TDZD-8 specifically induces cell death of primary leukemia specimens. (A) Primary AML (n = 37), CLL (n = 12), ALL (n = 6), bcCML (n = 6), and normal mononuclear cells (n = 13, obtained from BM [n = 3], CB [n = 7], or MPB [n = 3]) were cultured for 18 to 24 hours in the presence of 20 μM TDZD-8. Cell viability was assessed by Annexin V/7-AAD staining. Percent viability is represented relative to untreated control. Leukemia specimens were significantly (P < .001) more sensitive to TDZD-8 than were normal specimens. Error bars represent the SEM. All assays were performed in triplicate. (B) Representative dot plots for the 24-hour flow cytometric analysis with Annexin V/7-AAD stain in the presence or the absence of 20 μM TDZD-8.

TDZD-8 treatment induces oxidative stress. (A) Flow cytometric overlays for mBBr fluorescence from primary AML, CLL, ALL, or normal mononuclear cells treated for 30 minutes with 20 μM TDZD-8 (black line) versus untreated controls (black line/gray filling). (B) Percentage of viability of primary AML cells pretreated with NAC (gray bars) for 1 hour before treatment with 20 μM TDZD-8. Viability was determined 24 hours after the addition of each drug. Error bars represent the SEM.

TDZD-8 inhibits PKC and FLT3 in primary AML specimens. (A) Immunoblots for CD34⁺ AML and normal BM specimens to determine PKC phosphorylation. Actin is shown as a loading control. (B) Primary CD34⁺ AML and ALL specimens treated with TDZD-8 for 1 hour were processed to obtain membrane fractions. Immunoblots were performed to determine PKCα and PKCβ levels in the membrane. HSP70 is shown as a control. (C) Titration curve and IC₅₀ value for FLT3 kinase assay. (D) Overlays of flow cytometric analysis for phospho-FLT3 in primary AML specimens. Light gray solid line histograms represent untreated cells. Dotted line histograms represent TDZD-8–treated cells. Gray solid no line histogram represent controls. Cells were processed for analysis 30 minutes after the addition of drug. CD34⁺ (31% inhibition; left panel) and CD34⁺CD38⁻ (29.5% inhibition; right panel) populations.

TDZD-8 ablates leukemia progenitor and stem cells. (A) Primary AML (n = 10), ALL (n = 3), bcCML (n = 3), and normal specimens (n = 7, obtained from BM, CB, or MPB specimens) were cultured for 18 to 24 hours in the presence or absence of 20 μM TDZD-8. Cell viability was assessed by flow cytometry in CD34⁺CD38⁻ populations for AML and bcCML (CML) and CD34⁺CD10⁻ cells for ALL using Annexin V/7-AAD stain. Percent viability is represented relative to untreated control. Specificity to leukemia specimens was significant (**P < .01). Error bars represent the SEM. (B) Primary cells from AML (n = 11), bcCML (CML; n = 3), and normal specimens (n = 12) were treated for 18 hours in suspension culture, followed by plating in methylcellulose. Error bars represent the SEM. Percentage of colony-forming units (CFUs) are normalized to untreated controls. All assays were performed in triplicate. Specificity to leukemia specimens was significant (**P < .01, *P < .05). (C) Percentage of marrow engraftment for NOD/SCID mice that received a transplant with AML (top panels) or normal CB (bottom panels) cells after 18 hours of culture with or without 20 μM TDZD-8. Each circle or triangle represents a single animal analyzed at 6 weeks after transplantation. Each plot represents an independent AML or CB specimen. Mean engraftment is indicated by the horizontal bars. **P < .01; ***P < .001.

TDZD-8 induces cell death with extremely rapid cell death kinetics showing loss of membrane integrity. (A) Percent viability assessed at the indicated time points for CD34⁺CD38⁻ populations of primary AML specimens (n = 8) treated with TDZD-8 (■) or PTL (□). Percent viability is represented relative to untreated control. (B) Percent viability assessed at the indicated time points for unfractionated primary AML specimens (n = 17) treated with TDZD-8. Percent viability is shown relative to untreated controls. Error bars represent SEM. (C) Cells were treated with 20 μM TDZD-8 for the indicated periods of time, then washed and placed in culture until analysis at 24 hours. Percent viability represented relative to untreated control. (D) Percentage of CFUs relative to untreated control. Cells were washed and placed in methylcellulose culture medium at the indicated time points after the addition of TDZD-8. (E) Loss of membrane integrity assessed by YoPro-1 uptake after 15 minutes of TDZD-8 treatment. Multispectral imaging flow cytometry shows the internalization of YoPro-1. Cells were stained with CD45 to delineate the plasma membrane and with the cell-permeable DNA dye Draq5 to identify the nucleus. (F) Flow cytometric histograms for YoPro-1 and PI overlaying TDZD-8–treated (20 μM for 15 minutes) normal mononuclear cells or primary AML cells over untreated controls. Treated cells are represented with black line histograms and untreated controls with gray solid histograms. (G) Percent viability of primary AML cells pretreated with Z-VAD (□) for 1 hour before the treatment with TDZD-8 20 μM. Viability was determined 24 hours after the addition of TDZD-8. Error bars represent the SEM.