Oxidative Stress Response and Cytotoxicity of Electrophilic Compounds in Primary Human Chronic Lymphocytic Leukemia.

The transcription factor Nrf2 [NFE2L2] is the master regulator of the oxidative stress response pathway. At low oxidative states, Nrf2 is degraded by an E3 ubiquitin ligase complex containing KEAP1. Reactive oxygen species or certain electrophiles cause Nrf2 accumulation and nuclear translocation by inactivating Keap1 through oxidation or adduction of its free thiols. Recent work has found that various electrophilic natural products (parthenolide, isothiocyanates) and synthetic molecules (ethacrynic acid, EA and 2-methoxyestradiol, 2-ME) are selectively cytotoxic to PBMCs from Chronic Lymphocytic Leukemia (CLL) patients. The high oxidative stress state of CLL cells was hypothesized to be the mechanism of specificity. However, the effect, if any, of electrophilic compounds on Nrf2 signaling in CLL is unknown, because Nrf2 activation has never been assessed in this leukemia. The purpose of the present studies is to determine if the Nrf2 pathway is active in primary CLL, and if it can be modified by electrophilic agents that are cytotoxic to CLL cells.

Peripheral blood mononuclear cells (PBMCs) from normal donors and CLL patients were isolated by Ficoll density-gradient centrifugation. B cells were purified with the negative selection method using RosetteSep human B cell isolation kit (Stemcell Technologies). Steady-state levels of Nrf2 and its target gene heme oxygenase 1 (HO-1) were compared between PBMCs from normal donors and CLL patients by immunoblotting and quantitative PCR. Five structural classes of small molecules with known electrophilic or potential oxidant activity were tested, including β,β-unsaturated carbonyls, isothiocyanates, thiol reactive metals, flavones and polyphenols. An initial screen for Nrf2 induction by the compounds was performed using a HepG2 cell line expressing β-lactamase under the control of the Antioxidant Response Element, an element essential for Nrf-2 mediated transcription. Cytotoxicity to normal donor and primary human CLL PBMCs was tested by tetrazolium reduction assay. To identify the active chemical moiety we catalytically reduced the unsaturated group on two of these compounds and tested them for Nrf2 activation and CLL selective cytotoxicity. To confirm Keap1 adduction as the mechanism of Nrf2 activation, we synthesized EA conjugated to biotin. Biotin-EA treated CLL cells were lysed and the lysate was passed through streptavidin agarose resin to collect EA modified proteins. Western blot analysis was performed with antibodies specific to Keap1 protein to determine if Keap1 is one of the targets.

Comparison between normal and CLL PBMCs shows that the Nrf2 pathway is both more highly expressed and more active in the CLL cells. Thus, electrophilic molecules induced HO-1 synthesis in primary leukemia cells. Purified normal B lymphocytes and CLL cells had similar Nrf2 expression that was significantly higher than PBMCs from normal donors. Compounds containing α,β-unsaturated carbonyls and thiol reactive metals were strong activators of Nrf2, and were also found to be selectively cytotoxic to CLL PBMCs compared to normal PBMCs. Reduction of the α,β-unsaturation abrogated Nrf2 activation and CLL toxicity, suggesting that this functional group is critical for both functions. Furthermore, binding studies with biotin-EA demonstrated that Keap1 was the target of electrophilic modification in CLL PBMCs. To our knowledge this is the first study of Nrf2 signaling in primary human CLL. This work is supported by the Leukemia and Lymphoma Society and by the NIH.

No relevant conflicts of interest to declare.