Pharmacologic inhibition of histone demethylation as a therapy for pediatric brainstem glioma

Abstract

Pediatric brainstem gliomas often harbor oncogenic K27M mutation of histone H3.3. Here we show that GSKJ4 pharmacologic inhibition of K27 demethylase JMJD3 increases cellular H3K27 methylation in K27M tumor cells and demonstrate potent antitumor activity both in vitro against K27M cells and in vivo against K27M xenografts. Our results demonstrate that increasing H3K27 methylation by inhibiting K27 demethylase is a valid therapeutic strategy for treating K27M-expressing brainstem glioma.

Figure 1. GSKJ4 increases K27 methylation, inhibits K27M glioma cell growth and prevents K27M colony formation

(a) Immunoblot results showing K27M mutant (K27M mt), K27me3, K27me2, K27me1, histone H3.3 and total histone H3 expression in K27M brainstem glioma cell lines SF7761 and SF8628, wild-type (WT) H3.3 glioma cell lines SF9012, SF9402 and SF9427, and human astrocytes (HA). (b) Immunoblot results for K27me3, K27me2, K27me1, and total histone H3 in glioma cells either untreated or treated with 6 μM GSKJ4 for 48 h. (c) Temporal effect of GSKJ4 on H3K27 methylation in K27M SF8628 cells. (d) Proliferation response of glioma cells to increasing concentrations of GSKJ4. Values shown are the average (mean ± SEM) from quadruplicate samples for each incubation condition. (e) GSKJ4 colony-forming effect on cells with or without K27M.

Figure 2. GSKJ4 treatment shows K27M antitumor activity in vivo

(a) Mice with SF8628 subcutaneous tumor were treated with either vehicle (DMSO) or GSKJ4 (100 mg per kg per day) daily for 10 d. Left, tumor bioluminescence in mice at day 47 d after tumor cell injection. Horizontal bars indicate the mean from 3 mice for each treatment group. A two-tailed unpaired t-test was used to compare vehicle and GSKJ4 treatment. Right, tumor bioluminescence overlay images showing relative bioluminescence intensities from representative vehicle- vs. GSKJ4-treated mice. (b) Left, growth plots for subcutaneous tumors, established by injection of SF8628 cells transfected with scrambled (Control) or JMJD3 (KDM6B) siRNA. Tumor bioluminescence values were normalized against bioluminescence values obtained 3 d after tumor cell injection. Values are mean ± s.e.m. from 3 mice at each time after implantation. Right, corresponding tumor bioluminescence intensity overlay images for representative scrambled and JMJD3 siRNA–treated mice 29 d after tumor cell implantation. (c) Left, bioluminescence distributions of SF7761 (top; 106 d after tumor cell implantation), SF8628 (66 d after tumor cell implantation) and GBM43 (12 d after tumor cell implantation) brainstem xenografts, following 10 d (SF7761 and SF8628) or 7 d (GBM43) of treatment with GSKJ4 or vehicle. Horizontal bars indicate the mean from 4 mice for each treatment group in SF7761, 5 for each group in SF8628, and 6 for Control and 7 for GSKJ4 treatment in GBM43. A two-tailed unpaired t-test was used to compare vehicle and GSKJ4 treatment. Center, corresponding tumor bioluminescence intensity overlay images for representative vehicle- and GSKJ4-treated mice. Right, survival plots for each experiment. Statistical analysis was performed using a log-rank test. Pink shaded areas indicate the duration of treatment. (d) Representative Ki-67 and TUNEL staining for vehicle- vs. GSKJ4-treated SF8628 tumors, from mice euthanized at the end of treatment. Bottom, average percentage of positive cells in four high-powered fields for each tumor. Error bars, s.e.m. Two mice were analyzed in each treatment. A two-tailed unpaired t-test was used to compare vehicle and GSKJ4 treatment.