Dual lysosomal-mitochondrial targeting by antihistamines to eradicate leukaemic cells

Abstract

Background: Despite great efforts to identify druggable molecular targets for AML, there remains an unmet need for more effective therapies.

Methods: An in silico screening was performed using Connectivity Maps to identify FDA-approved drugs that may revert an early leukaemic transformation gene signature. Hit compounds were validated in AML cell lines. Cytotoxic effects were assessed both in primary AML patient samples and healthy donor blood cells. Xenotransplantation assays were undertaken to determine the effect on engraftment of hit compounds. The mechanism of action responsible for the antileukaemic effect was studied focussing on lysosomes and mitochondria.

Findings: We identified a group of antihistamines (termed ANHAs) with distinct physicochemical properties associated with their cationic-amphiphilic nature, that selectively killed leukaemic cells. ANHAs behaved as antileukaemic agents against primary AML samples ex vivo, sparing healthy cells. Moreover, ANHAs severely impaired the in vivo leukaemia regeneration capacity. ANHAs' cytotoxicity relied on simultaneous mitochondrial and lysosomal disruption and induction of autophagy and apoptosis. The pharmacological effect was exerted based on their physicochemical properties that permitted the passive targeting of both organelles, without the involvement of active molecular recognition.

Interpretation: Dual targeting of lysosomes and mitochondria constitutes a new promising therapeutic approach for leukaemia treatment, supporting the further clinical development. FUND: This work was funded by the Fundación Mutua Madrileña (RMR), CaixaImpulse (RMR), the Spanish Ministry of Economy (RMR), the Josep Carreras International Leukaemia Foundation (RMR), l'Obra Social "La Caixa" (RMR), and Generalitat de Catalunya (IJC).

Keywords: Antihistamines; Ebastine; Leukaemia; Lysosomes; Mitochondria.

Fig. 1

A subgroup of antihistamines displays antileukaemic effects in AML cell lines. (A) Viability of KG-1 cells after 72 h-treatment with histamine-related ligands (10 μM). Agonist: histamine (H, yellow). HRH1 antagonists (blue): acrivastine (Ac), clemastine (Cl), cetirizine (Ce), cyproheptadine (Cy), ketotifen (Ke), fexofenadine (Fe), loratadine (Lo), rupatadine (Ru), terfenadine (Te), ebastine (Eb). HRH2 antagonists (dark green): ranitidine (Ra), cimetidine (Ci), famotidine (Fa). HRH3 antagonists (light green): JNJ5207852 (J2). HRH4 antagonists (green): JNJ7777120 (J0), JNJ10191584 (J4). Bars show mean ± SEM of triplicates (2 experiments). (B) Cytotoxicity dose-response curves of HL60 (grey), KG1 (orange), MM (green) and THP1 (blue) cells treated 48 h with ebastine or rupatadine. (C). CFSE proliferation assay in KG1 cells treated with loratadine 10 μM, rupatadine 10 μM, ebastine 10 μM or terfenadine 10 μM. CFSE MFI at days 1, 3, 5 and 7. (D). Clonogenicity assays in AML cell lines treated with loratadine 10 μM, rupatadine 10 μM, terfenadine 10 μM or ebastine 10 μM. CFUs counted at day 10. (E). Expression of monocytic (CD14) and granulocytic (CD11b) markers in KG1 cells treated with 10 μM loratadine, 10 μM rupatadine or 5 μM ebastine for 72 h. Bars show mean ± SEM of triplicates (2 experiments). (F). Cell cycle analysis of KG1 cells treated with loratadine 10 μM, rupatadine 10 μM, or ebastine 10 μM. Left: Frequencies of cell cycle phases referred to control. Bars show mean ± SEM of triplicates (2 experiments). Right: representative cytometric histograms (left: vehicle-treated, right: ebastine-treated; green: G0/G1, yellow: S, blue: G2/M). (G) Viability of KG1 cells treated for 48 h with loratadine 10 μM, rupatadine 10 μM, or ebastine 5 μM in the presence (right) or absence (left) of bone marrow stroma (HS-5). Bars show mean ± SEM of triplicates (3 experiments). (H). 48 h-viability of KG-1 cells treated with ebastine and/or ara-C at the indicated concentration. Bars show mean ± SEM of triplicates of representative experiment (3 experiments). *or# p < .05; **or## p < .01; *** p < .001; **** p < .0001 (t-tests). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

ANHAs selectively target AML cells ex vivo. (A) AML patient samples were treated with ANHAs loratadine (dark blue; 5, 10 and 20 μM), rupatadine (orange; 5, 10 and 20 μM), ebastine (light blue; 1, 5 and 10 μM) or terfenadine (green; 1, 5 and 10 μM). Blast viability was assessed at day 3 and 6. Each symbol corresponds to an AML patient sample (n = 16), and each point to a measure. (B). Clonogenicity assay (day 14) in AML patient samples treated with loratadine 10 and 20 μM (Lora), rupatadine 10 and 20 μM (Rupa), ebastine 5 and 10 μM (Ebas) or terfenadine 5 μM (Terfe). Each symbol corresponds to an AML represents a sample (n = 8), each point to a measure. (C). Viability of mononuclear cells from healthy-donor buffy coats was assessed after 3- or 6-day treatment with loratadine 20 μM, rupatadine 20 μM, ebastine 10 μM or terfenadine 5 μM. Bars show mean ± SEM of 5 healthy samples assayed in triplicates. (D). Lineage-depleted UCB cells were applied for 18 h the same treatment as in (C) and cultured as in (B). Left panel: total number of CFUs normalized to control; right panel: frequency of colony subtypes. 3 different samples are represented in triplicates. Bars show mean ± SEM. (E). Ex vivo effects of ANHAs on healthy and AML samples were compared in terms of viability (left), and clonogenicity (right). Data represent viability or clonogenicity reduction of treatments with ebastine 5 and 10 μM (Eb), loratadine 20 μM (Lo) or rupatadine 20 μM (Ru). Bars show superimposed mean + SEM of data from Figs. 2A to 2D. Tables show mean percent reduction in viability or CFU formation, as well as its fold of change (FC; effect on AML/ healthy samples). *p < .05; ** p < .01; *** p < .001; **** p < .0001 (Mann-Whitney tests). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

ANHAs selectively target AML cells in vivo. (A).Conditioned NSG mice (n = 25) were intravenously transplanted with AML patient samples (AML#2, AML#3 and AML#14) previously treated for 18 h with ebastine 10 μM (Ebas, light blue), rupatadine 10 μM (Rupa, orange) or loratadine 10 μM (Lora, dark blue). After 8 weeks, engraftment was analysed in bone marrow (BM). Frequency of human AML blasts in BM is represented as mean value normalized to vehicle-treated control ± SEM. A representative flow plot from each experimental mouse group is shown. (B). Conditioned NSG mice (n = 36) were intravenously transplanted with lineage-depleted cord blood samples (n = 3) previously treated for 18 h with ebastine 10 μM (Ebas, light blue) or rupatadine 10 μM (Rupa, orange). After 8 weeks, engraftment was analysed in bone marrow (BM). Frequency of human blood cells in BM is represented as mean value normalized to vehicle-treated control ± SEM. A representative flow plot from each experimental mouse group is shown. (C). Engrafted cell subpopulations from (B) were analysed according to CD34, CD13 and CD19 positivity to assess changes in the frequency of progenitors, myeloid and B cells respectively. Bars show frequency of each population normalized to vehicle-treated control ± SEM. (D). In vivo effects of ANHAs on healthy and AML samples were compared in terms of engrafttment. Data represent engraftment after treatment with ebastine 10 μM (Eb, light blue) or rupatadine 20 μM (Ru, orange). Bars show superimposed mean + SEM of data from Fig. 3A and B. Tables show mean percent reduction of engraftment in AML and cord blood (CB) samples, as well as its fold of change (FC; effect on AML/effect on CB). *p < .05; ** p < .01; *** p < .001; **** p < .0001 (t-tests). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

ANHA's antileukemic effects are independent of HRH1. (A). Representative flow cytometry histograms of HRH1 surface expression on AML cell lines. Grey shadow, control; red line, HL-60; blue line, KG-1; green line, MM. (B). Representative HRH1 surface staining (green) on HL-60 and KG-1 cells by immunofluorescence. Nuclei are stained with Hoechst33342 (blue). (C). KG-1 cells were treated with ANHAs alone or in combination with histamine 200 μM. Viability was measured by flow cytometry (7AAD-, Hoechst+). Bars show mean ± SEM of triplicates from a representative experiment (three independent experiments were performed). (D). 72 h cytotoxicity data from Fig. 1A (antihistamine-treated KG-1 cells) were correlated with the corresponding pKi value for each drug. Left panel includes a regression line and its corresponding R². Right panel shows pKis for ANHAs and non-ANHAs. Horizontal lines represent means. (E). Frequency of HRH1 expression on the surface of cell lines from haematological neoplasias, either myeloid (yellow), T (green) or B (blue) lineage, as detected by flow cytometry. (F). Cell lines from different haematological neoplasias were treated for 72 h with ebastine at 10 μM and viability was assessed by flow cytometry. Bars show mean ± SEM of triplicates from 2 independent experiments. (G). 72 h cytotoxicity data from Fig. 4F (Ebastine 10 μM -treated KG-1 cells, Y axis) were correlated with the corresponding %HRH1 expression of each cell line (X axis, data from Fig. 4E). Graphs include regression lines and their corresponding R². Right panel shows pKis for ANHAs and non-ANHAs. Error bars represent SEM. *p < .05; ** p < .01; *** p < .001; **** p < .0001 (t-tests). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

ANHAs induce an expansion in lysosomal and autophagic compartments. (A). Confocal microscopy images of KG-1 cells treated 18 h with control or ebastine 5 μM, stained with LysoTracker DeepRed 50 nM and Hoechst33342. Left: 630× (scale = 25 μm); right: 1260× (scale = 5 μm). (B). KG-1 cells treated 24 h with control (Ø) ebastine 5 μM (Eb), rupatadine10μM (Ru), loratadine10μM (Lo) or chloroquine20μM (Ch), stained with LysoTracker 50 nM and analysed by cytometry. Bars represent mean ± SEM of triplicates (2 experiments). (C). KG-1 cells were treated 18 h with control or ebastine 5/7.5 μM; galectin-1 (LGALS1, green) distribution was assessed by immunofluorescence. Representative images shown (1260×; scale = 5 μm). Right: frequency of cells with galectin-1 puncta (mean ± SEM, 2 experiments). (D). KG-1 cells were treated 18 h with vehicle control or ebastine 7.5 μM, and cathepsinB (CTSB, green) distribution was assessed by immunofluorescence. Representative images shown (1260×; scale = 5 μm). (E). Viability of KG-1 after 1 h-treatment with α-tocopherol (500, 1000 and 2000 μM) followed by 48 h-treatment with ebastine 10 μM (Ebas), loratadine 10 μM (Lora), or rupatadine 10 μM (Rupa). Bars represent mean ± SEM of triplicates from representative experiment (2 experiments). (F). KG-1 cells treated 48 h with control (Ø) ebastine 5 μM (Eb), rupatadine 10 μM (Ru), loratadine 10 μM (Lo) or chloroquine 20 μM (Ch), stained with CYTO-ID and analysed by flow cytometry. Bars represent mean ± SEM of triplicates (2 experiments). (G). HL-60, KG-1, MonoMac-1 and THP-1 cells were treated 24 h with control (Ø) ebastine 10 μM (Eb), rupatadine 10 μM (Ru), loratadine 10 μM (Lo) or chloroquine 20 μM (Ch). Proteins were extracted and run in a western blot using LC3 and GAPDH antibodies. Representative membranes shown. Data in dot plot; each symbol corresponds to a cell line, each point to a replicate. (H). Viability of KG-1 cells after 48 h-treatment with control (Cont), 3-MA 500 μM, ebastine 7 μM (Ebas) or a combination of both (Combo). Bars show mean ± SEM of triplicates from a representative experiment (2 experiments). *p < .05; ** p < .01; *** p < .001; **** p < .0001 (t-tests). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

ANHA-induced cell death partly depends on mitochondrial homeostasis disruption. (A). MitoSOX Red superoxide indicator staining in KG-1 cells were treated 6 h with vehicle control (Ø) ebastine 10 μM (Eb), rupatadine 10 μM (Ru), loratadine 10 μM (Lo) or rotenone 0,1 μM (Ro). Bars show mean ± SEM of triplicates from two independent experiments. (B). THP-1 cells were treated for 48 with increasing concentrations of N-acetyl cysteine (NAC; 2, 5 and 10 mM) and ebastine 5 or 10 μM (Ebas). Cell viability was assessed by flow cytometry. Bars represent mean ± SEM of triplicates from a representative experiment (3 independent experiments). (C). KG-1 cells were treated for 18 h with ebastine 7.5 μM (Eb5, Eb10), loratadine 10 μM (Lora) or rupatadine 10 μM (Rupa). After 18 h, cells were stained with rhodamine 123 and analysed by flow cytometry. Bars show mean ± SEM of triplicates from two independent experiments (D). KG-1 cells were treated for 6 or 24 h with vehicle control (grey), loratadine 10 μM (dark blue), rupatadine 10 μM (orange), ebastine 10 μM (light blue) or etoposide 5 μM (red). Caspase activation was analysed using Caspase-Glo ® 3/7 assay. Bars show mean ± SEM of triplicates from two independent experiments (E). KG-1 cells were treated with ebastine 5 or 10 μM (Eb), loratadine 10 μM (Lora) or rupatadine 10 μM (Rupa) in the presence or absence of Z-VAD-FMK 50 μM, and viability was assessed after 48 h. Bars show mean ± SEM of triplicates from a representative experiment (out of 3 independent experiments) (F). KG-1 cells were treated with ebastine 1, 5 or 10 μM and frequency of annexin V+ cells was assessed by flow cytometry at 48 h. Bars show mean ± SEM of triplicates from a representative experiment (3 independent experiments) *p < .05; ** p < .01; *** p < .001; **** p < .0001 (t-tests). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

ANHAs simultaneously target mitochondria and lysosomes (A).KG-1 cells were treated with ebastine 10 μM, loratadine 10 μM or rupatadine 10 μM and different parameters were analysed at the indicated times; caspase activation (caspaseGLO), autophagy (CYTO-ID), lysosomal volume (LysoTracker) and mitochondrial ROS (MitoSOX). Each point represents the mean of triplicates from two independent experiments. (B). Calculated logD_7,4 values obtained from ChEMBL (ACDLabs calculations) were plotted for each functional antihistamine group. Each point represents an antihistamine (C). Antihistamines were plotted according to their calculated logD_7,4 and their calculated topological polar surface area (TPSA, drugbank.ca). ANHAs are represented in green, non-ANHAs in red, and intermediate antihistamines in yellow. (D). Model depicting the hypothesized antileukaemic mechanism of action of antihistamines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)