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. 2019 Oct 14;129(11):5005–5019. doi: 10.1172/JCI128571

Figure 3. Tagraxofusp resistance is mediated by loss of the diphthamide synthesis pathway enzyme DPH1.

Figure 3

(A) Differentially expressed genes between CAL1 (BPDCN) and SHI1 (AML) parental cells compared to 3 independent tagraxofusp-resistant subclones each (n = 6 parental and 6 resistant total). Log2(fold change) expression in resistant compared with parental cells plotted (x axis) versus –log10 adjusted P (Padj) value (y axis). Genes names in blue (downregulated in resistant cells) or red (upregulated in resistant cells) for genes with –log10 Padj > 10. (B) Log2(fold change) in gene expression associated with tagraxofusp resistance (as from panel A), where negative values represent lower expression in resistant cells, plotted gene-by-gene versus a CRISPRi score for CTx-DTA resistance (19), where positive values represent genes that conferred CTX-DTA resistance when their expression was inhibited. DPH1 is highlighted in red. (C) Western blotting for DPH1 and actin in parental and tagraxofusp-resistant CAL1 cells. (D) In vitro ADP-ribosylation assay with or without tagraxofusp (top row) and Western blotting for eEF2, DPH1, and actin (bottom rows) for parental THP1 and tagraxofusp-resistant (R1–R3) subclones. (E) Western blotting for DPH1 and actin in parental THP1 cells, and cells transduced with independent nontargeting (NTG1-2) and DPH1-targeted (g2, g3, g5, and g6) sgRNAs. (F) Percentage lentivirus-containing (GFP+) cells plotted over time after treatment with tagraxofusp in CAL1, NOMO1, and THP1 cells transduced with the CRISPR sgRNA-containing lentiviruses as in panel E, each coexpressing GFP. (G) In vitro ADP-ribosylation in the presence of tagraxofusp (top row) and Western blotting for eEF2, DPH1, and actin (bottom rows) for parental CAL1 cells and tagraxofusp-resistant cells expressing a doxycycline-inducible full-length DPH1 cDNA, an N-terminally truncated, enzymatically inactive DPH1, or empty vector. (H) Viability after treatment with serial dilutions of tagraxofusp in parental and tagraxofusp-resistant cells expressing doxycycline-inducible DPH1 or variants as in panel G. Triplicate points plotted relative to cells in vehicle alone.