The oncogenic MAF-associated transcriptome. (A) MAF expression by RNA sequencing (RNA-seq) (top) and MAF messenger RNA quantification via qPCR (bottom) in the same primary myeloma PC samples (dark) compared with myeloma cell lines (light) and healthy donor PCs (boxed). Shaded in gray are the MAF-translocated cells. Expression via qPCR normalized to GAPDH, using 2 delta CT method relative to a housekeeping gene: MF; MAF, MS; MMSET, CD1; and CCND1. (B) Volcano plot showing differential gene expression in MAF primary myeloma cells as compared with healthy donor (ND) bone marrow plasma cells (Padj < .05; log2FC ≥1.5). Genes highlighted for illustration represent previously validated MAF target genes in myeloma (NUAK1, CCR1, and ITGB7) or genes previously associated with the MF molecular group in gene expression studies. (C) GSEA of the genes upregulated in MAF primary samples (MAF s1 and MAF s2) compared with healthy PCs (ND1, ND2, and ND3). Only pathways with false discovery rate q value < .05 are shown. (D) Heatmap of selected genes depicting significant differential expression compared with healthy PCs across genetic subgroups. (E) Heatmaps showing shared upregulated and downregulated genes in the MM.1S and JJN3 MAF-translocated cell lines transduced with anti-MAF short hairpin 1 RNA (sh1-RNA) and sh4-RNA, compared with the scrbl control. A selective list of differentially downregulated genes common to both cell lines is shown and includes genes previously associated with the MAF molecular group (CCND2, NUAK1, CCR1, and ITGB7) as well as genes not previously linked specifically to MAF in myeloma (FLI1, MYB, CD28, IRF4, and ITGB5). (F) GSEA of genes differentially downregulated by MAF knockdown in both cell lines, against the MF subgroup top upregulated genes, as previously identified by Zhan et al (G–H). Cell cycle analysis performed on transduced MM.1S cells on day 6 of the experiment. Cells were gated on green fluorescent transduced live cells that are double negative for annexin V and propidium iodide staining. Hoechst stain was used to assess the cell cycle. In each panel, an overlay of representative flow cytometry histograms is shown (G) along with bar graphs showing the mean percentage (and standard deviation) of cells found at each cell cycle stage (H). ∗∗∗∗P < .0001; 2-way analysis of variance (ANOVA; Tukey post hoc multiple comparisons correction) for n = 3 independent experiments. HY, hyperdiploid, ns, nonsignificant.

The oncogenic MAF-associated transcriptome. (A) MAF expression by RNA sequencing (RNA-seq) (top) and MAF messenger RNA quantification via qPCR (bottom) in the same primary myeloma PC samples (dark) compared with myeloma cell lines (light) and healthy donor PCs (boxed). Shaded in gray are the MAF-translocated cells. Expression via qPCR normalized to GAPDH, using 2 delta CT method relative to a housekeeping gene: MF; MAF, MS; MMSET, CD1; and CCND1. (B) Volcano plot showing differential gene expression in MAF primary myeloma cells as compared with healthy donor (ND) bone marrow plasma cells (Padj < .05; log2FC ≥1.5). Genes highlighted for illustration represent previously validated MAF target genes in myeloma (NUAK1, CCR1, and ITGB7) or genes previously associated with the MF molecular group in gene expression studies. (C) GSEA of the genes upregulated in MAF primary samples (MAF s1 and MAF s2) compared with healthy PCs (ND1, ND2, and ND3). Only pathways with false discovery rate q value < .05 are shown. (D) Heatmap of selected genes depicting significant differential expression compared with healthy PCs across genetic subgroups. (E) Heatmaps showing shared upregulated and downregulated genes in the MM.1S and JJN3 MAF-translocated cell lines transduced with anti-MAF short hairpin 1 RNA (sh1-RNA) and sh4-RNA, compared with the scrbl control. A selective list of differentially downregulated genes common to both cell lines is shown and includes genes previously associated with the MAF molecular group (CCND2, NUAK1, CCR1, and ITGB7) as well as genes not previously linked specifically to MAF in myeloma (FLI1, MYB, CD28, IRF4, and ITGB5). (F) GSEA of genes differentially downregulated by MAF knockdown in both cell lines, against the MF subgroup top upregulated genes, as previously identified by Zhan et al (G–H). Cell cycle analysis performed on transduced MM.1S cells on day 6 of the experiment. Cells were gated on green fluorescent transduced live cells that are double negative for annexin V and propidium iodide staining. Hoechst stain was used to assess the cell cycle. In each panel, an overlay of representative flow cytometry histograms is shown (G) along with bar graphs showing the mean percentage (and standard deviation) of cells found at each cell cycle stage (H). ∗∗∗∗P < .0001; 2-way analysis of variance (ANOVA; Tukey post hoc multiple comparisons correction) for n = 3 independent experiments. HY, hyperdiploid, ns, nonsignificant.

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