Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency

Abstract

Autophagy is a lysosome-dependent degradative process that protects cancer cells from multiple stresses. In preclinical models, autophagy inhibition with chloroquine (CQ) derivatives augments the efficacy of many anticancer therapies, but CQ has limited activity as a single agent. Clinical trials are underway combining anticancer agents with hydroxychloroquine (HCQ), but concentrations of HCQ required to inhibit autophagy are not consistently achievable in the clinic. We report the synthesis and characterization of bisaminoquinoline autophagy inhibitors that potently inhibit autophagy and impair tumor growth in vivo. The structural motifs that are necessary for improved autophagy inhibition compared with CQ include the presence of two aminoquinoline rings and a triamine linker and C-7 chlorine. The lead compound, Lys01, is a 10-fold more potent autophagy inhibitor than HCQ. Compared with HCQ, Lys05, a water-soluble salt of Lys01, more potently accumulates within and deacidifies the lysosome, resulting in impaired autophagy and tumor growth. At the highest dose administered, some mice develop Paneth cell dysfunction that resembles the intestinal phenotype of mice and humans with genetic defects in the autophagy gene ATG16L1, providing in vivo evidence that Lys05 targets autophagy. Unlike HCQ, significant single-agent antitumor activity is observed without toxicity in mice treated with lower doses of Lys05, establishing the therapeutic potential of this compound in cancer.

Fig. 1.

Chemical structure of mono- and bisaminoquinolines.

Fig. 2.

Effects of Lys01–Lys04 on LC3 immunoblotting. Immunoblotting and quantification of LC3II/LC3I ratio in lysates from LN229 cells treated for 4 h. The graphs show (mean ± SD) LC3II/LC3I ratios of each treatment normalized to the LC3II/LC3I ratio of control-treated cells for each experiment.

Fig. 3.

Autophagy inhibition and cytotoxicity of Lys01 compared with HCQ. (A) Representative images of LN229 GFP-LC3 cells treated as indicated for 4 h. White arrows: small puncta; red arrows: dense puncta. Graph shows mean ± SEM puncta per cell. (B) Representative electron micrographs of LN229-GFP-LC3 cells treated (4 h) with DMSO, HCQ 10 μM, or Lys01 10 μM. Arrows: autophagic vesicles. (C) (Left) LC3 immunoblotting of LN229 cells treated for 24 h as indicated. (Right) Calculated ratio of LC3II/LC3I ratios for bafilomycin vs. control cotreatment. Ratios higher than 1 (dashed line) indicate an autophagy inducer or control; ratios lower than 1 indicate an autophagy inhibitor. (D) MTT assay (72 h) for four cell lines. Red: Lys01; blue: Lys02; purple: Lys03; green: Lys04; orange: HCQ. Values presented are means ± SEM with five replicates per treatment.

Fig. 4.

In vivo autophagy inhibition and antitumor activity of Lys05. (A) Representative electron micrographs (12,000×) of c8161 xenograft tumors harvested after 2 d of daily i.p. treatment with PBS, HCQ 60 mg/kg, or Lys05 76 mg/kg. Arrows: autophagic vesicles. (Scale bar: 2 μm.) (B) Quantification of mean ± SEM number of autophagic vesicles per cell from two representative tumors from each treatment group. (C and D) 1205Lu xenografts were treated with PBS (blue), HCQ 60 mg/kg i.p. (green), or Lys05 76 mg/kg (red) i.p. every 3/5 d. (C) Tumor volumes over 14 d. (D) Daily tumor growth rate. (E–G) HT29 xenografts were generated in the flanks of nude mice and treated with PBS, Lys05 10 mg/kg i.p. daily, Lys05 40 mg/kg i.p. daily, or Lys05 80 mg/kg i.p. every 3/5 d. (E) Average daily tumor growth rate. (F) Tumor volumes over 14 d. (G) Weight of excised tumors, *P < 0.05.

Fig. 5.

Lys05 treatment at the highest dose reproduces the intestinal phenotype of a genetic autophagy deficiency. (A–F) Weight and intestines were analyzed for mice bearing HT29 xenografts treated with PBS and Lys05 10–80 mg/kg. (A) Daily weight. (B) Representative excised gastrointestinal tracts after 14 d of treatment. (C) Representative images (40×) of H&E-stained ileal crypts from mice bearing HT29 xenografts (14 d). Arrows: Paneth cells. (D) Paneth cell number per crypt. (E) Paneth cell dysfunction score, *P < 0.05. (F) Scoring of lysozyme-positive cells, *P = 0.001. Representative images of lysozyme immunofluorescence (green) of ileum in mice treated with PBS and Lys05 80 mg/kg i.p. 3/5 d.

Fig. 6.

Lys05 inhibits autophagy by accumulating in and deacidifying the lysosome. (A) 1205Lu cells (treated with PBS, HCQ 10 μM, or Lys05 10 μM for 24 h) and harvested 1205Lu xenograft tumors (treated with PBS, HCQ 60 mg/kg i.p. 3/5 d, or Lys05 76 mg/kg i.p. 3/5 d for 14 d) were homogenized and fractionated into whole-cell (WC) and lysosomal (L) fractions. LAMP2 immunoblotting confirmed isolation of concentrated lysosomes for analysis. (B) Concentrations of HCQ or Lys05 in cells and tumor WC and L homogenates. (C) Fluorescence imaging of 1205Lu cells treated as indicated for 30 min and stained with LysoTracker Red. LysoTracker puncta (red) per cell was scored for three high-powered fields. Blue: nuclear DAPI staining. Data presented is mean ± SEM. (D) Fluorescence imaging of c8161 cells treated as indicated for 24 h and stained with AO. Orange: aggregated AO; green: diffuse AO. Graph shows mean +/− SD %acidic vesicles.