Targeting IDH1-Mutated Malignancies with NRF2 Blockade

Abstract

Background: Neomorphic IDH1 mutations disrupt the redox balance by promoting reactive oxygen species (ROS) production. However, the mechanism by which IDH1-mutant cells maintain ROS homeostasis remains elusive. It is also not known whether reprogrammed ROS homeostasis establishes targetable vulnerability in IDH1-mutated cancers.

Methods: We investigated ROS homeostasis in wild-type (GSC827, GSC923, GSC627, and GSC711) and IDH1-mutated cells (IDH1R132C- and IDH1R132H-transduced U87, U251; MGG152, and TS603 cells). We analyzed the stability and transcriptional activity of NRF2 in IDH1-mutated cells. The oxidative DNA damage was analyzed using NRF2-targeting small interfering RNA. Moreover, we evaluated the effect of the NRF2 inhibitor brusatol in an IDH1-mutated subcutaneous xenograft nude mouse model (control group, n = 5; brusatol-treated group, n = 6). All statistical tests were two-sided.

Results: We showed that IDH1-mutated cells develop a dependency on the NRF2 antioxidative pathway. Genetic or pharmacologic blockade of NRF2 not only disrupted ROS homeostasis (mean [SD] ROS levels increased by 317 [42.1]%, P = .001, in IDH1R132C and by 286. 5 [48.7]%, P = .003, in IDH1R132H cells) but also enhanced oxidative DNA damage and decreased proliferation of IDH1-mutated cells. Brusatol selectively suppressed IDH1-mutated cancer progression in vivo (mean [SD] final tumor volume was 761.6 [391.6] mm3 in the control and 246.2 [215] mm3 in the brusatol-treated group, P = .02).

Conclusions: IDH1 mutation reprograms ROS homeostasis in cancer cells, which leads to dependency on the NRF2 antioxidant pathway for ROS scavenging. NRF2 blockade might be a novel therapeutic approach to treat malignancies with IDH1 mutation.

Figure 1.

Redox homeostasis in IDH1-mutated cells. A) Quantification of reactive oxygen species (ROS) levels by ROS-Glo H₂O₂ assay is shown for IDH1-mutated U251 cells (left panel) or doxycycline (DOX)-induced mutated-IDH1 expression U251 model (right panel). All groups were normalized to IDH1^WT dimethylsulfoxide group. N-acetylcysteine (NAC) was used as a ROS scavenger. B) Flow cytometry analysis of ROS level using CM-H₂DCFDA staining in IDH1-mutated U251 cells is shown. C) Confocal microscopy of mitochondrial ROS in IDH1-mutated cells was performed using MitoSOX staining (red). Cell nuclei were labeled with Hoechst 33342 (blue). MitoSOX signal is highlighted in the binary image (lower panel). Scale bar = 10 μm. D) Protein carboxylation analysis was done in lysate from IDH1-mutated U251 cells (upper panel). Coomassie blue staining was used as loading control (lower panel). E) Gene expression assay quantified ROS generating and scavenging genes from IDH1^WT and IDH1^R132H U251 cells. F) Immunoblot analysis for expression of NRF2 and ROS scavenging genes in IDH1-mutated glioma cells is shown. β-actin was used as loading control. G) Expression of NFE2L2 and NQO1 was measured by real-time polymerase chain reaction after 10–20 passages of DOX-induced mutated-IDH1^R132H expression in U251 cells. All groups were normalized to passage 0 group. H) Expression level of NFE2L2, HMOX1, and NQO1 were measured by real-time polymerase chain reaction after removing mutated-IDH1^R132H expression in U251 cells. All groups were normalized to DOX group. I) Cox regression analysis for the association between IDH1-mutation status and patient outcome (n = 366) is shown for lower-grade gliomas. P values were calculated using a two-sided Student t test. Error bars represent standard deviation. DMSO = dimethylsulfoxide; LGG = low-grade glioma; NAC = N-acetylcysteine.

Figure 2.

Activity of NRF2 in IDH1-mutated cells. A) Antioxidant response elements transcriptional activity was measured using luciferase reporter assay in IDH1-mutated U251 cells with the presence of exogenous reactive oxygen species (ROS) scavenger catalase (left panel) or N-acetylcysteine (right panel). All groups were normalized to IDH1^WT dimethylsulfoxide group. B) The affinity of NRF2 to the promoter region of HMOX1 and NQO1 was measured using chromatin immunoprecipitation quantitative polymerase chain reaction (PCR) assay in IDH1-mutated U251 cells. C) NRF2 protein half-lives in IDH1-mutated U251 cells were shown by cycloheximide pulse chase assay and Immunoblot (left panel). Quantification of Immunoblot band was performed using gray levels with Image J (right panel). β-actin was used as loading control. This experiment was repeated three times. D) NRF2 ubiquitination in IDH1-mutated U251 cells was shown by immunoprecipitation assay and Immunoblot. E) The mRNA levels of NFE2L2 and NQO1 were measured using real-time PCR with genetic silencing of NRF2 in IDH1^WT and IDH1^R132H U251 cells. F) ROS quantification using ROS-Glo H₂O₂ assay was performed with genetic silencing of NRF2 in IDH1-mutated U251 cells. G) Quantification of 8-hydroxydeoxyguanosine was performed using oxidative DNA damage ELISA assay in IDH1-mutated U251 cells at baseline level and with genetic silencing of NRF2 (siNRF2-1). H) DNA fragmentation was measured by electrophoresis in IDH1-mutated U251 cells with genetic silencing of NRF2. I) Cell proliferation was measured using bromodeoxyuridine (BrdU) incorporation assay in IDH1-mutated U251 cells with NRF2 silencing (siNRF2-1). BrdU was labeled with fluorescent-conjugated antibody (green). Cell nuclei were labeled with Hoechst 33342 (blue). Bar = 10 μm. J) Quantification of BrdU incorporation assay in Figure 2I using cell count with Image J was shown. For all the quantification of siRNA assay, all groups were normalized to IDH1^WT siControl group. P values were calculated using a two-sided Student t test. Error bars represent standard deviation. ARE = antioxidant response elements; 8-OH-dG = 8-hydroxydeoxyguanosine; CHX = cycloheximide; DMSO = dimethylsulfoxide; NAC = N-acetylcysteine.

Figure 3.

Effects of brusatol in NRF2 biology and reactive oxygen species (ROS) homeostasis. A) Antioxidant response elements transcriptional activity was measured using luciferase reporter assay in IDH1-mutated U251 cells after brusatol treatment. B) The affinity of NRF2 to the promoter regions of HMOX1 and NQO1 was measured using chromatin immunoprecipitation quantitative polymerase chain reaction assay quantifies in IDH1-mutated U251 cells with brusatol treatment. C) The expression of NRF2 downstream gene GCLC and NQO1 was measured using immunoblot in IDH1-mutated U251 cells after brusatol treatment. β-actin was used as loading control. D) NRF2 ubiquitination was measured by immunoprecipitation assay and immunoblot in IDH1-mutated U251 cells after brusatol treatment. E) Quantification of NRF2 half-lives were measured by cycloheximide pulse chase assay and immunoblot in IDH1-mutated U251 cells with brusatol treatment. This experiment was repeated three times. Data was quantified using gray levels with Image J. F) ROS quantification using ROS-Glo H₂O₂ assay was performed in IDH1-mutated U251 cells with brusatol treatment. G) Flow cytometry analysis using 2', 7'-dichlorodihydrofluorescein diacetate and MitoSOX staining was performed in IDH1-mutated U251 cells with brusatol treatment. All groups were normalized to IDH1^WT dimethylsulfoxide group. P values were calculated using a two-sided Student t test. Error bars represent standard deviation. ARE = antioxidant response elements; CHX = cycloheximide; CM-H₂DCFDA = 2', 7'-dichlorodihydrofluorescein diacetate; DMSO = dimethylsulfoxide; IB=immunoblotting.

Figure 4.

Effect of Brusatol to the genetic integrity in IDH1-mutated cells. A) 8-hydroxydeoxyguanosine level was measured using oxidative DNA damage ELISA assay in IDH1-mutated U251 cells with brusatol treatment. B) 8-oxoguanine (8-oxoG) level was measured using dot blot assay in IDH1-mutated U251 cells with brusatol treatment. C) DNA fragmentation was measured by comet assay in IDH1-mutated U251 cells with brusatol treatment (left panel). Quantification of the tail moments is shown (right panel). D) DNA fragmentation was measured by electrophoresis in IDH1-mutated U251 cells with brusatol treatment. E) Quantification of immunofluorescent staining was performed by mean fluorescent intensity of 8-oxoG (left panel) and ɣH2A.X (right panel) in brusatol treated U251 cells. F) Reactive oxygen species (ROS) level was measured using ROS-Glo H₂O₂ assay in U251 cells treated with brusatol and exogenous ROS scavenger catalase. G) DNA damage (ɣH2A.X expression) was measured using Immunoblot in IDH1-mutated U251 cells treated with brusatol and ROS scavenger catalase. β-actin was used as loading control. All groups were normalized to IDH1^WT dimethylsulfoxide group. P values were calculated using a two-sided Student t test. Error bars represent standard deviation. 8-OH-dG = 8-hydroxydeoxyguanosine; DMSO = dimethylsulfoxide.

Figure 5.

Cytotoxic effect of brusatol to IDH1-mutated cells. A) Dose-response curve of cell viability was measured by CCK8 assay in IDH1-mutated U251 cells with brusatol treatment. Data was fit to nonlinear regression. B) Cell apoptosis was measured by Annexin V/PI flow cytometry analysis in IDH1-mutated U251 cells with brusatol treatment. C) Quantification of apoptotic cells percentage in Figure 5B is shown. D) Cleaved poly (ADP-ribose) polymerase was measured using Immunoblot in IDH1-mutated U251 cells treated with brusatol. β-actin was used as loading control. E) Caspase 3/7 activity was measured using Caspase 3/7-Glo assay in IDH1-mutated U251 cells with brusatol treatment. Luminescence was measured and normalized to protein quantification. All groups were normalized to IDH1^WT dimethylsulfoxide group. F) Cell proliferation was measured using CCK-8 assay in IDH1-mutated U251 cells with brusatol treatment. G) Cell proliferation was measured using bromodeoxyuridine (BrdU) incorporation immunofluorescence staining in IDH1-mutated cells with brusatol treatment. BrdU was labeled with antibody (green). Cell nuclei were labeled with Hoechst 33342 (blue). Bar = 10 μm. H) Quantification of BrdU staining in Figure 5G is shown by cell counting with Image J. P values were calculated using a two-sided Student t test. Error bars represent standard deviation. DMSO = dimethylsulfoxide; PARP = poly (ADP-ribose) polymerase; PI = propidium iodide.

Figure 6.

Effect of brusatol to IDH1-mutated xenografts. A) Sphere formation assay was performed in brain tumor-initiating cell (BTIC) lines with brusatol treatment. (B) The quantification of spheres numbers in BTIC lines after culture. For each group, 7 to 14 regions of interest were quantified. C) The quantification of spheres diameter in BTIC lines after culture. For each group, 7 to 50 spheres were quantified. D) Sphere formation capability was measured by cell-limiting dilution assay in IDH1^WT GSC927 and IDH1^R132H TS603 cells with brusatol treatment. The 0.37 intercept was labeled in dash lines. E) Tumor growth curve of TS603 xenograft is shown. n = 5 for dimethylsulfoxide group, n = 6 for brusatol group. F) Tumor size and weight of TS603 xenograft is shown. Tumors were excised and weighed at the end of the experiment (25 days after treatment). G) The expression of NRF2, GCLC, and NQO1 was measured by immunohistochemistry staining in TS603 xenografts. Bar = 100 μm. H) The expression of tumor proliferation markers Ki67 and PCNA was measured by immunohistochemistry staining in TS603 xenografts. Bar = 100 μm. I) The level of DNA damage markers 8-oxoguanine and ɣH2A.X, as well as TUNEL staining, was measured by immunohistochemistry staining in TS603 xenografts. Bar = 100 μm. P values were calculated using a two-sided Student t test. Error bars represent standard deviation. 8-oxoG = 8-oxoguanine; Bru = brusatol; DMSO = dimethylsulfoxide.

Figure 7.

Targeting NRF2 antioxidant pathway for IDH1-mutated cancers. IDH^WT cells exhibit normal reactive oxygen species (ROS) burden. NRF2 is recognized by KEAP1 and constitutively removed through proteasomal degradation. In IDH1^R132H cells, elevated ROS burden leads to NRF2 stabilization, nuclear translocation, and transcriptional activation of antioxidant genes. NRF2 downstream genes not only assist ROS homeostasis in IDH1-mutated cancer but also serve as potential resistance mechanism for chemotherapies.