Functionally defined therapeutic targets in diffuse intrinsic pontine glioma

Abstract

Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer. We performed a chemical screen in patient-derived DIPG cultures along with RNA-seq analyses and integrated computational modeling to identify potentially effective therapeutic strategies. The multi-histone deacetylase inhibitor panobinostat demonstrated therapeutic efficacy both in vitro and in DIPG orthotopic xenograft models. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4 revealed that the two had synergistic effects. Together, these data suggest a promising therapeutic strategy for DIPG.

Figure 1. Functionally-defined targets in DIPG therapy

(a) Chemical screen of 14 patient-derived DIPG cell cultures. Heatmap demonstrating DIPG cell line sensitivity to each of the 83 agents tested. The values shown are the absolute IC₅₀ divided by the maximum dose. All drugs had a maximum dose of 10 µM except for vismodegib, sodium butyrate, pazopanib, alisertib, and vemurafenib, which had a maximum dose of 100 µM. Values are shown as gradations of red to white, with red representing submicromolar IC₅₀ values, white indicating IC₅₀ greater than the maximum dose for that drug (i.e. 10 µM or 100 µM), and pink showing the range in between. Grey boxes indicate those drugs not included in the screen for that cell line. Numbers corresponding to the drug names in the key below the heatmap are listed on the horizontal axis, culture IDs listed on the vertical axis. The histone status of each culture used in the screen is indicated by green (wild type, WT), yellow (H3.3K27M, H3F3A-K27M) or blue (H3.1K27M, HIST1H3B-K27M); see also Supplementary Table 1. Recurrent “hits” are visualized as a column of red or pink. (b) Dose-response curves: Patient-derived DIPG lines (SU-DIPG IV, SU-DIPG-VI, SU-DIPG-XIII, JHH-DIPG1, SF7761³) were treated with the indicated drugs at 0.001/0.01/0.1/1/10 µM or 0.1% DMSO control in at least triplicate (n = 3 wells) and cell viabilities were assessed at 72 hr. Data are expressed as relative to the 0.1% DMSO control values. A pediatric cortical GBM line (SU-pcGBM2; histone WT; orange curves) was treated in parallel for comparison in a subset. Data are shown as mean ± SD. (c) Panobinostat time course: DIPG cells were treated with panobinostat in quadruplicate (n = 4 wells) at indicated concentrations (25 nM – 500 nM) or 0.1% DMSO vehicle control. Cell viabilities were assessed at 0, 24, 48 and 72 hrs of treatment. Data are shown as mean ± SD. **P < 0.01, ***P < 0.001 (two-tailed t test results shown for the lowest concentration to reveal a significant difference at 48 or 72 hours).

Figure 2. Panobinostat is a promising therapy for DIPG

(a) FACS analysis of DIPG tumor cell proliferation and cell death: Top row: Overlapping histogram plots of EdU FACS analyses are shown on the left; quantifications of EdU⁺ cell population levels from each condition are shown in bar plots on the right for DIPG cell cultures SU-DIPG-VI and SU-DIPG-XIII (both H3.3K27M mutant cell lines). Bottom row: Left, Overlapping plots of Annexin V, DAPI FACS analyses; Right, bar plots show early apoptotic (AV⁺DAPI⁺) or late apoptotic (AV⁺DAPI⁺) cell population levels from each condition for each cell line as above. (b) HDAC1 and HDAC2 knock-down in DIPG cells using shRNA verify panobinostat mechanism of action in four DIPG cell lines. Cell viability assays at each time point for each cell line were performed in triplicate (n = 3 wells); data are expressed relative to Day 0 and are shown as mean ± SD. Note the varying growth rates of cell cultures result in varying y-axes depicting relative change in cell viability. **P < 0.01; ***P<0.001 (Two-way ANOVA). (c) Panobinostat increases histone-3 acetylation and restores H3K27 trimethylation. Western blot analyses of histone-3 acetylation and H3K27 trimethylation (H3K27me3) in H3K27M mutant DIPG cell lines SU-DIPG-VI and SU-DIPG-XIII (left blots) and in 293T cells expressing a mutant H3.3K27M-HA tagged construct (293-H3.3-K27M-FH; right blots). Controls included total protein levels of H3, HDAC1, HDAC2 and EZH2. Expression of the HA tag in the 293T cells confirms expression of the H3.3K27M-FH construct. (d) Schematic illustrating convection enhanced delivery strategy to infuse drug into brainstem. Blue illustrates approximate distribution of the infused solution. (e) Distribution of infusate illustrated by delivering blue dye to the brainstem by CED. Ventral side of a mouse brain is shown immediately following CED delivery of Coomassie Blue dye. Scale bar = 3 mm (f) in vivo bioluminescent imaging of DIPG xenografts 7 days following CED delivery of panobinostat (T = treated with panobinostat) or vehicle control (C = control). The heat map superimposed over the mouse head represents the degree of photon emission by DIPG cells expressing firefly luciferase. Scale bar = 3.5 cm. (g) in vivo DIPG xenograft tumor growth as measured by change in bioluminescent photon emission over the seven days following (g) CED delivery of panobinostat. panobinostat = red squares (n = 5 mice) and vehicle control = blue circles (n = 4 mice). Data points represent the change in maximum photon flux (percent of baseline) between Day 0 and Day 7 for each mouse. (h) As in (g), with systemic administration of panobibostat. Three systemic dose levels were used, 1 mg/kg (n = 6 control, 8 treated mice) or 10 mg/kg (n = 5 mice per group) delivered IP on M,W,F or 20 mg/kg (n = 7 mice per group) delivered once per week. Error bars, s.e.m. *P < 0. 0.5; **P < 0.01; N.S. indicates P > 0.05 (two-tailed t test). (i) Systemic delivery of panobinostat prolongs survival in a histone H3 wild type DIPG orthotopic xenograft model IBs-W0128DIPG. Panobinostat 10 mg/kg I.P. doses given as indicated by arrows. n = 10 per group; P = 0.0179 (log-rank analysis).