Fig 1.
Panobinostat shows efficacy against brainstem glioma cells in vitro.
(A-C) Mouse brainstem glioma cells driven by PDGF-B, H3.3-K27M, and Cre-induced p53 loss from three separate tumors were cultured as neurospheres and treated with varying doses of panobinostat for 48 h. (A) Proliferation assessment using a BrdU assay (**** p < 0.0001). (B) Cell viability assessment using a Celltiter-Glo assay (* p = 0.0252, **** p < 0.0001). (C) Apoptosis assessment using a Caspase-Glo 3/7 assay (* p = 0.0149, *** p = 0.0005, **** p < 0.0001). Values from each drug concentration were normalized to the control (0 nM panobinostat, 0.1% DMSO). IC50 values for each tumor cell line are shown in the legends. Each experiment was performed in triplicate and independently repeated three times for each tumor cell line. For all panels, error bars represent mean with SEM. Statistical significance to compare drug concentration groups to controls were determined using an unpaired two-tailed t-test.
Fig 2.
Panobinostat decreases proliferation and viability and induces apoptosis independent of H3.3-K27M status in vitro.
Three independent murine brainstem glioma H3.3-K27M and H3.3-WT cell lines (driven by PDGF-B, Cre-induced p53 loss, and either H3.3-K27M or H3.3-WT, respectively) were treated with varying doses of panobinostat for 48 h. (A) Proliferation assessment using a BrdU assay. (B) Cell viability assessment using a CellTiter-Glo assay. (C) Apoptosis assessment using a Caspase-Glo 3/7 assay. Results from the different drug concentrations were normalized to the control (0 nM Panobinostat, 0.1% DMSO). Each experiment was performed in triplicate and independently repeated three times for each tumor cell line. Error bars represent mean with SEM. Statistical significance to compare H3.3-K27M and H3.3-WT cells at each drug concentration were determined using an unpaired two-tailed t-test (* p = 0.035).
Fig 3.
Panobinostat inhibits survival and clonogenicity of human DIPG cells in vitro (A) Human glioma cell models were treated with panobinostat for 72 h and then assayed for cell survival with an MTS Assay. Data are presented as the mean (with SD) of percent survival of control (untreated) cells. For each model, n = 6 replicates. (B-C) Human DIPG cells (HSJD-DIPG-007) were incubated with the indicated concentrations of panobinostat in soft agar for 2 weeks, and then colonies were stained with MTT and counted. Data in B are the mean with SEM, and statistical significance to compare drug concentration groups to controls were determined using an unpaired two-tailed t-test. * p = 0.02, **** p < 0.0001. Representative pictures of colonies are shown in C. (D) The indicated human glioma cell models were treated with either 0, 0.1, or 0.5 μM panobinostat for 48 h and then harvested for histone extraction. Histone lysates were separated via SDS-PAGE and blotted for the indicated antibodies.
Table 1.
IC50s of human DIPG cell models with Panobinostat treatment.
Fig 4.
Pharmacokinetic studies reveal in vivo delivery of panobinostat into brain tissue of a murine brainstem glioma model, greater in the brainstem tumor than in the normal cerebral cortex.
Neonatal Ntv-a;p53-fl/fl mice were injected with RCAS-PDGF-B, -H3.3-K27M, and -Cre viruses to induce tumor formation. (A) H&E (top panels) and IHC staining for HA (lower panels, brown nuclei, to identify the HA tag on the RCAS-H3.3-K27M construct) in tumors arising in these mice. Left panels, 100x, scale bar = 250 μm; right panels, 400x, scale bar = 50 μm). Note increased cellular density, pseudopalisading necrosis (white arrow heads), and invasion of HA+ tumor cells into normal brain (black arrow heads). (B) Upon the appearance of brain tumor symptoms (3–5 weeks post-injection), mice were treated with three doses of 20 mg/kg panobinostat (vehicle, 25% DMSO, 0.25x PBS, 5% glucose, n = 3) once daily by i.p. injections. Mice were then sacrificed 4 h after the final treatment. Pharmacokinetic analysis was performed to determine the concentration of panobinostat in the normal cerebral cortex and brainstem glioma tissue. Statistical significance was determined using unpaired two-tailed t-test to compare groups. CC = normal cerebral cortex, BSG = brainstem glioma.
Fig 5.
Short-term in vivo treatments with panobinostat reduces tumor cell proliferation and increases H3 acetylation without evidence of apoptosis.
Neonatal Ntv-a;p53-fl/fl mice were injected with RCAS-PDGF-B, -H3.3-K27M, and–Cre viruses to induce tumor formation. Upon the first appearance of brain tumor symptoms (3–5 weeks post-injection), mice were treated with five doses of 20 mg/kg panobinostat (Drug) or vehicle (Veh, 25% DMSO, 0.25x PBS, 5% glucose, n = 6 in each group) once daily via i.p. injections and then sacrificed 1 hour after the final treatment. Shown is immunohistochemistry of brain tumor tissue for cell proliferation (phospho histone H3, pH3) (A), cell apoptosis (cleaved caspase-3, cc3) (B), and H3 acetylation (AcH3) (C), 400x, scale bar = 50 μm, with quantification of total positive nuclear staining as a percentage of total nuclear area below in each panel. Statistical significance was determined using unpaired two-tailed t-test to compare groups. (ns: nonsignificant).
Fig 6.
No overall survival benefit with panobinostat treatments in vivo.
(A) Mice with tumors driven by PDGF-B and p53 loss harboring H3.3-K27M mutations (generated as described in Materials and Methods) were treated with 20 mg/kg panobinostat (n = 11) or vehicle (25% DMSO, 0.25x PBS, 5% glucose, n = 8) administered via intraperitoneal (i.p.) injection once per day twice a week beginning 21 days post-virus injection and continuing until mice reached humane endpoints. The mice were monitored daily and sacrificed upon moribund condition (lethargy, enlarged head circumference, ataxia, and/or > 25% weight loss) (p = 0.756, log rank test). (B-D) Effect of panobinostat treatment on the survival of mice-bearing H3.3-K27M HSJD-DIPG-007 orthotopic xenografts. NOD-SCID mice (7 weeks old) were orthotopically injected with HSJD-DIPG-007 cells (passage 39) into the brainstem via stereotactic coordinates. (B) Representative H&E (top panel) and IHC staining for Ki67 (middle panel) and Vimentin (bottom panel) of control mice (treated with vehicle and sacrificed immediately after the last dose, as described in the Materials and Methods). 400x magnification, scale bar = 50 μm. (C-D) Starting on day 28 post-implantation, mice were treated with panobinostat prepared in a vehicle containing 5% dextrose (C) or 2.5% DMSO, 5% PEG400 and 5% Tween80 in 0.9% saline (D) via intraperitoneal (i.p.) injection at 10 mg/kg, three times a week for four weeks (p>0.05, log-rank test). Shaded areas under the curves in A, C-D indicate treatment duration.