IFN-1 signaling potentiates Flt3^ITD/NHD13 AML. (A) UMAP plots demonstrating clusters from ICGS2 for the indicated genotypes. In this case, cluster 19 demarcates an NHD13- and Flt3^ITD/NHD13-specific cluster based on marker genes (supplemental Table 6). The heatmap is shown in supplemental Figure 12. (B) Bar graph showing percentages of cells for each genotype that populate clusters 19. Ifnar1 deletion depletes cluster 19 cells in NHD13 and Flt3^ITD/NHD13 mice. ∗∗∗P < .0001 for Ifnar1^+/− vs Ifnar1^−/− by χ² test. (C-D) Surface marker phenotypes within cluster 19 are Ifnar1-dependent. In the absence of Ifnar1, cells lose MPP phenotypes and acquire pGM and GMP phenotypes despite very similar gene expression profiles. This is evident in both NHD13 and Flt3^ITD/NHD13 mice, indicating that conventional cell phenotypes do not adequately describe the transcriptional identities of cells that carry these mutations. ∗P < .05, ∗∗∗P < .0001 for Flt3^ITD/NHD13/Ifnar1^+/− vs Flt3^ITD/NHD13/Ifnar1^−/− by χ² test. (E) Violin plots showing expression of several genes that are differentially expressed between Flt3^ITD/NHD13/Ifnar1^+/− and Flt3^ITD/NHD13/Ifnar1^−/− cluster 19 cells. ∗∗∗P < .001 by Wilcoxon Rank Sum test. (F) Survival curves for Vav1-Cre; Flt3^ITD; NHD13; Ifnar1^+/− and Vav1-Cre; Flt3^ITD; NHD13; Ifnar1^−/− mice. (G) Schematic overview of survival experiments for an adoptive transfer model. LK cells were isolated from NHD13 mice on Ifnar1^+/− or Ifnar1^−/− backgrounds and transduced with Flt3^ITD-expressing retrovirus. The cells were transplanted, and recipient survival was monitored. (H) mCherry chimerism for the indicated Ifnar1 genotypes at 4 weeks after transplantation. (I) Survival curves for recipient mice that were transplanted with cells of the indicated genotypes. For panels F and I, survival curves were compared by the log-rank test. Group sizes are indicated in the panels. Adoptive transfer data reflect 4 independent experiments. ICGS2, Iterative Clustering and Guide Gene Selection version 2.