Targeting PDGFRα-activated glioblastoma through specific inhibition of SHP-2-mediated signaling

Abstract

Background: Glioblastoma (GBM) is the most malignant primary brain tumor, with dismal median survival. Treatment of GBM is particularly challenging given the intrinsic resistance to chemotherapy and difficulty of drugs to reach the tumor beds due to the blood-brain barrier. Here, we examined the efficacy of SHP099, a potent, selective, and oral SHP-2 inhibitor for treating GBM with activated platelet derived growth factor receptor alpha (PDGFRα) signaling.

Methods: The effects of SHP099 on cell survival of neural progenitor cells (NPCs), GBM cell lines, and patient-derived glioma stem-like cells (GSCs) were evaluated. Brain and plasma pharmacokinetics of SHP099 and its ability to inhibit SHP-2 signaling were assessed. SHP099 efficacy as a single agent or in combination with temozolomide (TMZ) was assessed using transformed mouse astrocyte and GSC orthotopic xenograft models.

Results: Activated PDGFRα signaling in established GBM cells, GSCs, and transformed mouse astrocytes was significantly inhibited by SHP099 compared with NPCs in vitro and in vivo through targeting SHP-2-stimulated activation of extracellular signal-regulated protein kinases 1 and 2 in GBM. SHP099 treatment specifically inhibited expression of JUN, a downstream effector of PDGFR signaling, thereby attenuating cell cycle progression in GBM cells with activated PDGFRα. Moreover, SHP099 accumulated at efficacious concentrations in the brain and effectively inhibited orthotopic GBM tumor xenograft growth. SHP099 exhibited antitumor activity either as a single agent or in combination with TMZ and provided significant survival benefits for GBM tumor xenograft-bearing animals.

Conclusions: Our data demonstrate the utility and feasibility of SHP099 as a potential therapeutic option for improving the clinical treatment of GBM in combination with TMZ.

Keywords: PDGFRα; SHP-2; SHP099; cell cycle; glioma stem-like cell.

Fig. 1

GSCs are more responsive to SHP099 treatment than NPCs. (A) Viability of NPC, GSC 1123, R83, R39, 528, 157, and AC17 cells at 72 h after treatment with SHP099. (B) Comparison of SHP099 IC₅₀ values in A. (C) Effects of SHP099 in limiting dilution glioma sphere-forming assay of GSC R83, R39, and 157 cells. (D) Western blotting (WB) analysis of expression levels of EGFR, p-EGFR, PDGFRα, p-PDGFRα, SHP-2, and p-SHP-2 in NPCs and GSCs. β-actin was used as a loading control. Arrow, EGFR; arrowhead, EGFRvIII. *P < 0.05, **P < 0.01, by one-way ANOVA.

Fig. 2

Glioma cells with PDGFRα activation are more responsive to SHP099. (A) Viability of GSC 1123, R83, 157, and AC17 cells with PDGF-A stimulation after SHP099 treatment. GSCs were pre-cultured for 24 h in Dulbecco’s modified Eagle’s medium/F12 with EGF (2 ng/mL) and basic fibroblast growth factor (2 ng/mL) and then followed by co-culturing with or without 100 ng/mL PDGF-A and the indicated SHP099 concentrations for 72 h. (B and G) Viability of LN444 cells with ectopic expression of an EV or PDGF-A (B) or Ink4a/Arf^−/− mAsts with overexpression of an EV or PDGFRα plus PDGF-A (G) at 72 h after treatment with SHP099. (C and H) Comparison of SHP099 IC₅₀ in (B) or (G). (D and I) WB assays of expression of PDGF-A, PDGFRα, and p-PDGFRα in LN444 or Ink4a/Arf^−/− mAsts. (E and J) Representative images of drug-resistant colony formation in LN444 cells (E) or Ink4a/Arf^−/− mAsts (J) at day 7 post SHP099 treatment. (F and K) Quantification of drug-resistant colony formation in (E) and (J), respectively. Scale bars, 400 μm. *P < 0.05, **P < 0.01, by one-way ANOVA or two-tailed Student’s t-test.

Fig. 3

SHP099 specifically inhibits PDGFRα‒SHP-2-stimulated ERK1/2 activity. (A) WB assays of effects of SHP099 on PDGF-A stimulated p-PDGFRα, ERK1/2 phosphorylation (p-ERK1/2), and Akt phosphorylation (p-Akt) in Ink4a/Arf^−/− mAsts overexpressing an EV, PDGFRα wild-type (WT), or kinase-dead mutant (R627). (B) Viability of Ink4a/Arf^−/− mAsts with an EV, PDGFRα WT, or R627 mutant at 72 h after treatment with SHP099 in combination with or without PDGF-A stimulation. (C) Schematics of various PDGFRα mutants. (D) Effects of ectopic expression of PDGFRα WT and F720 mutant on SHP099 inhibition of ERK1/2 phosphorylation in Ink4a/Arf^−/− mAsts. (E) Effects of expression of PDGFRα F7, Y720, and Y1018 mutants on SHP099 inhibition of ERK1/2 phosphorylation. (F) Viability of PDGF-A-stimulated Ink4a/Arf^−/− mAsts with ectopic expression of an EV, PDGFRα WT, or indicated mutants at 72 h after treatment with SHP099. (G) Effects of SHP-2 knockdown on SHP099 inhibition of ERK1/2 phosphorylation in LN444 with PDGF-A overexpression. (H) Viability of LN444/PDGF-A/shSHP-2 and LN444/PDGF-A/shC cells at 72 h after treatment with SHP099. ShSHP-2, SHP-2 shRNAs. shC, a control shRNA. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA or two-tailed Student’s t-test.

Fig. 4

SHP099 specifically inhibits cell cycle pathways in glioma cells with PDGFRα activation. (A) Heatmap of RNA-Seq analysis of differentially expressed genes (2-fold change and false discovery rate < 0.05) in Ink4a/Arf^−/− mAsts with ectopic expression of PDGFRα and PDGF-A treated with 0, 5, or 10 μM SHP099 in duplicate samples. (B) Expression of JUN after SHP099 treatment using RNA-Seq (left) and qRT-PCR (right) assays. (C) Gene Ontology analysis indicated that genes downregulated by SHP099 were associated with cell cycle pathways. (D) GSEA of SHP099-inhibited pathways using ranked gene expression changes in Ink4a/Arf^−/− mAsts with ectopic expression of PDGFRα and PDGF-A treated with 5 or 10 μM SHP099 compared with a vehicle control (0 μM SHP099). NES, normalized enrichment score. (E and G) Representative images from flow cytometric analysis of the influence of SHP099 treatment on cell cycle in Ink4a/Arf^−/− mAsts with ectopic expression of PDGFRα and PDGF-A (E) or LN444 cells with ectopic expression of an EV or PDGF-A (G). (F and H) Percentage of cells in G0/G1 phase in (E) and (G), respectively. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA.

Fig. 4

SHP099 specifically inhibits cell cycle pathways in glioma cells with PDGFRα activation. (A) Heatmap of RNA-Seq analysis of differentially expressed genes (2-fold change and false discovery rate < 0.05) in Ink4a/Arf^−/− mAsts with ectopic expression of PDGFRα and PDGF-A treated with 0, 5, or 10 μM SHP099 in duplicate samples. (B) Expression of JUN after SHP099 treatment using RNA-Seq (left) and qRT-PCR (right) assays. (C) Gene Ontology analysis indicated that genes downregulated by SHP099 were associated with cell cycle pathways. (D) GSEA of SHP099-inhibited pathways using ranked gene expression changes in Ink4a/Arf^−/− mAsts with ectopic expression of PDGFRα and PDGF-A treated with 5 or 10 μM SHP099 compared with a vehicle control (0 μM SHP099). NES, normalized enrichment score. (E and G) Representative images from flow cytometric analysis of the influence of SHP099 treatment on cell cycle in Ink4a/Arf^−/− mAsts with ectopic expression of PDGFRα and PDGF-A (E) or LN444 cells with ectopic expression of an EV or PDGF-A (G). (F and H) Percentage of cells in G0/G1 phase in (E) and (G), respectively. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA.

Fig. 5

SHP099 can effectively cross the BBB and inhibits PDGFRα-driven glioma tumor growth as a single agent. (A) Pharmacokinetics of SHP099 in plasma and brain tissue of immunocompetent mice. Six-week-old C57BL/6J female mice were administered by oral gavage a single dose of SHP099 (100 mg/kg in a total volume of 400 μL). Brain tissue and plasma were harvested at indicated time points post oral gavage (0 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h) and subjected to UHPLC-MS analysis. Three mice were used for each point. See also Supplementary Table 3B and G) Treatment schemes for the evaluation of in vivo efficacy of SHP099 in Ink4a/Arf^−/− mAst (B) or GSC 157 (G) xenografts. Animals were treated with the indicated SHP099 doses from Monday to Friday within 2 weeks. (C and D) Representative images (C) at day 20 and quantitation of BLI (D) of Ink4a/Arf^−/− mAst xenografts with ectopic expression of PDGFRα and PDGF-A from SHP099 treated and control mice. (E) Kaplan–Meier survival analysis of animals with Ink4a/Arf^−/− mAst tumors (n = 10 per group). (F) Upper panel, representative images of IHC analysis of xenografts in C using anti-p-ERK1/2 and anti-CD31 antibodies. Lower panel, quantitation of p-ERK1/2 and CD31 positive cells. (H, I) Representative images at day 50 (H) and quantitation of BLI (I) of GSC 157 xenografts from SHP099 treated and control mice. (J) Kaplan–Meier survival analysis of mice with GSC 157 tumors (n = 10 per group). (K) Representative images of IHC analysis of xenografts in H using anti-p-ERK1/2 and anti-CD31 antibodies. Lower, quantitation of p-ERK1/2 and CD31 positive cells. In F and K, scale bars, 50 μm. *P < 0.05, **P < 0.01, by two-tailed Student’s t-test or log-rank analysis.

Fig. 5

SHP099 can effectively cross the BBB and inhibits PDGFRα-driven glioma tumor growth as a single agent. (A) Pharmacokinetics of SHP099 in plasma and brain tissue of immunocompetent mice. Six-week-old C57BL/6J female mice were administered by oral gavage a single dose of SHP099 (100 mg/kg in a total volume of 400 μL). Brain tissue and plasma were harvested at indicated time points post oral gavage (0 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h) and subjected to UHPLC-MS analysis. Three mice were used for each point. See also Supplementary Table 3B and G) Treatment schemes for the evaluation of in vivo efficacy of SHP099 in Ink4a/Arf^−/− mAst (B) or GSC 157 (G) xenografts. Animals were treated with the indicated SHP099 doses from Monday to Friday within 2 weeks. (C and D) Representative images (C) at day 20 and quantitation of BLI (D) of Ink4a/Arf^−/− mAst xenografts with ectopic expression of PDGFRα and PDGF-A from SHP099 treated and control mice. (E) Kaplan–Meier survival analysis of animals with Ink4a/Arf^−/− mAst tumors (n = 10 per group). (F) Upper panel, representative images of IHC analysis of xenografts in C using anti-p-ERK1/2 and anti-CD31 antibodies. Lower panel, quantitation of p-ERK1/2 and CD31 positive cells. (H, I) Representative images at day 50 (H) and quantitation of BLI (I) of GSC 157 xenografts from SHP099 treated and control mice. (J) Kaplan–Meier survival analysis of mice with GSC 157 tumors (n = 10 per group). (K) Representative images of IHC analysis of xenografts in H using anti-p-ERK1/2 and anti-CD31 antibodies. Lower, quantitation of p-ERK1/2 and CD31 positive cells. In F and K, scale bars, 50 μm. *P < 0.05, **P < 0.01, by two-tailed Student’s t-test or log-rank analysis.

Fig. 6

SHP099 in combination with TMZ extends the survival of GBM-bearing animal. (A and E) Treatment scheme for the evaluation of in vivo efficacy of SHP099 in combination with TMZ in Ink4a/Arf^−/− mAsts (A) or GSC 157 (E) tumor xenografts. The mice were treated with indicated 100 mg/kg SHP099 with or without 8 mg/kg TMZ from Monday to Friday within 2 weeks. (B and C) Representative images (B) on day 20 and quantitation of BLI at the indicated date (C) of Ink4a/Arf^−/− mAst xenografts with ectopic expression of PDGFRα and PDGF-A from SHP099 treated and control mice. (D) Kaplan–Meier survival analysis of animals with Ink4a/Arf^−/− mAst tumors (n = 10 per group). (F and G) Representative images on day 50 (F) and quantitation of BLI at the indicated date (G) of GSC 157 tumor xenografts from SHP099 treated and control mice. (H) Kaplan–Meier survival analysis of mice with GSC 157 tumor xenografts (n = 6 per group). **P < 0.01, ***P < 0.001, by two-tailed Student’s t-test, one-way ANOVA, or log-rank analysis.