scRNA-seq analysis of CD34⁺ AML patient cells identifies differentially expressed gene signatures and that SLC-391, alone or with venetoclax, inhibits expression/activity of key signaling proteins. (A) CD34⁺ AML cells (n = 3, patients #2, #3, and #14) were pretreated with the indicated control and inhibitors (alone or in combination) for 16 hours and were then sorted for propidium iodide⁻Annexin V⁻CD34⁺CD33⁺ cells. Three thousand and three hundred cells per treatment group and per patient sample were used for barcoding and library construction for scRNA-seq with 10× Genomics. Heat map of enriched up/downregulated gene signatures based on adjusted P values and NESs derived via GSEA of scRNA-seq data from 3 AML patient samples after the indicated treatments (relative to DMSO control). (B) GSEA enrichment plots for significant gene signatures from panel A with corresponding NESs. Negative values indicate downregulation of gene sets, whereas positive values indicate upregulation of gene sets, relative to DMSO-treated control cells. (C) MV4-11 cells were treated with 0.25 µM SLC-391 (SLC) and 5 nM venetoclax (VEN), alone or in combination, for 3 hours, and cell pellets were harvested for western blotting analysis. Antibodies used are indicated. (D) MV4-11 cells were treated as in panel C for 72 hours, and cell lysates were subjected to western blotting analysis for BCL-2 family proteins, as indicated. (E) MV4-11 cells were treated with SLC and VEN, alone or in combination, for 16 hours. BIM was immunoprecipitated from whole-cell lysates and then subjected to western blotting analysis. Antibodies used are indicated. Phosphorylation or total protein expression changes were quantified relative to actin and normalized to no drug treatment control, with fold changes indicated.