Impact of Npm1^cA/+on the transcriptome of Nras^G12D/+- and Flt3^ITD/+-mutant hematopoietic progenitors. (A) Overlap of differentially expressed mRNAs reveals that Npm1^cA/+ has a dramatic impact on Lin⁻ progenitor GEPs when combined with Flt3^ITD/+ but only a modest impact when combined with Nras^G12D/+. Nonetheless, the characteristic hallmarks of Npm1^cA/+, namely overexpression of Hoxa genes and of the homeobox genes Hopx and Nkx2-3, are retained in both types of double-mutant progenitors. Gene Set Enrichment Analysis reveals enrichment of differentially expressed genes from these models in human AMLs harboring mutant NPM1 or MLL gene fusions. (B) Comparison of human NPM1-mutant (NPM1^c) vs NPM1-wildtype (NPM1^WT) normal karyotype AML also shows marked overexpression of HOXA and HOXB genes as well as of NKX2.3, raising the possibility that the latter may mediate some of the effect of NPM1^c. (C) Effects of Nkx2-3 and Hoxa9 overexpression on mouse hematopoietic progenitors. (i) Lin⁻ bone marrow progenitors from wild-type and Flt3^ITD/+ mice were transduced with MSCV-Nkx2.3-CFP or MSCV-Hoxa9-GFP constructs or both, maintained in liquid culture for 7 days, FACS sorted for CFP and GFP single- and double-transfected cells, and plated in semisolid media. (ii) Colony assays of 2,500 transduced cells show that both MSCV-Hoxa9 and MSCV-Nkx2-3 conferred an increase in self-renewal of both wild-type and Flt3^ITD/+ cells. However, double MSCV-Hoxa9/MSCV-Nkx2-3 transfected cells showed no further changes in self-renewal when compared with MSCV-Hoxa9 alone. Mean ± SEM (n = 3). *P < .05; **P < .01; ***P < .001; Student t test. (D) Sorting strategy for LSK/CD34⁺/Flt3⁺/CD48⁺ progenitor cells and overlap of differentially expressed genes (Illumina MouseWG-6 version 2 Expression BeadChip) for Nras^G12D/+ vs (i) Npm1^cA/+;Nras^G12D/+ and (ii) Flt3^ITD/+ vs Npm1^cA/+;Flt3^ITD/+ MPP datasets. (E) Heat maps of normalized Hox gene expression in (i) MPP and (ii) Lin⁻ progenitor populations reveal that, unlike Lin⁻ cells, Npm1^cA/+-mutant (single or double) MPPs have similar patterns of Hox gene expression to Npm1-WT MPPs (normalized average expression values are used to generate heat map values). (F) Differentially expressed genes in Npm1^cA/+;Flt3^ITD/+ MPPs vs WT controls. Adj., adjusted; FC, fold change; NK, normal karyotype.