Dimethyl fumarate and vitamin D derivatives cooperatively enhance VDR and Nrf2 signaling in differentiating AML cells in vitro and inhibit leukemia progression in a xenograft mouse model

•Distinct Nrf2 activators synergistically enhance differentiation of AML cells induced by vitamin D derivatives (VDDs).•This synergy is associated with enhancement of VDR and Nrf2 protein expression and transcriptional activity.•PRI-5202 (a 19-nor analog of 1,25D2) is 5-fold less toxic in mice than its direct precursor (PRI-1907) or 1,25D3.•PRI-5202 is more resistant than 1,25D2 and 1,25D3 to metabolic degradation by human CYP24 A1.•The Nrf2 activator dimethyl fumarate and PRI-5202 cooperate in inhibiting AML tumor growth in mice without toxicity. Acute myeloid leukemia (AML) is one of the deadliest hematological malignancies without effective treatment for most patients. Vitamin D derivatives (VDDs) - active metabolites 1α,25-dihydroxyvitamin D2 (1,25D2) and 1α,25-dihydroxyvitamin D3 (1,25D3) and their analogs - are differentiation-inducing agents which have potential for the therapy of AML. However, calcemic toxicity of VDDs limits their clinical use at doses effective against cancer cells in vivo. Here, we demonstrate that in AML cell cultures, moderate pro-differentiation effects of low concentrations of VDDs can be synergistically enhanced by structurally distinct compounds known to activate the transcription factor Nuclear Factor (Erythroid-derived 2)-Like 2 (NFE2L2 or Nrf2). Particularly, dimethyl fumarate (DMF), which is clinically approved for the treatment of multiple sclerosis and psoriasis, strongly cooperated with 1,25D3, PRI-5100 (19-nor-1,25D2; paricalcitol) and PRI-5202 (a double-point modified 19-nor analog of 1,25D2). The pro-differentiation synergy between VDDs (1,25D3 or PRI-5202) and Nrf2 activators (DMF, tert-butylhydroquinone or carnosic acid) was associated with a cooperative upregulation of the protein levels of the vitamin D receptor (VDR) and Nrf2 as well as increased mRNA expression of their respective target genes. These data support the notion that VDDs and Nrf2 activators synergize in inducing myeloid cell differentiation through the cooperative activation of the VDR and Nrf2/antioxidant response element signaling pathways. We have previously reported that PRI-5202 is more potent by approximately two orders of magnitude than 1,25D3 as a differentiation inducer in AML cell lines. In this study, we found that PRI-5202 was also at least 5-fold less calcemic in healthy mice compared to both its direct precursor PRI-1907 and 1,25D3. In addition, PRI-5202 was remarkably more resistant against degradation by the human 25-hydroxyvita