MEK inhibition exhibits efficacy in human and mouse neurofibromatosis tumors

Abstract

Neurofibromatosis type 1 (NF1) patients develop benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). These incurable peripheral nerve tumors result from loss of NF1 tumor suppressor gene function, causing hyperactive Ras signaling. Activated Ras controls numerous downstream effectors, but specific pathways mediating the effects of hyperactive Ras in NF1 tumors are unknown. We performed cross-species transcriptome analyses of mouse and human neurofibromas and MPNSTs and identified global negative feedback of genes that regulate Ras/Raf/MEK/ERK signaling in both species. Nonetheless, ERK activation was sustained in mouse and human neurofibromas and MPNST. We used a highly selective pharmacological inhibitor of MEK, PD0325901, to test whether sustained Ras/Raf/MEK/ERK signaling contributes to neurofibroma growth in a neurofibromatosis mouse model (Nf1(fl/fl);Dhh-Cre) or in NF1 patient MPNST cell xenografts. PD0325901 treatment reduced aberrantly proliferating cells in neurofibroma and MPNST, prolonged survival of mice implanted with human MPNST cells, and shrank neurofibromas in more than 80% of mice tested. Our data demonstrate that deregulated Ras/ERK signaling is critical for the growth of NF1 peripheral nerve tumors and provide a strong rationale for testing MEK inhibitors in NF1 clinical trials.

Figure 1. PD0325901 reduces p-ERK and inhibits MPNST cell growth.

(A–D) Brown staining indicates detection of p-ERK in paraffin tissue sections. p-ERK is robust in human S462TY MPNST xenografts (A), but is absent 30 minutes after treatment with 10 mg/kg PD0325901 (PD) (B). p-ERK remains detectable at low levels at 6 hours after treatment (C) and returns to pretreatment levels by 24 hours (D). Scale bar: 50 μm. (E) Dose-response analysis of PD0325901 on 5 MPNST cell lines. Effect on cell growth is expressed as percentage of control, and PD0325901 concentration shows nM concentrations on a log scale. (F) Tumor volume (mm³) was significantly reduced in S462TY MPNST xenografts treated with PD0325901 (n = 18) versus control (n = 12). Reduction in tumor volume observed by treatment day 5 was maintained through day 15 (P = 0.004), when many control mice required sacrifice. (E and F) Error bars represent mean ± SEM. (G) Survival of MPNST xenografted mice doubled with 3 months PD0325901 treatment (P < 0.0001).

Figure 2. PD0325901 inhibits neurofibroma growth.

(A–F) Serial MRI in Nf1^flox/flox;Dhh-Cre mice given vehicle (A–C) versus 10 mg/kg PD0325901 (D–F). MRI was conducted on 5-month-old pretreated mice (A and D) at treatment onset (B and E, day 0; 7 months old), and at the end of treatment (C and F, day 60; 9 months old). Images show representative tumor-bearing mice given vehicle control (A–C) or PD0325901 (D–F). Note the reduction in size and intensity of bright bilateral neurofibromas treated with PD0325901. (G) Volumetric measurements of vehicle-treated or PD0325901-treated Nf1^flox/flox;Dhh-Cre mice indicate a decrease in neurofibroma volume treated with 1.5 mg/kg, 5 mg/kg, or 10 mg/kg PD0325901 for 2 months. The y axis shows tumor volume in mm³ quantified by measurements of MRI scans. Each bar represents the difference in tumor volume in an individual animal from day 0 (7 months) to day 60 (9 months). Mixed effects model analysis indicated statistical significance for each dose (P < 0.001; see Supplemental Figure 4).

Figure 3. Molecular mechanism of PD0325901 in Nf1^fl/fl;Dhh-Cre neurofibromas and MPNST xenografts.

(A and B) Assessment of proliferation in neurofibromas and MPNSTs by quantification of Ki67⁺ cells as compared with hematoxylin-stained nuclei. (A) Neurofibromas in Nf1^fl/fl;Dhh-Cre mice treated with 1.5, 5.0, or 10 mg/kg PD0325901 for 60 days showed a significant (***P < 0.001) reduction in the percentage of Ki67⁺ cells relative to mice treated with control vehicle. (B) Mice harboring MPNST xenografts treated with PD0325901 for 11 days and 28 days show a significant (*P < 0.05) reduction in the percentage of Ki67⁺ cells relative to mice treated with control vehicle. (C and D) Labeling of Ki67⁺ proliferating cells (green), CNPase⁺ (red) Schwann cells, and DAPI⁺ (blue) cell nuclei in Nf1^fl/fl;Dhh-Cre neurofibromas (C) and MPNST xenografts (D). Arrows represent double-label cells (Ki67⁺; CNPase⁺). Scale bar: 50 μm. (E and F) Assessment of vasculature in neurofibromas and MPNSTs by quantification of MECA⁺ endothelial cells. Number of blood vessels per high-powered field was significantly (**P < 0.01) reduced in both neurofibromas (E) and MPNSTs (F) in response to PD0325901. (G–J) p-S6K is detected in mouse neurofibromas (G and H) and human MPNST xenografts (I and J); p-S6K levels decrease in response to treatment with PD0325901 (H and J) relative to control (G and I). (K–N) p-AKT is detected in mouse neurofibromas (K and L) and human MPNST xenografts (M and N), and p-AKT levels do not change in response to treatment with PD0325901 (L and N) relative to control (K and M). Scale bars: 50 μm.

Figure 4. Negative feedback regulation of p-ERK in Dhh-Cre neurofibromas.

(A–H) Brown staining indicates detection of p -ERK in paraffin tissue sections. Nf1^fl/fl;Dhh-Cre mouse p-ERK staining is robust in carrier-treated neurofibroma (A and E), but is absent 30 minutes after treatment with 10 mg/kg (B) or 1.5 mg/kg (F) PD0325901. p-ERK becomes detectable 6 hours (C) after treatment with 10 mg/kg PD0325901 and returns to pretreatment levels by 24 hours (D). p-ERK also becomes detectable 6 hours (G) after treatment with 1.5 mg/kg PD0325901, but does not return to pretreatment levels by 24 hours (H). Scale bars: 50 μm. (I–K) qRT-PCR assessment of Ras pathway negative feedback. (I) Independent qRT-PCR confirmation of microarray data (Supplemental Figure 1B) showing overexpression of SPRY4 and DUSP6 in Nf1^fl/fl;Dhh-Cre neurofibromas relative to WT mouse nerve. (J) Fold-change in SPRY4 and DUSP6 gene expression relative to pretreatment (control) at 6 and 24 hours after treatment with 10 mg/kg PD0325901, reflecting changes in p-ERK observed in (A–D). (K) Fold change in SPRY4 and DUSP6 gene expression relative to pretreatment (control) at 6 and 24 hours after treatment with 1.5 mg/kg PD0325901, reflecting changes in p-ERK observed in E and F. Error bars represent mean ± SD.