Setbp1 overexpression drives a gene expression signature that overlaps, but is distinct, from that associated with mutant NPM1 and MLL gene fusions. (A) Comparative gene-expression profiles of differentially expressed genes between Flt3^ITD/+/Setbp1^IM+and Flt3^ITD/+/Setbp1^IM− leukemias, highlighting a Hoxa-rich gene signature that overlaps with that seen with NPM1 mutations and the MLL-AF9 gene fusion. (B) Comparative gene-expression profile of differentially expressed genes between Flt3^ITD/+/Setbp1^IM+ and Flt3^ITD/+/Npm1^cA/+ AMLs (adjusted to average log₁₀ FPKM for each group). (A-B) Adjusted P values (Padj). (C) Gene expression of selected genes in WT lin⁻ cells and Flt3^ITD/+/Setbp1^IM−, Flt3^ITD/+/Npm1^cA/+, and Flt3^ITD/+/Setbp1^IM+ leukemias. (D) NKX2.3 and HOXA9 expression levels in AMLs in Leucegene cohort showing that NPM1-mutant leukemias uniformly express high HOXA9 and NKX2.3 (P = 2.08E-43; Fisher’s exact test), whereas the majority of SETBP1^high (log₁₀ FPKM >1) leukemias express only high HOXA9 and significantly associate with lower NKX2.3 expression (P = 1.72E-15; Fisher’s exact test). The pie chart shows that SETBP1 overexpression and mutant NPM1 are almost completely mutually exclusive and associated with relatively similar proportions of HOXA9^high AMLs. (E) Impact of CRISPR gRNA gene knockouts in competitive coculture in Flt3^ITD/+/Npm1^cA and Flt3^ITD/+/Setbp1^IM+ AML cell lines (n = 3 per cell line). The results show that, although both types of AML are vulnerable to loss of Hoxa9 and Hoxa10, only Flt3^ITD/+/Setbp1^IM+ cells are vulnerable to loss of Setbp1, and only Flt3^ITD/+/Npm1^cA AMLs are vulnerable to loss of Nkx2.3.