A primary goal of this study was to identify and evaluate gene expression signatures shared between human NF1 tumors and GEM Nf1 models. We posited that shared signatures would reveal critical mechanisms of tumorigenesis and key therapeutic targets to facilitate preclinical studies and development of NF1 therapeutics. Functional enrichment analysis identified a prominent theme, Raf/MEK/ERK suppression, shared between mouse and human. Despite this transcriptional signature, ERK signaling remained activated in NF1 tumors and inhibiting Raf/MEK/ERK signaling with a MEK inhibitor diminished tumor cell growth. Our data set provides a wealth of gene expression information and supports MEK signaling as an important clinical target in NF1.
Our results identify a transcriptional response to suppress Raf/MEK/ERK activity in neurofibromas and MPNST, including overexpression of DUSP
family members, feedback inhibitors of Raf/MEK/ERK signaling. Expression of DUSP
family members was reported in response to NF1 ablation in human fibroblasts (17
) and KRAS2
-activating mutations in lung tumors (45
), suggesting a common mechanism of suppression in Ras-driven tumors. Elevated SPRY4
expression was also reported in response to NF1 deficiency in fibroblasts, and exogenous expression of SPRY2
induced senescence in NF1-deficient fibroblasts (17
genes are members of a 52-gene transcriptional profile that is specifically downstream of Raf/MEK/ERK signaling; these genes are expressed at high levels in tumor cell lines with an activating Raf mutation (V600EBRAF
) but not tumor cell lines with receptor tyrosine kinase (RTK) activation (46
). Consistent with our results in Nf1fl/fl;Dhh-Cre
neurofibromas, PD0325901 MEK inhibition reduces expression of these genes and blocks cell proliferation in tumor cell lines with an activating Raf mutation (40
The PD0325901-induced reduction of neurofibroma volume in 39/49 (80%) of neurofibroma-bearing Nf1fl/fl;Dhh-Cre
mice represents the most dramatic result described to date for treatment of neurofibroma-bearing mice. In contrast, RAD001 did not decrease neurofibroma volume, and sorafenib, a multikinase inhibitor, decreased tumor volume in only 5 of 9 (56%) mice (30
). Imatinib, a tyrosine kinase inhibitor, showed an un-quantified effect on tumor burden in another neurofibroma mouse model (29
). Plexiform neurofibromas have a slow growth rate and a complex nonspherical shape. Therefore, standard response criteria for malignant solid tumors have limited applicability. A more sensitive and reproducible method of response evaluation for human NF1 plexiform neurofibromas was developed, evaluating response and progression using volumetric MRI analysis. Response is defined as 20% or greater decrease in tumor volume compared with baseline, and progression is defined as 20% or greater increase in tumor volume compared with baseline (32
). Plexiform neurofibroma shrinkage has not been observed in clinical trials, with the exception of up to 22% in response to pegintron (47
) and rarely in response to imatinib (48
). The method of volumetric MRI analysis used in our preclinical studies is identical to the method used in clinical trials, and we used identical response criteria.
Similar to our results, a previous study reported variable effects on MPNST cell survival in vitro with the MEK inhibitor from which PD0325901 was derived, PD184352 (CI-1040) (49
). In contrast, the PD0325901 MEK inhibitor showed a robust, yet transient, in vivo effect on survival, possibly due to effects on tumor vasculature. We did not observe persistent apoptosis following PD0325901 treatment in vivo, in contrast with effects in vitro (49
). Due to the multiplicity of Ras effectors and complexity of negative feedback regulation, therapeutic strategies against more aggressive Ras-related tumors are likely to include combinations of compounds that target multiple points in the Ras signaling network (40
). These studies support the investigation of combinatorial effects of PD0325901 with additional Ras pathway inhibitors in NF1 tumors.
Our results provide preclinical evidence implicating PD0325901 as a candidate NF1 therapeutic agent. This was unexpected, as the MEK inhibitor from which PD0325901 was derived failed to show efficacy in a GEM model of Nf1
). Recently, PD0325901 was found to be effective in reversing myeloproliferative disease in an activated KRas mouse model (54
) and in Nf1
-driven JMML (33
), likely due to the more persistent inhibition of MEK by PD0325901, a second-generation MEK inhibitor modified to improve efficacy for clinical cancer trials (55
). Collectively, these studies suggest that Raf/MEK/ERK is a critical pathway in NF1.
Preliminary studies reported adverse effects after prolonged treatment of patients with advanced cancers with 10 mg or more BID PD0325901 (35
). MPNST xenografts were only moderately sensitive to PD0325901 treatment at this dose, likely requiring combination therapies for MPNST treatment. However, the results of our experiments suggest that the lowest dose of 1.5 mg/kg in our preclinical neurofibroma mouse model, equivalent to 8 mg BID in humans, is as effective in inhibiting tumor growth as the higher dose of 10 mg/kg, supporting evaluation of new dosing schedules in clinical trials of PD0325901. Furthermore, the lower dose (1.5 mg/kg) appeared more effective in maintaining inhibition of MEK, with the higher dose activating the negative feedback response and elevating p-ERK to pretreatment levels. Fine-tuning the long-term maximal effective dose below the threshold of negative feedback may be relevant to monitoring PD0325901 in the clinic.
In summary, NF1
mutation causing neurofibromatosis results in hyperactive Ras, potentially activating numerous downstream signaling pathways. However, a successful targeted therapy in humans has not yet been developed. Combining mouse and human transcriptome data focused attention on increased expression of genes that suppress the Raf/MEK/ERK arm of Ras signaling. This transcriptional repression does not apparently compensate, as ERK phosphorylation was detected in neurofibromas and MPNST. Furthermore, inhibition of neurofibroma and MPNST growth with PD0325901 indicated dependence of both tumor types on sustained Raf/MEK/ERK signaling. The results of our preclinical tests of PD0325901 in MPNST and neurofibroma support investigation of MEK inhibitors as candidate therapeutics in the treatment of Ras-related diseases, including NF1 and other “RASopathies” (56
). In addition, NF1
mutations are frequently found in tumor types other than neurofibroma and MPNST, including glioblastoma (57
), lung adenocarcinoma (58
), and ovarian cancer (59
). Thus, PD0325901 may be a potential molecular-targeted treatment for a wide variety of disorders.