Fig 2: NAMPT activates EMT and stem pathway effectors(A) Q-RT-PCR analysis of NAMPT overexpression in empty vector [**p < 0.01 with t-test]. Q-RT-PCR analysis of stem cell gene expression in empty vector: NANOG [***p < 0.001 with t-test], SOX2 [*p < 0.05; **p < 0.01 with t-test], OCT4 [**p < 0.01; ***, p < 0.001 with t-test] and CD133 [*p < 0.05; **p < 0.01 with t-test]. (B) RT-qPCR analysis of EMT gene expression in empty vector: FOXC2 [**p < 0.01 with t-test], TWIST1 [*p < 0.05; **p <0.01 with t-test], VIM [*p <0.05; **p <0.01 with t-test] and SNAI1 [**p < 0.01 with t-test]. (C) Analysis of the Yamanaka dataset (GSM241846) with respect to induced pluripotent stem cell (iPSC) generation from human dermal fibroblasts (hDF) demonstrates an increase in NAMPT levels with reprogramming [*p = 0.01; **p < 0.01 with ANOVA compared to human dermal fibroblasts]. (D) Analysis of the Yamanaka dataset (GSE15148) with respect to iPSC generation from mouse embryonic fibroblasts (mEF) demonstrates a significant increase in NAMPT expression with reprogramming. Mouse embryonic stem cells (mESC) also express high levels of NAMPT compared to fibroblasts [*p = 0.01; **p < 0.01 with ANOVA compared to mouse embryonic fibroblasts]. (E) Analysis of the Thomson dataset (GSE5259) with respect to iPSC generation from human foreskin fibroblasts (parent) demonstrates an increase in NAMPT levels with reprogramming. Human embryonic stem cell lines (H1L and H7) also express high levels of NAMPT compared to fibroblasts [*p = 0.01; **p < 0.01 with ANOVA compared to human foreskin fibroblasts]. V: cells expressing vector only. NAMPT: Cells overexpressing ectopic NAMPT cDNA.

Fig 3: Response to NAMPT inhibition in MM and THCA cell lines according to BRAF mutations. A Short-term proliferation assay comparing sensitivity to NAMPTi (FK866 and GMX1778 at 25 nM, 72 h of treatment) of MM BRAF-mutated vs. WT cell lines (upper graphs) and THCA BRAF-mutated vs. WT cell lines (lower graphs). Data represent the average and SEM of at least three biological replicates. Unpaired t test was used to calculate the statistical difference of sensitivity between BRAF WT vs. mutated cell lines. B, C Proliferation assay comparing sensitivity to NAMPTi in MEL-505 (B) and Mewo (C) infected with a CTRL or BRAF V600E construct. Data represent the average and SEM of at least three biological replicates. Significance was represented as: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Fig 4: NAMPT protein levels are increased in BRAF-mutated vs. BRAF WT melanoma and thyroid carcinomas. A Dot plots representing NAMPT protein levels [H-score, defined by immunohistochemistry (IHC) staining] on tissue microarrays (TMAs) in metastatic melanoma (MM) grouped in BRAF WT, BRAF mutated (MUT), NRAS MUT and C-KIT MUT. N of samples in each group are indicated. Statistical significance was calculated using Mann–Whitney test. The line in the dot plot defines the median and the error bars define the interquartile range. Below the graphs, representative immunohistochemistry (IHC) stainings for NAMPT in BRAF WT, BRAF V600E, and NRAS Q61R MM cases. Scale bars are reported under the images. B Dot plot representing H-score of NAMPT protein levels on THCA whole slide sections grouped in BRAF WT and BRAF V600E. N of samples in each group are indicated. Statistical significance was calculated using Mann–Whitney test. The line in the dot plot defines the median and the error bars define the interquartile range. On the right, representative IHC staining for NAMPT in THCA cases. Scale bars are reported under images. Significance was represented as: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Fig 5: NAMPT expression correlates with tumor grade(A) Analysis of Normal Brain [n = 216], Glioma [n = 577], Pilocytic Astrocytoma [n = 41] and N euroblastoma [n = 45] datasets available through the R2 database indicates that NAMPT is significantly upregulated in tumors derived from the ectoderm, such as pilocytic astrocytoma [*p = 0.01 with ANOVA compared to normal brain] and glioma [*p = 0.01 with ANOVA compared to normal brain]. (B) Analysis of normal brain [n = 23] and glioblastoma [n = 81] datasets from the Sun database available in Oncomine indicates that NAMPT is highly overexpressed in glioblastoma [**p < 0.01 with ANOVA compared to normal brain]. (C) New retrospective analyses of new databases, comparing normal brain GSE13564, GSE11882 [n = 216], Glioblastoma [GSE4290, GSE16011 Glioblastoma series taken together; n = 159], Anaplastic Astrocytoma - glioma grade III [GSE4290, Astrocytoma grade III GSM series; n = 16] and Glioblastoma [n = 9; GSM379855, GSM379856, GSM379857, GSM379858, GSM379870, GSM379871, GSM379872, GSM379873 and GSM379874] cell lines shows that NAMPT is upregulated in the mRNA pools of these cell lines [GSE15209 ****p < 0.0001 with ANOVA compared to normal brain] and that its expression is upregulated at baseline in glioma grade III cells [***p < 0.001 with ANOVA compared to normal brain]. (D–E) The Oncomine glioma dataset shows a correlation between NAMPT expression and tumor grade. (D) Analysis of the Oncomine Glioma dataset stratifying NAMPT according to Glioma grade with Grade II [n = 45] Grade III [n = 31] and grade IV [n = 81] tumors shows a significant difference in NAMPT expression between grade II and grade IV tumors [**p < 0.001]. (E) A second database analyzed in Oncomine strengthens the data previously shown: NAMPT expression correlating with tumor grade: Grade II [n = 50], Grade III [n = 26], Grade IV [n = 77] shows a significant difference in NAMPT expression between grade II and grade IV tumors [**p = 0.0052] (F) Analysis of TCGA Glioblastoma molecular subtypes Proneural, Neural, Classical and Mesenchymal.