Figure 5.
Differential regulome and thiostrepton cytotoxicity profiling of primary chr1q-amp vs nonamplified MM cells. (A) Schematic representation of experimental strategy. Myeloma PCs were isolated via magnetic beads selection (CD138+) from bone marrow aspirates derived from 6 patients with chr1q-amp [chr1q-amp(+)] MM and 6 patients with nonamplified [chr1q-amp(-)] MM. Differential regulome (TF expression and wiring) analysis was performed via parallel chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) profiling. (B) Volcano plot displaying differentially expressed genes [chr1q-amp(+), green; chr1q-amp(-), orange]. Genes implicated in chr1q-amp pathogenesis in this study (pink) or previous studies (black) are indicated. (C) Enrichment analysis (NCI-Nature pathways) of differentially expressed genes in 2 patient subgroups. (D) Differential ATAC-seq analysis between chr1q-amp(+) and chr1q-amp(-) myeloma PCs. Increased accessibility was found on genetic loci of genes of interest upon chr1q amplification (as indicated here). (E) Differential ATAC-seq footprinting analysis of expressed TFs in chr1q-amp(+) vs chr1q-amp(-) cells (ΔP, differential regulatory potential). TFs of interest are indicated. (F) Scatter plot representation of differential expression (x-axis) and differential regulatory potential (y-axis) of 63 TFs displaying significant differences in both dimensions. Green quartile: TFs with increased expression and ΔP in chr1q-amp(+) cells; orange quartile: TFs with decreased expression and ΔP in chr1q-amp(+) cells. Key TFs are also highlighted. (G) Selective sensitivity of chr1q-amp(+) (n = 3, green) vs chr1q-amp(-) (n = 3, orange) primary myeloma PCs to thiostrepton at 48 hours after treatment. Fifty percent inhibitory concentration (IC50) values were calculated for each patient sample using a nonlinear fitting model (fitting line shown here). (H) Transcriptional profiling (RT-qPCR) of FOXM1 and NEK2 mRNA levels in chr1q-amp(+) (green) and chr1q-amp(-) (orange) primary samples 24 hours after thiostrepton (1μM) or mock (0nM) treatment. The (%) decrease in FOXM1 and NEK2 mRNA levels is also indicated. ***P < .001; ****P < .0001. n/a, not applicable.

Differential regulome and thiostrepton cytotoxicity profiling of primary chr1q-amp vs nonamplified MM cells. (A) Schematic representation of experimental strategy. Myeloma PCs were isolated via magnetic beads selection (CD138+) from bone marrow aspirates derived from 6 patients with chr1q-amp [chr1q-amp(+)] MM and 6 patients with nonamplified [chr1q-amp(-)] MM. Differential regulome (TF expression and wiring) analysis was performed via parallel chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) profiling. (B) Volcano plot displaying differentially expressed genes [chr1q-amp(+), green; chr1q-amp(-), orange]. Genes implicated in chr1q-amp pathogenesis in this study (pink) or previous studies (black) are indicated. (C) Enrichment analysis (NCI-Nature pathways) of differentially expressed genes in 2 patient subgroups. (D) Differential ATAC-seq analysis between chr1q-amp(+) and chr1q-amp(-) myeloma PCs. Increased accessibility was found on genetic loci of genes of interest upon chr1q amplification (as indicated here). (E) Differential ATAC-seq footprinting analysis of expressed TFs in chr1q-amp(+) vs chr1q-amp(-) cells (ΔP, differential regulatory potential). TFs of interest are indicated. (F) Scatter plot representation of differential expression (x-axis) and differential regulatory potential (y-axis) of 63 TFs displaying significant differences in both dimensions. Green quartile: TFs with increased expression and ΔP in chr1q-amp(+) cells; orange quartile: TFs with decreased expression and ΔP in chr1q-amp(+) cells. Key TFs are also highlighted. (G) Selective sensitivity of chr1q-amp(+) (n = 3, green) vs chr1q-amp(-) (n = 3, orange) primary myeloma PCs to thiostrepton at 48 hours after treatment. Fifty percent inhibitory concentration (IC50) values were calculated for each patient sample using a nonlinear fitting model (fitting line shown here). (H) Transcriptional profiling (RT-qPCR) of FOXM1 and NEK2 mRNA levels in chr1q-amp(+) (green) and chr1q-amp(-) (orange) primary samples 24 hours after thiostrepton (1μM) or mock (0nM) treatment. The (%) decrease in FOXM1 and NEK2 mRNA levels is also indicated. ***P < .001; ****P < .0001. n/a, not applicable.

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