Flt3^ITD/NHD13 cooperation results in IFN-1 target gene hyperactivation, in contrast to Flt3^ITD/Runx1^DELcooperation. (A) Reactome pathway analysis of Flt3^ITD/NHD13 target genes. (B) Almost half of all genes induced in Flt3^ITD/NHD13 MPPs were IFN-1 targets, based on a previous comparison of wild-type and Ifnar1^−/− MPPs.³⁰ Only ∼3% of genes induced in Flt3^ITD/Runx1^DEL MPPs were IFN-1 targets. (C) Heatmap showing expression of IFN-1 targets from panel B in MPPs with the indicated genotypes. (D) Gene set enrichment analysis showing enrichment of IFN-1 targets in Flt3^ITD/NHD13 and Flt3^ITD/Runx1^DEL MPPs relative to indicated wild-type and single-mutant MPPs. Both Flt3^ITD and NHD13 alone activated IFN-1 targets (top row), but the effects were greatly amplified in Flt3^ITD/NHD13 MPPs (bottom row). In contrast, the IFN-1 signature was repressed in Flt3^ITD/Runx1^DEL MPPs relative to Flt3^ITD alone. (E) IFNα levels in the serum of P14 mice of the indicated genotypes, as measured by ELISA. n = 3 to 4. Error bars reflect the standard deviation. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 by one-way ANOVA with Holm-Sidak posthoc test. (F) GSEA showing activation of IFN-1 targets in Flt3^ITD/NHD13 MPPs at P0. ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; GSEA, gene set enrichment analysis.