(A) Percentage of methylated CpGs in the DPH1 locus are shown for the indicated genomic positions in parental THP1 cells and 2 independent tagraxofusp-resistant subclones, before and after 2 weeks of pulsatile treatment with noncytotoxic doses of azacitidine. (B) Quantitative RT-PCR for DPH1 expression in parental THP1 cells and a tagraxofusp-resistant subclone treated with vehicle or azacitidine (n = 3 replicates each). Dots represent relative expression, bars are ±SD. Conditions compared by 1-way ANOVA with Dunnett’s multiple-comparisons correction, adjusted P values shown. Data are representative of 2 independent resistant subclones with similar results. (C) In vitro ADP-ribosylation assay in the presence of tagraxofusp (top row) and Western blotting for eEF2, DPH1, and actin (bottom rows) are shown for parental THP1 and 3 independent tagraxofusp-resistant subclones (R1–R3) after 2 weeks of pulsatile treatment with noncytotoxic doses of azacitidine or vehicle. (D) Tagraxofusp cytotoxicity assays in parental and tagraxofusp-resistant AML (THP1) and BPDCN (CAL1) cells after 2 weeks of pulsatile treatment with noncytotoxic doses of azacitidine or vehicle, or with weekly exposure to 1 μg/mL tagraxofusp. Each point was assessed in triplicate and plotted relative to cells growing in vehicle alone.