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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

Re-activation of Akt and induction of Erk occur within 24 hours of buparlisib treatment.(A) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in ES (TC-174, A4573), OS (KHOS), and RMS (RD) cells after treatment with buparlisib for periods of 5 minutes to 24 hours. TC-174, KHOS, and RD cells were treated with 1 μM buparlisib. A4573 cells were treated with 3 μM buparlisib. (B) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in KHOS cells after treatment with 3 μM buparlisib for periods of 5 minutes to 24 hours. (C) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), and total Erk in TC-32 and A4573 cells with increasing concentrations of buparlisib for 24 hours. (D) Immunoblot analysis of phospho-STAT3 (Y705), total STAT3, phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in TC-174 and A4573 parental cells and cells passaged in increasing concentrations of buparlisib. Phospho-Akt levels were quantitated based on Odyssey software integrated intensity values. Values are listed below each band.
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pone.0133610.g003: Re-activation of Akt and induction of Erk occur within 24 hours of buparlisib treatment.(A) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in ES (TC-174, A4573), OS (KHOS), and RMS (RD) cells after treatment with buparlisib for periods of 5 minutes to 24 hours. TC-174, KHOS, and RD cells were treated with 1 μM buparlisib. A4573 cells were treated with 3 μM buparlisib. (B) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in KHOS cells after treatment with 3 μM buparlisib for periods of 5 minutes to 24 hours. (C) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), and total Erk in TC-32 and A4573 cells with increasing concentrations of buparlisib for 24 hours. (D) Immunoblot analysis of phospho-STAT3 (Y705), total STAT3, phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in TC-174 and A4573 parental cells and cells passaged in increasing concentrations of buparlisib. Phospho-Akt levels were quantitated based on Odyssey software integrated intensity values. Values are listed below each band.

Mentions: We also performed a time course of buparlisib treatment to examine the dynamics of Akt inhibition. Cells were treated with buparlisib for periods of five minutes up to 24 hours and immunoblot analysis was utilized to measure levels of Akt and Erk phosphorylation. Akt was inhibited quickly and displayed maximum inhibition after approximately one hour of buparlisib treatment. After three hours, phospho-Akt levels increased despite continued treatment with buparlisib (Fig 3A). This pattern of Akt reactivation was also observed MCF-7 cells (S1A Fig). Increasing the concentration of buparlisib lengthened the duration of time prior to Akt reactivation and reduced the amount of phosphorylation (Fig 3B). Akt phosphorylation increased at threonine 308 and serine 473 (Fig 3A), indicating a reactivation mechanism that is upstream of both PDK1 and mTORC2. Additionally, we did not observe an increase in phospho-S6K levels over time after buparlisib treatment (S2 Fig). In some cell lines, the increase in Akt phosphorylation corresponded with an increase in phospho-Erk levels (Fig 3A). Additionally, elevated Erk phosphorylation was observed at higher concentrations of buparlisib in TC-32 and A4573 cells after 24 hours of treatment (Fig 3C).


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)

Re-activation of Akt and induction of Erk occur within 24 hours of buparlisib treatment.(A) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in ES (TC-174, A4573), OS (KHOS), and RMS (RD) cells after treatment with buparlisib for periods of 5 minutes to 24 hours. TC-174, KHOS, and RD cells were treated with 1 μM buparlisib. A4573 cells were treated with 3 μM buparlisib. (B) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in KHOS cells after treatment with 3 μM buparlisib for periods of 5 minutes to 24 hours. (C) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), and total Erk in TC-32 and A4573 cells with increasing concentrations of buparlisib for 24 hours. (D) Immunoblot analysis of phospho-STAT3 (Y705), total STAT3, phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in TC-174 and A4573 parental cells and cells passaged in increasing concentrations of buparlisib. Phospho-Akt levels were quantitated based on Odyssey software integrated intensity values. Values are listed below each band.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133610.g003: Re-activation of Akt and induction of Erk occur within 24 hours of buparlisib treatment.(A) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in ES (TC-174, A4573), OS (KHOS), and RMS (RD) cells after treatment with buparlisib for periods of 5 minutes to 24 hours. TC-174, KHOS, and RD cells were treated with 1 μM buparlisib. A4573 cells were treated with 3 μM buparlisib. (B) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in KHOS cells after treatment with 3 μM buparlisib for periods of 5 minutes to 24 hours. (C) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), and total Erk in TC-32 and A4573 cells with increasing concentrations of buparlisib for 24 hours. (D) Immunoblot analysis of phospho-STAT3 (Y705), total STAT3, phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/T204 on Erk1, T185/T187 on Erk2), total Erk, and actin in TC-174 and A4573 parental cells and cells passaged in increasing concentrations of buparlisib. Phospho-Akt levels were quantitated based on Odyssey software integrated intensity values. Values are listed below each band.
Mentions: We also performed a time course of buparlisib treatment to examine the dynamics of Akt inhibition. Cells were treated with buparlisib for periods of five minutes up to 24 hours and immunoblot analysis was utilized to measure levels of Akt and Erk phosphorylation. Akt was inhibited quickly and displayed maximum inhibition after approximately one hour of buparlisib treatment. After three hours, phospho-Akt levels increased despite continued treatment with buparlisib (Fig 3A). This pattern of Akt reactivation was also observed MCF-7 cells (S1A Fig). Increasing the concentration of buparlisib lengthened the duration of time prior to Akt reactivation and reduced the amount of phosphorylation (Fig 3B). Akt phosphorylation increased at threonine 308 and serine 473 (Fig 3A), indicating a reactivation mechanism that is upstream of both PDK1 and mTORC2. Additionally, we did not observe an increase in phospho-S6K levels over time after buparlisib treatment (S2 Fig). In some cell lines, the increase in Akt phosphorylation corresponded with an increase in phospho-Erk levels (Fig 3A). Additionally, elevated Erk phosphorylation was observed at higher concentrations of buparlisib in TC-32 and A4573 cells after 24 hours of treatment (Fig 3C).

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