LSD1 inhibitors normalize aberrant SETBP1 transcriptional programs. (A) A medium-throughput inhibitor screen was performed on the CSF3R^T618I plus SETBP1^D868N cell line, and both LSD1 inhibitors and JAK inhibitors were among the top hits. The 175 inhibitors evaluated have known sensitivity in patient samples (BeatAML cohort²⁹). The inhibitors were ranked for this analysis by dividing the median IC₅₀ of all samples previously screened by our cell line IC₅₀ to determine a fold change. (B) A luciferase E-box activity assay was performed with 4 concentrations of the LSD1 inhibitor GSK2879552. In 293T17 cells expressing CSF3R^T618I and SETBP1^D868N, LSD1 inhibition reduced MYC activity by 24% at 250 nM. (C) In our cell line where SETBP1^D868N expression was regulated by doxycycline (DOX), we evaluated whether LSD1 inhibitors would reduce Myc gene expression to the level of DOX⁻ cells. The LSD1 inhibitors GSK2879552 (1000 nM) and GSK-LSD1 (100 nM) both reduced Myc expression in CSF3R^T618I plus SETBP1^D868N cells, but JQ1 (200 nM) did not. (D) qPCR for Myc was performed after treatment of the CSF3R^T618I plus SETBP1^D868N cell line with 1 of 3 LSD1 inhibitors at 100 nM (GSK2879552) or 30 nM (GSK-LSD1 and ORY-1001) for 48 hours. (E) qPCR for Myb. (F) qPCR for Meis1. (G) qPCR for Hoxa9, which is not modulated by LSD1 inhibition at these concentrations. (H) RNA-seq was performed after treatment of the cell line with 100 nM of GSK2879552 or 30 nM of ORY-1001 for 24 hours. GSEA demonstrated that this treatment was associated with a reversal of MYC amplification with both inhibitors. (I) A CSF3R^T618I- and SETBP1^G870S-mutated patient sample was treated with 100 nM of ORY-1001 for 24 hours, and CITE-seq (single-cell RNA-seq with barcoded antibody labeling) was performed. Treatment significantly decreased Myc expression in hematopoietic progenitor clusters expressing high levels of CD34. *P < .05, **P < .01, ***P < .001, ****P < .0001. FDR, false-discovery rate; NES, normalized enrichment score.