ANKL cells are vulnerable to the inhibition of TfR1 function. (A) Schema of the in vivo competitive assay using the CRISPR-Cas9 system. ANKL1-Cas9 cells are harvested from PDX mice and then transduced with sgRNA-mOrange using a lentiviral vector. After 48 hours, hCD45⁺mOrange⁺ and hCD45⁺mOrange⁻ cells are sorted, mixed at a ratio of 2000 to 1000 cells, and transplanted into mice. (B) The percentage of mOrange⁺ cells in the liver at day 14 after transplantation are compared between sgNT and sgTFRC transduced ANKL1-Cas9 cells (n = 3). (C) Viability of NK-cell leukemia cell lines after treatment with various concentrations of PPMX-T003 for 96 hours in vitro, evaluated by propidium iodide and hCD45 staining and flow cytometry. (D) Viability of primary ANKL cells obtained from ANKL1, ANKL3, and ANKL5 PDX mice after treatment with various concentrations of PPMX-T003 for 48 hours in vitro. (E) Schema for testing the efficacy of PPMX-T003 in vivo using ANKL-PDX mice. (F) Comparing the ratio of human CD45⁺ cells to mouse CD45⁺ cells in PB on days 11 and 14 after transplantation among ANKL1-PDX mice treated with 0, 1, 3, and 10 mg/kg of PPMX-T003 (left). Comparing the liver weight per body weight among 0, 1, 3, and 10 mg/kg of PPMX-T003 treatments in ANKL1-PDX mice (right) (n = 4). (G) Schema for RNA-seq to analyze the global gene expression changes in ANKL1 cells upon PPMX-T003 or phosphate-buffered saline (PBS) treatment in vivo (n = 3). (H) Volcano plot of RNA-seq data. (I) Intracellular ferrous staining of ANKL3 cells 48 hours after treatment with PPMX-T003 in vitro, stained with FerroOrange, and analyzed using confocal microscopy. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.