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.

Utilizing CD44v6 and V600EBRAF-mutation for in vitro targeted combination therapy of thyroid carcinomas

“…Wild-type written as wt. Cell line Cancer origin BRAF status Sorafenib IC 50 8305c ATC V600E [ 16 ] 12 μM 8505c ATC V600E [ 16 ] 5.3 μM ACT-1 ATC wt (NRAS mutant) [ 16 ] 15 μM B-CPAP PTC V600E [ 33 ] 0.4 μM CAL-62 ATC wt (KRAS mutant) [ 34 ] 9 μM FTC-238 FTC wt [ 35 ] 6.5 μM MDA-T32 PTC V600E [ 36 ] 4.5 μM SW1736 ATC V600E [ 15 ] 8.1 μM

Fig. 2 A) LigandTracer evaluation of CD44v6-binding with 3 nM and 10 nM of 131 I-AbN44v6 on ATC cell lines ACT-1, 8305c, SW1736 and CAL-62, followed by retention measurements (0 nM).

…”

NAD/NAMPT and mTOR Pathways in Melanoma: Drivers of Drug Resistance and Prospective Therapeutic Targets

“…Several transcriptional and post-transcriptional mechanisms regulate NAMPT expression and activity in tumors, as well as multiple signals, including hypoxia, stress conditions, DNA damage, and pro-inflammatory cytokines, can induce its release [ 160 ]. In addition, rather than being mutated, the NAMPT gene was found amplified in cancer [ 169 , 171 ].…”

“…Transcriptionally, NAMPT is regulated by the activation of the BRAF oncogenic signaling, and, in turn, NAMPT overexpression can amplify and sustain MAPK and downstream pathways activation [ 211 , 212 , 213 ]. Recently, Audrito et al showed that patients with BRAF-mutated melanoma have increased NAMPT mRNA and protein levels more than the BRAF-wild type ones [ 171 ], indicating a direct molecular link between a driver oncogenic signaling and the regulation of the expression of this enzyme. Functionally, NAMPT over-expression promotes signaling activation, proliferation and colony-formation capacity, phenotypic plasticity supporting mesenchymal/invasive transition and stem cell-like features, and supports tumorigenicity in vivo [ 212 ] ( Figure 2 ).…”

“…NAMPT over-expression induces also metabolic rewiring financing increased NAD levels, transcriptomic, and epigenetic reshuffling that steer melanoma cells toward an invasive phenotype associated with the onset of resistance to targeted therapies [ 179 , 212 , 214 ]. In BRAF-mutated melanoma cells, the inhibition of NAMPT, using FK866 and GMX1778, or its genetic knock-down, reduced cancer cell proliferation, both in vitro and in vivo [ 179 , 212 , 214 ], highlighting NAMPT as an actionable target for melanoma patients with BRAF mutations [ 171 ] ( Figure 2 ).…”

Mitochondrial rewiring drives metabolic adaptation to NAD(H) shortage in triple negative breast cancer cells

“…Due to its central role in NAD + synthesis and bioenergetics, NAMPT has been found to be upregulated in many solid tumors, revealing the significance of the development of NAMPT inhibitors for cancer treatment [18] , [19] , [20] , [21] . Moreover, NAMPT inhibitors like FK866 (APO866) have been extensively used in preclinical cancer research to mimic cellular NAD(H) shortage, characteristic of the high energetic demand of rapidly proliferating cancer cells [22] .…”