervisor) & Michela Garofalo (Supervisor)