Fig. 5: Alteration of NFκB-mediated transcriptional signatures by RSK1 inhibition.

a Spearman correlation of CD14 and RPS6KA1 expression from TCGA LAML (n = 165) and WashU Cohort CD14 + monocytes (n = 68). Correlation (blue line) and two-tailed P-values of the WashU Cohort CD14 + monocytes represent values across all MPN patients (n = 54). b Spearman correlation of percent PB monocyte and RPS6KA1 expression from the BeatAML cohort (n = 451). Correlation (blue line) and two-tailed P-value presented. c Top altered Hallmark pathways by enrichment from top 500 DEGs from RNA-seq analysis of CD14 + cells isolated from 3 MF patients treated with 5 μM PMD-026 compared to control for 6 h. Gene count represents the number of genes enriched across each pertinent gene set. Gene ratio represents the number of genes enriched/total number of genes in each gene set. d Heatmap showing changes to RNA-seq expression of key effectors from (d) after PMD-026 treatment relative to control. e qRT-PCR of key effectors from CD14+ monocytes seven additional MPN patients after 6 h 5 μM PMD-026 treatment relative to control. n = 7 patients for all effectors except CXCL10 (n = 6). Statistics were assessed by a two-tailed paired Student’s t test compared to control. Data are presented as mean values ± SD. f Luciferase assay of THP-1 NFκB-Luc2 cells treated with 5 μM PMD-026 or control for 24 h. n = 6 for each group. Statistics were assessed by a two-tailed Student’s t test. Data are presented as mean values ± SD. g Immunoblot of RELA localization upon TNF and/or PMD-026 treatment. THP-1 cells were pre-treated with 10 μM PMD-026 for 15 min and then stimulated with 20 ng/mL TNF for 15 min. h Transcription start site plot and profile heatmap of annotated peaks within 2000 base pairs from ATAC-seq analysis of THP-1 cells treated with 5 μM PMD-026 or DMSO control for 24 h. i Pie charts of gene section breakdown of annotated peaks from (h). j Enrichment bubble plots of top altered pathways from differentially annotated regions with PMD-026 treatment. Cut-off was denoted as p-value ≤ 0.05 and false discovery rate (FDR) step up ≤ 0.05. k Enrichment bubble plots of top-downregulated pathways from differentially annotated TSS regions with PMD-026 treatment. Cut-off was denoted as TSS annotation with p-value ≤ 0.05 and false discovery rate (FDR) step up ≤ 0.05. l Known vertebrate motifs from the JASPAR database are over-represented in the list of sequences in the significant regions list from j). Each motif name, matrix ID, and p-value are denoted. Analysis performed using Partek Flow pipeline. m CUT&Tag H3K27ac histone tracks at promoter regions of genes of interest. THP-1 cells were treated with 5 μM PMD-026 or DMSO control for 24 h. Genomic tracks were visualized using the WashU EpiGenome Browser.