Role of Glutathione in the Synergistic Induction of Differentiation of AML Cells by the Vitamin D Analog Paricalcitol and Nrf2 Activators

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, mainly in older adults, characterized by uncontrolled growth of immature myeloid blasts. Despite initial responses to standard chemotherapy, prognosis remains grim for most patients. Differentiation therapy of AML is an alternative to cytotoxic chemotherapy. Natural and synthetic vitamin D derivatives (VDDs) are powerful inducers of monocytic differentiation of AML cells in culture; however, their differentiation-inducing concentrations can be lethal in vivo due to severe hypercalcemia. We have previously shown that fumaric acid esters (FAEs), such as the clinically approved drug dimethyl fumarate and its in-vivo metabolite monomethyl fumarate (MMF), can synergistically enhance the prodifferentiation effects of near-physiologic concentrations of different VDDs [PMID: 30508646]. Since FAEs are known activators of the transcription factor Nrf2, we hypothesized that Nrf2 may mediate the enhancing effects of these agents on the differentiation of AML cells induced by 19-nor-1,25-(OH)2-vitamin D2 (paricalcitol). Here, we demonstrate that in non-transfected and empty vector-transfected HL60 human AML cells, the differentiation-inducing effect of paricalcitol was markedly potentiated by MMF, monoethyl fumarate (MEF) or the Nrf2-activating phenolic diterpene carnosic acid (CA). This potentiation was associated with a marked upregulation of the vitamin D receptor (VDR) protein levels and mRNA expression of VDR target genes, e.g. CAMP and CYP24A1. However, these enhancing effects of the Nrf2 activators were dramatically reduced in HL60 cells stably expressing a dominant-negative Nrf2 (dnNrf2) mutant that lacks the transactivation domain. Notably, co-treatment of dnNrf2-expressing cells with the glutathione precursor N-acetyl cysteine or cell-permeant glutathione ethyl ester partially restored the synergy between paricalcitol and Nrf2 activators. These data suggest that the differentiation-enhancing effects of FAEs and CA are mediated by the transcriptionally active Nrf2, possibly through the Nrf2-dependent elevation of cellular glutathione levels.