Mutation-specific RAS oncogenicity explains NRAS codon 61 selection in melanoma

Abstract

NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways.

Significance: This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional "knock-in" mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation.

Figure 1. Activation of LSL-N-Ras^Q61R or LSL-N-RAS^G12D slows melanocyte proliferation

(A) Diagrammatic representation of the LSL-N-RAS^G12D (17) and LSL-N-Ras^Q61R alleles in the presence or absence of CRE recombinase. Green triangles denote lox P sites and unnumbered black boxes represent FRT sites. Blue boxes indicate the location of a puromycin (PURO) resistance cassette. SA-splice acceptor; SD-splice donor; 3×STOP- transcription and translational stop sequence. (B) PCR genotyping of melanocytes treated for 6 days with ethanol vehicle (E; lane 7) or 1.0 µM 4-OHT (lane 7). DNA from mice of the indicated genotypes is included as a control (lanes 3–5). (C) Melanocytes cultured as in ‘B’ were incubated with EdU, a thymidine analog, for 16 hours and then analyzed by flow cytometry. EdU incorporation in vehicle treated cells was set to 100%. Each dot represents a biological replicate with the mean and standard error of the mean indicated by black lines.

Figure 2. In vivo melanomagenesis is isoform and mutation-specific

(A) Kaplan-Meier curve of melanoma-free survival. Animals were followed for a total of 80 weeks. Survival of each strain was compared pairwise to that of TpN^61R/61R animals using a log-rank (Mantel-Cox) test. The following significant p-values were determined: Tp and TpN^12D/12D - p<0.0001; TpK^12D/WT- p<0.0027. (B) Representative images of the amelanotic tumors found throughout the skin of TpN^12D/12D, TpN^61R/61R and TpK^12D/WT animals. White arrowheads indicate the tumor location. (C) Hematoxylin and eosin staining of representative tumors of the indicated genotypes. Black bars represent a length of 20µm. White arrowheads indicate areas of melanin deposition.

Figure 3. Lkb1 deficiency promotes metastasis in TpN^61R/61R tumors

(A) Kaplan-Meier curve comparing the melanoma-free survival of TpN^61R/61R and TpLN^61R/61Rmice treated neonatally with 4-OHT. A log-rank (Mantel-Cox) test revealed no significant difference in disease onset in these models (p>0.05). (B) Representative photographs showing severe melanocytic hyperproliferation in the ears, tails and paws of TpLN^61R/61Rmice. (C) Images of macrometastases in the lung, spleen and lymph nodes (LN) of TpLN^61R/61Rmice. (D) Representative hematoxylin and eosin stained TpLN^61R/61Rtissues showing tumor invasion into the liver, spleen and lymph node. (E) Expression of TRP1 in the spleen and liver of TpLN^61R mice with primary melanomas. The aqua line designates tissue from a wild type animal and the red line shows TRP1 expression in a TpLN^61R/61Rmouse with observed macrometastases. For splenic tissues, CD45+ cells were excluded prior to analysis.

Figure 4. Activation of ERK and AKT in human melanoma cell lines is not codon-specific

Shown are immunoblots for total and phosphorylated ERK (A), or AKT (B) in established human melanoma cell lines harboring the indicated NRAS mutations. Protein expression levels were quantified using LI-COR ImageStudio software. Each dot represents a single cell line. The mean is indicated by a line, and the standard error is shown as whiskers. * p<0.05