Rapid Loss of Membrane Integrity and Irreversible Commitment to Cell Death of Primary AML Stem Cells: A Novel Activity for 4-benzyl, 2-methyl,1,2,4-Thiadiazolidine,3,5 Dione (TDZD-8).
Authors: Guzman Monica L. , Li Xiaojie , Corbett Cheryl , Rossi Randall M. , Bushnell Timothy , Jordan Craig T. 
Published: October 2019

Abstract
Leukemia stem cells (LSCs) have been shown to play a crucial role in the pathogenesis of acute myelogenous leukemia (AML). However, current chemotherapy agents have not demonstrated effective targeting of the LSC population. Thus, failure to eradicate LSCs may lead to disease relapse and progression. Consequently, the identification and characterization of drugs that can efficiently eradicate the LSC population is required. We have identified a compound, 4-benzyl, 2-methyl,1,2,4-thiadiazolidine,3,5 dione (TDZD-8), with the novel characteristic of rapidly inducing cell death of different types of leukemia cells, including AML progenitor and stem cell populations. After 18–24 hours of treatment with 20 uM TDZD-8, the mean survival for primary AML cells was 7% (n=30), blast crisis CML 5% (n= 5), CLL 3% (n=13) and ALL 6% (n= 4). In contrast, normal specimens treated in the same fashion showed 80% mean survival (n=10). Functional assays were performed to determine the effect of TDZD-8 on primitive populations. NOD/SCID xenotransplant analysis showed a 93% decrease of engraftment for AML specimens, while normal cell engraftment was only reduced by 11%. Furthermore, colony-forming assays demonstrated a 93% decrease in CFU formation for AML cells (n=11, where 6 of the specimens showed no CFUs), in contrast to a 30% decrease in normal cells. Strikingly, cell death occurred with extremely rapid kinetics. Time course studies from the different primary AML specimens demonstrated that most of the cell death can be observed within 2 hours after exposure to TDZD-8 (where 10% of the samples tested appear dead with as little as 15–30 minutes of drug exposure). Furthermore, a 30 minute exposure to the drug was sufficient to irreversibly induce cell death of primitive CD34+CD38- cells and CFUs. Interestingly, TDZD-8 was originally described as a GSK-3b inhibitor and analogs to this molecule have also been found to activate PPAR-gamma. However, when we tested different known GSK-3b inhibitors (n=12) and PPAR-gamma activators (containing a thioazolidine ring) we did not observe the toxicity, specificity, or kinetics observed with TDZD-8. Molecular studies suggest that the mechanism by which TDZD-8 induces cell death involves a rapid thiol depletion, Nrf2 activation, and NF-kappaB inhibition. Moreover, a rapid loss of membrane integrity was observed using imaging flow cytometry (Amnis ImageStream) to determine permeability of different viability dyes. Furthermore, TDZD-8 induced cytochrome c release, but cell death was not blocked by pan-caspase inhibitors, suggesting a non-apoptotic cell death mechanism. Finally, preliminary in vivo experiments using mice transplanted with primary AML cells showed that Nrf2 was induced as soon as 1h after dosing animals with 4.4 mg/kg of TDZD-8 (i.p.). Together, these data suggest that TDZD-8 represents a novel class of anti-leukemia specific molecules capable of destroying leukemia progenitor and stem cells with extremely rapid kinetics.