Tumors carrying BRAF-mutations over-express NAMPT that is genetically amplified and possesses oncogenic properties.

ABSTRACT

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD) biosynthesis, is up-regulated in several cancers, including metastatic melanoma (MM). The BRAF oncogene is mutated in different cancer types, among which MM and thyroid carcinoma (THCA) are prominent. Drugs targeting mutant BRAF are effective, especially in MM patients, even though resistance rapidly develops. Previous data have linked NAMPT over-expression to the acquisition of BRAF resistance, paving the way for therapeutic strategies targeting the two pathways. METHODS: Exploiting the TCGA database and a collection of MM and THCA tissue microarrays we studied the association between BRAF mutations and NAMPT expression. BRAF wild-type (wt) cell lines were genetically engineered to over-express the BRAF V600E construct to demonstrate a direct relationship between over-activation of the BRAF pathway and NAMPT expression. Responses of different cell line models to NAMPT (i)nhibitors were studied using dose-response proliferation assays. Analysis of NAMPT copy number variation was performed in the TCGA dataset. Lastly, growth and colony forming assays were used to study the tumorigenic functions of NAMPT itself. RESULTS: The first finding of this work is that tumor samples carrying BRAF-mutations over-express NAMPT, as demonstrated by analyzing the TCGA dataset, and MM and THC tissue microarrays. Importantly, BRAF wt MM and THCA cell lines modified to over-express the BRAF V600E construct up-regulated NAMPT, confirming a transcriptional regulation of NAMPT following BRAF oncogenic signaling activation. Treatment of BRAF-mutated cell lines with two different NAMPTi was followed by significant reduction of tumor growth, indicating NAMPT addiction in these cells. Lastly, we found that several tumors over-expressing the enzyme, display NAMPT gene amplification. Over-expression of NAMPT in BRAF wt MM cell line and in fibroblasts resulted in increased growth capacity, arguing in favor of oncogenic properties of NAMPT. CONCLUSIONS: Overall, the association between BRAF mutations and NAMPT expression identifies a subset of tumors more sensitive to NAMPT inhibition opening the way for novel combination therapies including NAMPTi with BRAFi/MEKi, to postpone and/or overcome drug resistance. Lastly, the over-expression of NAMPT in several tumors could be a key and broad event in tumorigenesis, substantiated by the finding of NAMPT gene amplification.