Activation of Chloride Channels with the Anti-Parasitic Agent Ivermectin Induces Membrane Hyperpolarization and Cell Death in Leukemia Cells.
Authors: Sharmeen Sumaiya , Skrtic Marko , Sukhai Mahadeo , Sun Joyce , Hurren Rose , Gronda Marcela , Wang Xiaoming , Wood Tabitha E , Batey Robert A. , Fonesca Sonali , Kelley Shana , Datti Alessandro , Wrana Jeff , Schimmer Aaron D. 
Published: October 2019

Abstract
Abstract 410
FDA-approved drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication. To identify such compounds, we compiled a library of known drugs with high maximal tolerated doses and well-known toxicity profiles. We screened this library in a dose-response manner in 4 leukemia cell lines to identify cytotoxic compounds as measured by the MTS assay. From these screens, we identified the anti-parasitic agent ivermectin (IVM) that induced cell death at low micromolar concentrations in all four leukemia lines tested. IVM is a derivative of avermecin B1 and licensed for the treatment of strongyloidiasis and onchocerciasis parasitic infections, but also effective against other worm infestations (e.g., ascariasis, trichuriasis and enterobiasis). To evaluate the effects of IVM as a potential anti-cancer agent, leukemia and myeloma (n = 9) cell lines were treated with increasing concentrations of IVM. 72 hours after incubation, cell viability was determined by the MTS assay. IVM decreased the viability of 3/5 leukemia cell lines with an LD50 < 5uM and all other tested malignant cell lines with an LD50 < 10uM. Cell death was confirmed by Trypan blue staining and Annexin V staining. In clonogenic growth assays, 6uM IVM reduced clonogenic growth by ≥ 40% in 3/6 primary AML samples, but <15% in 3/3 samples of normal hematopoietic mononuclear cells. Given the effects of IVM in cell lines, we evaluated the drug in mouse models of leukemia. Here, K562, OCI-AML2, and MDAY-D2 leukemia cells were implanted subcutaneously into the flanks of sublethally irradiated NOD/SCID mice. One week after implantation, when the tumors were palpable, mice were treated with IVM at 3mg/kg/day by oral gavage or buffer control. Compared to control, IVM decreased tumor volume and weight in all 3 xenograft models by up to 72% without observable toxicity. Of note, a dose of 3mg/kg in mice translates to a dose of 0.24 mg/kg in humans based on scaling of body weight and surface area. This dose is readily achievable in human as patients routinely receive 0.2mg/kg for the treatment of parasitic disease and overdoses of up to 6g were not toxic. As an anti-parasitic, IVM binds and activates chloride ion channels, so we tested the effects of IVM on chloride flux in leukemia cells. OCI AML2 cells were treated with increasing concentrations of IVM and changes in intracellular chloride were measured using the fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium and flow cytometry. IVM increased intracellular chloride ion concentrations within 30 minutes of treatment. Chloride influx was accompanied by plasma membrane hyperpolarization within 1 hr of treatment, but no change in mitochondrial membrane potential was noted up to 24 hours after treatment. Plasma membrane hyperpolarization was dependent on chloride influx, as treatment with IVM in chloride-free media did not induce membrane hyperpolarization. Alternations in intracellular chloride and membrane hyperpolarization can lead to increased reactive oxygen species (ROS) generation. Therefore, we measured changes in ROS after treatment with IVM. IVM (6uM) increased ROS generation in OCI-AML2 cells up to 2 ± 0.2 fold within 4 hours of treatment. Increased ROS generation appeared functionally important for IVM-induced cell death as pre-treatment with the antioxidant N-acetyl-L-cysteine (NAC) inhibited IVM-induced cell death. Further supporting a mechanism of cell death related to increased ROS, IVM treatment increased expression of STAT1, IFIT3, OAS1, and TRIM22, members of the STAT1 signaling pathway that are known to be upregulated upon ROS generation. Cytarabine and daunorubicin are used in the treatment of AML and increase ROS production through mechanisms related to DNA damage. Therefore, we evaluated the combination of IVM and cytarabine and daunorubicin. By isobologram analysis, IVM synergized with cyatarabine (CI=0.51, 0.58, 0.65 at ED25, ED50, ED75, respectively) and daunorubicin (CI=0.48, 0.51, 0.54 at ED25, ED50, ED75, respectively). Thus, in summary, IVM activates chloride channels in leukemia cells leading to membrane hyperpolarization and increased ROS generation. In addition, it demonstrated preclinical activity in this disease at pharmacologically achievable concentrations. Therefore, IVM could be rapidly repurposed for the treatment of leukemia and highlights a potential new therapeutic strategy for this disease.

Disclosures:
Off Label Use: Ivermectin is an antiparasitic agent, licensed for the treatment of strongyloidiasis and onchocerciasis parasitic infections, but also effective against other worm infestations (e.g., ascariasis, trichuriasis and enterobiasis).