Figure 7.

Endothelial and AML cells exhibit overlap in surface protein expression that is influenced by cytokines. A, TIME cells were either untreated or treated for 24 hours (red) or 48 hours (green) with IFNγ and TNFα (100 ng/mL). CD93 expression patterns on endothelial cells were compared with other common AML targets (CD123, CD38, and CD33) by flow cytometric analysis under both conditions. B, Quantification of endothelial surface protein expression, measured by fold MFI over untreated control. TIME cells were incubated with IFNγ and TNFα at varying doses (+++ = 100 ng/mL, ++ = 10 ng/mL, + = 1 ng/mL) at 24 hours (red) and 48 hours (green), compared with untreated and FMO as negative controls. C, Bulk RNA-seq analysis was performed on two endothelial cell lines (iHUVEC and TIME) that were either untreated or treated with IFNγ, TNFα, or IFNγ and TNFα at 10 ng/mL for 24 hours. Transcriptional levels for selected AML targets on endothelial cells are reported as log₂TPM+1. D, RNA was isolated from two endothelial cell lines (iHUVEC and TIME) and three AML cell lines (THP-1, NOMO-1, and Kasumi-1) that were untreated or treated with IFNγ and TNFα (10 ng/mL). E, Heat map of genes that meet criteria for differential expression in AML or endothelial cells reveals clusters of genes with or without exposure to IFNγ and TNFα. Z-scores highlight differences among the cell types and treatment groups. F, Proposed model for gene expression analysis for rational combinatorial targeting (with NOT- or AND-gated CARs) to overcome endothelial toxicity from an AML-directed CAR. DEG, differentially expressed gene.