TLR2 signaling induces AML cell transcriptional reprogramming. (A) UMAP projection of iterative clustering and guide-gene selection (ICGS2) of CD45.2⁺Dnmt3a^+/−Flt3^ITD AML cells single-cell RNA sequencing on sorted BM of transplanted CD45.1⁺ WT mice 24 hours after treatment with PBS or Pam₃CSK₄ (n = 2/group). Experimental schematic supplemental Figure 2A. (B-C) Fraction of cells in each cluster from sorted CD45.2⁺Lin⁻cKit⁺ cells (B) and CD45.2⁺CD11b⁺ cells (C). (D) Density plot depicting pseudotime (URD) on the x-axis and relative cell abundance on the y-axis. The density is scaled to total number of cells within a given cell type and treatment condition (PBS: gray, Pam₃CSK₄: red). (E-F) Transcription factor activity across pseudotime of Klf4 (left) and Irf8 (right) (E), C/ebpa (left) and Gfi1 (right) (F), in PBS (gray) and Pam₃CSK₄ (red) treatment groups. Vertical bars at the top of each graph indicate median pseudotime value for each cell-type pseudotime, to provide context for cell type (B-C). Bracketed pseudotime m1 and m2 indicate cell types with increased monocyte-specifying Klf4 and Irf8 activity and g1 indicates cell types with decreased granulocyte-specifying C/ebpa and Gfi1 activity in Pam3CSK4 treatment. (G) Average ± SEM percent proNeu1 (left) and proNeu2 (right) of CD45.2⁺Dnmt3a^+/−Flt3^ITD AML cells in WT recipient mice 24 hours after PBS or Pam₃CSK₄ treatment (n = 6/group). (H-I) Average ± SEM percent CD11b⁺F4/80⁺ (H) and CD11b⁺Gr1⁺ (I) of CD45.2⁺ BM. (H-I) Replicates for donor-recipient transplants include AML-WT (n = 9/group) and Tlr2^−/−AML-WT (n = 12/group). Statistical significance was calculated by unpaired t test (G) and ordinary 1-way ANOVA using Tukey multiple comparison test (H-I). ∗∗∗∗P < .0001, ∗∗∗P < .001, ∗∗P < .01, ∗P < .05. BM neu, BM neutrophil; DC, dendritic cell; gran, granulocytic; HSCP, hematopoietic stem cell progenitor; Mac, macrophage; Mono, monocyte; MP, myeloid progenitor.