Figure 3.
YKL-5-124 treatment disrupts oncogenic gene expression programs in MM. (A) Scatter plot visualizing gene set enrichment analysis normalized enrichment score comparisons between H929 and AMO1 cells treated with DMSO or YKL-5-124 (100 nM) for 24 hours. The bar graph shows NES for the top 18 gene signatures in both cell lines after treatment with YKL-5-124. (B) Biological upstream regulators associated with CDK7 inhibition were identified using ingenuity pathway analysis (IPA). (C) Whole-cell lysates from AMO1 and H929 cells treated with YKL-5-124 for 0.5 to 4 hours were subjected to WB analysis and probed with MYC antibody, with GAPDH as a loading control. The ratio of MYC/GAPDH was analyzed with Image J software and represented as fold change from untreated cells. Mean values ± SD in 2 MM cell lines is shown in the graph. (D) The diagram depicts the intermediates of glycolysis, and the enzymes regulated by CDK7 (red bars). Mean of log2-fold change for H929 and AMO1 cells after treatment with YKL-5-124 are shown (∗P < .05). (E) Bubble plot showing the differentially expressed proteins involved in the glycolytic pathway after treatment with YKL-5-124 for 24 hours. The x-axis displays the log2 (fold change), and the y-axis represents the negative log of the adjusted P value. (F) A panel of cells treated with YKL-5-124 for 24 hours was subjected to western blot analysis and probed with antibodies against HK2 and β-actin as a loading control (upper panel). The ratio of HK2/actin was analyzed with Image J software and represented as fold change from untreated cells (lower panel). Mean values ± SD in 5 MM cell lines are shown in the graph. (G) ChIP-seq tracks showing MYC signal on individual loci for LDHA and HK2. The x-axis shows genomic coordinates with gene model depicted below. The y-axis shows signal in units of rpm/bp. (H) MM1S cells were treated with YKL-5-124 for 6 hours and subjected to ChIP with a MYC or IgG antibody. HK2, LDHA, and a negative control region were amplified by polymerase chain reaction. Data are shown as mean ± SD of triplicates and represented as the percentage of input. bp, base pair; ChIP, chromatin immunoprecipitation; CHIP, clonal hematopoiesis of indeterminate potential; DMSO, dimethyl sulfoxide; FDR, false discovery rate; IgG, immunoglobulin G; NES, normalized enrichment score; rpm, units of reads per million; SD, standard deviation.

YKL-5-124 treatment disrupts oncogenic gene expression programs in MM. (A) Scatter plot visualizing gene set enrichment analysis normalized enrichment score comparisons between H929 and AMO1 cells treated with DMSO or YKL-5-124 (100 nM) for 24 hours. The bar graph shows NES for the top 18 gene signatures in both cell lines after treatment with YKL-5-124. (B) Biological upstream regulators associated with CDK7 inhibition were identified using ingenuity pathway analysis (IPA). (C) Whole-cell lysates from AMO1 and H929 cells treated with YKL-5-124 for 0.5 to 4 hours were subjected to WB analysis and probed with MYC antibody, with GAPDH as a loading control. The ratio of MYC/GAPDH was analyzed with Image J software and represented as fold change from untreated cells. Mean values ± SD in 2 MM cell lines is shown in the graph. (D) The diagram depicts the intermediates of glycolysis, and the enzymes regulated by CDK7 (red bars). Mean of log2-fold change for H929 and AMO1 cells after treatment with YKL-5-124 are shown (∗P < .05). (E) Bubble plot showing the differentially expressed proteins involved in the glycolytic pathway after treatment with YKL-5-124 for 24 hours. The x-axis displays the log2 (fold change), and the y-axis represents the negative log of the adjusted P value. (F) A panel of cells treated with YKL-5-124 for 24 hours was subjected to western blot analysis and probed with antibodies against HK2 and β-actin as a loading control (upper panel). The ratio of HK2/actin was analyzed with Image J software and represented as fold change from untreated cells (lower panel). Mean values ± SD in 5 MM cell lines are shown in the graph. (G) ChIP-seq tracks showing MYC signal on individual loci for LDHA and HK2. The x-axis shows genomic coordinates with gene model depicted below. The y-axis shows signal in units of rpm/bp. (H) MM1S cells were treated with YKL-5-124 for 6 hours and subjected to ChIP with a MYC or IgG antibody. HK2, LDHA, and a negative control region were amplified by polymerase chain reaction. Data are shown as mean ± SD of triplicates and represented as the percentage of input. bp, base pair; ChIP, chromatin immunoprecipitation; CHIP, clonal hematopoiesis of indeterminate potential; DMSO, dimethyl sulfoxide; FDR, false discovery rate; IgG, immunoglobulin G; NES, normalized enrichment score; rpm, units of reads per million; SD, standard deviation.

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