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. 2023 May 4;25(5):857-870.
doi: 10.1093/neuonc/noac242.

A novel compound EPIC-0412 reverses temozolomide resistance via inhibiting DNA repair/MGMT in glioblastoma

Jixing Zhao  1 , Shixue Yang  1 , Xiaoteng Cui  1 , Qixue Wang  1 , Eryan Yang  1 , Fei Tong  1 , Biao Hong  1 , Menglin Xiao  2   3 , Lei Xin  2   3 , Can Xu  2   3 , Yanli Tan  4   3 , Chunsheng Kang  1
Affiliations

A novel compound EPIC-0412 reverses temozolomide resistance via inhibiting DNA repair/MGMT in glioblastoma

Jixing Zhao et al. Neuro Oncol. .

Abstract

Background: Temozolomide (TMZ) resistance has become an important obstacle affecting its therapeutic benefits. O6-methylguanine DNA methyltransferase (MGMT) is primarily responsible for the TMZ resistance in Glioblastoma multiforme (GBM) patients. In addition, active DNA damage repair pathways can also lead to TMZ resistance. Here, we reported a novel small-molecule inhibitor EPIC-0412 that improved the therapeutic efficacy of TMZ by inhibiting the DNA damage repair pathway and MGMT in GBM via epigenetic pathways.

Methods: The small-molecule compound EPIC-0412 was obtained through high-throughput screening. RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), and chromatin immunoprecipitation (ChIP) assays were used to verify the effect of EPIC-0412. Co-immunoprecipitation (Co-IP) was used to elucidate the interactions of transcription factors at the MGMT promoter region. Animal experiments using a mouse model were performed to verify the efficacy of EPIC-0412 in sensitizing GBM cells to TMZ.

Results: EPIC-0412 physically interrupts the binding of HOTAIR and EZH2, leading to the upregulation of CDKN1A and BBC3, causing cell cycle arrest and apoptosis in GBM cells. EPIC-0412 inhibits DNA damage response in GBM cells through the p21-E2F1 DNA damage repair axis. EPIC-0412 epigenetically silences MGMT through its interaction with the ATF3-p-p65-HADC1 axis at the MGMT promoter region. The application of EPIC-0412 restored the TMZ sensitivity in GBM in vivo experiments.

Conclusion: This study discovered a small-molecule inhibitor EPIC-0412, which enhanced the chemotherapeutic effect of TMZ by acting on the p21-E2F1 DNA damage repair axis and ATF3-p-p65-MGMT axis, providing evidence for combining epigenetic drugs to increase the sensitization toward TMZ in GBM patients.

Keywords: DNA repair; MGMT; glioblastoma; small-molecule inhibitor; temozolomide.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Design and development of EPIC-0412. (A) Drug screening process based on the lead-molecule AQB. (B) IC50 assay for all candidates in GBM and breast cancer cell lines, data representing a mean of 3 independent experiments. (C) Molecular formula of EPIC-0412 and its interaction with HOTAIR-EZH2. (D) RIP-qPCR of cells treated with DMSO, AQB (20 µM), or EPIC-0412(5 µM) for 48 h. (E) ChIRP-western blotting to detect EZH2 level bound to HOTAIR after treatment with DMSO, AQB (20 µM), or EPIC-0412 (5 µM) for 48 h. To help eliminate non-specific signals two different pools of probes complementary to HOTAIR are used (even and odd probe sets). (F) Tumor-bearing mice were treated with DMSO, AQB (50 mg/kg), or EPIC-0412 (15 mg/kg) by oral gavage. (G) Representative bioluminescence images of mice (n = 6). (H) Bioluminescence quantitative analysis of all treatment groups. P value was determined by two-way ANOVA. (I) Kaplan–Meier survival curve of nude mice. P was determined by Log–rank test or Gehan–Breslow–Wilcoxon test. (J) Representative H&E staining images of tumor volume in nude mice. Scale bar, 1 mm. Data are represented as mean ± standard deviation (s.d.); n = 3 independent experiments. ***P < .001, **P < .01, *P < .05.
Figure 2.
Figure 2.
EPIC-0412 causes apoptosis by upregulating BBC3. (A) Top 10 gene sets from GO-BP analysis of overlapping genes. (B) Heatmap of gene set-positive regulation of cell death. (C) The qRT-PCR assay of BBC3 mRNA level after treatment with AQB or EPIC-0412. (D) Western blot of PUMA, Caspase-3/7, and cleaved-Caspase-3/7 in cells treated with AQB or EPIC-0412. (E and F) The effect of AQB and EPIC-0412 on cell apoptosis were analyzed by flow cytometry. In (C–E) cells were treated with 80 µM of AQB or 20 µM of EPIC-0412 for 48 h. (G) IF images of cleaved-Caspase-3/7 in TBD0220 xenografts from DMSO, AQB- or EPIC-0412-treated mice. Scale bar, 100 µm. Data are represented as mean ± s.d.; n = 3 independent experiments. ***P < .001, **P < .01, *P < .05.
Figure 3.
Figure 3.
EPIC-0412 causes cell cycle arrest via upregulating CDKN1A. (A) Heatmap analysis of gene set-negative regulation of cell population proliferation. (B) The CDKN1A mRNA level in cells treated with AQB or EPIC-0412. (C) Protein levels of p21 and its downstream modules in AQB- or EPIC-0412-treated cells. (D) Rb, E2F1, and the downstream targets were evaluated by WB. (E) Cell cycle detection of GBM cells. (F and G) Colony formation assay of GBM cells. (H) IHC of tumor tissues from TBD0220 xenografts revealing p21 and Ki-67 expressions. Scale bar, 50 µm. In (B–F) cells were treated with 80 µM of AQB or 20 µM of EPIC-0412 for 48 h. Data represented as mean ± s.d.; n = 3 independent experiments. ***P < .001, **P < .01, *P < .05.
Figure 4.
Figure 4.
EPIC-0412 enhances the sensitization of GBM cells to TMZ via the p21-E2F1 DNA repair axis. GSEA analysis of DEGs between EPIC-0412-group and DMSO-group using (A) GO-BP and KEGG gene sets (B) Hallmark gene sets. (C) Combination matrices of cell inhibition and synergy scores by EPIC-0412 and TMZ. Data represent a mean of 3 independent experiments. (D) mRNA levels of DDR genes in cells treated with 20 µM of EPIC-0412 for 48 h. (E) mRNA levels of DDR genes in cells treated with 20 µM EPIC-0412 or shE2F1 for 48 h. (F) ChIP at promotor regions of DDR genes in cells treated with 20 µM EPIC-0412 for 48 h using an anti-E2F1 antibody. (G) Western blotting of γ-H2AX and E2F1-downstream modules. (H) IF images of γ-H2AX in TBD0220 and U87-MG cells. Scale bar, 40 µm. (I) Western blot analysis of Caspase-3/7 and cleaved-Caspase-3/7. (J) Colony formation assays of TBD0220 and U87-MG. In (G–J), cells were treated with 200 µM of TMZ, 20 µM of EPIC-0412, or 200 µM of TMZ + 20 µM of EPIC-0412. Data represents mean ± s.d.; n = 3 independent experiments. **P < .01, ***P < .001, ****P < .0001, ns = not significant.
Figure 5.
Figure 5.
EPIC-0412 epigenetically silences MGMT expression through ATF3-p-p65-HADC1 axis. (A) Western blotting of p21 and its downstream proteins in T98G cells. (B) qRT-PCR analysis of E2F1-targeting mRNA levels in T98G cells. (C) Western blot analysis of γ-H2AX and E2F1 downstream DNA damage repair modules in T98G cells. (D) IF images of γ-H2AX in T98G cells. Scale bar, 30 µm. (E) Relative MGMT mRNA levels and (F) Relative ATF3 mRNA levels in T98G cells treated with indicated concentrations of EPIC-0412 for 48 h or 20 µM of EPIC-0412 for indicated hours. (G) Western blot analysis of UBXN1, p65, ATF3, and MGMT. (H) ChIP at the ATF3 promotor region in T98G cells using an anti-H3K27me3 antibody. (I) Co-IP assays were performed with anti-p-p65 antibody, followed by immunoblotting with p-p65, HDAC1, and ATF3 antibodies. (J) ChIP at the MGMT promotor region in T98G cells using an anti-H3K27ac antibody. (K) IF images of O6-MetG in T98G cells. Scale bar, 100 µm. In (A, B, H, I, and J), T98G cells were treated with 20 µM of EPIC-0412 for 48 h. In (C, D and K) T98G cells were treated with 800 µM of TMZ, 20 µM of EPIC-0412 or 800 µM of TMZ + 20 µM of EPIC-0412 for 48 h. Data are represented as mean ± s.d.; n = 3 independent experiments. ****P < .0001, ***P < .001, **P < .01, *P < .05, ns = not significant.
Figure 6.
Figure 6.
EPIC-0412 increases TMZ sensitivity in vivo. (A) Tumor-bearing nude mice were treated with DMSO, TMZ (5 mg/kg), EPIC-0412 (7.5 mg/kg) or TMZ (5 mg/kg) + EPIC-0412 (7.5 mg/kg) by gavage. (B) Bioluminescence images from representative mice of all groups. (C) Quantification of bioluminescence intensity from all groups. P, two-way ANOVA. (D) Kaplan–Meier survival curve of nude mice. P, Log-rank test. (E) Representative images of H&E staining from all groups of mice. Scale bar, 1 mm. (F) IHC of tumor tissues showing Ki-67 expression. Scale bar, 50 µm. (G) IF images of cleaved-Caspase-3/7 in xenografts from all groups of mice. Scale bar, 150 µm. (H) γ-H2AX and E2F1-downstream DDR modulators’ levels are shown in IHC images. Scale bars, 50 µm. (I) Mechanism of action of EPIC-0412. Data are represented as the mean ± s.d.; n = 6 mice. ***P < .001, **P < .01.
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