Preclinical assessments of the MEK inhibitor PD-0325901 in a mouse model of Neurofibromatosis type 1

Abstract

Background: Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes affected individuals to formation of benign neurofibromas, peripheral nerve tumors that can be associated with significant morbidity. Loss of the NF1 Ras-GAP protein causes increased Ras-GTP, and we previously found that inhibiting MEK signaling downstream of Ras can shrink established neurofibromas in a genetically engineered murine model.

Procedures: We studied effects of MEK inhibition using 1.5 mg/kg/day PD-0325901 prior to neurofibroma onset in the Nf1 (flox/flox); Dhh-Cre mouse model. We also treated mice with established tumors at 0.5 and 1.5 mg/kg/day doses of PD-0325901. We monitored tumor volumes using MRI and volumetric measurements, and measured pharmacokinetic and pharmacodynamic endpoints.

Results: Early administration significantly delayed neurofibroma development as compared to vehicle controls. When treatment was discontinued neurofibromas grew, but no rebound effect was observed and neurofibromas remained significantly smaller than controls. Low dose treatment of mice with PD-0325901 resulted in neurofibroma shrinkage equivalent to that observed at higher doses. Tumor cell proliferation decreased, although less than at higher doses with drug. Tumor blood vessels per area correlated with tumor shrinkage.

Conclusions: Neurofibroma development was not prevented by MEK inhibition, beginning at 1 month of age, but tumor size was controlled by early treatment. Moreover, treatment with PD-0325901 at very low doses may shrink neurofibromas while minimizing toxicity. These studies highlight how genetically engineered mouse models can guide clinical trial design.

Keywords: MEK; NF1; nerve; neurofibroma; therapy.

© 2015 Wiley Periodicals, Inc.

Figures

Fig. 1. Early exposure to MEK inhibitor delays neurofibroma growth

(A) Treatment paradigm. Mice were treated from 1 to 4 months of age with either vehicle or PD-0325901 1.5 mg/kg, left untreated from 4 to 7 months of age and for a brief period (12 days) some mice were re-exposed to PD-0325901 or vehicle for pharmacodynamics (PD) assessment. (B) Waterfall plot showing neurofibroma volume in mice at 4 months of age. Volumes of neurofibromas in mice treated with PD-0325901 are on average significantly smaller than controls. Each bar represents the volume of neurofibroma in an individual mouse. The y-axis shows the neurofibroma volume in mm3. (C, D) Immunohistochemistry at 4 months of age; Blood vessel recruitment was not affected by PD-0325901 when compared to vehicle treated neurofibroma (C). A significant decrease in cell proliferation was found after 90 days of treatment with PD-0325901 (D).

Fig. 2. Representative electron micrograph of saphenous nerves

Untreated wild type (A), vehicle treated Nf1 flox/flox; Dhh-Cre (B) and PD-0325901 treated Nf1 flox/flox; Dhh-Cre (C) mice. The higher magnification in (A) indicates a well-organized un-myelinated Schwann cell ensheathing multiple small axons, cut in cross section. The low magnification micrographs in (B) and (C) show unorganized non-myelinated Schwann cells in Nf1 flox/flox; Dhh-Cre mice treated with vehicle (B) or PD-0325901 (C). At higher magnification (C, bottom) one can appreciate a rare bundle with improved Remak bundle organization. Percentage of grouped axons in wild type control, Nf1 flox/flox; Dhh-Cre vehicle treated control mice and Nf1 flox/flox; Dhh-Cre PD-0325901 treated mice at 4 months of age (D). A high percentage (6 or more) of grouped axons per Schwann cell signifies normal Remak bundle organization.

Fig. 3. Neurofibromas resume growing when PD-01325901 is removed

(A) Neurofibroma volumes at 4 and 7 months of age in mice either treated with PD-0325901 from 1 – 4 months of age (n=8), or vehicle treated/control mice (n=8). (B) Neurofibroma volume at 7 months of age, from mice treated from 1 – 4 months of age. Each bar represents the volume of neurofibromas in an individual mouse. Volumes of treated mice differ from those of untreated mice (p=0.0001). (C) Pharmacodynamic readout. Tumors lysates were prepared from mice treated with PD-0325901 for 90 days at 1.5 mg/kg, taken off drug for 90 days and subsequently treated daily for 12 days. Tumors were harvested 3–4 hours after final dose. Top: phosphorylated ERK (pERK), bottom: total Erk (ERK), V= vehicle treated tumor, PD = PD-0325901 1.5 mg/kg treated tumors.

Fig. 4. Low Dose PD-0325901 shrinks neurofibromas

(A) Mice were MRI scanned at 5 and 7 months of age to determine neurofibroma growth rate. Mice were then treated for 2 months with vehicle, or PD-0325901 (0.5 mg/kg or 1.5 mg/kg), followed by a final scan at 9 months of age. (B) Change in neurofibroma volume from 7 to 9 months of age in mice exposed to vehicle, PD-0325901 0.5 mg/kg or PD-0325901 1.5 mg/kg. Each bar displays the absolute difference in neurofibroma volume between 7 and 9 months in a single mouse based on serial MRI imaging. A positive value indicates neurofibroma size increase; a negative value indicates neurofibroma shrinkage. Neurofibroma volume remained significantly lower both PD-0325901 treatment groups compared to vehicle treated controls.

Fig. 5. MEK inhibitor retains efficacy at low doses

(A) Blood vessels per field. Immunohistochemistry with the Meca 32 antibody was used to highlight blood vessels. Vessels per field were reduced in the 0.5 mg/kg, showing a significant difference from wild type (pNf fl/+; DhhCre) mice collected at 0.5, 2, 6, and 24 hours after a single dose of drug (n=3 per time point/dose). Blood was also collected from tumor bearing (Nf1 flox/flox; DhhCre) mice 0.5, 2, 6, and 24 hours after a final dose of drug (n=3 per time point/dose). Differences in the PK values between wild type and knockout mice may be explained by the number of doses received or genotype (1 vs. 60 respectively). (F) Pharmacodynamic Analysis. Phosphorylated ERK (pERK): total Erk (ERK), V= vehicle treated tumor, 0.5 and 1.5 correspond to the doses in mg/kg. Numbers are the ratio of P-ERK to total ERK for each sample.