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DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance
Katsuhiro Togami, … , Cory M. Johannessen, Andrew A. Lane
Katsuhiro Togami, … , Cory M. Johannessen, Andrew A. Lane
Published August 22, 2019
Citation Information: J Clin Invest. 2019;129(11):5005-5019. https://doi.org/10.1172/JCI128571.
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Research Article Hematology Oncology

DNA methyltransferase inhibition overcomes diphthamide pathway deficiencies underlying CD123-targeted treatment resistance

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Abstract

The interleukin-3 receptor α subunit, CD123, is expressed in many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp’s ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.

Authors

Katsuhiro Togami, Timothy Pastika, Jason Stephansky, Mahmoud Ghandi, Amanda L. Christie, Kristen L. Jones, Carl A. Johnson, Ross W. Lindsay, Christopher L. Brooks, Anthony Letai, Jeffrey W. Craig, Olga Pozdnyakova, David M. Weinstock, Joan Montero, Jon C. Aster, Cory M. Johannessen, Andrew A. Lane

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Figure 3

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

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Tagraxofusp resistance is mediated by loss of the diphthamide synthesis ...
(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.
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