Pb1757: novel therapeutic strategy targeting cellular redox systems using dimethyl fumarate and venetoclax in acute myeloid leukemia

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Dimethyl fumarate (DMF) has been shown to induce apoptosis of lymphocytes, and thus used for treating relapsing-remitting multiple sclerosis and psoriasis. It is of interest that this drug also has a potent anti-tumor activity against a range of cancer cells. To date, glycolytic Inhibition and accumulation of oxidative stress through glutathione (GSH) oxidation have been proposed as potential mechanisms, but less data is available about leukemia cells. The Keap1-Nrf2 pathway is a major elimination system of reactive oxygen species (ROS) other than GSH-glutathione disulfide (GSSG) redox balance. Intracellular ROS induces nuclear translocation of Nrf2, which can subsequently bind to antioxidant response elements (ARE), and in turn promotes the expression of antioxidant genes. Previous studies indicate that Nrf2 inhibition can downregulate antioxidant gene levels, thereby leading to oxidative stress-derived apoptosis of cancer cells. Interestingly, a novel Bcl-2 inhibitor venetoclax (VEN) appeared to inhibit the Keap1-Nrf2 pathway by dissociating BCL-2/Nrf-2 interaction and promoting Nrf2 ubiquitination. This led us to hypothesize that DMF and VEN may have a synergistic anti-tumor effect against leukemia cells by dual inhibition of cellular redox systems. Aims: The first aim of this study is to examine the anti-leukemic effect of DMF in terms of oxidative stress and cellular metabolism. The second aim is to investigate the synergistic effect of DMF in combination with VEN against leukemia cells. Methods: Human acute myeloid leukemia cell lines (MOLM-14 and U937) were used to investigate the anti-leukemia effects of DMF in combination with VEN. Intracellular GSH and GSSG levels were quantified by luminescence-based assay. ROS accumulation and apoptosis rate were measured by flow cytometry. Nuclear translocation of Nrf2 in leukemia cells was determined by Western blotting. Quantitative real-time PCR was used to determine the expression levels of antioxidant genes. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured by Seahorse XFe96 analyzer. Results: Consistent with previous studies on cancer cells, DMF decreased the GSH/GSSG ratio in both MOLM-14 and U-937 cells. The ROS accumulation and cellular apoptosis rate were significantly increased by DMF treatment in a time- and dose-dependent manner. After N-acetylcysteine (NAC) treatment, GSH/GSSG ratio, ROS levels, and cell viabilities were all reverted to the basal level. The Western blotting revealed that DMF promotes nuclear translocation of Nrf2, suggesting a compensatory response to excess oxidative stress in leukemia cells. In fact, the mRNA expression levels of HO-1 and NQO-1 were upregulated, whereas they were almost completely restored by NAC or specific Nrf2 inhibitor ML385. DMF increased the ECAR in a dose-dependent manner, but decreased the coupled OCR levels, suggesting metabolic reprogramming to attenuate mitochondrial ROS generation. As has been shown in cancer cells, VEN hampered nuclear translocation of Nrf2 in leukemia cells to the same extent of ML385. VEN did not affect ROS levels when used as single agents, but significantly enhanced DMF-mediated oxidative stress. Furthermore, this combination induced more apoptosis of leukemia cells than did single agents. Summary/Conclusion: Our data demonstrate that DMF in combination with VEN can promote significant apoptosis of leukemia cells by inhibiting GSH activity and Nrf2-dependent anti-oxidant response. DMF/VEN may have the potential to become a novel therapeutic alternative to conventional VEN-based regimens.Keywords: Reactive oxygen species, Venetoclax, Acute myeloid leukemia, Antioxidants