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A high-throughput in vitro drug screen in a genetically engineered mouse model of diffuse intrinsic pontine glioma identifies BMS-754807 as a promising therapeutic agent.

Halvorson KG, Barton KL, Schroeder K, Misuraca KL, Hoeman C, Chung A, Crabtree DM, Cordero FJ, Singh R, Spasojevic I, Berlow N, Pal R, Becher OJ - PLoS ONE (2015)

Bottom Line: In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM, significant inhibition of proliferation at a concentration of 1.5 μM, as well as inhibition of AKT activation.Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R.Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50, suggesting that inadequate drug delivery may limit in vivo efficacy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Duke University Medical Center, Durham, NC, United States of America; Department of Pathology, Duke University Medical Center, Durham, NC, United States of America; Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, United States of America; Department of Surgery, Duke University, Durham, NC, United States of America.

ABSTRACT
Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system, including a model driven by PDGF-B, H3.3K27M, and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study, an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R, IR, MET, TRKA, TRKB, AURKA, AURKB. In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM, significant inhibition of proliferation at a concentration of 1.5 μM, as well as inhibition of AKT activation. Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo, systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50, suggesting that inadequate drug delivery may limit in vivo efficacy. In summary, an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG, but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice.

No MeSH data available.


Related in: MedlinePlus

BMS-754807 decreases AKT phosphorylation in DIPG cell-lines cultured in serum-free media.Western blot for (A) pAKT (Ser473, 60 kDa) and total AKT (60 kDa), (B) pPDGFRA (195 kDa) and total PDGFRA (195 kDa) after four hours of treatment with BMS-754807 with or without IGF ligand stimulation for 15 minutes in DIPG cell lines induced by PDGF-B, H3.3K27M, and p53 loss. A representative blot from three independent experiments is shown. Actin (43 kDa) is shown as a loading control.
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pone.0118926.g003: BMS-754807 decreases AKT phosphorylation in DIPG cell-lines cultured in serum-free media.Western blot for (A) pAKT (Ser473, 60 kDa) and total AKT (60 kDa), (B) pPDGFRA (195 kDa) and total PDGFRA (195 kDa) after four hours of treatment with BMS-754807 with or without IGF ligand stimulation for 15 minutes in DIPG cell lines induced by PDGF-B, H3.3K27M, and p53 loss. A representative blot from three independent experiments is shown. Actin (43 kDa) is shown as a loading control.

Mentions: To investigate the mechanism of BMS-754807 in this DIPG model, we treated neurospheres derived from mouse PDGF-B; H3.3K27M; p53-deficient tumors with 1μM and 10μM of drug in the presence or absence of IGF ligand. There was no phosphorylated IGF-1R in the neurospheres, with or without IGF-1 ligand stimulation (S1 Fig.). We did however note phosphorylated AKT (Serine 473), which is downstream of several known BMS-754807 targets such as MET, TRKA, TRKB, IR (Fig. 3A). Phosphorylated AKT was inhibited after 4 hours of BMS-754807 treatment in vitro, both with and without stimulation with IGF-1 ligand with the exception of the 10μM dose in the stimulated cells (Fig. 3A). In addition, we investigated the possibility that BMS-754807 is targeting PDGFRA (which is activated in our mouse model, as shown in Fig. 2B). However, we observed no reduction in phosphorylated PDGFRA expression with BMS-754807 treatment (Fig. 3B).


A high-throughput in vitro drug screen in a genetically engineered mouse model of diffuse intrinsic pontine glioma identifies BMS-754807 as a promising therapeutic agent.

Halvorson KG, Barton KL, Schroeder K, Misuraca KL, Hoeman C, Chung A, Crabtree DM, Cordero FJ, Singh R, Spasojevic I, Berlow N, Pal R, Becher OJ - PLoS ONE (2015)

BMS-754807 decreases AKT phosphorylation in DIPG cell-lines cultured in serum-free media.Western blot for (A) pAKT (Ser473, 60 kDa) and total AKT (60 kDa), (B) pPDGFRA (195 kDa) and total PDGFRA (195 kDa) after four hours of treatment with BMS-754807 with or without IGF ligand stimulation for 15 minutes in DIPG cell lines induced by PDGF-B, H3.3K27M, and p53 loss. A representative blot from three independent experiments is shown. Actin (43 kDa) is shown as a loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352073&req=5

pone.0118926.g003: BMS-754807 decreases AKT phosphorylation in DIPG cell-lines cultured in serum-free media.Western blot for (A) pAKT (Ser473, 60 kDa) and total AKT (60 kDa), (B) pPDGFRA (195 kDa) and total PDGFRA (195 kDa) after four hours of treatment with BMS-754807 with or without IGF ligand stimulation for 15 minutes in DIPG cell lines induced by PDGF-B, H3.3K27M, and p53 loss. A representative blot from three independent experiments is shown. Actin (43 kDa) is shown as a loading control.
Mentions: To investigate the mechanism of BMS-754807 in this DIPG model, we treated neurospheres derived from mouse PDGF-B; H3.3K27M; p53-deficient tumors with 1μM and 10μM of drug in the presence or absence of IGF ligand. There was no phosphorylated IGF-1R in the neurospheres, with or without IGF-1 ligand stimulation (S1 Fig.). We did however note phosphorylated AKT (Serine 473), which is downstream of several known BMS-754807 targets such as MET, TRKA, TRKB, IR (Fig. 3A). Phosphorylated AKT was inhibited after 4 hours of BMS-754807 treatment in vitro, both with and without stimulation with IGF-1 ligand with the exception of the 10μM dose in the stimulated cells (Fig. 3A). In addition, we investigated the possibility that BMS-754807 is targeting PDGFRA (which is activated in our mouse model, as shown in Fig. 2B). However, we observed no reduction in phosphorylated PDGFRA expression with BMS-754807 treatment (Fig. 3B).

Bottom Line: In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM, significant inhibition of proliferation at a concentration of 1.5 μM, as well as inhibition of AKT activation.Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R.Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50, suggesting that inadequate drug delivery may limit in vivo efficacy.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Duke University Medical Center, Durham, NC, United States of America; Department of Pathology, Duke University Medical Center, Durham, NC, United States of America; Preston Robert Tisch Brain Tumor Center, Duke University Medical Center, Durham, NC, United States of America; Department of Surgery, Duke University, Durham, NC, United States of America.

ABSTRACT
Diffuse intrinsic pontine gliomas (DIPGs) represent a particularly lethal type of pediatric brain cancer with no effective therapeutic options. Our laboratory has previously reported the development of genetically engineered DIPG mouse models using the RCAS/tv-a system, including a model driven by PDGF-B, H3.3K27M, and p53 loss. These models can serve as a platform in which to test novel therapeutics prior to the initiation of human clinical trials. In this study, an in vitro high-throughput drug screen as part of the DIPG preclinical consortium using cell-lines derived from our DIPG models identified BMS-754807 as a drug of interest in DIPG. BMS-754807 is a potent and reversible small molecule multi-kinase inhibitor with many targets including IGF-1R, IR, MET, TRKA, TRKB, AURKA, AURKB. In vitro evaluation showed significant cytotoxic effects with an IC50 of 0.13 μM, significant inhibition of proliferation at a concentration of 1.5 μM, as well as inhibition of AKT activation. Interestingly, IGF-1R signaling was absent in serum-free cultures from the PDGF-B; H3.3K27M; p53 deficient model suggesting that the antitumor activity of BMS-754807 in this model is independent of IGF-1R. In vivo, systemic administration of BMS-754807 to DIPG-bearing mice did not prolong survival. Pharmacokinetic analysis demonstrated that tumor tissue drug concentrations of BMS-754807 were well below the identified IC50, suggesting that inadequate drug delivery may limit in vivo efficacy. In summary, an unbiased in vitro drug screen identified BMS-754807 as a potential therapeutic agent in DIPG, but BMS-754807 treatment in vivo by systemic delivery did not significantly prolong survival of DIPG-bearing mice.

No MeSH data available.


Related in: MedlinePlus