Targeting Redox Homeostasis As a Means to Selectively Eradicate Primary Human Leukemia Cells,
Authors: Pei Shanshan , Callahan Kevin , Minhajuddin Mohammad , Balys Marlene , Neering Sarah , Corbett Cheryl , Becker Michael W. , Liesveld Jane L. , Hagen Fred , Friedman Alan E , Crooks Peter , Jordan Craig T. 
Published: October 2019

Abstract
Abstract 3506
Many studies have indicated that induction of oxidative stress is one component of the mechanism by which anti-leukemia agents function. However, little is known about the differences between leukemic and normal redox homeostasis, and whether these agents achieve their selective toxicity to leukemia through specifically targeting leukemic redox homeostasis. Hence, the present study performed a detailed analysis of CD34+ cells derived from normal bone marrow (NBM) or primary acute myelogenous leukemia (AML) specimens. By evaluating the expression profiles of redox regulatory genes, we observed that glutathione peroxidase (GPX1), glutathione reductase (GSR), and several other genes are significantly dysregulated in AML cells. Consistent with these findings, we found a lower GSH/GSSG ratio as well as a lower amount of total glutathione in AML cells compared to normal. In addition, we also found that AML cells display a different spectrum of cell surface thiols. Together, these data suggest that leukemia cells have a distinct redox state maintained by a set of differentially regulated genes in comparison to normal cells.
We next tested if parthenolide (PTL), a known ROS-inducer and an effective anti-leukemia agent, targets any of these differences to achieve its leukemia selectivity. We found that PTL directly depletes glutathione in a time and dose dependent manner. However, the amount of glutathione remaining after PTL treatment was less in primary CD34+ AML cells (∼1.5nmol/mg lysate) compared to CD34+ NBM cells (∼5nmol/mg). Thus, the net consequence on glutathione levels is much more severe in AML cells. Importantly, PTL, at the tested dose, selectively kills both AML bulk and stem/progenitor populations but spares normal cells. Given the central role of glutathione in redox homeostasis, we reason the selective toxicity of PTL to leukemia is at least partially because AML cells has a limited pool of glutathione compared to normal. Furthermore, the active moiety of PTL, a α-metheylene-γ-lactone group, predicts it will alkylate cysteine (Cys) residues required for the critical function and stability of several redox proteins. To identify these interactions, we used a biotinylated analogue of PTL to perform a streptavidin-based pull-down assays followed by liquid phase chromatography-mass spectrometry (LC-MS) analysis. We demonstrate that thioredoxin (TXN) and glutathione peroxidase (GPX1), key regulators of cell surface and intracellular redox balance, are direct binding targets of PTL. These binding events were found to at least partially inhibit the enzymatic function of these proteins, contributing to PTL-induced oxidative stress in AML cells. Knowing that GPX1 is already dysregulated in AML cells compared to normal, and considering the increased surface thiol level on AML cells, we hypothesize that these interactions are also important for the selectivity of PTL to AML cells. This is in addition to its ability to exploit the differential pool of glutathione in AML cells. Lastly, PTL induces ROS stress in AML cells as evidenced by redox dye staining, and robust Nrf2-mediated anti-oxidant responses in AML cells. Notably, SLC7A11, a gene that is required for importing cysteine into AML cells for glutathione synthesis, and GCLM which is directly involved in glutathione synthesis are also both up-regulated by PTL.
In summary, our studies identify altered GSH/GSSG ratio, decreased glutathione levels, dysregulated expression of GPX1, GSR and other redox regulatory genes, and increased cell surface thiols as critical differences between leukemic and normal redox homeostasis. Our study of PTL indicates that leukemia-specific agents of this nature function by exploiting the differences between glutathione system in leukemic vs. normal cells, and further acting on redox proteins that are already dysregulated in AML cells. These findings have ramifications for the rational design of regimens that manipulate oxidative stress as a means to selectively kill malignant cells.

Disclosures:
No relevant conflicts of interest to declare.