Auranofin induces lethal oxidative and endoplasmic reticulum stress and exerts potent preclinical activity against chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) exhibits high remission rates after initial chemoimmunotherapy, but with relapses with treatment, refractory disease is the most common outcome, especially in CLL with the deletion of chromosome 11q or 17p. In addressing the need of treatments for relapsed disease, we report the identification of an existing U.S. Food and Drug Administration-approved small-molecule drug to repurpose for CLL treatment. Auranofin (Ridaura) is approved for use in treating rheumatoid arthritis, but it exhibited preclinical efficacy in CLL cells. By inhibiting thioredoxin reductase activity and increasing intracellular reactive oxygen species levels, auranofin induced a lethal endoplasmic reticulum stress response in cultured and primary CLL cells. In addition, auranofin displayed synergistic lethality with heme oxygenase-1 and glutamate-cysteine ligase inhibitors against CLL cells. Auranofin overcame apoptosis resistance mediated by protective stromal cells, and it also killed primary CLL cells with deletion of chromosome 11q or 17p. In TCL-1 transgenic mice, an in vivo model of CLL, auranofin treatment markedly reduced tumor cell burden and improved mouse survival. Our results provide a rationale to reposition the approved drug auranofin for clinical evaluation in the therapy of CLL.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

©2014 AACR.

Figures

Figure 1

Treatment with auranofin (AF) induces apoptosis of primary CLL CD19+ but not CD3+ cells or normal CD19+ or CD34+ cells. A, cell viability of primary CLL (n = 50) following treatment with auranofin for 24 hours. Columns, mean loss of viability; bars, SEM. *, cell viability values significantly less in auranofin-treated cells than untreated cells (P < 0.05). B, percentage of apoptotic normal CD34+ cord blood cells (n = 3) and primary CLL cells (n = 5) following exposure to 1.0 µmol/L of auranofin for 48 hours. Columns, mean apoptosis from the CD34+ cord blood cells or primary CLL cells; bars, SEM. C, schematic of the flow cytometry gating method used to determine the percentage of apoptotic CD3+ or CD19+ cells following treatment with 1.0 µmol/L of auranofin for 24 hours. D, individual reduction in viability (relative to untreated control cells) of CD3+ and CD19+ cells from patients with CLL treated with 1.0 µmol/L of auranofin for 24 hours. E, the % reduction in viability induced by 24-hour treatment with 1 µmol/L of auranofin in CD3+ and CD19+ cells from patients with CLL (n = 8). The horizontal black bar indicates the median reduction in cell viability ± SE. *, significantly greater reduction in viability of auranofin-treated CD19+ cells compared with CD3+ cells (P < 0.05).

Figure 2

Treatment with auranofin (AF) selectively induces apoptosis of cultured and primary CLL CD19+ cells independent of cytogenetics or IGHV mutation status, despite coculture with NLCs and reduced in vivo leukemia burden. A, IC50 values for the primary CLL patient cells treated with auranofin for 24 hours subgrouped by IGHV mutational (U, unmutated; M, mutated) status. Black line, median IC50 value. B, IC50 values for primary CLL patient cells treated with auranofin for 24 hours subgrouped by ZAP70 status. Black line, median IC50 value. C, cell viability of primary CLL cells from patients subgrouped by ZAP70 status (ZAP70+n = 12; ZAP70−n = 15) following treatment with the indicated concentrations of auranofin for 24 hours. Each point on the line represents the mean viability of the CLL cells at each concentration of auranofin; bars, SEM. *, cell viability values significantly less in ZAP70+ auranofin-treated cells than ZAP70− auranofin-treated cells (P < 0.05). D, percent apoptosis of MEC-1 cells and CD19+ primary CLL cells (11q deleted, 13q deleted, and 17p deleted) exposed to the indicated doses of auranofin for 48 hours. *, apoptosis values significantly greater in 17p-deleted CLL than in 11q- and 13q-deleted CLL cells at 0.25 µmol/L of auranofin (P < 0.05). **, apoptosis values significantly greater in 17p-deleted CLL than in 11q-and 13q-deleted CLL cells at 0.5 µmol/L of auranofin (P < 0.05). E, the percentage of sub-G1 cells and percentage of Annexin V-positive, apoptotic MEC-1 cells following 24-hour exposure to auranofin. Immunoblot analyses were also conducted as indicated. F, IC50 values for the primary CLL patient cells treated with auranofin for 24 hours, subgrouped by adverse (ADV) or favorable (FAV) cytogenetics. Black line, median IC50 value. G, percent apoptosis of MEC-1 cells with and without coculture on HK cells following treatment with auranofin for 48 hours. *, values significantly less in MEC-1 cells cocultured with HK cells compared with MEC-1 without coculture (P < 0.05). H, primary CLL cells (n = 4) were cultured with or without NLCs and exposed to two times the IC50 of auranofin for 24 hours. At the end of treatment, the percentage of nonviable cells was determined by flow cytometry. The absolute viability of the CLL cells in each condition is shown. I, treatment of TCL-1 mice (n = 5) with auranofin (10 mg/kg, 5 days per week for 2 weeks) significantly reduced the leukemia cell burden. Columns represent the mean ± SEM of the leukemia cell burden in all 5 mice tested. J, Kaplan-Meier survival plot of TCL-1 mice treated with auranofin compared with untreated mice. (P = 0.001, log-rank (Mantel-Cox) test.

Figure 3

Treatment with auranofin (AF) induces an oxidative stress response in cultured and primary CLL cells. A, the percent induction of ROS and the percent inhibition of TRR activity in MEC-1 and CD19+ primary CLL cells exposed to 1.0 µmol/L of auranofin for 1 or 8 hours, respectively. B, percentage increase of ROS in MEC-1 cells exposed to auranofin with or without 1 mmol/L of NAC for one hour. Columns, mean of three independent experiments; bars, SEM. C, localization of Nrf2 in primary CLL cells treated with auranofin for 6 hours. Cells were also stained with phalloidin and DAPI. D, immunoblot analyses of MEC-1 cells and CD19+ primary CLL cells treated with auranofin for 16 hours. E, immunoblot analyses of CD19+ primary CLL cells treated with auranofin and NAC for 24 hours. F, induction of ROS, loss of mitochondrial membrane potential, and apoptosis of primary CLL cells (n = 8) treated with auranofin as indicated. Values represent the percent induction corrected for the untreated, control cells. G, percent apoptosis of CD19+ primary CLL cells following treatment with auranofin and/or NAC for 48 hours. *, apoptosis values significantly less in cells treated with auranofin and NAC than those treated with auranofin alone (P = 0.00062). H, percentage of sub-G1 and Annexin V-positive MEC-1 cells following treatment with auranofin and/or NAC for 24 hours. *, values significantly less in cells treated with auranofin and NAC than those treated with auranofin alone (P < 0.001). Immunoblot analyses were also conducted as indicated.

Figure 4

mRNA expression profiling of auranofin (AF)-treated primary CLL cells. A, heatmap representing the relative expression of 98 probe sets (81 genes) in CLL cells from two patients treated with 1.0 µmol/L of auranofin for 4 and 10 hours in vitro compared with untreated cells (>2-fold change; P < 0.01 by ANOVA). Patient samples are arranged in columns. Gene symbols highlight select genes. Gene expression is median centered and scaled as indicated. B, graphical representation of the top thirty-nine up- and downregulated genes in primary CLL cells following treatment with 1.0 µmol/L of auranofin for 8 hours compared with untreated control cells. C, relative mRNA expression of HMOX-1, GCLM, and CHOP in primary CLL cells treated with 1.0 µmol/L of auranofin for 8 hours compared with untreated control cells. D, relative mRNA expression of HMOX-1, GCLM, and CHOP in 17p-deleted primary CLL cells treated with 1.0 µmol/L of auranofin for 8 hours compared with untreated control cells.

Figure 5

Inhibition of HMOX-1 by shRNA or cotreatment with auranofin (AF) and ZnBG or BSO synergistically induces apoptosis of cultured and primary CLL cells. A, immunoblot analyses of MEC-1 cells transduced with nontargeted shRNA(sh-NT) or sh-HMOX1 for 48 hours, then treated with auranofin for 8 hours. B, percent apoptosis of sh-NT and sh-HMOX-1-transduced MEC-1 cells following treatment with auranofin for 48 hours. Columns, mean of three independent experiments; bars, SEM. *, apoptosis values significantly greater in sh-HMOX1 -transduced cells compared with sh-NT-transduced cells (P < 0.001). C, MEC-1 cells (black line) and primary CLL cells (gray line) were exposed to of auranofin (100–500 nmol/L) and ZnBG (10–20 µmol/L) for 48 hours. After treatment, the percentage of apoptotic cells was determined by flow cytometry. Median dose effect and isobologram analyses were performed utilizing Calcusyn software. CI values less than 1.0 indicate a synergistic interaction between the two agents. D, MEC-1 cells (black line) and primary CLL cells (gray line) were treated with auranofin (10–250 nmol/L) and BSO (1–10 µmol/L) for 48 hours. Following this, the percentage of apoptotic cells was determined by flow cytometry. Isobologram analyses were performed as described above.

Figure 6

Treatment with auranofin (AF) induces ER stress in CLL cells. A, fold induction of GRP78 and CHOP mRNA in primary CLL (n = 4) treated with 1.0 µmol/L of auranofin for 8 hours is presented. B, Western blot analyses of CHOP and GRP78 in primary CLL cells treated with auranofin for 24 hours (top). Ratio of induction of CHOP to GRP78 in the primary CLL cells (bottom). C, relative mRNA expression of ATF4 and CHOP in MEC-1 and primary CLL cells following treatment auranofin and/or NAC for 8 hours. D, immunoblot analyses of GRP78, CHOP, and (3-actin in the cell lysates from CD19+ primary CLL cells treated with auranofin and/or NAC for 24 hours. E, immunoblot analyses of MEC-1 cells following treatment with auranofin and/or NAC for 24 hours. F, relative CHOP mRNA expression 48 hours posttransduction in nontargeted (sh-NT) or CHOP shRNA-transduced MEC-1 cells (top). Western blot analysis of CHOP and (3-actin in MEC-1 cells before auranofin treatment (bottom). G, percent apoptosis of stably transfected sh-NT or sh-CHOP#2 MEC-1 cells treated with 0.25 µmol/L of auranofin for 48 hours. *, apoptosis values significantly less in MEC-1 sh-CHOP cells treated with auranofin compared with sh-NT cells (P < 0.02).

Figure 7

Treatment with auranofin (AF) inhibits hsp90 chaperone function and induces a heat shock response in CLL cells. A, representative immunoblot analyses of polyubiquitinated proteins (poly Ub) and β-actin in the cell lysates from three primary CLL samples treated with auranofin alone (1.0 µmol/L) or auranofin and NAC (2.5 mmol/L) for 24 hours. B, immunoblot analysis of HDAC6, HSF1, and p97 expression in hsp90 immunoprecipitates following treatment with 1.0 µmol/L of auranofin for 8 hours. The blot was stripped and reprobed for hsp90. C, representative immunoblot analysis of HDAC6 expression from primary CLL cells (n = 10) exposed to 1.0 µmol/L of auranofin for 16 hours. The values in the graph underneath the blot represent quantitation of the levels of HDAC6 (mean ± SE) relative to the untreated control (arbitrarily set as 1.0) as determined by densitometry analysis. The right panel shows the levels of acetylated hsp90 using anti-acetyl-K69 hsp90 antibody, following exposure of a representative primary CLL sample to 1.0 µmol/L of auranofin for 16 hours. D, localization of HSF1 in primary CLL cells exposed to auranofin for 6 hours. A representative mage is shown. E, immunoblot analyses of hsp40, hsp27, hsp70, and β-actin in the cell lysates from primary CLL cells exposed to auranofin for 16 hours. F, immunoblot analyses of ZAP70, c-RAF, AKT, and β-actin in the cell lysates from primary CLL cells exposed to auranofin for 16 hours. G, mmunoblot analyses of c-RAF, ZAP70, HDAC6, and β-actin in the lysates from primary CLL cells treated with 1.0 µmol/L of auranofin and/or 20 nmol/L of carfilzomib (CZ) for 8 hours. The numbers underneath the bands represent densitometry analysis performed on representative immunoblot analyses.