Antitumor effects of chloroquine/hydroxychloroquine mediated by inhibition of the NF-κB signaling pathway through abrogation of autophagic p47 degradation in adult T-cell leukemia/lymphoma cells

Abstract

Adult T-cell leukemia/lymphoma (ATLL) originates from human T-cell leukemia virus type 1 (HTLV-1) infection due to the activation of the nuclear factor-κB (NF-κB) signaling pathway to maintain proliferation and survival. An important mechanism of the activated NF-κB signaling pathway in ATLL is the activation of the macroautophagy (herafter referred to as autophagy in the remainder of this manuscript)-lysosomal degradation of p47 (NSFL1C), a negative regulator of the NF-κB pathway. Therefore, we considered the use of chloroquine (CQ) or hydroxychloroquine (HCQ) (CQ/HCQ) as an autophagy inhibitor to treat ATLL; these drugs were originally approved by the FDA as antimalarial drugs and have recently been used to treat autoimmune diseases, such as systemic lupus erythematosus (SLE). In this paper, we determined the therapeutic efficacy of CQ/HCQ, as NF-κB inhibitors, in ATLL mediated by blockade of p47 degradation. Administration of CQ/HCQ to ATLL cell lines and primary ATLL cells induced cell growth inhibition in a dose-dependent manner, and the majority of cells underwent apoptosis after CQ administration. As to the molecular mechanism, autophagy was inhibited in CQ-treated ATLL cells, and activation of the NF-κB pathway was suppressed with the restoration of the p47 level. When the antitumor effect of CQ/HCQ was examined using immunodeficient mice transplanted with ATLL cell lines, CQ/HCQ significantly suppressed tumor growth and improved the survival rate in the ATLL xenograft mouse model. Importantly, HCQ selectively induced ATLL cell death in the ATLL xenograft mouse model at the dose used to treat SLE. Taken together, our results suggest that the inhibition of autophagy by CQ/HCQ may become a novel and effective strategy for the treatment of ATLL.

Fig 1. CQ and HCQ inhibit ATLL cell growth in vitro.

(A) Four ATLL cell lines (Su9T01, KK1, S1T, and ST1), two T-ALL cell lines (MOLT4 and JURKAT), and PBMCs from a healthy donor were treated with varying concentrations of CQ or HCQ for 48 hours. Cell viability was determined by an MTT assay. Relative cell viability was calculated based on the percentage of untreated cells (0 μM). Data are presented as the mean ± SD (n = 3). *, #, +, ‡, $, or ¥ p < 0.05 for Su9T01, KK1, S1T, ST1, MOLT4, or JURKAT cells compared to healthy PBMCs, respectively. (B) Primary ATLL cells from four acute-type ATLL patients were treated with varying concentrations of CQ or HCQ for 48 hours. Cell viability was determined with the MTT assay. Relative cell viability was calculated based on the percentage of untreated cells (0 μM). Data are presented as the mean ± SD (n = 3). *, #, or + p < 0.05 for Pt #1, Pt #2, or Pt #3 compared to healthy PBMCs, respectively. (C) S1T and KK1 cell lines were treated with 50 μM CQ or 25 μM HCQ for 6, 12, 18, or 24 hours. The expression of cleaved caspase-3 was examined by Western blot analysis. β-actin was used as a loading control. (D) S1T and KK1 cell lines were treated with 50 μM CQ or 25 μM HCQ for 24 hours, double stained with Annexin-V and DAPI, and then analyzed by flow cytometry.

Fig 2. CQ and HCQ inhibited autophagy and the canonical NF-κB pathway in ATLL cell lines.

(A) Representative confocal immunofluorescence of LC3 in KK1 and S1T cells after treatment with either 50 μM CQ or 25 μM HCQ for 24 hours. Scale bar 10μm. (B) Western blot analysis of CADM1, p47, and the indicated NF-κB and autophagic signaling proteins in S1T cell lines was performed after treatment with either 50 μM CQ or 25 μM HCQ for 6, 12, 18, or 24 hours. β-actin was used as a loading control. The cropped gels/blots are shown in the figure, and the full-length gels/blots are presented in S1 Raw images. (C) LC3-II turnover is presented as the mean ± SD (n = 2 independent experiments, representative blot was shown at Fig 2A). *p < 0.05, **p<0.01 compared to the control.

Fig 3. CQ/HCQ inhibited tumor growth and extended the lifespan of ATLL cells-xenografted mice.

(A) Tumor volume of Su9T01 xenograft tumors in NOG mice intraperitoneally administered water (control) or 50 mg/kg bw CQ daily for 16 days. (B, C) Tumor volume of MT2 (B) or Su9T01 (C) xenograft tumors in NOG mice orally administered water (control), 6.5 mg/kg bw HCQ, or 60 mg/kg bw HCQ daily for 16 days. (D, E) Representative H&E (D) and IHC images of Caspase-3 (E) in tumor tissues from NOG mice orally administered water (1), 6.5 mg/kg bw HCQ (2), or 60 mg/kg bw HCQ (3) daily for 16 days. Scale bars: 300 μm (D), 30 μm (E). Higher magnification images of the boxed areas are shown on the right (Scale bars: 30 μm). The arrows indicate dead tumor cells. (F) Quantification Caspase3 stained cells is presented as the mean ± SD (n = 10 HPF, representative image was shown at Fig 3E). (G) Kaplan-Meier survival curves of the intravenous Su9T01 leukemia model orally treated with HCQ or water daily for 16 days. *p < 0.05, **p<0.01 compared to the control.

Fig 4. Model illustrating the mechanism of CQ/HCQ-induced ATLL cell apoptosis by autophagy and NF-κB pathway inhibition.

In ATLL cells, p47 is degraded by the autophagy-lysosome pathway, leading to increased NEMO protein level and activation of IKK, which result in the constitutive activation of the NF-κB pathways. CQ and HCQ inhibit autophagy and increase p47 protein level, which inhibit NF-κB pathway activation along with loss of CADM1 expression, leading to caspase-3 related apoptosis.