Fig 2: Loss of NACC1 or FKBP10 attenuates tumor growth in mice with an intact immune system.(A and B) Expression of indicated genes was assessed in NR2F6 KO (CRISPR-based) (A) or KD (shRNA-based) (B) B16F10 cells by qPCR. n = 3 for each group. (C) ReMap peaks predicted transcriptional regulatory regions of Nacc1. R1 included promoter and a part of intron1, and the other candidates (R2, R3, and R4) were predicted within intron1. (D) Abundance of NR2F6, RNA polymerase II, and H3K27 acetylation on each candidate region was assessed by ChIP-qPCR using corresponding antibodies and primers. Relative abundance to input (5% of pre-pulldown material) was calculated. n = 3 for each group. (E) Abundance of NR2F6, RNA polymerase II, and H3K27 acetylation on R4 was assessed in control and NR2F6 KO B16F10 cells. n = 3 for each group. (F) B16F10 cells were transduced with control scrambled (Scr) shRNA or two shRNAs (sh334 and sh336) targeting NACC1. mRNA and protein expression was assessed by qPCR and immunoblotting, respectively. n = 3 for each group. (G and H) Cells were then used to inoculate C57BL/6 (G) or NSG (H) mice. Tumor volumes were monitored at indicated time points. n = 9 mice (G) and n = 5 mice (H) for each group. (I to K) As in (F) and (H), B16F10 cells were transduced with control scrambled shRNA or two (sh486 and sh876) shRNAs targeting FKBP10. FKBP10 expression was assessed (I). n = 3 for each group. The growth of tumors emerging from transduced cells was monitored in C57BL/6 (J) or NSG (K) mice. n = 10 mice (J) and n = 5 mice (K) for each group. Data are presented as means ± SD. Statistical significance was assessed by one-way ANOVA with Dunnett’s test (A, B, E, F, and I) or two-way ANOVA with Dunnett’s test (G, H, J, and K).

Fig 3: RNA-seq identifies NACC1 and FKBP10 as NR2F6 effectors.(A) DEGs between control and CRISPR-KO B16F10 were identified on the basis of RNA-seq analysis of bulk tumors, MACS-sorted tumor cells, or cultured cells. Unique or common genes up-regulated (top) or down-regulated (bottom) are plotted in Venn diagrams. (B and C) Activated or repressed pathways (B) and upstream regulators (C) of identified DEGs were analyzed using Ingenuity Pathway Analysis. (D) Heatmaps show the Spearman’s correlation coefficient (top) and corresponding P values (bottom) in expression analysis of 15 genes commonly up- or down-regulated in RNA-seq data from the three samples described in (A). x and y axes indicate up-/down-regulated genes and 10 IFN-? signature genes, respectively. CXCL10, FKBP10, and NACC1 expression were significantly correlated with NR2F6 and the IFN-? signature. (E) Overall survival of melanoma patients with relatively high expression (z score > 2.0) of NACC1, FKBP10, or CXCL10 was compared with all other patients (z score < 2.0) based on Kaplan-Meier analysis. (F) NR2F6 expression was assessed in melanoma specimens from patients with low or high expression of NACC1, FKBP10, or CXCL10 in (E). Data are presented as means ± SD. Statistical significance was assessed by log-rank test (E) or one-way ANOVA with Sidak’s test (F). ERK, extracellular signal–regulated kinase; MAPK, mitogen-activated protein kinase; HIF1, hypoxia-inducible factor 1; IRF, interferon regulatory factor; TH1, T helper 1; FLT3, fms-like tyrosine kinase 3; BEX2, brain-expressed X-linked 2; PD-L1, programmed death-ligand 1; LPS, lipopolysaccharide; NAD, nicotinamide adenine dinucleotide.

Fig 4: Loss of NACC1 and FKBP10 phenocopies NR2F6 loss.(A) B16F10 cells were transduced with scrambled shRNAs [Scr (puromycin resistant) and B-Scr (blasticidin resistant)] and one of two shRNAs (sh334 for NACC1 KD and B-sh876 for FKBP10 KD). mRNA and protein expression was assessed by qPCR and immunoblotting, respectively. n = 3 for each group. (B) Growth of transduced cells in vitro, as assessed by CellTiter-Glo. n = 6 for each group. (C and D) Transduced cells were used to inoculate C57BL/6 (C) or NSG (D) mice. Tumor growth was monitored at indicated time points. n = 9 and 10 mice for Scr/B-Scr and sh334/B-876 group, respectively. (E) Overall survival was assessed in patients with melanoma (SKCM in TCGA, n = 428) whose specimens showed both high NACC1 and high FKBP10 expression, high expression of just one, or low expression of both. (F) NR2F6 expression was assessed in groups of specimens in (E). (G and H) Scrambled (Scr/B-Scr) and NACC1/FKBP10 KD (sh334/B-sh876) B16F10 cells were engrafted into C57BL/6 mice. Tumors were collected 12 days later, and their volume was assessed (G). Different shapes of data points indicate the pooled cells for FACS analysis. n = 7 and 10 mice for Scr/B-Scr and sh334/B-876 group, respectively. (H) Shown is the abundance of T cell subtypes within all CD45+ cells based on FACS. n = 5 pooled samples for each group. Data are presented as means ± SD. Statistical significance was assessed by Student’s t test (G) with Holm-Sidak’s test (A and H), one-way ANOVA with Dunnett’s test (F), two-way ANOVA with Sidak’s test (B to D), or log-rank test (E).