Plexiform neurofibroma is one of the most debilitating complications of NF1 and is associated with substantial significant morbidity [2
]. A preclinical model predicting activity would be useful to prioritize clinical trials for investigational targeted agents in patients with NF1 and plexiform neurofibroma. In the Nf1flox/flox;DhhCre
mouse model GEM grade I neurofibromas form in 100% of mice and recapitulate the histology and imaging characteristics of human neurofibromas [13
]. In patients, neurofibromas develop along nerve roots and adjacent peripheral nerves, paraspinally, and in deep or superficial locations. Our use of 7 Tesla small-animal MRI enabled our conclusion that the Nf1flox/flox;DhhCre
mouse model mimics mainly the paraspinal phenotype, with tumors predominantly related to the cervical and thoracic spine. We assessed tumor growth rate in the Nf1flox/flox;DhhCre
mouse model using volumetric MRI analysis. The same volumetric measurement technique is in use in ongoing clinical trials and has been proven to sensitively detect small changes in tumor size over time [3
]. The reproducibility of this method is similar for tumors in mice and humans, and thus the response criteria used in human trials can be applied to the preclinical evaluation in mice. In humans, growth rate varies between patients but appears to be consistent within an individual. Similarly, in the mouse model we identified fast and slow growing tumors, and steady growth for individual tumors. However, in patients with NF1 enrolling in clinical trials most rapid plexiform neurofibroma growth was in young children; older patients typically had little or no growth [4
]. In contrast, in the Nf1flox/flox;DhhCre
mouse model, tumors are visible by 4 months on MRI and continue to grow until mice require sacrifice due to spinal cord compression at around 1 year.
We scanned untreated and carrier treated mice at numerous intervals. Based on tumor natural history, we suggest that future preclinical trials using this model will best be accomplished by imaging mice at 5 and 7 months, then using a 2 months treatment followed by a final scan. This paradigm takes into account both the continuous growth of tumors in the model and the time of significant death of Nf1flox/flox;DhhCre mice, occurring mainly after 9 months of age. Because in individual mice tumor size and growth rate differ, another possible paradigm would be to measure tumor growth rate and only treat mice with large tumors, or tumors of roughly the same size. The fact that we have no evidence that large and small tumors respond differently to drugs argues against this approach, and such a restriction would not reflect the heterogeneity of patients seen in clinical settings.
The predictable neurofibroma growth rate in the Nf1flox/flox;DhhCre
mouse model enabled pre-clinical drug screening. We did not detect discernable effects on tumor growth, tumor cell proliferation, or cell apoptosis on RAD001 treated mice. Similarly, sirolimus (rapamycin) was not effective in shrinking non-progressive plexiform neurofibromas in a Phase 2 trial in children and adults with NF1 and inoperable plexiform neurofibromas. Whether sirolimus prolongs time to progression in subjects with progressive plexiform neurofibromas remains to be determined, and we await trial results with interest [25
]. Mouse tumor cells had adequate exposure to RAD001, as neurofibroma pS6 kinase was blocked by exposure to RAD001. It is known that in some systems mTOR blockade can cause feedback activation of Akt activity [26
], and it remains possible that this or alternative compensatory mechanisms might account for the failure of RAD001 to block neurofibroma growth. Mechanisms of drug resistance (cell autonomous and/or non-cell autonomous) in many tumors will be an interesting avenue for follow-up studies.
Sorafenib is a multi-targeted kinase inhibitor being tested in a Phase I trial in pediatric patients with NF1 and plexiform neurofibroma. Mice exposed to Sorafenib with tumor growth inhibition also showed decreased expression of the cell cycle regulator cyclin D1, consistent with an effect on tumor growth. Sorafenib inhibited tumor cell proliferation, as attested by immunostaining. The target(s) of Sorafenib in this model are not clear. Raf is predicted to be activated downstream of Ras activation caused by NF1
loss. Tumor lysates showed increased pERK expression, likely due to a negative feedback loop caused by Raf kinase inhibition [26
]. Sorafenib also inhibits activity of receptors implicated in neurofibroma cells including c-kit, VGFR2, VGFR3, platelet-derived growth factor receptor ß, and Flt-3, one or more of which might account for some effects of Sorafenib on individual tumors [15
]. The reason that 5 of 9 mice responded to Sorafenib exposure by tumor shrinkage while 4 of 9 did not is unknown. Because the mouse strain is a mixed genetic background, there may be co-modifier genes that differ among the animals that alter drug metabolism or target sensitivity, possibilities supported by the variability seen in our individual pharmacodynamic and pharmacokinetic data (, respectively). Drug penetration into different tumor sites may also vary among mice because of the blood-tumor barrier, or interstitial pressure on selected tumors. Tissue drug levels and pharmacodynamic studies of tumor tissue will be of interest in future preclinical neurofibroma trial design.
Our data demonstrate that Sorafenib can reduce the growth of certain neurofibromas and suggest that Sorafenib might have clinical therapeutic effects on neurofibromas. More importantly, our data show that the Nf1flox/flox;DhhCre mice are useful to monitor tumor response to targeted therapeutics, indicating the potential usage of this model for preclinical drug screening.