Figure 2
Figure 2. Pharmacologic inhibition of PI3K catalytic subunit p110δ reduces proliferation of GOF-Shp2-expressing cells and JMML primary cells. (A) Proliferation of bone marrow LDMNCs from polyI:polyC-treated Shp2D61Y/+;Mx1Cre− (D61Y;Cre−) and Shp2D61Y/+;Mx1Cre+ (D61Y;Cre+) mice in response to GM-CSF 1 ng/mL in the presence of increasing concentrations of the p110δ-specific inhibitor, GS-9820; n = 4. *P < .05 for D61Y;Cre+ cells treated with GS-9820 compared with no drug; statistics performed using unpaired, 2-tailed student’s t test. (B) Immunoblot demonstrating reduced phospho-Akt and phospho-Erk in response to increasing concentrations of GS-9820. (C) Proliferation of bone marrow LDMNCs from polyI:polyC-treated Shp2D61Y/+;Mx1Cre− (D61Y;Cre−) and Shp2D61Y/+;Mx1Cre+ (D61Y;Cre+) mice in response to GM-CSF 1 ng/mL in the presence of the MEK inhibitor, PD-0325901, and increasing concentrations of the p110δ-specific inhibitor, GS-9820; n = 4. *P = .0002 for D61Y;Cre+ cells treated with 0.05 μM PD0325901 vs no PD0325901. **P = .01 or **P = .0002 for D61Y;Cre+ cells treated with either 0.05 μM PD0325901 + 0.1 μM GS-9820 or 0.05 μM PD0325901 + 1 μM GS-9820, respectively, compared with 0.05 μM PD-0325901, statistics performed using unpaired, 2-tailed student’s t test. (D) Immunoblot demonstrating maximal inhibition of Erk and Akt activation in the presence of both PD0325901 and GS-9820. (E) Control human bone marrow LDMNCs or primary JMML LDMNCs (2 independent patient samples) were plated in duplicate in methylcellulose-based progenitor assays in the absence or presence of human GM-CSF 10 ng/mL and increasing concentrations of the PI3K p110δ-specific inhibitor, idelalisib. Data are represented as percentage maximal colony formation, calculated by dividing the number of colonies in each condition by the average number of colonies in the presence of 10 ng/mL GM-CSF. (F) Myelomonocytic cell line U937 or primary JMML LDMNCs (3 independent samples) were treated with GM-CSF, idelalisib alone, or GM-CSF plus idelalisib, followed by examination of AKT (S473) and ERK activation by immunoblot.

Pharmacologic inhibition of PI3K catalytic subunit p110δ reduces proliferation of GOF-Shp2-expressing cells and JMML primary cells. (A) Proliferation of bone marrow LDMNCs from polyI:polyC-treated Shp2D61Y/+;Mx1Cre− (D61Y;Cre−) and Shp2D61Y/+;Mx1Cre+ (D61Y;Cre+) mice in response to GM-CSF 1 ng/mL in the presence of increasing concentrations of the p110δ-specific inhibitor, GS-9820; n = 4. *P < .05 for D61Y;Cre+ cells treated with GS-9820 compared with no drug; statistics performed using unpaired, 2-tailed student’s t test. (B) Immunoblot demonstrating reduced phospho-Akt and phospho-Erk in response to increasing concentrations of GS-9820. (C) Proliferation of bone marrow LDMNCs from polyI:polyC-treated Shp2D61Y/+;Mx1Cre− (D61Y;Cre−) and Shp2D61Y/+;Mx1Cre+ (D61Y;Cre+) mice in response to GM-CSF 1 ng/mL in the presence of the MEK inhibitor, PD-0325901, and increasing concentrations of the p110δ-specific inhibitor, GS-9820; n = 4. *P = .0002 for D61Y;Cre+ cells treated with 0.05 μM PD0325901 vs no PD0325901. **P = .01 or **P = .0002 for D61Y;Cre+ cells treated with either 0.05 μM PD0325901 + 0.1 μM GS-9820 or 0.05 μM PD0325901 + 1 μM GS-9820, respectively, compared with 0.05 μM PD-0325901, statistics performed using unpaired, 2-tailed student’s t test. (D) Immunoblot demonstrating maximal inhibition of Erk and Akt activation in the presence of both PD0325901 and GS-9820. (E) Control human bone marrow LDMNCs or primary JMML LDMNCs (2 independent patient samples) were plated in duplicate in methylcellulose-based progenitor assays in the absence or presence of human GM-CSF 10 ng/mL and increasing concentrations of the PI3K p110δ-specific inhibitor, idelalisib. Data are represented as percentage maximal colony formation, calculated by dividing the number of colonies in each condition by the average number of colonies in the presence of 10 ng/mL GM-CSF. (F) Myelomonocytic cell line U937 or primary JMML LDMNCs (3 independent samples) were treated with GM-CSF, idelalisib alone, or GM-CSF plus idelalisib, followed by examination of AKT (S473) and ERK activation by immunoblot.

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