Identification of NRAS isoform 2 overexpression as a mechanism facilitating BRAF inhibitor resistance in malignant melanoma

Abstract

Activating mutations in BRAF are found in 50% of melanomas and although treatment with BRAF inhibitors (BRAFi) is effective, resistance often develops. We now show that recently discovered NRAS isoform 2 is up-regulated in the setting of BRAF inhibitor resistance in melanoma, in both cell lines and patient tumor tissues. When isoform 2 was overexpressed in BRAF mutant melanoma cell lines, melanoma cell proliferation and in vivo tumor growth were significantly increased in the presence of BRAFi treatment. shRNA-mediated knockdown of isoform 2 in BRAFi resistant cells restored sensitivity to BRAFi compared with controls. Signaling analysis indicated decreased mitogen-activated protein kinase (MAPK) pathway signaling and increased phosphoinositol-3-kinase (PI3K) pathway signaling in isoform 2 overexpressing cells compared with isoform 1 overexpressing cells. Immunoprecipitation of isoform 2 validated a binding affinity of this isoform to both PI3K and BRAF/RAF1. The addition of an AKT inhibitor to BRAFi treatment resulted in a partial restoration of BRAFi sensitivity in cells expressing high levels of isoform 2. NRAS isoform 2 may contribute to resistance to BRAFi by facilitating PI3K pathway activation.

Synergy from gene expression and network mining (SynGeNet) method predicts synergistic drug combinations for diverse melanoma genomic subtypes

“…We also chose the maximum tolerated dose of 50 mg/kg for vemurafenib for a treatment period of 2 weeks that we have shown to be efficacious in A375 mouse xenograft models previously. 40 To create melanoma mouse models, 11-week old athymic nude mice were inoculated with the A375 melanoma cell line (1.0 × 10 6 cells suspended in PBS) via subcutaneous injection, and tumors were grown to 1000 mm 3 . Mice were randomized to drug treatment groups ( n = 8 mice per group).…”

Genetic Target Modulation Employing CRISPR/Cas9 Identifies Glyoxalase 1 as a Novel Molecular Determinant of Invasion and Metastasis in A375 Human Malignant Melanoma Cells In Vitro and In Vivo

“…It is important to note that our studies (employing CRISPR/Cas9-based generation of isogenic A375_ GLO1 _KO clones together with CMV-driven rescue clones) represent a limited yet powerful prototype approach towards a more stringent elucidation of GLO1 function in melanomagenesis. Likewise, the use of isogenic NRAS mutant-A375 melanoma cell lines is firmly established, and the commercial availability of these lines enables the explorative molecular examination of genetic mechanisms underlying BRAF kinase inhibitor resistance and the BRAF RAS–RAF–MEK–ERK (MAPK) signaling pathway in melanoma [ 44 ]. However, more detailed follow up studies will have to examine the role of GLO1 deletion in a genetically diverse set of representative human melanoma cell lines to further substantiate our prototype data.…”

The Tubulin Inhibitor VERU-111 in Combination With Vemurafenib Provides an Effective Treatment of Vemurafenib-Resistant A375 Melanoma

“…Several mechanisms have been documented to mediate Vem-resistance, for example, overexpression of P-glycoprotein (P-gp), BRAF mutation, aberrant expression of miRNA, translocation of E3 ligase, or PI3K/AKT pathway ( Johnson et al, 2014 ; Duggan et al, 2017 ; Lim et al, 2017 ; Thang et al, 2017 ; Diaz-Martinez et al, 2018 ). Such mechanistic understandings have led to a number of exciting synergistic combinations to re-sensitize Vem against metastatic melanoma.…”