Preclincial testing of sorafenib and RAD001 in the Nf(flox/flox) ;DhhCre mouse model of plexiform neurofibroma using magnetic resonance imaging

Abstract

Background: Neurofibromatosis type 1 (NF1) is an inherited disease predisposing affected patients to variable numbers of benign neurofibromas. To date there are no effective chemotherapeutic drugs available for this slow growing tumor. Molecularly targeted agents that aim to slow neurofibroma growth are being tested in clinical trials. So preclinical models for testing potential therapies are urgently needed to prioritize drugs for clinical trials of neurofibromas.

Procedure: We used magnetic resonance imaging (MRI) to monitor neurofibroma development in the Nf1(flox/flox) ;DhhCre mouse model of GEM grade I neurofibroma. Based on studies implicating mTOR and Raf signaling in NF1 mutant cells, we tested the therapeutic effect of RAD001 and Sorafenib in this model. Mice were scanned to establish growth rate followed by 8 weeks of drug treatment, then re-imaged after the last dose of drug treatment. Tumor volumes were determined by volumetric measurement.

Results: We found that rate of tumor growth varied among mice, as it does in human patients. RAD001 inhibited its predicted target pS6K, yet there was no significant decrease in the tumor volume in RAD001 treated mice compared to the vehicle control group. Sorafenib inhibited cyclinD1 expression and cell proliferation in tumors, and volumetric measurements identified significant decreases in tumor volume in some mice.

Conclusion: The data demonstrate that volumetric MRI analysis can be used to monitor the therapeutic effect in the preclinical neurofibroma drug screening, and suggest that Sorafenib might have clinical activity in some neurofibromas.

Copyright © 2011 Wiley Periodicals, Inc.

Figures

Figure 1. Mouse paraspinal plexiform neurofibromas are similar to human neurofibromas on MRI

Left panels show coronal (top) and axial (bottom) mouse cervical neurofibromas. Arrows denote hyperintense plexiform neurofibroma. Right panels show coronal (top) and axial (bottom) human brachial plexus neurofibroma. For volumetric measurements, tumors were outlined as shown in the bottom panels.

Figure 2. Plexiform neurofibroma development in the Nf1 flox/flox;DhhCre mouse monitored by MRI scanning

A: Schema of sequential MRI scans on Nf1 flox/flox;DhhCre mice. Mice (n=5) were MRI scanned at 4, 6, 8, 10, 12, 14 months of age. B. Top row shows coronal and bottom row shows axial images of cervical spine taken from the same mouse at 6, 8 and 12 months of age. Tumors are outlined. Arrows denote plexiform tumors. C. Quantification of tumor volume (in mm3) measured by volumetric measurement on Nf1 flox/flox;DhhCre mice. The data showed are pooled from this natural history study, the RAD001 trial, Sorafenib trial, and other trials. The x-axis is time in months and y-axis is plexiform neurofibroma tumor volume in mL (mm3). We defined 0 as the time at which drug treatment started, time before the treatment as minus, after the treatment as plus (in months). Tumor volumes for each mouse at time 0 were adjusted to 0 on the y axis. Thus the y axis reflects changes from time 0 volume in mm3.

Figure 3

Volumetric measurements of effects of RAD001 on the Nf1flox/flox;DhhCre mouse. A. Diagram of Nf1 flox/flox;DhhCre mouse MRI scanning and RAD001 treatment schedule. B. Western blots showed that RAD001 inhibited phospho-S6 kinase (pS6K) and 4E-BP1 phosphorylation in Nf1 flox/flox;DhhCre mouse neurofibromas at the end of the 2 months treatment trial. Total S6K was used as a loading control. C, D. Volumetric measurement on mice treated with vehicle (C, n=4) or RAD001 (D, n=10).

Figure 4. Volumetric measurements show a significant effect of Sorafenib on the Nf1 flox/flox;DhhCre mouse tumor growth rate

A. Diagram of Nf1 flox/flox;DhhCre mouse MRI scanning and Sorafenib treatment schedule. B. Pharmacodynamic study of Sorafenib in Nf1 flox/flox;DhhCre mice. Western blots showed that Sorafenib inhibited cyclin D1 and induced pERK protein expression in Nf1 flox/flox;DhhCre mouse neurofibromas at the end of mouse treatment. β-actin and total ERK were used as loading controls. C. Volumetric measurements of mice treated with vehicle (left, n=4) or Sorafenib (right, n=9). D. Pharmacokinetics of Sorafenib in mouse. Sorafenib concentration in mouse plasma samples after oral administration of 45mg/kg Sorafenib by Nf1 flox/flox;DhhCre mouse wild type littermates performed at times 0.5, 1, 2, 4, and 8 hours (n=3 per time point, ±SEM).

Figure 5. Sorafenib treatment results in decreased tumor cell proliferation

A. Immunostaining with Ki-67 (MIB-1) shows significantly decreased proliferation (Ki-67+ cells; blue) in the GEM-grade neurofibromas from Sorafenib-treated mice (right) compared with vehicle treated mice (left). B. Quantification shows that there is a significant decrease in cell proliferation in Sorafenib treated GEM-grade neurofibromas (n=4, right) compared to vehicle treated mice (n=4, left). Bar, 20 μm.