Re-activation of Akt and induction of Erk occur within 24 hours of buparlisib treatment.

<p>(<b>A</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 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>B</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. (<b>C</b>) 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. (<b>D</b>) 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.</p

Buparlisib synergizes with trametinib in cell lines harboring MAPK pathway mutations.

<p>(<b>A</b>) Cell lines with (A673, RD) and without (TC-32, A4573) MAPK pathway mutations were exposed to a series of 1.5-fold dilutions of buparlisib and trametinib alone or in combination at a constant ratio of 1:15 for 72 hours, then cell viability was determined by MTT assay. Columns represent the average of three independent experiments, error bars represent standard deviation. Combination index values greater than 1, equal to 1, or less than one indicate antagonism, additivity, or synergy. (<b>B</b>) Isobologram plot of the effect of buparlisib combined with trametinib. The effective doses of trametinib and buparlisib are plotted on the x- and y-axis, respectively. Points for combination treatment above, on, or below the lines indicate antagonism, additivity, or synergy with lines of linear additivity connecting the ED₅₀, ED₇₅, and E₉₀ for individual treatments. Isobologram could not be generated for TC-32 cells since no tramenitib cytotoxicity was observed. (<b>C</b>) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/204, T185/187), and total Erk1/2 in A673 cells after 1 hour of treatment with buparlisib, trametinib, or both. (<b>D</b>) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-Erk1/2 (T202/204 on Erk1, T185/187 on Erk2), total Erk1/2, cleaved PARP, and actin in A673 cells after 24 hours of treatment with buparlisib, trametinib, or both.</p

Buparlisib inhibits proliferation of pediatric sarcoma cell lines.

<p>(<b>A</b>) Cells were treated with media containing 0.1% DMSO or concentrations of buparlisib ranging from 10 nM to 10 μM for 72 hours. Cell viability was determined by MTT assay. Percent cell viability was 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 point is the average of at least three independent experiments. (<b>B</b>) Cell viability IC50 values for pediatric sarcoma cell lines. Columns represent the average of at least three independent experiments, error bars represent 95% confidence intervals. Dashed line indicates median IC50. (<b>C</b>) Scatter plot showing the low correlation (R² = 0.07311) between cell viability and phospho-Akt IC50 values. The cell lines that possess the lowest (A204) and highest (A4573) phospho-Akt IC50s are indicated on the graph. Dot colors indicate sarcoma type: gray—ES, blue—OS, burgundy—RMS.</p

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

<div><p>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 <i>in vitro</i> 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.</p></div

PTEN and basal phospho-Akt levels in a panel of tumor cell lines.

<p>(<b>A</b>) Immunoblot analysis of PTEN, phospho-Akt (S473), total Akt, and actin in ES, OS, RMS, breast cancer (MCF-7), and prostate cancer (LNCaP) cell lines. (<b>B</b>) Known PI3K and MAPK pathway mutations in cell lines evaluated in A. ES, Ewing sarcoma; ERMS, embryonal rhabdomyosarcoma; DEL, deletion; INS, insertion.</p

Buparlisib treatment reduces phosphorylation of signaling molecules downstream of PI3K and mTORC1.

<p>(<b>A</b>) 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>B</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.</p

Buparlisib demonstrates increased synergy with rapamycin in PTEN deficient cell lines.

<p>(<b>A</b>) PTEN wild type (TC-32, A673, RD) or PTEN null (A4573) cell lines were exposed to a series of 1.5-fold dilutions of buparlisib and rapamycin alone or in combination at a constant ratio of 20:3 (TC-32, A673, RD) or 100:3 (A4573) for 72 hours, then cell viability was determined by MTT assay. Columns represent the average of three independent experiments, error bars represent standard deviation. Combination index values greater than 1, equal to 1, or less than one indicate antagonism, additivity, or synergy. (<b>B</b>) Isobologram plot of the effect of buparlisib combined with rapamycin. The effective doses of rapamycin and buparlisib are plotted on the x- and y-axis with lines of linear additivity connecting the ED₅₀, ED₇₅, and E₉₀ for individual treatments. Points for combination treatment above, on, or below the lines indicate antagonism, additivity, or synergy, respectively. (<b>C</b>) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-4E-BP1 (T37/46), total 4E-BP1, and actin in A4573 cells after 1 hour of treatment with buparlisib, rapamycin, or both. (<b>D</b>) Immunoblot analysis of phospho-Akt (S473), total Akt, phospho-4E-BP1 (T37/46), total 4E-BP1, cleaved PARP, and actin in A4573 cells after 24 hours of treatment with buparlisib, rapamycin, or both.</p