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Evaluation of In Vitro Activity of the Class I PI3K Inhibitor Buparlisib (BKM120) in Pediatric Bone and Soft Tissue Sarcomas.

Anderson JL, Park A, Akiyama R, Tap WD, Denny CT, Federman N - PLoS ONE (2015)

Bottom Line: Anti-proliferative effects were observed in both anchorage dependent and independent conditions and apoptosis was induced within 24 hours of drug treatment.Buparlisib demonstrated cytotoxicity as a single agent, but was found to be more effective when used in combination.The addition of NVP-AEW541 also further reduced phospho-Akt levels and more potently induced apoptosis compared to buparlisib treatment alone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Division of Hematology/Oncology, Gwynne Hazen Cherry Memorial Laboratories, University of California, Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Pediatric bone and soft tissue sarcomas often display increased Akt phosphorylation through up regulation of insulin-like growth factor (IGF1) signaling. Additionally, Akt signaling has been linked to resistance to IGF1 receptor (IGF1R) and mTOR (mammalian target of rapamycin) inhibitors in sarcoma, further demonstrating the role of Akt in tumor survival. This suggests targeting components of the PI3K/Akt pathway may be an effective therapeutic strategy. Here, we investigated the in vitro activity of the pan-class I PI3K inhibitor buparlisib (BKM120) in pediatric bone and soft tissue sarcomas. Buparlisib inhibited activation of Akt and signaling molecules downstream of mTORC1 (mTOR complex 1) in Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines. Anti-proliferative effects were observed in both anchorage dependent and independent conditions and apoptosis was induced within 24 hours of drug treatment. Buparlisib demonstrated cytotoxicity as a single agent, but was found to be more effective when used in combination. Synergy was observed when buparlisib was combined with the IGF1R inhibitor NVP-AEW541 and the mTORC1 inhibitor rapamycin. The addition of NVP-AEW541 also further reduced phospho-Akt levels and more potently induced apoptosis compared to buparlisib treatment alone. Additionally, the combination of buparlisib with the MEK1/2 inhibitor trametinib resulted in synergy in sarcoma cell lines possessing MAPK pathway mutations. Taken together, these data indicate buparlisib could be a novel therapy for the treatment of pediatric bone and soft tissue sarcomas.

No MeSH data available.


Related in: MedlinePlus

Buparlisib treatment reduces phosphorylation of signaling molecules downstream of PI3K and mTORC1.(A) Immunoblot analysis of phospho-Akt (S473 and T308), total Akt, phospho-p70 S6K (T389), total p70 S6K, phospho-4E-BP1 (T37/46), total 4E-BP1, and actin in ES (TC-32), OS (KHOS), and RMS (RD) cells treated with increasing concentrations of buparlisib for one hour. (B) Phospho-Akt (S473) and total Akt levels were quantitated based on Odyssey software integrated intensity values. Phospho-Akt levels were normalized to total Akt levels, then normalized to cells treated with DMSO in order to determine relative phospho-Akt levels. Relative phospho-Akt levels were plotted against log buparlisib concentration and IC50 values were calculated by fitting this data to a four-parameter, variable slope sigmoid dose-response model. Each data point represents the average of at least three independent experiments.
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pone.0133610.g002: Buparlisib treatment reduces phosphorylation of signaling molecules downstream of PI3K and mTORC1.(A) Immunoblot analysis of phospho-Akt (S473 and T308), total Akt, phospho-p70 S6K (T389), total p70 S6K, phospho-4E-BP1 (T37/46), total 4E-BP1, and actin in ES (TC-32), OS (KHOS), and RMS (RD) cells treated with increasing concentrations of buparlisib for one hour. (B) Phospho-Akt (S473) and total Akt levels were quantitated based on Odyssey software integrated intensity values. Phospho-Akt levels were normalized to total Akt levels, then normalized to cells treated with DMSO in order to determine relative phospho-Akt levels. Relative phospho-Akt levels were plotted against log buparlisib concentration and IC50 values were calculated by fitting this data to a four-parameter, variable slope sigmoid dose-response model. Each data point represents the average of at least three independent experiments.

Mentions: We next evaluated the ability of buparlisib to inhibit cellular signaling downstream of PI3K. Increasing doses were added to cells and phosphorylation of signaling molecules was examined by immunoblot after one hour of treatment (Fig 2A). Phosphorylation of Akt was inhibited at both serine 473 and threonine 308. Quantitative immunoblot analysis was used to measure levels of Akt phosphorylation at serine 473 (Fig 2A). Phospho-Akt levels were normalized to levels of total Akt and fit to dose-response curves to calculate the half maximal inhibitory concentration (IC50) (Fig 2B). IC50 values for ranged from 64 to 916 nM, with the largest values occurring in cell lines that lack PTEN expression (S1 Table). Buparlisib also inhibited the phosphorylation of proteins downstream from mTORC1, such as S6K and 4E-BP1 (Fig 2A). Phosphorylation of S6K was more potently inhibited by buparlisib. Inhibition of phospho-4E-BP1 was only observed at high buparlisib concentrations.


Evaluation of In Vitro Activity of the Class I PI3K Inhibitor Buparlisib (BKM120) in Pediatric Bone and Soft Tissue Sarcomas.

Anderson JL, Park A, Akiyama R, Tap WD, Denny CT, Federman N - PLoS ONE (2015)

Buparlisib treatment reduces phosphorylation of signaling molecules downstream of PI3K and mTORC1.(A) Immunoblot analysis of phospho-Akt (S473 and T308), total Akt, phospho-p70 S6K (T389), total p70 S6K, phospho-4E-BP1 (T37/46), total 4E-BP1, and actin in ES (TC-32), OS (KHOS), and RMS (RD) cells treated with increasing concentrations of buparlisib for one hour. (B) Phospho-Akt (S473) and total Akt levels were quantitated based on Odyssey software integrated intensity values. Phospho-Akt levels were normalized to total Akt levels, then normalized to cells treated with DMSO in order to determine relative phospho-Akt levels. Relative phospho-Akt levels were plotted against log buparlisib concentration and IC50 values were calculated by fitting this data to a four-parameter, variable slope sigmoid dose-response model. Each data point represents the average of at least three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581723&req=5

pone.0133610.g002: Buparlisib treatment reduces phosphorylation of signaling molecules downstream of PI3K and mTORC1.(A) Immunoblot analysis of phospho-Akt (S473 and T308), total Akt, phospho-p70 S6K (T389), total p70 S6K, phospho-4E-BP1 (T37/46), total 4E-BP1, and actin in ES (TC-32), OS (KHOS), and RMS (RD) cells treated with increasing concentrations of buparlisib for one hour. (B) Phospho-Akt (S473) and total Akt levels were quantitated based on Odyssey software integrated intensity values. Phospho-Akt levels were normalized to total Akt levels, then normalized to cells treated with DMSO in order to determine relative phospho-Akt levels. Relative phospho-Akt levels were plotted against log buparlisib concentration and IC50 values were calculated by fitting this data to a four-parameter, variable slope sigmoid dose-response model. Each data point represents the average of at least three independent experiments.
Mentions: We next evaluated the ability of buparlisib to inhibit cellular signaling downstream of PI3K. Increasing doses were added to cells and phosphorylation of signaling molecules was examined by immunoblot after one hour of treatment (Fig 2A). Phosphorylation of Akt was inhibited at both serine 473 and threonine 308. Quantitative immunoblot analysis was used to measure levels of Akt phosphorylation at serine 473 (Fig 2A). Phospho-Akt levels were normalized to levels of total Akt and fit to dose-response curves to calculate the half maximal inhibitory concentration (IC50) (Fig 2B). IC50 values for ranged from 64 to 916 nM, with the largest values occurring in cell lines that lack PTEN expression (S1 Table). Buparlisib also inhibited the phosphorylation of proteins downstream from mTORC1, such as S6K and 4E-BP1 (Fig 2A). Phosphorylation of S6K was more potently inhibited by buparlisib. Inhibition of phospho-4E-BP1 was only observed at high buparlisib concentrations.

Bottom Line: Anti-proliferative effects were observed in both anchorage dependent and independent conditions and apoptosis was induced within 24 hours of drug treatment.Buparlisib demonstrated cytotoxicity as a single agent, but was found to be more effective when used in combination.The addition of NVP-AEW541 also further reduced phospho-Akt levels and more potently induced apoptosis compared to buparlisib treatment alone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Division of Hematology/Oncology, Gwynne Hazen Cherry Memorial Laboratories, University of California, Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Pediatric bone and soft tissue sarcomas often display increased Akt phosphorylation through up regulation of insulin-like growth factor (IGF1) signaling. Additionally, Akt signaling has been linked to resistance to IGF1 receptor (IGF1R) and mTOR (mammalian target of rapamycin) inhibitors in sarcoma, further demonstrating the role of Akt in tumor survival. This suggests targeting components of the PI3K/Akt pathway may be an effective therapeutic strategy. Here, we investigated the in vitro activity of the pan-class I PI3K inhibitor buparlisib (BKM120) in pediatric bone and soft tissue sarcomas. Buparlisib inhibited activation of Akt and signaling molecules downstream of mTORC1 (mTOR complex 1) in Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma cell lines. Anti-proliferative effects were observed in both anchorage dependent and independent conditions and apoptosis was induced within 24 hours of drug treatment. Buparlisib demonstrated cytotoxicity as a single agent, but was found to be more effective when used in combination. Synergy was observed when buparlisib was combined with the IGF1R inhibitor NVP-AEW541 and the mTORC1 inhibitor rapamycin. The addition of NVP-AEW541 also further reduced phospho-Akt levels and more potently induced apoptosis compared to buparlisib treatment alone. Additionally, the combination of buparlisib with the MEK1/2 inhibitor trametinib resulted in synergy in sarcoma cell lines possessing MAPK pathway mutations. Taken together, these data indicate buparlisib could be a novel therapy for the treatment of pediatric bone and soft tissue sarcomas.

No MeSH data available.


Related in: MedlinePlus