Figure 7
Figure 7. SHP2 inhibitor II-B08 enhances the efficacy of PI3K inhibitor in repressing oncogenic KITD814V-induced growth. (A) The 32D cells bearing WT KIT or KITD814V were starved for 6 hours followed by treatment with or without II-B08 (20μM) for 1 hour. After incubation, cells were lysed and equal amounts of protein lysates were subjected to Western blot analysis using an anti–phospho-KIT (Y719), total KIT, anti–phospho-AKT, total AKT, anti–phospho-ERK, or total ERK antibody as indicated. Similar results were observed in 3 independent experiments. (B) The 32D cells bearing WT KIT or KITD814V were starved of serum and growth factors for 6 hours and subjected to proliferation assay in the presence or absence of PI-3K inhibitor (LY294002, 2μM) and SHP2 inhibitor (II-B08, 5μM) alone or in combination. Bars represent the mean thymidine incorporation (CPM ± SD) from 1 of 3 independent experiments performed in quadruplicate. *P < .05, no GF vs LY294002 or II-B08. **P < .05, LY294002 or II-B08 vs LY294002 and II-B08. (C) Kaplan-Meier survival analysis of MPD mice treated with II-B08 or LY294002 alone or in combination. The 32D cells bearing oncogenic KITD814V were injected into syngeneic C3H/HeJ mice through tail vein. After 48 hours, mice were treated with either vehicle DMSO or II-B08 (50 mg/kg body weight) or LY294002 (10 mg/kg body weight) or a combination of II-B08 and LY294002 (50 + 10 mg/kg body weight, respectively) at 24-hour intervals for 21 days (n = 5). Significantly prolonged survival of mice treated with II-B08 or LY294002 alone or in combination was observed compared with mice treated with DMSO. *P < .05, DMSO vs II-B08 or LY294002. **P < .05, II-B08 or LY294002 vs II-B08 and LY294002. (D-E) Reduced splenomegaly and hepatomegaly in mice treated with II-B08 or LY294002 or combination. Average weights (D) and pictures (E) of spleens and livers from mice transplanted with cells bearing oncogenic KITD814V and treated with DMSO or II-B08 or LY294002 or combination of II-B08 and LY294002. Significant reduction in spleen and liver weights was observed in mice treated with II-B08 or LY294002 or combination of II-B08 and LY294002 compared with mice treated with DMSO. n = 4. *P < .05, DMSO vs II-B08 or LY294002. **P < .05, II-B08 or LY294002 vs II-B08 and LY294002.

SHP2 inhibitor II-B08 enhances the efficacy of PI3K inhibitor in repressing oncogenic KITD814V-induced growth. (A) The 32D cells bearing WT KIT or KITD814V were starved for 6 hours followed by treatment with or without II-B08 (20μM) for 1 hour. After incubation, cells were lysed and equal amounts of protein lysates were subjected to Western blot analysis using an anti–phospho-KIT (Y719), total KIT, anti–phospho-AKT, total AKT, anti–phospho-ERK, or total ERK antibody as indicated. Similar results were observed in 3 independent experiments. (B) The 32D cells bearing WT KIT or KITD814V were starved of serum and growth factors for 6 hours and subjected to proliferation assay in the presence or absence of PI-3K inhibitor (LY294002, 2μM) and SHP2 inhibitor (II-B08, 5μM) alone or in combination. Bars represent the mean thymidine incorporation (CPM ± SD) from 1 of 3 independent experiments performed in quadruplicate. *P < .05, no GF vs LY294002 or II-B08. **P < .05, LY294002 or II-B08 vs LY294002 and II-B08. (C) Kaplan-Meier survival analysis of MPD mice treated with II-B08 or LY294002 alone or in combination. The 32D cells bearing oncogenic KITD814V were injected into syngeneic C3H/HeJ mice through tail vein. After 48 hours, mice were treated with either vehicle DMSO or II-B08 (50 mg/kg body weight) or LY294002 (10 mg/kg body weight) or a combination of II-B08 and LY294002 (50 + 10 mg/kg body weight, respectively) at 24-hour intervals for 21 days (n = 5). Significantly prolonged survival of mice treated with II-B08 or LY294002 alone or in combination was observed compared with mice treated with DMSO. *P < .05, DMSO vs II-B08 or LY294002. **P < .05, II-B08 or LY294002 vs II-B08 and LY294002. (D-E) Reduced splenomegaly and hepatomegaly in mice treated with II-B08 or LY294002 or combination. Average weights (D) and pictures (E) of spleens and livers from mice transplanted with cells bearing oncogenic KITD814V and treated with DMSO or II-B08 or LY294002 or combination of II-B08 and LY294002. Significant reduction in spleen and liver weights was observed in mice treated with II-B08 or LY294002 or combination of II-B08 and LY294002 compared with mice treated with DMSO. n = 4. *P < .05, DMSO vs II-B08 or LY294002. **P < .05, II-B08 or LY294002 vs II-B08 and LY294002.

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