Synergistic Effects of Crizotinib and Temozolomide in Experimental FIG-ROS1 Fusion-Positive Glioblastoma

Abstract

Glioblastoma (GB) is the most common malignant brain tumor. Drug resistance frequently develops in these tumors during chemotherapy. Therefore, predicting drug response in these patients remains a major challenge in the clinic. Thus, to improve the clinical outcome, more effective and tolerable combination treatment strategies are needed. Robust experimental evidence has shown that the main reason for failure of treatments is signal redundancy due to coactivation of several functionally linked receptor tyrosine kinases (RTKs), including anaplastic lymphoma kinase (ALK), c-Met (hepatocyte growth factor receptor), and oncogenic c-ros oncogene1 (ROS1: RTK class orphan) fusion kinase FIG (fused in GB)-ROS1. As such, these could be attractive targets for GB therapy. The study subjects consisted of 19 patients who underwent neurosurgical resection of GB tissues. Our in vitro and ex vivo models promisingly demonstrated that treatments with crizotinib (PF-02341066: dual ALK/c-Met inhibitor) and temozolomide in combination induced synergistic antitumor activity on FIG-ROS1-positive GB cells. Our results also showed that ex vivo FIG-ROS1+ slices (obtained from GB patients) when cultured were able to preserve tissue architecture, cell viability, and global gene-expression profiles for up to 14 days. Both in vitro and ex vivo studies indicated that combination blockade of FIG, p-ROS1, p-ALK, and p-Met augmented apoptosis, which mechanistically involves activation of Bim and inhibition of survivin, p-Akt, and Mcl-1 expression. However, it is important to note that we did not see any significant synergistic effect of crizotinib and temozolomide on FIG-ROS1-negative GB cells. Thus, these ex vivo culture results will have a significant impact on patient selection for clinical trials and in predicting response to crizotinib and temozolomide therapy. Further studies in different animal models of FIG-ROS1-positive GB cells are warranted to determine useful therapies for the management of human GBs.

Keywords: ALK; FIG; ROS1; c-Met inhibitor; crizotinib; glioblastoma; temozolomide.

Figure 1

Analysis of pre- and postoperative T1-weighted MRI with gadolinium (A), IHC (A), and Western blots (B) of collected FIG-ROS1 ± GB tumor samples. Tissue samples from the tumor regions of 15 patients reveal distinct FIG-ROS1, p-ALK, and p-Met expression patterns. GB tumor (white arrows; A) stained for astrocytic (GFAP) and proliferation (Ki-67) markers (Ki-67-red; GFAP-green; DAPI-blue). (B) Representative Western blots for FIG-R0S1, p-ALK, p-Met, and GAPDH are shown in the surgically collected GB tumor samples.

Figure 2

Synergistic effects of crizotinib and temozolomide in FIG-ROS1 fusion-positive GB cells. Quantification analyses of viabilities (MTT assay), ROS production (DCF-DA fluorometric assay), and caspase-3 activity (colorimetric assay) in in vitro (A) and ex vivo (C) FIG-ROS1-positive GB. Cell death was also confirmed by flow cytometry (B) in in vitro (U118) and DNA fragmentation (D) in ex vivo (slice culture) models. Treatments: Con, crizotinib, temozolomide, and crizotinib + temozolomide (^*P < 0.05 and ^**P < 0.01).

Figure 3

Combinatorial blockade of FIG, ROS1, p-ALK, and p-Met by crizotinib and temozolomide suppresses GB anti-apototic gene and activates pro-apoptotic gene. Representative Western blots for FIG, ROS1, p-ALK, p-Met, survivin, p-Akt, Mcl-1, and Bim are shown in in vitro (A) and ex vivo (D) models. Quantification of blot signals relative to GAPDH confirmed significant downregulation of FIG, ROS1, p-ALK, p-Met, survivin, p-Akt, and Mcl-1 concurrent with a significant activation of Bim in in vitro (U118) (B, C) and in ex vivo (slice culture) (E, F) models. Data taken from FIG-ROS1-positive U118 (in vitro) and 15 GB (ex vivo) samples. Treatments: Con, crizotinib, temozolomide, and crizotinib + temozolomide (^*P < 0.05 and ^**P < 0.01).

Figure 4

Partial effects of crizotinib and temozolomide on FIG-ROS1-negative GB cells and tissue slices. Quantification analysis of viabilities (MTT assay) and caspase-3 activity (colorimetric assay) in in vitro (U87MG: A) and ex vivo (slice cultures: B) FIG-ROS1-negative GB. Data taken from FIG-ROS1-negative U87MG (in vitro) and four GB samples. Treatments: Con, crizotinib, temozolomide, and crizotinib + temozolomide (^*P < 0.05).