Treatment with HHT alters the transcriptome in mtRUNX1-expressing AML cells. (A) OCI-AML2 and OCI-AML2 RUNX1^R174*/wt cells were treated with 100 nM of HHT for 8 hours in biologic triplicates and RNA-Seq analysis was performed. The Venn diagram shows the overlap of up- and downregulated genes in the HHT-treated OCI-AML2 and OCI-AML2 RUNX1^R174*/wt cells. (B) GSEA of HHT-treated OCI-AML2 RUNX1^R174*/wt cells against HALLMARK and Reactome pathways. (C) PD mtRUNX1 AML #11 cells were treated with 100 nM of HHT for 8 hours and single-cell RNA-Seq analysis was conducted. Aggregated Uniform Manifold Approximation and Projection plots showing cluster composition of control and HHT-treated PD mtRUNX1 AML #11 cells as determined by SingleR. (D) GSVA analysis of mRNA expression changes due to HHT treatment compared with HALLMARK pathways. (E) Log2 fold-change in MYC, PIM, elongation factor and ribosomal protein genes in the HSC cluster of PD mtRUNX1 AML #11 cells treated with 100 nM of HHT for 8 hours and analyzed by single-cell RNA-Seq analysis. (F) Violin plot of per-cell mRNA expression of MYC, EIF4A1 and PIM1 in the HSC cluster of control and HHT-treated PD, mtRUNX1 AML #11 cells. (G-H) PD mtRUNX1 AML samples 11, 12, and 13 were treated with the indicated concentrations of HHT or OTX015 for 16 hours. Cells were fixed, permeabilized and incubated with rare-metal-tagged antibodies for CyTOF analysis. Data were analyzed by Astrolabe. Fold-change in protein expressions in the stem cell population of PD mtRUNX1 AML samples 11, 12, and 13 following treatment with 100 nM of HHT or 1 µM of OTX015 for 16 hours is shown. (I) PD mtRUNX1 AML samples 11, 12, and 13 were treated with the indicated concentrations of HHT, OTX015, or venetoclax for 16 hours. Cells were fixed, permeabilized and incubated with rare metal tagged antibodies for CyTOF analysis. Data were analyzed by Astrolabe. Graphic shows the percentage of stem cells (relative to the total) in each AML sample pre- and posttreatment.