Nrf2 Regulates Sensitivity of Leukemias to Artemisinins

The artemisinins are a class of drugs widely used to treat severe malaria due to their potency and few adverse effects. In addition to antimalarial activity, artemisinins have been shown to have potent anti-cancer activity, specifically in leukemia. Therefore, artesunate (AS), the WHO-recommended treatment for severe malaria, and a new dimeric artemisinin derivative (ART-838) are promising agents to repurpose for leukemia treatment. Reactive oxygen species (ROS) have been implicated in the mechanism of artemisinin activity in both malaria and cancer; however, the full mechanisms of artemisinin activity in cancer have not yet been elucidated. In 2014, Ariey et al, published a paper on malaria identifying the Kelch (K13) propeller domain as a molecular marker for artemisinin resistance. Since Keap1 ̶ the human homolog of K13 ̶ is involved in ROS regulation, it may also play a role in artemisinin action in cancer. To investigate the role of Keap1-Nrf2 signaling cascades in mediating artemisinin action and resistance in leukemia, qPCRs and Western blots were performed to quantify endogenous levels of selected Keap1 pathway molecules (Keap1, Nrf2, Nqo1, and Gsta1) in 23 human acute leukemia cell lines. However no significant correlation was observed between the levels of these molecules (mRNA or protein) and AS/ART-838 sensitivity. Using an antioxidant response element (ARE) luciferase reporter that can be activated by Nrf2, a significant increase in ARE activity was observed in K562 cells upon AS or ART-838 treatment. In addition to increased transcription upon drug treatment, the ARE downstream proteins Nqo1 and Gsta1 were upregulated upon AS or ART-838 treatment. K562 cell lines with Keap1 and Nrf2 stably knocked down (KD) were generated. The Nrf2 KD K562 cell line had increased sensitivity to AS and ART-838. In contrast, KD of Keap1 did not result in a substantial change in drug sensitivity to either artemisinin. In conclusion, these studies suggest involvement of Nrf2 in human leukemia cell resistance to artemisinins. Further studies on Nrf2 may help us better understand the molecular actions of the artemisinins in order to optimize their efficacy as single agents or in drug combinations for leukemia treatment.