Niclosamide Inhibits Proliferation of Leukemia Cells and Synergizes with Chemotherapy

Abstract

There have been significant advances in the treatment of acute childhood leukemia, which accounts for 25% of all childhood cancer cases. The 5-year survival rate for children with acute lymphoblastic leukemia (ALL) is 90%; however, there are few therapies available for relapsed disease. Pediatric acute myeloid leukemia (AML) has a 5-year survival rate of 65%, but current treatments are associated with significant morbidity and mortality. Less toxic and more effective therapies are needed to improve outcomes for patients with acute childhood leukemia. We previously showed that the cAMP-responsive element binding protein (CREB) plays an important role in leukemia cell proliferation and is overexpressed in blast cells from both ALL and AML patients. Niclosamide, an FDA-approved oral drug that has been used for decades to safely treat tapeworm infections, suppresses AML cell proliferation through inhibition of CREB activation and oxidative phosphorylation (Chae, et al. 2016). As a result of our preclinical studies, a Phase I clinical trial with niclosamide and cytarabine for relapsed/refractory pediatric AML is in progress. The effects of niclosamide in ALL cells or mechanisms of resistance to niclosamide have not been previously studied. The purpose of this study was to determine the antileukemic effects of niclosamide in ALL and to investigate additional synergistic effects of niclosamide in combination with drugs that are known to be effective against ALL or AML. To determine the effects of niclosamide on ALL proliferation, we treated two human B-ALL cell lines, Nalm6 and REH, and two human T-ALL cell lines, Jurkat and Loucy, with niclosamide alone or in combination with vincristine. Cells (2x104) were treated with 0.01-10µM niclosamide or 0.1% DMSO control. After 72 hours, CellTiter-Glo assays were performed to assess the concentration of drug that inhibited cell viability by 50% (IC50). The IC50 values for Nalm6 and REH were 0.45±0.13µM and 0.51±0.11µM, respectively (n=3). IC50 values for Jurkat and Loucy were 0.21±0.30µM and 0.58±0.11µM, respectively (n=3). Next, we investigated the potential synergistic effects between niclosamide and the chemotherapy drug, vincristine. The combination index (CI) values were calculated using the CompuSyn software (Chou-Talalay method). Simultaneous treatment of Nalm6 cells (ED75, 0.547; ED90, 0.316; ED95, 0.219; n=3) and Jurkat cells (ED75, 0.790; ED90, 0.686; ED95, 0.625; n=3) with niclosamide and vincristine had synergistic effects on cellular viability, indicating activity against both B-ALL and T-ALL. To study potential mechanisms of resistance to niclosamide, we performed a CRISPR/Cas9 library screen with HL60 human AML cells. Downregulation of BCL-2, an anti-apoptotic gene, sensitized cells to niclosamide treatment. Therefore, we investigated the effects of combining niclosamide with the BCL-2 inhibitor venetoclax in human AML cell lines, HL60 and U937, and in primary AML cells. CI values indicated synergy between niclosamide and venetoclax in HL60 cells (ED75, 0.251; ED90, 0.103; ED95, 0.063; n=5), U937 cells (ED75, 0.628; ED90, 0.614; ED95, 0.614; n=3), and patient sample 186 (ED75, 0.226; ED90, 0.301; ED95, 0.383; n=3). To investigate the effects of pretreatment with niclosamide, HL60 cells were cultured with niclosamide or vehicle for 3 days, followed by venetoclax for 3 days. Pretreatment with 300nM niclosamide significantly decreased the IC50 of venetoclax from 17.7±4.5nM to 1.87±0.46nM (n=3, p<0.05), indicating that niclosamide pretreatment sensitized HL60 cells to venetoclax. Our study established that both B-ALL and T-ALL cells are sensitive to niclosamide. Furthermore, niclosamide has synergistic effects when combined with standard chemotherapeutic agents such as vincristine and venetoclax. Our results demonstrate that niclosamide is a promising therapeutic agent for both AML and ALL and should be considered in future clinical trials.