Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Dec 1;22(12):1413-1421.
doi: 10.1158/1535-7163.MCT-23-0179.

Microtubule-Targeting Combined with HDAC Inhibition Is a Novel Therapeutic Strategy for Diffuse Intrinsic Pontine Gliomas

Anahid Ehteda #  1 , Aaminah Khan #  1   2 , Gayathiri Rajakumar  2 , Anne S Vanniasinghe  2 , Anjana Gopalakrishnan  2 , Jie Liu  2 , Maria Tsoli #  1   2 , David S Ziegler #  1   2   3
Affiliations

Microtubule-Targeting Combined with HDAC Inhibition Is a Novel Therapeutic Strategy for Diffuse Intrinsic Pontine Gliomas

Anahid Ehteda et al. Mol Cancer Ther. .

Abstract

Diffuse intrinsic pontine gliomas (DIPG) are an incurable childhood brain cancer for which novel treatments are needed. DIPGs are characterized by a mutation in the H3 histone (H3K27M), resulting in loss of H3K27 methylation and global gene dysregulation. TRX-E-009-1 is a novel anticancer agent with preclinical activity demonstrated against a range of cancers. We examined the antitumor activity of TRX-E-009-1 against DIPG neurosphere cultures and observed tumor-specific activity with IC50s ranging from 20 to 100 nmol/L, whereas no activity was observed against normal human astrocyte cells. TRX-E-009-1 exerted its anti-proliferative effect through the induction of apoptotic pathways, with marked increases in cleaved caspase 3 and cleaved PARP levels, while also restoring histone H3K27me3 methylation. Co-administration of TRX-E-009-1 and the histone deacetylase (HDAC) inhibitor SAHA extended survival in DIPG orthotopic animal models. This antitumor effect was further enhanced with irradiation. Our findings indicate that TRX-E-009-1, combined with HDAC inhibition, represents a novel, potent therapy for children with DIPG.

PubMed Disclaimer

Figures

Figure 1. TRX-E-009–1 has anticancer effect against patient-derived DIPG cells. A, Patient-derived DIPG cell lines are sensitive to TRX-E-009–1 compared with normal healthy astrocytes (NHA) and normal lung fibroblast (MRC5) cells. TRX-E-009–1 impairs TRX-E-009–1 impairs clonogenic activity in three DIPG cell lines (B), which is further potentiated in HSJD-DIPG007 cells (C) when combined with irradiation in a dose-dependent manner. B and C, n = 4 independent experiments performed in duplicate. Statistical analysis was calculated using one-way ANOVA between cohorts and for treated and untreated samples (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). C, Synergy scores were calculated by Calcusyn software.
Figure 1.
TRX-E-009–1 has anticancer effect against patient-derived DIPG cells. A, Patient-derived DIPG cell lines are sensitive to TRX-E-009–1 compared with normal healthy astrocytes (NHA) and normal lung fibroblast (MRC5) cells. TRX-E-009–1 impairs TRX-E-009–1 impairs clonogenic activity in three DIPG cell lines (B), which is further potentiated in HSJD-DIPG007 cells (C) when combined with irradiation in a dose-dependent manner. B and C,n = 4 independent experiments performed in duplicate. Statistical analysis was calculated using one-way ANOVA between cohorts and for treated and untreated samples (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). C, Synergy scores were calculated by Calcusyn software.
Figure 2. Mechanism of action of TRX-E-009-1 on DIPG cells. A, Treatment of HSJD-DIPG007 cells with 1 and 5 μmol/L TRX-E-009-1 leads to increased protein expression of p21, c-PARP, and c-Caspase 3. Western blot analysis is representative of two independent experiments. B, HSJD-DIPG007 cells were treated with TRX-E-009-1 at 1, 5, and 10 μmol/L to observe the effect on polymerized and depolymerized α-tubulin levels. Western blot analysis is representative of three independent experiments. C, TRX-E-009-1 restores H3K27 trimethylation and increases H3K27 acetylation in treated HSJD-DIPG007 cells. Western blot analysis is representative of two independent experiments. D, TRX-E-009–1 significantly extends survival in the orthotopic mouse model of HSJD-DIPG007. Treatment commenced 4 weeks after intracranial injection of HSJD-DIPG007 cells 50 mg/kg TRX-E-009–1 intravenously three times a week (M, W, F) for 4 weeks. Statistical analysis has been performed using the log-rank (Mantel–Cox) test. E, TRX-E-009–1 drug concentration in the brainstem region of treated mice 2 hours posttreatment. Data are presented as mean values ± SEM from brain samples collected from n = 2 in each cohort.
Figure 2.
Mechanism of action of TRX-E-009-1 on DIPG cells. A, Treatment of HSJD-DIPG007 cells with 1 and 5 μmol/L TRX-E-009-1 leads to increased protein expression of p21, c-PARP, and c-Caspase 3. Western blot analysis is representative of two independent experiments. B, HSJD-DIPG007 cells were treated with TRX-E-009-1 at 1, 5, and 10 μmol/L to observe the effect on polymerized and depolymerized α-tubulin levels. Western blot analysis is representative of three independent experiments. C, TRX-E-009-1 restores H3K27 trimethylation and increases H3K27 acetylation in treated HSJD-DIPG007 cells. Western blot analysis is representative of two independent experiments. D, TRX-E-009–1 significantly extends survival in the orthotopic mouse model of HSJD-DIPG007. Treatment commenced 4 weeks after intracranial injection of HSJD-DIPG007 cells 50 mg/kg TRX-E-009–1 intravenously three times a week (M, W, F) for 4 weeks. Statistical analysis has been performed using the log-rank (Mantel–Cox) test. E, TRX-E-009–1 drug concentration in the brainstem region of treated mice 2 hours posttreatment. Data are presented as mean values ± SEM from brain samples collected from n = 2 in each cohort.
Figure 3. Combination of TRX-E-009–1 and SAHA is synergistic against DIPG. Combination of TRX-E-009–1 and SAHA is synergistic against HSJD-DIPG007 and SU-DIPGXVII neurospheres, resulting in decreased (A) cell survival and (B) fewer colonies formed upon combination treatment. Data are presented as mean values ± SEM. A, Single well examined over five independent experiments. B, n = 4 independent experiments performed in duplicate. Synergy scores were calculated by Calcusyn software.
Figure 3.
Combination of TRX-E-009–1 and SAHA is synergistic against DIPG. Combination of TRX-E-009–1 and SAHA is synergistic against HSJD-DIPG007 and SU-DIPGXVII neurospheres, resulting in decreased (A) cell survival and (B) fewer colonies formed upon combination treatment. Data are presented as mean values ± SEM. A, Single well examined over five independent experiments. B,n = 4 independent experiments performed in duplicate. Synergy scores were calculated by Calcusyn software.
Figure 4. Combination of TRX-E-009–1 and SAHA induces apoptosis and restores H3K27 trimethylation and acetylation in DIPG cells. Protein expression in HSJD-DIPG007 cells when treated with TRX-E-009–1 (1 μmol/L) and SAHA (5 μmol/L) of (A) c-PARP, c-caspase 3, γ-H2AX and (B) H3K27 trimethylation and acetylation. Representative blot from two independent experiments.
Figure 4.
Combination of TRX-E-009–1 and SAHA induces apoptosis and restores H3K27 trimethylation and acetylation in DIPG cells. Protein expression in HSJD-DIPG007 cells when treated with TRX-E-009–1 (1 μmol/L) and SAHA (5 μmol/L) of (A) c-PARP, c-caspase 3, γ-H2AX and (B) H3K27 trimethylation and acetylation. Representative blot from two independent experiments.
Figure 5. TRX-E-009–1 synergizes with SAHA and irradiation in orthotopic models of DIPG. A, Triple combination treatment of TRX-E-009–1 (0.005 μmol/L), SAHA (0.01 μmol/L), and irradiation (2Gy) significantly reduced the clonogenic activity of HSJD-DIPG007 cells. Data are presented as mean values ±SEM. n = 4 independent experiments performed in duplicate. Synergy scores were calculated by Calcusyn software (*, P < 0.05; ****, P < 0.0001). B, Mice were intracranially injected with DIPG cells and 4 weeks post-injection, treatments commenced. Mice were humanely euthanized when they displayed severe neurological decline and/or weight loss or reached maximum holding time (MHT). Survival curve of HSJD-DIPG007 with TRX-E-009–1/SAHA/Irradiation treatment. Median survival of cohorts is indicated in brackets. Statistical analysis has been performed using the log-rank (Mantel–Cox) test.
Figure 5.
TRX-E-009–1 synergizes with SAHA and irradiation in orthotopic models of DIPG. A, Triple combination treatment of TRX-E-009–1 (0.005 μmol/L), SAHA (0.01 μmol/L), and irradiation (2Gy) significantly reduced the clonogenic activity of HSJD-DIPG007 cells. Data are presented as mean values ±SEM. n = 4 independent experiments performed in duplicate. Synergy scores were calculated by Calcusyn software (*, P < 0.05; ****, P < 0.0001). B, Mice were intracranially injected with DIPG cells and 4 weeks post-injection, treatments commenced. Mice were humanely euthanized when they displayed severe neurological decline and/or weight loss or reached maximum holding time (MHT). Survival curve of HSJD-DIPG007 with TRX-E-009–1/SAHA/Irradiation treatment. Median survival of cohorts is indicated in brackets. Statistical analysis has been performed using the log-rank (Mantel–Cox) test.
Figure 6. Triple combination of TRX-E-009–1, SAHA, and irradiation inhibits tumour cell proliferation, induces DNA damage response and restores H3K27 trimethylation and acetylation in orthotopic tumors. Ki67, γ-H2AX. H3K27ac and H3K27me3 staining of DIPG tumors after 4 weeks of treatment in HSJD-DIPG007 model. Three images were taken from brain samples collected from two mice in each cohort. Statistical analysis was calculated using one-way ANOVA between cohorts and for treated and untreated samples (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Specific P values are listed in Supplementary Table S1.
Figure 6.
Triple combination of TRX-E-009–1, SAHA, and irradiation inhibits tumour cell proliferation, induces DNA damage response and restores H3K27 trimethylation and acetylation in orthotopic tumors. Ki67, γ-H2AX. H3K27ac and H3K27me3 staining of DIPG tumors after 4 weeks of treatment in HSJD-DIPG007 model. Three images were taken from brain samples collected from two mice in each cohort. Statistical analysis was calculated using one-way ANOVA between cohorts and for treated and untreated samples (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Specific P values are listed in Supplementary Table S1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Lin GL, Wilson KM, Ceribelli M, Stanton BZ, Woo PJ, Kreimer S, et al. . Therapeutic strategies for diffuse midline glioma from high-throughput combination drug screening. Sci Transl Med 2019;11:eaaw0064. - PMC - PubMed
    1. Donaldson SS, Laningham F, Fisher PG. Advances toward an understanding of brainstem gliomas. J Clin Oncol 2006;24:1266–72. - PubMed
    1. Johung TB, Monje M. Diffuse intrinsic pontine glioma: new pathophysiological insights and emerging therapeutic targets. Curr Neuropharmacol 2017;15:88–97. - PMC - PubMed
    1. Jansen MHA, van Vuurden DG, Vandertop WP, Kaspers GJL. Diffuse intrinsic pontine gliomas: a systematic update on clinical trials and biology. Cancer Treat Rev 2012;38:27–35. - PubMed
    1. Upton DH, Ung C, George SM, Tsoli M, Kavallaris M, Ziegler DS. Challenges and opportunities to penetrate the blood-brain barrier for brain cancer therapy. Theranostics 2022;12:4734–52. - PMC - PubMed