Combination Treatment with the GSK-3 Inhibitor 9-ING-41 and CCNU Cures Orthotopic Chemoresistant Glioblastoma in Patient-Derived Xenograft Models

Abstract

Resistance to chemotherapy remains a major challenge in the treatment of human glioblastoma (GBM). Glycogen synthase kinase-3β (GSK-3β), a positive regulator of NF-κB-mediated survival and chemoresistance of cancer cells, has been identified as a potential therapeutic target in human GBM. Our objective was to determine the antitumor effect of GSK-3 inhibitor 9-ING-41 in combination with chemotherapy in patient-derived xenograft (PDX) models of human GBM. We utilized chemoresistant PDX models of GBM, GBM6 and GBM12, to study the effect of 9-ING-41 used alone and in combination with chemotherapy on tumor progression and survival. GBM6 and GBM12 were transfected by reporter constructs to enable bioluminescence imaging, which was used to stage animals prior to treatment and to follow intracranial GBM tumor growth. Immunohistochemical staining, apoptosis assay, and immunoblotting were used to assess the expression of GSK-3β and the effects of treatment in these models. We found that 9-ING-41 significantly enhanced 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) antitumor activity in staged orthotopic GBM12 (no response to CCNU) and GBM6 (partial response to CCNU) PDX models, as indicated by a decrease in tumor bioluminescence in mouse brain and a significant increase in overall survival. Treatment with the combination of CCNU and 9-ING-41 resulted in histologically confirmed cures in these studies. Our results demonstrate that the GSK-3 inhibitor 9-ING-41, a clinical candidate currently in Investigational New Drug (IND)-enabling development, significantly enhances the efficacy of CCNU therapy for human GBM and warrants consideration for clinical evaluation in this difficult-to-treat patient population.

Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Treatment with GSK-3 inhibitor 9-ING-41 enhances the antitumor effect of CCNU and CPT-11 in an SC model of GBM6 PDX tumor. (A) Tumor proteins were extracted from fresh GBM PDX SC tumor tissues as indicated; tumor lysates were separated by SDS-PAGE (50 μg/well) transferred to PVDF membrane and probed with indicated antibodies. GS, glycogen synthase. (B) Serial tissue sections from GBM6 SC tumor were stained for GSK-3β and p-GS. Scale bar = 200 μm. (C-E) GBM6 PDX tumor pieces were engrafted SC to nude mice. Tumors were size matched, and mice were randomized into treatment groups (five mice per group). Vehicle (DMSO), 70 mg/kg 9-ING-41, 1 mg/kg CCNU (C), 5 mg/kg CPT-11 (D), or 1 mg/kg TMZ (E) was injected i.p. at indicated doses as shown by arrows (C-E, left panel). Mean tumor volumes are plotted; bars, SE. Mice were sacrificed 2 weeks after initiation of treatment, and the weight of resected tumors was determined (C-E, middle panel). Bar graphs: mean tumor weight; SE is indicated. Representative pictures of GBM6 PDX SC tumors from each group of animals (C-E, right panel).

Figure 2

Treatment with CCNU + 9-ING-41 leads to regression of intracranial GBM6 PDX tumors. (A) Kaplan-Meier survival analysis of treated mice bearing intracranial human GBM6 PDX-Tom-Luc tumors. GBM6-bearing mice were staged and randomized based on BLI. Mice were treated two times a week with vehicle control (DMSO; n = 5), 2 mg/kg CCNU (n = 5), 70 mg/kg 9-ING-41 (n = 5), and CCNU + 9-ING-41 (n = 5) as indicated. The median survival in the vehicle control, 9ING41, and CCNU groups was 30, 42, and 85 days, respectively. All of the 9-ING-41 + CCNU–treated animals were intentionally euthanized (censored) for histological analysis of brain at day 142 despite being healthy and luciferase-signal free. The combination therapy of CCNU and 9-ING-41 significantly prolonged survival of animals as compared to CCNU-treated group (P < .05). (B) Representative IVIS images of GBM6-bearing animals treated as indicated.

Figure 3

Treatment with CCNU + 9-ING-41 resulted in complete regression of intracranial GBM6 PDX tumor and recovery of mouse brain structures. CCNU (2 mg/kg) + 9-ING-41 (70 mg/kg) treatment was started in 3 weeks after intracranial transplantation of GBM6 PDX tumor. Mouse was treated by i.p. injections twice a week for 3 weeks. MR images were taken every week after the initiation of the treatment as indicated. Direct invasion of the tumor and significant deformation of olfactory bulb are indicated by red arrow. Green arrow indicates an absence of the tumor and a complete recovery of olfactory bulb and other brain structures by week 3 of the treatment. T, tumor.

Figure 4

Treatment with CCNU + 9-ING-41 leads to regression of intracranial GBM12 PDX tumors. (A) Kaplan-Meier survival analysis of treated mice bearing intracranial human GBM12 PDX-Tom-Luc tumors. Mice were staged and randomized based on BLI. Mice were treated two times a week with vehicle control (DMSO; n = 5), 5 mg/kg CCNU (n = 5), 70 mg/kg 9-ING-41 (n = 5), and CCNU + 9-ING-41 (n = 5) as indicated. The median survival in the vehicle control, 9-ING-41, and CCNU groups was 24, 24, and 26 days, respectively. Four of five 9-ING-41 + CCNU–treated animals were intentionally euthanized (censored) for histological analysis of brain at day 74 (m1618) and day 105 (m1603, m1616, m1617) despite being healthy and luciferase-signal free. The combination of CCNU and 9-ING-41 significantly prolonged survival of animals as compared to CCNU-treated group (P < .05). (B) Animal weight was measured weekly. Graph, mean animal weight; bars, SE. (C) Representative IVIS images of GBM12-bearing animals treated as indicated.

Figure 5

Treatment with CCNU + 9-ING-41 leads to regression of intracranial GBM12 PDX tumors and recovery of cachectic mice. (A) Kaplan-Meier survival analysis of treated mice bearing intracranial human GBM12 PDX-Tom-Luc tumors. Mice were staged and randomized based on IVIS imaging. Mice were treated with vehicle control (DMSO; n = 10) or 40 mg/kg 9-ING-41 (n = 10) daily for 2 weeks as indicated by red arrow. Next, mice were treated two times a week with DMSO (n = 5), 5 mg/kg CCNU (n = 5), and CCNU + 9-ING-41 (n = 10) as indicated by blue arrow. The median survival (from the start of vehicle, CCNU and CCNU + 9-ING-41 treatment) in the vehicle control and CCNU was 11 and 13 days, respectively. Six of ten 9-ING-41 + CCNU–treated animals were intentionally euthanized (censored) for histological analysis of brain at day 66 despite being healthy and luciferase-signal free. The combination of CCNU and 9-ING-41 significantly prolonged survival of cachectic animals as compared to CCNU-treated group (P < .05). (B) Representative IVIS images of GBM12-bearing animals treated i.p. with combination of 5 mg/kg CCNU and 40 mg/kg 9-ING-41 twice a week as indicated. (C) Animal weight was measured weekly. Graph, mean animal weight; bars, SE. (D) Representative IVIS images of GBM12-bearing animals treated i.p. with 5 mg/kg CCNU twice a week.

Figure 6

Treatment with CCNU + 9-ING-41 leads to regression of TMZ-resistant intracranial GBM12 tumors. (A) Mice were staged and randomized based on BLI. Mice were treated (red arrow) daily, Monday to Friday, with TMZ (n = 5; 50 mg/kg, oral administration) or TMZ + 9-ING-41 (n = 5; 40 mg/kg of 9-ING-41, i.p.) for 1 week. The treatment was continued (purple arrow) with CCNU (5 mg/kg, i.p., TMZ-treated group) and CCNU + 9-ING-41 (TMZ + 9-ING-41-treated group) twice a week for 4 weeks. (B) Kaplan-Meier survival analysis of GBM12-bearing mice treated as described in panel A. The median survival in the CCNU group was 87 days. Four of five CCNU + 9-ING-41–treated animals were intentionally euthanized (censored) for histological analysis of brain at day 130 despite being healthy and luciferase-signal free. Combination of CCNU and 9-ING-41 significantly prolonged survival of animals as compared to CCNU-treated group (P < .05).