Abstract
A distinct profile of NRAS mutants is observed in each tumor type. It is unclear whether these profiles are determined by mutagenic events or functional differences between NRAS oncoproteins. Here, we establish functional hallmarks of NRAS mutants enriched in human melanoma. We generate eight conditional, knock-in mouse models and show that rare melanoma mutants (NRAS G12D, G13D, G13R, Q61H, and Q61P) are poor drivers of spontaneous melanoma formation, whereas common melanoma mutants (NRAS Q61R, Q61K, or Q61L) induce rapid tumor onset with high penetrance. Molecular dynamics simulations, combined with cell-based protein–protein interaction studies, reveal that melanomagenic NRAS mutants form intramolecular contacts that enhance BRAF binding affinity, BRAF-CRAF heterodimer formation, and MAPK > ERK signaling. Along with the allelic series of conditional mouse models we describe, these results establish a mechanistic basis for the enrichment of specific NRAS mutants in human melanoma.
Introduction
It is unclear why the profile of oncogenic RAS mutations differs between tumor types. It was once thought that differences in tumor etiology determined the preferred location (codon 12, 13, or 61) and amino acid identity of oncogenic mutations in RAS. However, apart from KRAS12C mutations which are linked to cigarette carcinogens in lung cancer1, tumor type-specific mutational processes do not explain the enrichment of specific RAS mutations in many cancers. This trend is particularly evident in melanoma where the most common NRAS mutations (Q61R and Q61K) are not caused by direct damage from ultraviolet (UVB) light2. These observations suggest that each RAS mutant may fulfill different requirements for tumor initiation.
Emerging evidence shows that RAS mutants have distinct biochemical and tumorigenic properties. While all oncogenic RAS mutants are constitutively active, differential positioning of the switch I and II domains leads to variations in GTP binding and hydrolysis3. These structural differences can also influence effector interactions as evidenced by the positioning of switch II in KRAS12R, which prevents PI3Kα binding and the subsequent induction of micropinocytosis4,5. Such mechanistic differences may also explain the tissue-specific potential of RAS mutants to initiate tumorigenesis in genetically engineered mouse models (GEMMs). For example, we have shown that endogenous levels of NRAS61R or NRAS12D exhibit distinct tumorigenic potential in GEMMs of melanoma and leukemia6. Finally, mutation-specific functions of oncogenic RAS may influence patient outcomes as the efficacy of targeted therapies in colorectal and non-small cell lung cancer is dependent upon the underlying KRAS mutant7,8,9. Therefore, understanding functional differences that drive the selection of specific RAS mutants in each cancer type may identify pharmacologically tractable targets required for tumor initiation.
Technical challenges have made it hard to identify differences between RAS alleles that drive tumorigenesis. For example, exogenous gene expression is a commonly used tool, yet RAS gene dosage has been shown to affect signaling10, localization11 and in vivo functionality12,13. The biological consequences of mutant RAS expression also differ based on the isoform (H-, K- or N-RAS) and cell-type examined6,14,15,16,17. Therefore, it is essential to assess the differences between endogenous RAS mutants under physiologically relevant conditions.
Here, we report the development of eight NRAS–mutant mouse alleles, each of which enables the conditional expression of a distinct NRAS mutant from the endogenous gene locus. Crossing these alleles to a melanocyte-specific Cre, we find that the melanomagenic potential of NRAS mutants parallels their frequency in human melanoma. We link the melanomagenic potential of NRAS mutants to enhanced BRAF binding, dimerization, and MAPK > ERK signaling.
