Redeployment-based drug screening identifies the anti-helminthic niclosamide as anti-myeloma therapy that also reduces free light chain production

Abstract

Despite recent therapeutic advancements, multiple myeloma (MM) remains incurable and new therapies are needed, especially for the treatment of elderly and relapsed/refractory patients. We have screened a panel of 100 off-patent licensed oral drugs for anti-myeloma activity and identified niclosamide, an anti-helminthic. Niclosamide, at clinically achievable non-toxic concentrations, killed MM cell lines and primary MM cells as efficiently as or better than standard chemotherapy and existing anti-myeloma drugs individually or in combinations, with little impact on normal donor cells. Cell death was associated with markers of both apoptosis and autophagy. Importantly, niclosamide rapidly reduced free light chain (FLC) production by MM cell lines and primary MM. FLCs are a major cause of renal impairment in MM patients and light chain amyloid and FLC reduction is associated with reversal of tissue damage. Our data indicate that niclosamides anti-MM activity was mediated through the mitochondria with rapid loss of mitochondrial membrane potential, uncoupling of oxidative phosphorylation and production of mitochondrial superoxide. Niclosamide also modulated the nuclear factor-κB and STAT3 pathways in MM cells. In conclusion, our data indicate that MM cells can be selectively targeted using niclosamide while also reducing FLC secretion. Importantly, niclosamide is widely used at these concentrations with minimal toxicity.

Figure 1

Niclosamide has potent anti-myeloma activity at peak serum concentrations. (a) H929, JJN3 and U266 cells were treated with peak serum concentrations of a library of 100 drugs for 72 h. Viability was assessed using CellTiter Blue (Promega) and calculated as percentage of control. Myeloma cell line responses to the commonly used anti-myeloma therapeutics prednisolone (71.6 μ), oral methotrexate (1 μ), dexamethasone (0.43 μ), cyclophosphamide (200 μ), chlorambucil (1.62 μ), thalidomide (10.9 μ) and the anti-helminthic niclosamide (3.2 μ) are shown. Data are the mean of three experiments±s.e.m. (b) A panel of eight myeloma cell lines were treated for 72 h with two currently used anti-myeloma combinations MPT (melphalan 10 μ/prednisolone 71.6 μ/thalidomide 10.9 μ) or CTD (cyclophosphamide 200 μ/thalidomide 10.9 μ/dexamethasone 0.43 μ), and niclosamide at 3.2 μ. Viability was assessed using CellTiter Blue (Promega) and calculated as percentage of control. Data are the mean of a minimum of three experiments±s.e.m. Primary myeloma bone marrow samples (MM1-4) were also treated as for the cell lines but for 48 h. Viability was assessed by CD138/38 and annexin V staining and flow cytometry. (c) H929, JJJN3 and U266 cells were treated with different doses of niclosamide at different timepoints and viability assessed by flow cytometry using viable cells gates and Cytocount beads. Data are the mean of three experiments±s.e.m.; ^*P<0.05.

Figure 2

Niclosamide-treated myeloma cells die with markers of apoptosis and autophagy. H929, JJN3 and U226 were treated with different concentrations of niclosamide (Nic) for 48 h. Cells were analysed by flow cytometry for (a) annexin V/propidium iodide positivity (b) and caspase activity using Caspatag assay according to manufacturer's instructions. Data shown are the mean of three experiments±s.e.m. Representative dotplots are shown for each assay; ^*P<0.05. (c) Cell cycle was analysed by staining cells with propidium iodide and flow cytometry after treatment with Nic for 24 h. Representative histograms are shown for H929 cells. (d) Representative images are shown of Jenner/Giemsa-stained cytospins from H929 and JJN3 cells treated with solvent control or Nic for 48 h. (e) A volume of 30 μg total protein from JJN3 cells treated with Nic for 24 h was immunoblotted for LC3-II protein. Image shown is representative of n=2 experiments.

Figure 3

Niclosamide reduces FLC from myeloma cell lines and primary MM samples. (a) MM cell lines were treated in 96-well plates for 18 h with either solvent control, 0.5–1.0 μ niclosamide or 5 μ dexamethasone. Supernatants were harvested and FLC measured using FreeLite assay. FLC secretion was normalised for cell viability (data not shown). Nm=not measured as there was significant loss of viability. Data shown are the mean of n=3 experiments±s.e.m. (b) JJN3 and L363 cells were treated for 18 h with either solvent control or 1.0 μ niclosamide. Cells were washed, fixed and permeabilised and intracellular Ig detected using indirect staining and flow cytometry. Isotype matched control antibodies were used to control for non-specific binding. Data shown are the mean of n=3 experiments±s.e.m. (c) Ig mRNA levels were measured after 18 h treatment using quantitative real time PCR and normalised to 18S internal standards. Data shown are the mean of n=4 experiments±s.e.m.; P<0.05. (d) Primary MM bone marrow-derived total mononuclear cells were treated in 96-well plates for 18 h with MPT (melphalan 10 μ/prednisolone 71.6 μ/thalidomide 10.9 μ) or CTD (cyclophosphamide 200 μ/thalidomide 10.9 μ/dexamethasone 0.43 μ), or niclosamide at 1 μ. The supernatant was harvested and FLC levels measured using Luminex analysis. ^*P<0.05.

Figure 4

Niclosamide treatment results in loss of mitochondrial membrane potential (Δψ) and an increase in respiration rate. (a) Myeloma cells treated with niclosamide for 4 h were stained with JC-1 and analysed by flow cytometry. Red is indicative of cells with polarised mitochondrial membranes. Green staining indicates cells that have lost Δψ. Data shown is representative of n=3 experiments. (b, c) H929 and JJN3 cells were prestained with 10 n TMRE and baseline fluorescence intensity (geometric mean GM) determined by flow cytometry. Niclosamide (3.2 μ) was added and readings taken at 15, 30 s and every minute for 5 min. Representative histograms (b) are shown together with mean data (c) from n=4 experiments±s.e.m. (d) Respiration rate in 5 × 10⁷ myeloma cells was measured using a Clark oxygen electrode. Baseline respiration rate was established as μmol O₂/min/10⁶ cells before the addition of either 3.2 μ niclosamide or 6.7 μ p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP). Representative plots are shown together with a histogram of mean data from a minimum of n=4 experiments±s.e.m.; ^*P<0.05.

Figure 5

Generation of mitochondrial superoxide correlates with niclosamide anti-myeloma activity. (a) JJN3 and H929 cells were stained with MitoSOX Red or carboxy H₂DCFDA (Molecular Probes) and analysed by flow cytometry. Baseline was set before the addition of 3.2 μ niclosamide and measurements taken at regular time intervals. Histograms show representative MitoSOX Red data and line graphs show mean data from n=3 experiments±s.e.m. for both MitoSOX Red and ca-H₂DCFDA for JJN3 and H929 cells. (b) Myeloma cell lines H929, JJN3 and U266 were treated with different doses of niclosamide for 18, 42 or 66 h. Aliquots of cells were removed, stained with MitoSOX Red or carboxy H₂DCFDA and analysed by flow cytometry. Data are shown as intensity of fluorescence (geometric mean (GM)) relative to solvent-treated controls and is the mean of n=3 experiments±s.e.m. (c) Viability was also assessed at 18, 42 and 66 h by flow cytometry of fixed cells using viable gates and Cytocount beads and is plotted against relative MitoSOX Red fluorescence intensity to demonstrate the relationship between niclosamide dose, viability and mitochondrial superoxide generation. ^*P<0.05.

Figure 6

Niclosamide inhibits both the NF-κB and STAT3 pathways in myeloma cells. (a) UM3 cells were treated with either solvent control or 1 μ niclosamide before the addition of 100 ng/ml TNFα for 15 min. Cytospins were prepared, fixed with paraformaldehyde, stained for NF-κB p65 (red) and counterstained with Hoechst 33342 for DNA. Images are representative of triplicate experiments. (b) JJN3 cells were treated overnight with either solvent control, 1 μ niclosamide and/or 10 ng/ml TNFα. RNA was extracted from cells and Igκ levels measured by quantitative real-time PCR. Data shown are mean from n=4 experiments±s.e.m. (c) Supernatant was also harvested from JJN3 cells and secreted FLC protein levels measured by Luminex. Data shown are mean from n=4 experiments±s.e.m.; ^*P<0.05. (d) JJN3 cells were pretreated with niclosamide at either 1 or 2 μ for 2 h before the addition of 1 μg/ml interleukin 6 (IL6) for 15 min. Total protein was extracted from cells and immunoblotted for total STAT3 protein, Y705 phosphorylated STAT3 with β-actin as a loading control. Images are representative of n=3 experiments.