Fig. 2: RSK1 inhibition is efficacious in MPN.

a Schematic of RSK1 as a key node along canonical oncogenic signaling pathways. b Pearson correlations of pRSK1 T573 and pRSK1 T359/S363 with other phosphorylated proteins (total n = 54 phosphorylated proteins) from RPPA data from 34 AML cell lines from CCLE. c KINOMEscan of PMD-026 across 398 kinases identifying specificity against RSK1-4. d Cell viability assay of HEL cells treated with RSK inhibitors BI-D1970 and PMD-026 at the indicated drug doses. Cells were treated for 5 days and with six biological replicates. Data are presented as mean values ± SD. e Immunoblot of HEL cells treated with 5 μM,10 μM PMD-026, or control for 48 h. f Cell viability assay of Ba/F3 cells ectopically expressing wild-type or mutant JAK2 and MPL treated with PMD-026 at the indicated drug doses. Cells were treated for 5 days and with six biological replicates. Data are presented as mean values ± SD. g Annexin V assay of HEL cells treated with 5 μM,10 μM PMD-026, or control for 48 h and 72 h. Cells were treated in triplicate (n = 3 independent experiments). Data are presented as mean values ± SD. h Cell cycle assay of HEL cells treated with 5 μM, 10 μM PMD-026, or control for 24 h and 48 h. Cells were treated in triplicate (n = 3 independent experiments). Data are presented as mean values ± SD. i Top altered Hallmark pathways by enrichment analysis from top 500 DEGs from RNA-seq analysis of HEL cells treated with 10 μM PMD-026 compared to control for 24 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. j Colony counts from CD34 + colony assays. Sorted CD34 + cells from five patients were seeded into MethoCult H4034 in indicated doses of PMD-026 at micromolar concentration. Cells were plated in triplicate per condition (n = 3 biological replicates). Colonies were enumerated after 14 days. Data are presented as mean values ± SD. k Schematic of the CD34 + 784981 sAML pMF patient-derived xenograft mouse model. Two shRNAs targeting RPS6KA1 or control vector were ectopically expressed in isolated CD34 + cells after which transformed cells were transplanted into NSGS mice by intra-tibial injection into the bone marrow and followed for 12 weeks. l Percentage of human CD45 (hCD45) in the peripheral blood and bone marrow of transplanted mice ectopically expressing control (n = 4), shRPS6KA1 #1 (sh #1; n = 5), or shRPS6KA1 #2 (sh #2; n = 5) across multiple timepoints, and spleen and liver weights of mice at endpoint normalized by mouse weight. %hCD45 in PB statistics assessed by two-way ANOVA with Dunnett’s multiple comparisons test with control. %hCD45 in BM, and normalized spleen and liver weights statistics were assessed by one-way ANOVA with Dunnett’s multiple comparisons test with control. Data are presented as mean values +/− SD.