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Functionally defined therapeutic targets in diffuse intrinsic pontine glioma.

Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily MA, Quist MJ, Davis LE, Huang EC, Woo PJ, Ponnuswami A, Chen S, Johung TB, Sun W, Kogiso M, Du Y, Qi L, Huang Y, Hütt-Cabezas M, Warren KE, Le Dret L, Meltzer PS, Mao H, Quezado M, van Vuurden DG, Abraham J, Fouladi M, Svalina MN, Wang N, Hawkins C, Nazarian J, Alonso MM, Raabe EH, Hulleman E, Spellman PT, Li XN, Keller C, Pal R, Grill J, Monje M - Nat. Med. (2015)

Bottom Line: Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Spatial Systems Biomedicine, Department of Molecular and Medical Genetics, Oregon Health &Science University (OHSU), Portland, Oregon, USA.

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.

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Related in: MedlinePlus

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-FH8; 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).
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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-FH8; 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).

Mentions: The multi-HDAC inhibitor panobinostat14, among the most effective agents (Fig. 1, Supplementary Fig. 2), was selected for further exploration. In all, 12/16 DIPG cultures demonstrated sensitivity (Fig. 1). Compared to vorinostat, panobinostat illustrated substantially greater potency (Fig. 1b, Supplementary Fig. 5). Testing cell viability at 24, 48 and 72 hours at various dose levels of panobinostat demonstrated decreased viability by 48 hours (Fig. 1c). Expression of proliferation-associated genes MKI67 and CCND1 decreased with time after panobinostat exposure (Supplementary Fig. 6a). FACS analysis of EdU incorporation and Annexin V staining illustrated dose-dependent decreased cell proliferation and increased cell death (Fig. 2a, Supplementary Fig. 6b). shRNA-mediated knock-down of HDAC1 or HDAC2 decreased cell viability (Fig. 2b, Supplementary Fig. 6c – e). Western blot analyses demonstrated a dose-dependent increase in H3 acetylation and H3K27-trimethylation following panobinostat (Fig. 2c), suggesting partial rescue of the H3K27M-induced global hypotrimethylation phenotype. Increased K27 trimethylation is an unexpected effect of the drug, but is consistent with recent findings that acetylated H3K27 can “detoxify” K27M-induced inhibition of PCR215. RNAseq performed on panobinostat- or vehicle-treated DIPG cells revealed sweeping changes in gene expression, including normalization of the K27M gene expression signature16 and decrease in the oncogenic MYC target gene signature (Supplementary Figs. 7–8).


Functionally defined therapeutic targets in diffuse intrinsic pontine glioma.

Grasso CS, Tang Y, Truffaux N, Berlow NE, Liu L, Debily MA, Quist MJ, Davis LE, Huang EC, Woo PJ, Ponnuswami A, Chen S, Johung TB, Sun W, Kogiso M, Du Y, Qi L, Huang Y, Hütt-Cabezas M, Warren KE, Le Dret L, Meltzer PS, Mao H, Quezado M, van Vuurden DG, Abraham J, Fouladi M, Svalina MN, Wang N, Hawkins C, Nazarian J, Alonso MM, Raabe EH, Hulleman E, Spellman PT, Li XN, Keller C, Pal R, Grill J, Monje M - Nat. Med. (2015)

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-FH8; 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).
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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-FH8; 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).
Mentions: The multi-HDAC inhibitor panobinostat14, among the most effective agents (Fig. 1, Supplementary Fig. 2), was selected for further exploration. In all, 12/16 DIPG cultures demonstrated sensitivity (Fig. 1). Compared to vorinostat, panobinostat illustrated substantially greater potency (Fig. 1b, Supplementary Fig. 5). Testing cell viability at 24, 48 and 72 hours at various dose levels of panobinostat demonstrated decreased viability by 48 hours (Fig. 1c). Expression of proliferation-associated genes MKI67 and CCND1 decreased with time after panobinostat exposure (Supplementary Fig. 6a). FACS analysis of EdU incorporation and Annexin V staining illustrated dose-dependent decreased cell proliferation and increased cell death (Fig. 2a, Supplementary Fig. 6b). shRNA-mediated knock-down of HDAC1 or HDAC2 decreased cell viability (Fig. 2b, Supplementary Fig. 6c – e). Western blot analyses demonstrated a dose-dependent increase in H3 acetylation and H3K27-trimethylation following panobinostat (Fig. 2c), suggesting partial rescue of the H3K27M-induced global hypotrimethylation phenotype. Increased K27 trimethylation is an unexpected effect of the drug, but is consistent with recent findings that acetylated H3K27 can “detoxify” K27M-induced inhibition of PCR215. RNAseq performed on panobinostat- or vehicle-treated DIPG cells revealed sweeping changes in gene expression, including normalization of the K27M gene expression signature16 and decrease in the oncogenic MYC target gene signature (Supplementary Figs. 7–8).

Bottom Line: Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Spatial Systems Biomedicine, Department of Molecular and Medical Genetics, Oregon Health &Science University (OHSU), Portland, Oregon, USA.

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.

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Related in: MedlinePlus