INTRODUCTION
Neurofibromatosis type 1 (NF1) is one of the most common autosomal
dominant neurological disorders and results in a multifaceted phenotype
which includes: bone dysplasia, learning disabilities, benign nerve
sheath tumors, and malignant tumors. NF1 is caused by pathogenic
variants in the neurofibromin (NF1) gene, and the clinical
phenotypes of individuals with NF1 can vary widely, even among family
members with the same genotype. NF1 functions as a GTPase-Activating
Protein (GAP), simulating conversion of active Ras-GTP to the inactive
form, Ras-GDP (Figure 1). Almost 2,900 pathogenic variants have been
reported in the Human Gene Mutation Database (http://www.hgmd.org); most
lead to lack of expression of the NF1 gene product. NF1 does not
exhibit a mutagenic hotspot, with variants occurring throughout the
gene. A subset (17%) of these variants consists of missense (MS)
variants which may result in an unstable or dysfunctional protein
(Koczkowska et al., 2019). MS variants in NF1 are not confined
to any specific region of the gene. Some occur in the GAP-related domain
(GRD) and might be expected to interfere with GAP function. MS variants
occurring outside of this domain may result in other NF1 dysfunction,
including protein instability, cellular mis-localization, or the
disruption of neurofibromin interaction with other proteins in the cell.
Another subset of variants (up to 29% (Kang et al., 2020)) includes
nonsense (NS) variants, which result in premature termination of
translation, and, in most cases, nonsense-mediated decay of the
transcript.
The most critical role of neurofibromin appears to be the regulation of
Ras signaling, with loss of neurofibromin function resulting in
increased signaling. NF1 tumor cells (e.g., Schwann cells in
neurofibromas) exhibit increased Ras signaling as a consequence of loss
of function of both NF1 alleles (one in the germ line and one
somatically-acquired (Figure 1; right) (Cichowski & Jacks, 2001).
Therapeutic interventions to date have focused on inhibition of
upregulated Ras signaling (e.g., MEK inhibition with selumetinib
(Figure 1, right)). While MEK inhibitors have demonstrated
effectiveness, not all patients benefit, plexiform neurofibromas do not
completely disappear, and there can be significant side effects (Baldo,
Magnolato, Barbi, & Bruno, 2021). Therefore, additional treatments that
can be used in conjunction with MEK inhibitors are needed.
Only a small portion of the NF1 protein, the GRD, directly interacts
with Ras. Some of the key NF1 residues involved in the NF1-Ras
interaction include R1276 and K1423 (Yan et al., 2020). R1276, a highly
conserved arginine residue, also termed the arginine “finger”, aids in
the stabilization of the GTP/GDP-NF1 complex (Scheffzek et al., 1997).
Mutation of this residue has little effect on Ras binding, but results
in complete loss of GAP activity (Ahmadian, Stege, Scheffzek, &
Wittinghofer, 1997; Sermon, Lowe, Strom, & Eccleston, 1998). NF1 K1423
forms a salt bridge with Ras D38 to stabilize the protein-protein
interaction. In addition to dysregulated Ras signaling due to variants
located in the GRD, interference with neurofibromin’s localization to
the cytoplasmic membrane can result in abnormal interaction with Ras
(Stowe et al., 2012). MS variants that occur within the SPRED1-binding
domain of NF1 can reduce the affinity for SPRED1 such that neurofibromin
fails to traffic to the membrane (Dunzendorfer-Matt, Mercado, Maly,
McCormick, & Scheffzek, 2016; Hirata et al., 2016; Yan et al., 2020).
However, some pathogenic NF1 variants fail to co-immunoprecipiate with
SPRED1, do not directly interfere with Ras binding, and do retain GAP
activity (Dunzendorfer-Matt et al., 2016).
An additional complexity to NF1 interactions is its dimerization
activity (Figure 1, left) (Carnes, Kesterson, Korf, Mobley, & Wallis,
2019; Sherekar et al., 2019). While the mechanistic significance has yet
to be determined, dimerization provides a potential explanation for the
phenotypes observed with many NF1 disease variants. Often heterozygous
NS or frameshift (FS) variants are observed in NF1, where there is still
a single copy of wild type (WT) NF1 allele present. The total
amount of full-length neurofibromin in some affected individuals with
these variants may be considerably less than the predicted 50% of WT
levels (Anastasaki, Woo, Messiaen, & Gutmann, 2015). Neurofibromin
levels could be drastically lowered if mutant protein dimerizes with WT
and then is targeted to the proteasome for degradation (Figure 1). The
ubiquitin-proteasome pathway (UPP) controls NF1 levels and both the
amplitude and duration of Ras-mediated signaling (Cichowski, Santiago,
Jardim, Johnson, & Jacks, 2003). Excessive proteasomal degradation and
genetic loss results in NF1 inactivation in sporadic gliomas
(McGillicuddy et al., 2009). Proteasomal degradation of NF1 is partially
regulated by the binding of both the SAG-SKP1-CUL1-FBXW7 and
RBX1/2-CUL3-KBTBD7 complexes with NF1 (Figure 1; left) (Hollstein &
Cichowski, 2013; Tan et al., 2011). The CUL3/KBTBD7 complex has been
implicated in the pathogenic destabilization of neurofibromin in
glioblastomas (Hollstein & Cichowski, 2013).
Here, we further validate our heterologous mNf1 cDNA expression
system and multiple assays for neurofibromin function and use them to
evaluate patient-specific variants (Wallis et al., 2018). We have
included many MS variants with known genotype-phenotype correlations to
assess their functional effects on NF1 levels as well as Ras signaling.
We demonstrate that effects on stability and Ras signaling can be
mutually exclusive functions. We suggest that such stratification of
variant effects will have implications for mutation-targeted
therapeutics for NF1, neurofibromin-driven breast cancers, and
potentially other phenotypes. These assays may also have utility in
classifying newly identified variants of unknown significance. As new
Ras-independent functions are discovered for neurofibromin, it will be
important to assay variants for their effects in new functional assays.