SETBP1 upregulates early progenitor gene expression pathways and is associated with increased activation of MYC targets. (A) Top Enrichr transcription factor perturbation followed by expression pathways for genes that are differentially expressed with SETBP1. We performed RNA-seq on the CSF3R^T618I plus SETBP1^D868N-dox cell line at 24 hours postdoxycycline (DOX) withdrawal, when the cells were still viable and dividing. Data are reported for cells with DOX (CSF3R^T618I plus SETBP1^D868N-ON) relative to those without DOX (CSF3R^T618I plus SETBP1^D868N-OFF). Enrichr calculates the combined score by multiplying the pathway z score and log(P value). (B) GSEA was performed to identify pathways that are enriched with SETBP1 expression. Three of the top GSEA plots, each with a false-discovery rate (FDR) q value of 0.00, are shown. (C) Glimma volcano plot showing differentially expressed genes with SETBP1^D868N, with several key genes annotated. (D) HOMER motif analysis was run to identify the top motifs enriched in the genes differentially upregulated and downregulated by SETBP1. (E) Coexpression of SETBP1^D868N with CSF3R^T618I drove a 3.7-fold increase in MYC activity over CSF3R^T618I alone. A luciferase reporter assay for the MYC E-box was used to measure if SETBP1^D868N modulates E-box activity. Using an MYC E-box reporter plasmid, E-box activity was measured in transfected 293T17 cells expressing CSF3R^T618I alone or CSF3R^T618I plus SETBP1^D868N. (F) In transfected 293T17 cells expressing only CSF3R^T618I, SETBP1^WT, or SETBP1^D868N, CSF3R did not increase E-box activity above baseline, whereas both WT and mutated SETBP1 significantly increased E-box activity. (G) Coexpression of MYC with CSF3R^T618I in a CFU assay resulted in an increase in colony formation over either oncogene alone. A colony forming unit assay was performed to assess whether expression of MYC is sufficient to increase CSF3R-driven colony formation. A total of 10 000 cells expressing either MYC, CSF3R^T618I, or both were plated in cytokine-free methylcellulose, and CFUs were counted after 7 days. (H) An inducible lentiviral Myb short hairpin RNA (shRNA) construct was stably expressed in the CSF3R^T618I plus SETBP1^D868N cell line. Addition of DOX to the media knocked down Myb expression by ∼52%. (I) Knockdown (KD) of Myb resulted in a 47% decrease in Myc expression and a 22% decrease in Hoxa10 but no significant changes to Meis1 or Hoxa9. *P < .05, **P < .01, ***P < .001, ****P < .0001. FC, fold change; KO, knockout; NES, normalized enrichment score; ns, not significant; OE, overexpression.