EBioMedicine 47 (2019) 221–234 Contents lists available at ScienceDirect EBioMedicine journal homepage: www.ebiomedicine.com Research paper Dual lysosomal-mitochondrial targeting by antihistamines to eradicate leukaemic cells Josep M. Cornet-Masana a,b,c, Antònia Banús-Mulet a,d, José M. Carbó a, Miguel Ángel Torrente c,e, Francesca Guijarro c,e,f, Laia Cuesta-Casanovas a,g, Jordi Esteve a,c,e,f, Ruth M. Risueño a,⁎ a Josep Carreras Leukaemia Research Institute (IJC). Barcelona, Spain b Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP). Badalona, Spain c Faculty of Medicine, University of Barcelona, Spain d Faculty of Pharmacy, University of Barcelona, Spain e Department of Haematology, Hospital Clínic, Barcelona, Spain f Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain g Faculty of Biosciences, Autonomous University of Barcelona, Spain a r t i c l e i n f o a b s t r a c t Article history: Background: Despite great efforts to identify druggable molecular targets for AML, there remains an unmet need Received 18 July 2019 for more effective therapies. Received in revised form 8 August 2019 Methods: An in silico screening was performed using Connectivity Maps to identify FDA-approved drugs that may Accepted 9 August 2019 revert an early leukaemic transformation gene signature. Hit compounds were validated in AML cell lines. Cyto- Available online 28 August 2019 toxic effects were assessed both in primary AML patient samples and healthy donor blood cells. Xenotransplan- tation assays were undertaken to determine the effect on engraftment of hit compounds. The mechanism of Keywords: Leukaemia action responsible for the antileukaemic effect was studied focussing on lysosomes and mitochondria. Antihistamines Findings: We identified a group of antihistamines (termed ANHAs) with distinct physicochemical properties as- Ebastine sociated with their cationic-amphiphilic nature, that selectively killed leukaemic cells. ANHAs behaved as Lysosomes antileukaemic agents against primary AML samples ex vivo, sparing healthy cells. Moreover, ANHAs severely im- Mitochondria paired 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 Min- istry of Economy (RMR), the Josep Carreras International Leukaemia Foundation (RMR), l'Obra Social “La Caixa” (RMR), and Generalitat de Catalunya (IJC). © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Acute myeloid leukaemia (AML) is a clinically and biologically het- Abbreviations: 3-MA, 3-methyladenine; ALL, Acute lymphoblastic leukaemia; ANHA, erogeneous disease characterized by the accumulation of immature AntiNeoplastic HRH1 inverse Agonist; AML, Acute Myeloid Leukaemia; BM, Bone transformed myeloid progenitors in bone marrow (BM). Although sig- Marrow; CAD, Cationic Amphiphilic Drug; CFU, Colony-forming Unit; CML, Chronic nificant research efforts have been invested in improving outcomes Myeloid Leukaemia; CMML, Chronic Myelomonocytic Leukaemia; EC50, Effective concen- tration 50; hERG, Potassium voltage-gated channel subfamily H member 2; HR, Histamine for AML patients, the standard therapy for most subtypes of newly diag- Receptor; LGALS1, Galectin-1; LMP, Lysosomal Membrane Permeabilization; LSC, nosed AML has remained practically unchanged over the past 4 decades, Leukemic Stem Cell; mAChR, Muscarinic AcetylCholine Receptor; NAC, N-acetylcysteine; and the prognosis is still poor [1]. Indeed, most patients with AML will NSG, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; ROS, Reactive Oxygen Species; TPSA, relapse after achieving complete remission, with treatment of refractory Topological Polar Surface Area; UCB, Umbilical Cord Blood. ⁎ Corresponding author at: Josep Carreras Leukaemia Research Institute, Campus ICO- and relapsed AML being challenging in clinics. Therefore, new therapeu- GTP, Crta Can Ruti. Camí de les Escoles, s/n, 08916 Badalona Barcelona, Spain. tic approaches with high specificity and effectiveness are urgently E-mail address: risueno@carrerasresearch.org (R.M. Risueño). needed. https://doi.org/10.1016/j.ebiom.2019.08.021 2352-3964/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 222 J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 2. Materials and methods Research in context Evidence before this study 2.1. AML cell lines and cell cultures Despite great efforts to identify druggable molecular targets for Cell lines HL-60 (ACC-3), KG-1 (ACC-14), MonoMac-1 (ACC-252) K- acute myeloid leukaemia, there remains an unmet need for more 562 (ACC-10), Jurkat (ACC-282), RPMI-8402 (ACC-290), CCRF-CEM effective therapies. In this quest, in silico identification of (ACC-240), RAMOS (ACC-603), GRANTA-519 (ACC-342), RPMI-8226 repurposing drug candidates has been proven useful in the search (ACC-402), JJN-3 (ACC-541) and U-266 (ACC-9) were obtained from for potential therapies. As such, several antihistamines have been DSMZ (Braunschweig, Germany). THP-1 cell line (TIB-202™) was ob- preclinically described as antineoplastic agents in non-AML tained from ATCC (Manassas, VA, USA). HBL-2 cell line was kindly sup- tumors. plied by Dr. Pérez-Galán. Added value of this study 2.2. Primary samples In our study, we described the antileukaemic potential of several Primary AML samples were obtained from patients diagnosed at antihistamines and demonstrated its histamine receptor- Hospital Clínic of Barcelona (Spain) and Hospital Germans Trias i Pujol independence. Rather than a specific molecular target recognition, (Badalona, Spain). AML diagnosis and classification were based on stan- the mechanism of action was found to rely on the simultaneous dard WHO criteria (70). Main AML patient's characteristics are summa- disruption of lysosomes and mitochondria, based on the physico- rized in Table S1. Mononuclear cells (MNCs) were isolated by Ficoll chemical properties of these drugs. density gradient centrifugation (GE, Chicago, IL, USA). All patients pro- vided written informed consent in accordance with the Declaration of Implications of all the available evidence Helsinki, and the study was approved by the corresponding Ethics Com- mittees. Blood mature MNCs were isolated from healthy-donor buffy The preclinical results presented in the study are in line with previ- coats provided by Banc de Sang i Teixits (BSiT, Barcelona, Spain). Umbil- ous results in lung cancer and constitute a stepping stone towards ical cord blood was provided by BSiT, and MNCs were depleted for line- the development of novel treatments for AML independent of ac- age marker-positive cells using magnetic separation with human tive molecular recognition and based on simultaneous lysosomal Lineage Cell Depletion kit (Miltenyi Biotec, Bergisch Gladbach, and mitochondrial functionality disruption. Germany) following manufacturer's recommendations. 2.3. Drugs and antibodies All drugs were resuspended in H2O (Thermo Fischer Scientific, Wal- AML displays a high degree of heterogeneity that evolves tham, MA, USA), DMSO (Sigma-Aldrich, St. Louis, MO, USA) or ethanol through disease progression and/or relapse, affecting both the geno- (VWR) according to manufacturer's specifications. Antibodies used in type and the phenotype of leukaemic subclones [2–6]. Cytogeneti- this study are summarized in Table S3. cally, 50% of AML patients present normal karyotypes, suggesting the existence of other molecular events in leukaemogenesis [7]. De- 2.4. Cytotoxicity assays spite this disease complexity, MLL fusion genes have been demon- strated capable of initiating human leukaemogenesis in vivo, as 2 × 105 (72 h assays), 3 × 105 (48 h assays) or 7.5 × 105 (primary their expression is sufficient to transform human haematopoietic AML) cells per mL were cultured in 96-well plates and all drugs were progenitors and mature cells into leukaemic cells [8–10]. Specifi- added at the indicated concentrations. In co-culture experiments, 2 cally, MLL-AF9 fusion protein does not require collaborating muta- × 105 CFSE-stained HS-5 cells per mL were seeded in 96-well plates. tions to induce leukaemia transformation [11]. Although MLL-AF9 AML cell lines at 2 × 105 cells per mL were subsequently added. In Z- rearrangement is rarely found in AML patients (below 1%) [12,13], VAD-FMK experiments cells were pretreated 20 min with Z-VAD-FMK MLL-AF9 AML model faithfully recapitulates key aspects for this dis- 50 μM. In α-tocopherol experiments, cells were pretreated for 24 h ease, including in vivo regeneration capacity, clonogenicity and phe- with growing concentrations of α-tocopherol and later incubated with notype [8,9]. antihistamines for 48 h. Cell viability was measured by 7-AAD Although the hypothesis that histamine might be involved in carci- (eBioscience, San Diego, CA, USA) exclusion and Hoechst33342 nogenesis was proposed several decades ago [14], it remains under dis- (Sigma-Aldrich) positivity staining by flow cytometry, and cell count cussion today. Indeed, the evidence linking antihistamines per se to was obtained by volume. In experiments with primary cells, analyses cancer is controversial and complex [15,16]. In the Haematology field, were performed inside the blast population as detected by blast gates the antileukaemia effect of terfenadine has been recently described, (CD45low SSCint). suggesting a non-canonical mechanism of action [17]. Similar results were obtained in other solid tumors when different antihistamines 2.5. Proliferation assay were studied for their antineoplastic activity [18–24], mainly through an HRH1-independent mechanism. HL-60 and KG-1 cells were stained with 2,5 μM CFSE (CellTrace™ Based on the gene expression profile associated with MLL-AF9- CFSE Cell Proliferation kit, Thermo Fisher). Drugs were added at the in- driven early transformation events in AML, a group of antihista- dicated concentrations. mines was identified as potent antileukaemic agents due to their histamine receptor-independent physicochemical properties allowing 2.6. Clonogenicity assay mitochondrial and lysosomal disruption. Simultaneously targeting both organelles constitutes a new therapeutic approach for haema- 1 × 103 cells of AML cell lines or 50 × 103 primary AML cells or lin- tological neoplasias, affecting both the bulk population and the depleted UCB cells were treated at the indicated concentration for most primitive cell fraction without significant effect on their 18 h and cultured in 1 mL of MethoCult H4034 Optimum (StemCell healthy counterparts. Technologies, Vancouver, BC, Canada). Colonies were screened based J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 223 on morphology and cellularity at day 7 (cell lines) or 14 (AML primary 2.13. Mitochondrial reactive oxygen species (ROS) cells and lineage-depleted UCB cells). 4 × 105 cells/ml were treated at the indicated concentration of drugs 2.7. In vivo studies for 6 h and subsequently incubated with mitochondrial ROS detector MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fischer Scien- 6–8 week-old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG, Jackson Lab- tific) 5 μM and Hoechst33342 for 10 min at 37 °C. Cells were washed oratories) mice were myeloablated with busulfan (30 mg/kg IP) at day twice with HBSS and acquired in a FACSCantoII cytometer. 0. CD3-depleted primary AML patient samples or lineage-depleted UCB cells were ex vivo treated in the indicated conditions, and 4–34 2.14. Rhodamine 123 assay × 106 (AML) or 12–14 × 104 (lin− UCB) cells were injected IV and left 8–10 weeks untreated. Engraftment was determined as the percentage Changes in mitochondrial membrane potential were assessed using of live human CD45-expressing cells in bone marrow as assessed by the rhodamine 123 dye. Briefly, cells were treated for 18 h, washed with flow cytometry. Mice were randomized and blind-coded at the begin- PBS and incubated with rhodamine-123 50 nM (15 min, 37 °C). Cells ning of the experiment. were then washed twice with PBS and acquired in a flow cytometer. 2.15. CaspaseGlo assay 2.8. HRH1 immunofluorescence 4 × 105 cells per mL were seeded in 96-well plates and treated in in- A total of 3 × 105 cells were attached to poly-L-lysine (50 μg/mL)- dicated conditions (50 μL final volume). 50 μL reconstituted coated coverslips and fixed with formaldehyde 4% (Sigma), blocked CaspaseGlo® 3/7 Assay substrate (Promega) were added, and the mix with γ-globulin (10 μg/mL) and stained with Hoechst33342 and HRH1 was incubated 30′ at room temperature. Luminescence was analysed antibody (Antibodies-online GmbH, Aachen, Germany). Samples were using a Synergy HT Multi-Detection Microplate Reader (BioTek, Winoo- observed in a Leica DMI6000 B fluorescence microscope (Leica, Wetzlar, ski, VT, USA) following manufacturer's recommendations. Germany) and the images were analysed with Image J. 2.16. Annexin V staining 2.9. Lysotracker 3 × 105 cells per mL were treated for 48 h with compounds at the in- 4 × 105 cells per mL were treated, washed in HBSS and incubated dicated concentrations. Annexin V membrane translocation was mea- with 50 nM Lysotracker DeepRed (Thermo Fisher) and Hoechst33342 sured using the Annexin V apoptosis detection kit (Becton Dickinson, for 30 min at 37 °C. Cells were washed twice with HBSS and acquired Franklin Lakes, NJ, USA) according to the manufacturer's instructions. in a flow cytometer. For the microscopic analysis, 2 × 105 cells per con- Data were collected by flow cytometry dition were treated, washed with complete medium and incubated 1 h with 100 nM LysoTracker DeepRed and Hoechst 33342 at 37 °C. Cells 2.17. Flow cytometry were attached to chambered coverslips (μ-Slide 8 well, Ibidi) previously treated with Poly-L-Lysine 50 μg/mL. Live cell images were acquired All flow cytometric experiments were performed with a FACSCanto with a Zeiss LSM880 confocal microscope (Zeiss, Jena, Germany). II cytometer (Becton Dickinson). All flow cytometric analysis was done using FlowJo software (TriStar, Scottsdale, AZ, USA). 2.10. Lysosomal membrane permeabilization 2.18. Statistics 3 × 105 cells previously treated for 18 or 48 h with selected drugs were attached to poly-L-lysine (50 μg/mL)-coated coverslips, fixed Statistical significance was determined using GraphPad Prism® 6.01 with formaldehyde 4% (cathepsin B) or cold methanol (galectin-1) (GraphPad software, La Jolla, CA, USA) by using the statistical tests spec- and permeabilized with Triton X100 0.2%. Cells were then blocked ified in figure legends. Error bars correspond to SEM. All experiments with γ-globulin (10 μg/mL) and incubated with cathepsin B were done at least 2 times in biological triplicates, unless otherwise (NBP1–86048, 1:100) or galectin-1 (ab25138, 1:1000) antibodies. specified in the figure legend. Cells were subsequently incubated with Hoechst33342 and Alexa Fluor 488 Goat Anti-Rabbit IgG. Images were acquired in a confocal mi- 2.19. Ethics approval croscope Zeiss LSM880 and analysed with Fiji software [25]. All patients provided written informed consent in accordance with the Declaration of Helsinki, and the study was approved by the corre- 2.11. Cyto-ID staining sponding Ethics Committees (Ethics Committee Hospital Clínic de Barcelona, Ethics Committee Hospital Germans Trias i Pujol). All exper- 4 × 105 cells per mL were treated for 18 h with drugs at the indicated iments involving mice were approved by the Catalan Ethical Committee concentrations, washed in PBS and incubated 30 min at 37 °C in PBS of Animal Experimentation (CCEEA). with 5% FBS containing Hoechst 33342, 7-AAD and Cyto-ID green fluo- rescent dye (CYTO-ID ® Autophagy detection kit, Enzo Life Sciences, 2.20. Availability of data and materials Farmingdale, NY, USA) at a 1:4000 dilution. Cells were washed with PBS and acquired in a flow cytometer. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. 2.12. LC3B immunoblotting 3. Results 10 × 106 AML cell lines or 25–50 × 106 primary AML cells per condi- tion were treated in indicated conditions and subsequently harvested 3.1. A group of antihistamines displayed antileukaemic effects and lysed in RIPA lysis buffer. Antibodies used for this technique are listed on supplementary table 2. Images were acquired in a LICOR Odys- To search for drugs that may revert early leukaemogenesis-related sey Imager and quantified using Fiji software. events, the MLL-AF9-induced AML model was chosen, as MLL-AF9 not 224 J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 only is capable of transforming human progenitor blood cells into AML, cardiotoxicity, its study as a candidate was discontinued [31]. The but also induces the aberrant expression of stem-cell genes that charac- ratio between viability reduction and total CFUs generated from healthy terize leukaemic stem cells capable of initiating, maintaining and prop- blood cells versus AML cells was calculated to reveal the higher selectiv- agating the leukaemia [26]. The gene expression profile induced upon ity of ANHAs for targeting AML (Fig. 2E). MLL-AF9 expression in CD34+ cord blood cells [27] was screened in the Connectivity Map database [28] in order to search for small bioac- 3.2. ANHA treatment selectively impaired the leukaemia regeneration ca- tive molecules that may revert the transformation-associated gene sig- pacity in vivo nature. Unexpectedly, four antihistamines were found among hit compounds (Fig. S1). A primary screening on AML cell lines was per- Next, the effect of ANHAs on the in vivo engraftment potential was formed to validate the results obtained from the in silico prediction. evaluated. Primary AML samples were treated ex vivo for 18 h and AML cell viability remained unaffected in the presence of histamine transplanted into adult conditioned immunocompromised NSG mice. (natural HR agonist), and HR type 2 (HRH2), HR type 3 (HRH3) and Transplanted mice were left untreated for 8–10 weeks in order to mea- HR type 4 (HRH4) antagonists. However, a subgroup of HRH1 inverse sure the leukaemia regeneration capacity of AML cells upon treatment. agonists (loratadine, rupatadine, terfenadine and ebastine) (ANHA: An- As shown in Fig. 3A, ebastine severely reduced the leukaemia burden tiNeoplastic HRH1 inverse Agonist) significantly decreased leukaemic in BM, diminishing the presence of human AML cells in BM by N90%. In- survival (Fig. 1A and S2A; Table S4). Indeed, the antileukaemic effect deed, 75% ebastine-treated mice displayed b3% AML blasts in BM as measured by the EC50 on AML cell lines was in the low micromolar compared to vehicle-treated control. The treatment with rupatadine range (Fig. 1B and S2B), being similar in MLL-AF9-positive (MonoMac- also induced a reduction of the engraftment capacity of AML cells in 1, THP-1) and in MLL-AF9-negative (HL-60, KG-1) cell lines (Fig. S3). N50%. However, no significant differences were detected in the leukae- At that concentration, ANHAs significantly reduced the proliferation mia regeneration capacity upon loratadine treatment. As healthy con- rate of AML cells (Fig. 1C and S2C). Moreover, the AML clonogenic ca- trol, lineage-depleted umbilical cord blood cells (lin− UCB cells) were pacity as measured by the colony-formation potential decreased in the treated similarly to AML cells and transplanted into NSG mice. In con- presence of ANHAs (Fig. 1D). As the block in differentiation is a hallmark cordance with the lack of cytotoxic effect in healthy blood cells demon- of AML, we interrogated the differentiation status of AML cells treated strated in Fig. 2, lin− UCB cells ex vivo treated with ebastine displayed a with ANHAs. Upregulation of both CD11b and CD14 differentiation- slight reduction in normal haematopoietic potential as measured by the associated granulocyte and monocyte surface markers was detected human regeneration capacity on BM (Fig. 3B). Indeed, the frequency of upon treatment (Fig. 1E and S2D). In concordance, treatment with the most primitive haematopoietic human cell fraction represented by ANHAs lead to a G2/M cell-cycle arrest (Fig. 1F and S2E). Moreover, CD34+ cells in BM was equivalent in all treated animals as compared ANHA treatment synergized with cytarabine, the backbone of chemo- to vehicle-treated mice (Fig. 3C). Similar results were obtained in the therapy regimen in AML (Fig. 1G), enabling the eradication of CD13+ myeloid and CD19+ lymphoid cell population (Fig. 3C). Again, cytarabine-resistant cells. Due to the well-described BM microenviron- the ratio between the engraftment reduction levels observed in AML ment protection on AML [29], the antineoplastic effect of ANHA-drug vs. healthy blood cells revealed the high specificity towards neoplastic treatment was interrogated in a more relevant in vitro co-culture system cells (Fig. 3D). Thus, treatment with ANHAs dramatically reduced the with HS-5 human BM stroma cells. Treatment of AML cells in direct con- regeneration potential of AML cells with little effect on normal HSCs. tact with HS-5 stromal monolayers did not overcome the cytotoxic ef- fect of ANHAs (Fig. 1H and S2F). Our results suggest that ANHA induced cytotoxicity on AML cells by promoting differentiation and 3.3. ANHA antineoplastic effect was not mediated by HRH1 G2/M accumulation, and this neoplastic effect is stroma-independent. The antileukaemic effect of ANHA was then evaluated ex vivo in 16 Since ANHAs are well-known antihistamines that specifically recog- primary AML patient samples. The reduction of cell viability upon treat- nize HRH1, the role of this membrane receptor in leukaemogenesis was ment in all AML samples tested was dose- and time-dependent studied. First, the cell-surface expression of HRH1 was evaluated on (Fig. 2A). In contrast to AML cell lines (Fig. 1) where a 50% cell death AML cell lines. HRH1s were uniformly expressed on all AML cells was achieved in the presence of 5–10 μM of each drug at 48 h post- (Fig. 4A and B). However, the cytotoxic effect observed upon treatment treatment, primary AML samples were more resistant, and a clinically of AML cells with ANHAs was not reverted in the presence of the natural significant cytotoxic effect was detected at day 6. Interestingly, all the ligand of HRH1, histamine (Figs. 4C and S4A), and no relationship was AML samples tested were sensitive to the treatment. In order to mea- found between the sensitivity of AML cells and the affinity of antihista- sure the proliferation and differentiation ability of individual AML cells mines for HRH1 as measured by the pKi value (Fig. 4D). To further study and, therefore, their ex vivo stem-cell properties, the clonogenic capacity the relationship between the antileukemic potential and HRH1, we was measured 18 h after treatment. Additionally, the total number of analysed both HRH1 expression and ANHA cytotoxicity in a broader colonies decreased to approximately 50% (Fig. 2B), suggesting that panel of leukemic cell lines. Although all the different leukaemic cell ANHAs affected the most primitive fraction of AML cells. lines tested were sensitive to ANHA treatment, HRH1 expression and As ANHAs are already FDA- and/or EMA-approved antihistamines, cytotoxicity were inconsistent (Figs. 4E-G and Figs. S4B and S4C); equiv- the therapeutic maximal concentration achieved in plasma is already alent sensitivity to ANHA treatment was found in leukaemic cells with known and ranges between 1 and 200 nM [30]. However, the cytotoxic dramatically different levels of HRH1 (0–100%). These results suggested effect observed on AML cells was achieved at a low microMolar range; that ANHAs exerted their effect in a HRH1-independent manner; thus, therefore, the safety of these treatments on healthy blood cells was alternative already described targets were explored. Although many tested at the same conditions as previously described for primary AML HRH1 antagonists also inhibit muscarinic acetylcholine receptors samples. The cell viability of each blood-cell subtype (T, B and myeloid (mAChR) [32–34], no effect on cell viability was detected upon treat- cells) remained largely unaffected upon treatment; only the myeloid ment with mAChR antagonists (Fig. S5A) neither acetylcholine reverted population was significantly reduced in the presence of rupatadine the cytotoxicity induced by ANHAs (Fig. S5B). Next, we investigated the and ebastine at day 6 (Fig. 2C). Remarkably, treatment with ANHAs role of the potassium voltage-gated channel subfamily H member 2 spared the clonogenic capacity of healthy haematopoietic progenitor/ (hERG, KCHNH2 or Kv11.1) as many antihistamines block this channel stem cells as shown in Fig. 2D, where no differences were observed in [35–37]. Yet, the survival of AML cells remained largely unaffected in the total number or relative frequency of each type of CFUs. the presence of hERG blockers (Fig. S5C). Therefore, the pharmacologi- Only terfenadine significantly reduced the total number of CFUs and, cal effect of ANHAs on AML cells was not exerted by HRH1 inhibition, also considering its withdrawal from the market due to potential mAChR modulation or hERG blockage. J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 225 226 J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 3.4. ANHAs perturbed lysosomes in AML cells treatment (Fig. 6D and S8D); although the inhibition of caspases by z- VAD-FMK had no significant effect on ANHA treatment induced cell Cationic amphiphilic drugs (CADs) are molecules with a hydropho- death (Fig. 6E and S8E-F), suggesting that several cell death programs bic ring structure and a side chain with a cationic amine group [38]. As might be activated. Regardless, the canonical apoptosis marker CADs are not ionized at a physiologic pH and have overall hydrophobic- Annexin-V was upregulated in a dose-response manner (Fig. 6F and ity, they can easily permeate through the plasma membrane [39]. S8G). Within the highly acidic lysosomal compartment, the amine is proton- Due to the cross-talk between mitochondrial and lysosomal com- ated, and the drug is then trapped and concentrated, inducing mem- partment during activation of the cell death program, the temporal in- brane structure perturbation and compromising cell viability [40,41]. duction of mitochondrial and lysosomal damages was studied. Their uptake is therefore controlled by two major mechanisms: non- Mitochondrial ROS generation peaked 4 h post-treatment; while cas- specific binding to membrane phospholipids and ion-trapping within pase 3/7 activation, lysosomal enlargement and autophagosome accu- acidic cellular compartment [18]. As ANHAs have been described as mulation were induced 6–12 h after treatment (Fig. 6G). Although CADs exerting cytotoxicity effects in solid tumors through lysosomal ac- ROS is a described LMP-inducer, the increase in lysosomal volume can- cumulation [18], the lysosome compartment was examined using the not be also attributed, suggesting that ANHAs might be exerting their acidotropic probe LysoTracker. As expected for CADs, AML cells treated antileukaemic effect simultaneously on both organelles and are both with ANHAs showed an enlargement of the lysosome mass, as detected relevant for cell death. by fluorescence microscopy (Fig. 5A and S6A) and flow cytometry Lysosomo- and mitochondriotropism require passive membrane (Fig. 5B and S6B); which constitutes a common event preceding the diffusion and sequestration inside these organelles. Passive permeabil- cell death-inducer lysosomal membrane permeabilization (LMP) [42]. ity across lipid membranes is correlated positively with lipophilicity This phenomenon was detected by the formation of a punctate pattern and negatively with polarity; thus, high logD (pH 7.4) and low TPSA (to- of Galectin 1 (LGALS1) (Fig. 5C and S6C), as a result of its translocation pological polar surface area) predicts high passive diffusion across bio- from the cytosol to damaged lysosomes. As a consequence of LMP, lyso- logical membranes [39]. Ion trapping is generally responsible for drug somal cathepsins are released to the cytoplasm [43], as shown in Fig. 5D accumulation in lysosomes and mitochondria, where protonation of and S6D. Likewise, α-tocopherol, a molecule reported to protect lyso- the amines present in CADs are driven by pH and/or ΔΨ [47]. In concor- somes against CADs based on their redistribution to non-lysosomal dance with the dual effect on lysosomes and mitochondria, ANHAs had membranes and the stabilization of the lysosome membrane itself high logD (Fig. 7A) and an intermediate-low TPSA value (Fig. 7B). In- [44,45], strongly reverted the cytotoxicity induced by ANHAs (Fig. 5E deed, ANHAs clustered when these two parameters were analysed, and S7A). In concordance with the increase of the lysosomal compart- showing a different profile as compared to non-ANHA antihistamines ment upon ANHA treatment, the autophagic flux augmented in the (Fig. 7B); further confirming the relevance of physicochemical proper- presence of these drugs as measured by CytoID staining by flow cytom- ties rather than specific molecular recognition parameters for their an- etry (Fig. 5F and S7B). Indeed, the amount of LC3-II, a canonical marker tineoplastic potential (Fig. 7C). for autophagic activity [46], was higher in cells treated with ANHAs, both in AML cell lines (Fig. 5G) and primary AML patient samples (Fig. S7C). However, the autophagy inhibitor 3-methyladenine (3-MA) 4. Discussion was unable to revert the antileukaemic effect of ANHAs (Fig. 5H and S7D). Thus, ANHAs present lysosomotropism and induce autophagy, al- Despite high complexity and heterogeneity, several alterations in though alternative cell death pathways might be involved. cell organelles are thought to be shared across AML patient cells as com- pared to their healthy counterparts, including an enlarged lysosomal size and frailty [40,49] and a plethora of mitochondrial changes, such 3.5. ANHAs damaged the mitochondrial compartment as an increased ROS content [50–53] and dysfunctional metabolism [54], rendering AML cells more vulnerable to drugs targeting those cel- Prior to lysosomes, CADs may accumulate in the mitochondrial com- lular compartments. In an in silico screening searching for small mole- partment [47]. Also, LMP can activate the intrinsic apoptosis pathway cules that might revert an early transformation signature in AML, [48]; consequently, the mitochondrial status was investigated. Mito- several antihistamines were identified as highly-effective and selective chondrial oxidative stress measured by the generation of reactive oxy- antileukaemic agents by a dual mitochondrial-lysosomal targeting. gen species (ROS) was induced by ANHAs (Fig. 6A and S8A) and the Indeed, this group of antihistamines (ANHAs) were effective against dif- ROS scavenger N-acetylcysteine (NAC) cytoprotected AML cells in ferent types of lymphoblastic and myeloid neoplasias, highlighting the their presence (Fig. 6B), in a ROS-dependent manner (Fig. S8B). The mi- potential clinical utility of mitochondrial-lysosomal disrupters for the tochondrial membrane potential (ΔΨm) was also severely dissipated treatment of leukaemia. upon treatment (Fig. 6C and S8C), suggesting that ANHAs also affected As the transformation process leads, in general, to an increase in the the mitochondrial functionality. Both ROS generation and loss of ΔΨm lysosome biogenesis and mass, an alteration in the sphingolipid metab- leads to the activation of the apoptosis-associated executioner caspases olism, and an augmentation of ROS [55,56], these antihistamines acted 3 and 7. Consequently, caspases 3/7 were activated after ANHA also against different types of leukaemia. Mitochondria can be 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 b .05; **or## p b .01; *** p b .001; **** p b .0001 (t-tests). J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 227 150 AML patients 150 AML patients DAY 3 #CFU-B refer to control *** **** *** ** **** **** **** *** **** **** 100 **** 100 **** **** **** **** **** **** 50 50 0 0 Ø Loratadine Rupatadine Ebastine Terfenadine Ø Lora Rupa Ebas Terfe 150 DAY 6 100 **** **** **** **** **** AML#1 AML#9 **** AML#2 AML#10 **** **** **** AML#3 AML#11 **** AML#4 AML#12 50 AML#5 AML#13 AML#6 AML#14 0 AML#7 AML#15 AML#8 AML#16 Ø Loratadine Rupatadine Ebastine Terfenadine d c Healthy buffy coat Cord Blood Cord Blood Control 200 150 60 * Lora #CFUs refer to control DAY 3 Rupa 150 %CFU subtype 100 40 Ebas Terfe 100 * ** * 50 20 50 * 0 0 0 Total CD13+ CD19+ CD3+ C L20 R E10 T5 GEMM GM G M BFU-E Control e 300 Lora 150 150 %viability reduction DAY 6 AML BC A/H #live cells refer to control Rupa #CFUs refer to control #live cells refer to control Buffy coat Cord Blood E5 63.1 3.9 16.2 Ebas AML AML E10 83.4 34.8 2.4 200 Terfe 100 100 L20 33.8 8.0 4.2 R20 57.7 27.1 2.1 %CFU reduction 100 * 50 50 AML CB A/H ** * ** E5 45.3 3.7 12.2 E10 90.9 7.8 11.7 L20 37.8 20.6 1.8 0 0 0 R20 37.7 4.1 9.2 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 b .05; ** p b .01; *** p b .001; **** p b .0001 (Mann-Whitney tests). 228 J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 150 AML patient %hCD45+ cells in BM Control Ebastine Rupatadine refer to control 100 28.0% 0.3% 2.7% **** SSC 50 **** 0 Ø Ebas Rupa Lora hCD45 b 150 ** Cord blood Control Ebastine Rupatadine %hCD45+ cells in BM 37.0% 45.3% 40.4% refer to control 100 ** SSC 50 0 hCD45 Ø Ebas Rupa c d Cord Blood Control 150 AML 150 Ebas %engraftment reduction % human marker positive cells refer to control %hCD45+ cells in BM Rupa AML CB A/H * refer to control 100 100 E10 93.4 26.6 3.5 *** R10 52.0 -34.7 N/A 50 50 0 0 Ebas Rupa 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 b .05; ** p b .01; *** p b .001; **** p b .0001 (t-tests). successfully targeted at different levels in acute [57,58] and chronic leu- effects. ANHAs showed an interesting collaborative effect with currently kaemias [59]. Indeed, the more primitive leukaemic cell fraction is usu- used cytarabine; thus, sensitizing leukaemic cells to conventional ally the most sensitive to mitochondria-targeted drugs [58–61]. therapy. Sensitivity to mitochondrial damage is thought to be a common prop- The role of antihistamines in cancer has been controversial mainly erty not only of LSCs but also of other cancer stem cell populations due to the lack of consistency across different HRH1 inhibitors and [62,63]. Similarly, different effective preclinical therapeutic approaches the absence of a defined role of its well-described HRH1 molecular tar- have been described for lysosome destabilization in leukaemia get on cancer cells [15]. In haematological neoplasias, several antihista- [40,64–66]. ANHAs damage mitochondria and lysosomes, targeting mines have been shown to affect leukaemia cell viability to different two fragile organelles in cancer cells. Although an extensive crosstalk extents [17,68–71]. All of them display CAD-associated properties between mitochondria and lysosomes is found in cancer cells [67], (high logD pH 7.4 and intermedium-low TPSA) compatible to the dual simultaneous targeting may increase efficacy and specificity, con- model proposed. Interestingly, those antihistamines that presented no stituting a clinical opportunity for selective therapeutic approaches. antileukaemic effect could be predicted by our model [17,68,69,71]. As Due to the inter- and intrapatient heterogeneity and the darwinian na- most CADs, ANHAs are lysosomotropic drugs that induce irreversible ture of leukaemia, combination therapies are likely to provide high damage to lysosomes [40,41]. Recently, certain tropism to mitochondria specificity in low concentration ranges, reducing toxicity and side has been reported for the CAD azidobupramine [47]. Here, a dual J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 229 b HL-60 HL-60 KG-1 MM Control KG-1 HRH1 c d 150 KG-1 150 non-ANHA 11 Control #live cells refer to control ANHA #live cells refer to control ns +Histamine 10 100 HRH1 pKi 100 ns 9 ns ns ns ns 8 50 50 R2=0.089 7 ns 0 0 6 C L5 L10 R5 R10 E5 E10 6 7 8 9 10 11 ANHA non-ANHA HRH1 pKi e f g 100 100 100 #live cells refer to control #live cells refer to control Myeloid Ebastine 10 µM Myeloid lineage R2=0.018 %HRH1+ cells T cell p=0.269 T lineage B lineage B cell 50 50 50 **** **** **** **** *** **** **** **** **** **** **** **** **** **** 0 0 0 0 20 40 60 80 T 2 3 6 S M M 1 02 6 A 60 1 -2 JN 6 2 26 %HRH1+ cells P A O T E G M 5 2 4 L H K N L M C U -8 I8 K K J B T H R A A H M U I R R M P J G P R R 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 b .05; ** p b .01; *** p b .001; **** p b .0001 (t-tests). lysosome/mitochondria targeting is described for ANHA in leukaemic were assayed [72]. ANHAs have high apparent volumes of distribu- cells, prompting the activation of the lysosome-mediated autophagy tion [44,72], which facilitates an extensive distribution through all and the intrinsic apoptosis, and probably multiplying the selectivity the organs of the body because of their liposolubility and non- windows given by tumoral alterations (Fig. 7), enabling the develop- ionization. However, low distribution volumes compatible with the ment of highly effective therapies with reduced side effects. therapeutic objective are desirable to avoid side effects and increase Clinical data from the Danish Cancer Register revealed that well- effectiveness. Additionally, ANHAs are quickly metabolized in the tolerated CAD antihistamines combined with the standard cancer che- body and metabolites are not predicted to be active against leukae- motherapy regimen improved survival of solid tumor patients. Remark- mia according to the proposed model. Thus, the clinical use of ably, no clinical benefit was observed when non-CAD antihistamines ANHAs will require an extensive pharmacological optimization to 230 J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 Lysotracker MFI refer to control 250 ** Control 200 *** * * 150 100 50 Ebas 0 Ø Eb Ru Lo Ch c Hoechst LGALS1 Merge d DIC Hoechst CTPSB Merge %cells with LGALS1 puncta 40 * Control Control 30 20 10 Ebas Ebas 0 Cont Ebas 5 Ebas 7.5 e 150 f g LC3B-II/GAPDH band intensity THP1 KG1 #live cells refer to control 250 64 CytoID MFI refer to control MM HL60 32 100 200 ** refer to control 16 Ø EB RU LO CHL 150 *** ** **** 8 LC3-I 15 kDa LC3-II 50 100 4 40 kDa 2 50 0 1 Toco 0 Control Ebas Lora Rupa 0.5 ANHA Ø Eb Ru Lo Ch Lora Rupa Ebas h 150 #live cells refer to control 100 ns ** ** 50 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 b .05; ** p b .01; *** p b .001; **** p b .0001 (t-tests). J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 231 Rhodamine 123I refer to control Mitosox MFI refer to control mitROS 800 150 150 #live cells refer to control *** 600 * * * 100 100 ** 400 **** *** *** *** ** **** **** 50 ** ** 50 **** 200 ******* ******** **** 0 0 0 Ø Eb Ru Lo Ro NAC Ø Eb Ru Lo + Control Ebas 5 Ebas 10 d e f 48h apoptosis Luminiscence (RLU) refer to control 600 Executioner caspases 150 100 **** #live cells refer to control Control %Annexin V+ cells +Z-VAD-FMK 400 100 *** **** ns ns **** ** 50 * 200 50 ns 0 0 0 Ø Lo Ru Eb Et Cont Eb 5 Eb 10 Lora Rupa Ø 1 µM 5 µM 10 µM g Marker induction refer to control Marker induction refer to control Marker induction refer to control Caspase activation 1024 512 Loratadine 10 µM 1024 Rupatadine 10 µM Ebastine 10 µM Autophagy Lysosomal mass 512 512 256 mtROS 256 256 128 128 128 64 64 64 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 cytom- etry. 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 fre- quency 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 b .05; ** p b .01; *** p b .001; **** p b .0001 (t-tests). improve biodistribution and pharmacokinetics (including metabolism the high metabolism rate, permitting a sustained presence of non- protection). Recently, a new formulation of two commonly used che- catabolized ANHA drugs able to widely reach leukaemic cells. motherapeutics has been approved for AML. CPX-351 (Vyxeos, Jazz Here, a group of physicochemical properties present in some antihis- Pharmaceuticals) is a new liposomal formulation that encapsulates tamines has been defined for specifically passive targeting of leukaemic cytarabine and daunorubicin in a fix molar rate. Cytarabine and dau- cells, particularly the most primitive cellular subpopulation of LSCs. The norubicin are rapidly metabolized after administration and become antineoplastic effect is mediated by the concurrent disruption of lyso- undetectable within the first hour. However, CPX-351-encapsulated somal and mitochondrial homeostasis in cancer cells, which are more cytarabine and daunorubicin administration produce sustained sensitive than their healthy counterparts. This therapeutic strategy plasma levels of both drugs for several hours, protecting both seems to be highly specific and widely safe. However, drug delivery op- drugs from metabolism and elimination. Moreover, leukaemic cells timization is needed for achievement of the therapeutic dose at the can- exhibit preferential uptake of CPX-351 liposomes when compared cer cell localization. Targeting the mitochondrial and lysosomal with healthy cells [73–75]. Therefore, future clinical development of compartment in leukaemic cells holds great promises in the fight a liposome-based encapsulated form of ANHA drugs might overcome against leukaemia as it provides an alternative pathway to currently 232 J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 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 logD7,4 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 logD7,4 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. used chemotherapy that engages alternative cell death programs to Bancària La Caixa, grants from CERCA Programme, during the conduct which leukaemic cells are sensitive. of the study; and RMR is a shareholder of Leukos Biotech. Author contributions Acknowledgements JMCM, designed and performed experiments, analysed the data, and We would like to thank all members of the Risueño Laboratory for wrote the manuscript. ABM and JMC, performed experiments and helpful discussions and technical support, and Martí Duran-Ferrer analysed the data. MAT, FG and JE, provided patient samples and clinical (IDIBAPS, Barcelona, Spain) for assistance with bioinformatics analysis. data. LCC, assisted with experimental work. RMR, designed the project, We would like to acknowledge the technical help of the Advanced Op- analysed the data, interpreted results and wrote the manuscript. All au- tical Microscopy Unit (Scientific and Technological Centers of the Uni- thors discussed the results and commented on the manuscript. versity of Barcelona-CCiTUB, Campus Medicine Clínic, Barcelona, Spain). RMR is supported by Ramón y Cajal program of the Spanish Min- Declaration of Competing Interest istry of Economy (RYC-2011-07998 and IEDI-2016-00740), and JMCM is supported by Fundación Mutua Madrileña. This work was funded by Dr. Muñoz Risueño reports grants from Ramón y Cajal program, the Fundación Mutua Madrileña (RMR), CaixaImpulse CI16-00003 grants from Fundación Mutua Madrileña, grants from CaixaImpulse, (RMR), SAF2015-66721-P from MINECO (RMR), the Josep Carreras grants from MINECO, grants from Josep Carreras International Leukae- International Leukaemia Foundation (RMR), l'Obra Social “La Caixa”- mia Research Foundation, grants from Obra Social La Caixa-Fundació Fundació Bancària “La Caixa” (RMR), and CERCA Programme/ J.M. Cornet-Masana et al. / EBioMedicine 47 (2019) 221–234 233 Generalitat de Catalunya (IJC). RMR had full access to all the data in the [24] Jangi SM, Díaz-Pérez JL, Ochoa-Lizarralde B, Martín-Ruiz I, Asumendi A, Pérez-Yarza G, et al. H1 histamine receptor antagonists induce genotoxic and caspase-2- study and had final responsibility for the decision to submit for publica- dependent apoptosis in human melanoma cells. Carcinogenesis 2006;27:1787–96. tion. 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