Ref-1/APE1 as a Transcriptional Regulator and Novel Therapeutic Target in Pediatric T-cell Leukemia

Abstract

The increasing characterization of childhood acute lymphoblastic leukemia (ALL) has led to the identification of multiple molecular targets, but have yet to translate into more effective targeted therapies, particularly for high-risk, relapsed T-cell ALL. Searching for master regulators controlling multiple signaling pathways in T-ALL, we investigated the multi-functional protein redox factor-1 (Ref-1/APE1), which acts as a signaling “node” by exerting redox regulatory control of transcription factors important in leukemia. Leukemia patients’ transcriptome databases showed increased expression in T-ALL of Ref-1 and other genes of the Ref-1/SET interactome. Validation studies demonstrated that Ref-1 is expressed in high-risk leukemia T-cells, including in patient biopsies. Ref-1 redox function is active in leukemia T-cells, regulating the Ref-1 target NF-κB, and inhibited by the redox-selective Ref-1 inhibitor E3330. Ref-1 expression is not regulated by Notch signaling, but is upregulated by glucocorticoid treatment. E3330 disrupted Ref-1 redox activity in functional studies and resulted in marked inhibition of leukemia cell viability, including T-ALL lines representing different genotypes and risk groups. Potent leukemia cell inhibition was seen in primary cells from ALL patients, relapsed and glucocorticoid-resistant T-ALL cells, and cells from a murine model of Notch-induced leukemia. Ref-1 redox inhibition triggered leukemia cell apoptosis and down-regulation of survival genes regulated by Ref-1 targets. For the first time, this work identifies Ref-1 as a novel molecular effector in T-ALL and demonstrates that Ref-1 redox inhibition results in potent inhibition of leukemia T-cells, including relapsed T-ALL. These data also support E3330 as a specific Ref-1 small molecule inhibitor for leukemia.

Physiological regulation of reactive oxygen species in organisms based on their physicochemical properties

“…ROS can directly or indirectly regulate the activity of redox factor‐1 (Ref‐1) by targeting Cys‐65 of Ref‐1 and target active activator protein‐1 (AP‐1), p53, nuclear factor‐kappa beta (NF‐κβ), hypoxia‐inducible factor (HIF‐1α), Nrf2 and other TFs. These activated TFs further delicately manipulate the “on” and “off” switches of target genes . Various human bZIP TFs have been demonstrated to be regulated in a redox‐dependent manner by Ref‐1 …”

Cleavage of the APE1 N-Terminal Domain in Acute Myeloid Leukemia Cells Is Associated with Proteasomal Activity

“…The pursuit of potential multi-pathway regulators in acute myeloid leukemia has led to the identification of APE1 as a potential therapeutic target [13,28]. Apart from its crucial role as a DNA-repair enzyme, APE1 is a multifunctional protein acting as a unique nuclear redox factor [29].…”

“…A peptide substrate was designed as corresponding to the portion (26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) present within the APE1 N-terminal region, having a FRET couple at N-[DABCYL, 4-(4-dimethylaminophenylazo) benzoic acid] and C-[EDANS, 5-(2-aminoethyl) aminonaphthalene-1-sulfonic acid] terminal ends. The peptide was synthesized and purified as already reported [16].…”

APE1 Upregulates MMP-14 via Redox-Sensitive ARF6-Mediated Recycling to Promote Cell Invasion of Esophageal Adenocarcinoma

“…E3330 (APE1 redox-specific inhibitor) was purchased from Novus Biologicals, and APE1-i3 (APE1 DNA repair-specific inhibitor) was purchased from Milli-poreSigma. The usage of inhibitors were following pharmacologic studies with recommended doses for the E3330 (25)(26)(27) and APE1-i3 (28). Transfection reagents (Polyjet and Lipojet) were obtained from SignaGen Laboratories.…”