ROS1 amplification mediates resistance to gefitinib in glioblastoma cells

Abstract

Glioblastoma (GBM) is the most aggressive brain tumor in adults and remains incurable despite multimodal intensive treatment regimens. The majority of GBM tumors show a mutated or overexpressed EGFR, however, tumors treated with tyrosine kinase inhibitors (TKIs) will inevitably recur highlighting the need to identify signalling pathways involved in GBM resistance to these drugs. To this end, we treated GBM cells that overexpress EGFR with increasing concentrations of gefitinib and isolated resistant clones. These resistant clones were subject to RNAseq and the expression of several genes was found to be upregulated. These genes are mainly tyrosine kinase receptors and include ROS1, DDR1 and PDGFRA and are known to control several downstream targets of EGFR. The upregulation of ROS1 and DDR1 was confirmed at the protein level by western blot. Treatment with a potent and highly specific pyrazole ROS1 inhibitor in ROS1 overexpressing clones led to a sensitization of these cells to low concentrations of gefitinib. Combined treatment with gefitinib and ROS1 inhibitor induces massive cell death by apoptosis following a prolonged S phase cell cycle arrest. Our current study led to the discovery of alternative pathways used by GBM cells to evade cell death following treatment with gefitinib and identifies new therapeutic targets to prevent GBM cell resistance to the drug.

Keywords: DDR1; EGFR; ROS1; gefitinib; tyrosine kinase inhibitors.

Figure 1. ROS1 and DDR1 mRNA is upregulated in gefitinib-resistant GBM cells

A. Outline of the experimental strategy used to isolate and characterize the gefitinib-resistant cells. B. A list of the most upregulated genes in the gefitinib-resistant clones. C. A ratio of ROS1 and GAPDH1 control and D. a ratio of DDR1 and GAPDH control.

Figure 2. ROS1 and DDR1 protein expression in gefitinib-resistant cells

To confirm that the increase in mRNA expression translates to an increase in protein expression, several clones were tested for the expression of ROS1 protein. A. Four resistant clones (RC) show the expression of different ROS1 fusion proteins (indicated by asterisk). The sensitive clone control (SC) did not show such fusions. B. Resistant clone #1 shows an overexpression of Vav 3, a target of ROS1 and a potential ROS1 ligand as well. C. The gefitinib-resistant cell pool was also tested for the upregulated expression of the DDR1 protein.

Figure 3. ROS1 inhibitor sensitizes gefitinib-resistant cells to the gefitinib

A. Structure of pyrazole ROS1 inhibitor. B. Gefitinib and ROS1 Inhibitor combined treatment of U87-EGFR clones that are either sensitive or resistant to gefetinib at 36 hrs post-treatment. C1, C3, C5 and C8 indicate the four resistant clones that overexpress ROS1 fusions.

Figure 4. Cell cycle profile of gefitinib-sensitive and gefitinib-resistant glioblastoma cell lines following treatment with gefitinib, pyrazole ROS1 inhibitor or a combination of both

A. Pyrazole ROS 1 inhibitor sensitizes gefitinib-resistant cells to gefitinib through a prolonged S phase checkpoint arrest followed by cell death by apoptosis as indicated by the accumulation of cells in sub-G1 phase of the cell cycle. B. Increased PARP cleavage in cells treated with ROS1 inhibitor and gefitinib. C. Inhibition of pAKT1 and p-p42MAPK proteins following treatment with gefitinib and ROS1 inhibitor.