Targeting the Cellular Schizophrenia. Likely Employment of the Antipsychotic Agent Pimozide in Treatment of Refractory Cancers A

Critical Reviews in Oncology / Hematology 128 (2018) 96–109

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Critical Reviews in Oncology / Hematology

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Targeting the cellular schizophrenia. Likely employment of the T antipsychotic agent pimozide in treatment of refractory cancers and glioblastoma ⁎ Ilhan Elmacia, Meric A. Altinozb,c, a Memorial University, Neuroacademy Group, Istanbul, Turkey b Neurooncology Branch, Neuroacademy Group, Istanbul, Turkey c Department of Psychiatry, Maastricht University, Holland, Turkey

ARTICLE INFO ABSTRACT

Keywords: Pimozide is currently being used in clinic as a neuroleptic and exerts versatile biological actions. Pimozide is a Pimozide cationic amphiphilic drug (CAD); CADs block the synthesis of neutral lipids, impair cholesterol homeostasis of Refractory cancers cancer cells and increase accumulation of diacylglycerol-3-phosphate. Pimozide exerts tumoricidal activity Glioblastoma which was first shown for melanoma and neuroblastoma via proposed anti- dopaminergic effects. Recently, pancreas cancers are shown to elevate dopamine receptor-2 synthesis, which is blocked by pimozide leading growth inhibition. Besides binding to inner mitochondrial membrane and reducing cellular respiration, pimozide also inhibits calmodulin, T-type calcium channels and σ-receptors which all correlate with tumor-inhibitory functions. Pimozide also exerts chemotherapy and radiotherapy-sensitizing effects in cancer cells and acts as an inhibitor of STAT-3 and STAT-5 signaling proteins with potential activity in leukemia, liver and prostate cancer. Pimozide also blocks stem cell features and Wnt-β/catenin signaling in liver cancer. Pimozide interferes with Fatty Acid Protein Binding-4 and activates PPAR-γ and it was proposed to alleviate cancer cachexia. Besides mechanisms of calmodulin and σ-receptor associated pathways, pimozide was proposed to inhibit glioblastoma via serotonin receptor 5-HT7. Pimozide is a selective inducer of autophagy and also inhibits ubiquitine specific protease (USP-1) which may associate with its chemosensizing potential in lung cancer and glioblastoma. Via versatile mechanisms of tumoricidal actions and due to its highly traversing capability through the blood-brain barrier, pimozide highly deserves to be studied in animal models of drug resistant refractory cancers and glioblastoma, which have very poor prognosis.

1. Introduction. The cancer problem and targeting cancer with et al., 2017). Hence, there is an urgent need to develop novel innovative Neuroleptic/ antipsychotic drugs strategies to treat metastatic and chemorefractory cancers, cancers with a generally poor prognosis (eg. pancreas and lung cancers) and glio- Despite immense efforts to discover novel anticancer agents; in blastomas. Interestingly, many studies showed decreased risk of cancer 2012, about 14.1 million new cases of cancer occurred globally, causing in schizophrenia patients; and despite most of these patients smoke around 8.2 million deaths (World Cancer heavily, metaanalysis studies also found that the pooled overall cancer Report 2014. World Health Organization (WHO) 2014. pp. Chapter incidence rates for patients with schizophrenia did not parallel their 1.1; The top 10 causes of death Fact sheet No:310. WHO. 2014). The cancer risk factor exposure (Wiklund et al., 2010). Very noteworthy, a financial costs of cancer were estimated at $1.16 trillion US dollars per comprehensive in vivo screen of 297 central nervous system acting year as of 2010 (World Cancer Report 2014. WHO. 2014. pp. Chapter compounds revealed 6.7). Many agents were developed to block the growth of malignancies, that psychoactive agents were 18-fold more likely to exert anti- yet metastatic cancers are systemic diseases which remain manageable proliferative potential when compared with a random population of but incurable. Glioblastomas have the highest incidence among primary molecules (Wiklund et al., 2010). Many subsequent investigations on brain tumors, second only to meningioma with a very poor prognosis (a cancer cell lines confirmed that especially neuroleptic agents as a class 5-year survival rate of about 4% from the time of diagnosis) (Altinoz possesed cytotoxic potential. Tumoricidal actions occurred in a wide

⁎ Corresponding author at: Nurtepe Mh. Guven Sk. No:5, D:6 Kagithane, Istanbul, Turkey. E-mail address: [email protected] (M.A. Altinoz). https://doi.org/10.1016/j.critrevonc.2018.06.004 Received 20 February 2018; Received in revised form 16 May 2018; Accepted 6 June 2018 1040-8428/ © 2018 Elsevier B.V. All rights reserved. I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109

dopamine D2 receptor with a high affinity, which causes both adverse effects (eg. extrapyramidal) and therapeuticity against the positive symptoms of schizophrenia. But as will be suggested, pimozide seems also to act beneficial on negative symptoms of schizophrenia in contrast to other typical neuroleptics. The side effects of pimozide include akathisia, tardive dyskinesia, prolongation of the QT interval, and rarely, neuroleptic malignant syndrome (Zemrak and Kenna, 2008). Diphenylbutylpiperidine class neuroleptics (Fig. 3) including pimozide, fluspirilene, penfluridol, and clopimozide, exert structural si- milarity to verapamil-like Ca+2-channel antagonists and inhibit 3H-ni-

trendipine binding with IC50 values of 13–30 nM (nitrendipine is a dihydropyridine class Ca+2-channel antagonist) (Gould et al., 1983). This inhibition involves receptors for the verapamil/prenylamine class of Ca+2-channel antagonists. These diphenylbutylpiperidines also inhibit K+-induced Ca+2-dependent contractions of rat vas deferens at concentrations of 40–350 nM. Other phenothiazine and butyrophenone neuroleptics lack such potent Ca+2-antagonist actions (Gould et al., 1983). As suggested, diphenylbutylpiperidines also differ from other typical neuroleptics in their efficacy to relieve negative symptoms of schizophrenia, such as emotional withdrawal, which were proposed to relate to an inhibition of voltage-operated Ca+2 channels (Gould et al., 1983). T-type (“T” for transient) Ca+2 channels are low-voltage activated Ca+2-channels that open during membrane depolarization and are ex- Fig. 1. Molecular Structure of Pimozide. pressed in diverse tissues, including neuronal, cardiovascular, and endocrine, which regulate the development, maintenance, and repair of range of human cancer cell lines, including those employed in the these tissues (Kopecky et al., 2014). Although low-voltage-activated +2 National Cancer Institute’s screening program, and first generation Ca -channels have limited amino acid homology to the high-voltage- +2 antipsychotics generally appeared to be more cytotoxic than second activated Ca -channel families, which include L-type channels, all +2 generation antipsychotics (Wiklund et al., 2010). The repurposing of voltage-gated Ca -channels are structurally related (Uebele et al., +2 “ ” non-cancer drugs by 2009). The L-type Ca -channel ( L for long-lasting) is part of the +2 screening for novel anti-cancer activity is a plausible and powerful high-voltage activated family of voltage-dependent Ca channels. L- 2+ strategy to fastly determine new cancer therapeutics from the known type Ca channels (Cav1) represent one of the three major classes – +2 pool of small molecules that have already undergone rigorous tests via (Cav1 3) of voltage-gated Ca -channels as the target of clinically used +2 clinical trials for human safety, thus potentially fast-tracking the de- Ca -channel blockers including dihydropyridines (Striessnig et al., velopment and approval process, while reducing life-threatening risks 2014). In almost all electrically excitable cells one or more of these L- 2+ (Fako et al., 2016). In this manuscript, we will discuss the powerful type Ca channels isoforms is expressed. Hence, diverse functions potentials of the neuroleptic pimozide (Fig. 1) as a novel anticancer including muscle, brain, endocrine, and sensory functions depend on 2+ agent against refractory cancers and glioblastoma. proper L-type Ca channels activity and even small changes of their expression or activity may be associated with psychiatric disease or cardiac arrhythmias (Striessnig et al., 2014). Pimozide is a potent in- 2. Pimozide. Chemical structure and interactions with cellular hibitor of T-type Ca2+ channels but with less selectivity than mibe- biology fradil; in pituitary and heart cells, pimozide inhibits L-type Ca2+ channels, whereas in adrenal glomerulosa and spermatogenic cells, it Antipsychotics also known as neuroleptics or major tranquilizers, blocks T channels and Ca2+ influx (Bertolesi et al., 2002). The actions are medicines primarily employed to treat psychosis principally in of pimozide and mibefradil are not restricted to Ca2+ channels but may − schizophrenia (the terms “neuroleptic” and “antipsychotic” are used also affect other ion channels, including K+ and Cl channels (Bertolesi interchangeably in this review). Pimozide (depicted in Fig. 1)isa et al., 2002). neuroleptic of the diphenylbutylpiperidine class which was discovered Pimozide is a strong inhibitor of dopamine as well as the release of in 1963 by the physician Paul Janssen (Awouters and Lewi, 2007; melanocyte stimulating hormone (MSH), corticotropin releasing factor Mothi and Sampson, 2013). Pimozide has neuropsychiatric indications and gonadotropin releasing hormone (GnRH) in rats (Krummel et al., for paranoid personality disorder, delusional disorder (Munro and Mok, 1982). In old clinical studies, pimozide was employed to treat gen- 1995), delusions of parasitosis (Driscoll et al., 1993) and for the eralized lipodystrophy. Following this treatment, corticotropin and Tourette syndrome and resistant tics (Termine et al., 2013; Yang et al., GnRH were markedly reduced; over a period of time cutaneous hy- 2016). Pimozide is an antagonist of the dopamine D2 receptor subfmily perpigmentation was eliminated (Krummel et al., 1982). Lipotropin is a (D2, D3 and D4 receptors) and the 5-HT7 serotonin receptor (Fig. 2). prohormone synthesized inside the adenohypophysis, which promotes

Pimozide exerts high affinity for dopamine receptors (Kis = 2.4, 0.2, both fat mobilization and skin darkening by stimulation of melanocytes. and 1.8 nM for D2,D3, and D4, respectively). As will be partly detailed Hence, pimozide induced beneficial effects in lipodystrophy can be below, pimozide also displays actions as an antagonist, inverse agonist, ascribed to alterations in lipotropin but it is also likely that the changes or channel blocker, with lower affinities, at serotonin, α-adrenergic, in releasing hormones were responsible (Krummel et al., 1982). Con- muscarinic cholinergic, and histamine receptors and calcium and so- genital generalized lipodystrophy (also called Berardinelli-Seip con- dium channels. A detailed description of pimozide’s relative affinities to genital lipodystrophy) is a rare disease manifested by an almost total diverse receptors is provided in Table 1 and in vivo antitumor actions of lack of adipose tissue in the body and a very muscular appearance. A pimozide are summarized in Table 2. Pimozide also inhibits hERG shortage of dermal adipose tissue leads to the storage of fat in the liver (human Ether-a-go-go-related) K+-Channel (Kongsamut et al., 2002). and muscles and causes insulin resistance. As will be suggested, pimo- As in the case for other typical antipsychotics, pimozide binds the zide is shown to stimulate PPAR-γ pathway (Fig. 4) (a feature of insulin

97 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109

Fig. 2. Pimozide binding and inhibition of Dopamine D2, D3 and D4 Receptors. Structure and Classiﬁcation (http://www.scienceofeds.org/2013/01/05/dopamine- and-anorexia-nervosa-tackling-the-myths-part-1-intro/).

Table 1 Relative binding aﬃnity of pimozide to different receptors. 5-HT: Serotonergic receptor, α-1: α-1 A Adrenergic receptor, M3: Muscarinic acetylcholine receptor, D: Dopaminergic Receptor, hERG: Ether-à-go- go-Related Channel, H1: Histamine H1 Receptor, σ: Sigma Receptor.

Protein Ki (nM)

5-HT1A 650

5-HT2A 48.4

5-HT2C 2,112

5-HT6 71

5-HT7 0.5 Fig. 3. General Molecular Structure of Diphenylbutylpiperidines.

α1A 197.7

α2A 1,593

α2B 821 prominent accumulation of phosphatidate (diacylglycerol 3-phosphate)

α2C 376.5 (Pappu and Hauser, 1981). Also in lymphocytes, a number of CADs M3 1,955 enhanced phosphatidylinositol biosynthesis at the expense of neutral D1 > 10,000 lipids. Chronic administration of 4,4’-bis (diethylaminoethoxy) α,β- D 0.33 2 diethyldiphenylethane caused formation of secondary lysosomes and D3 0.25

D4 1.8 increases in the hepatic lysosomal lipid with accumulation of an unu- hERG 18 sual phospholipid, bis(monoacylglycero) phosphate (Pappu and H1 692 Hauser, 1981). σ 508 CADs inhibit cytoplasmic phosphatidate phosphohydrolase, a key enzyme in the biosynthesis of lipids. CADs of varied clinical use in- fi fl Table 2 cluding pimozide were analyzed to de ne their in uence on the la- 32 In vivo antitumoral actions of pimozide in humans and experimental animals. beling with Pi of cerebral cortex phospholipids (Pappu and Hauser, 1981). The altered phospholipid labeling patterns were similar, the ff ’ Pimozide Induced Anticancer E ect. Patient s Findings, References dominant features being decreased incorporation into phosphati- Trials and Animal Experiments dylcholine and increased incorporation into phosphatidic acid. Relative Case Report – Regression of Melanoma with Pimozide Taub and Baker, 1979 potencies were: ( ± )-propranolol > chlorpromazine = 4,4’-bis(die- Phase II Clinical Trial – Activity of Pimozide in Human Neifeld et al., 1983 thy1aminoethoxy) α,β-diethyldiphenylethane > desipramine > Melanoma dibucaine > pimozide > oxymetazo- Inhibition of murine neuroblastoma growth by McGrath and Neifeld fl dopamine antagonists (1984) line = fen uramine = haloperidol = chloroquine > Inhibition of pancreatic cancer growth in mice by Jandaghi et al., 2016 amphetamine = control (Pappu and Hauser, 1981). In dopamine re- Dopamine Receptor D2 antagonism ceptor (D2)-negative platelets, pimozide exerted different effects on polyphosphoinositide cycle metabolites and it has been proposed that CADs affect the activity of membrane-bound proteins by intercalation sensitizing glitazone anti-diabetics) and interfere with fatty acid me- among anionic glycerophospholipids in the inner leaflet of the plasma fi tabolism, which may be of bene t in terms of managing cancer ca- membrane (Oruch et al., 2008). Lipidosis and cellular accumulation of chexia. phospholipids are also witnessed in antidepressant- and progesterone- Pimozide is also a cationic amphiphilic drug (CAD) and CADs in- treated glioblastoma cells and it has been proposed that lipidosis may ’ cluding chloroquine, amiodarone, perhexiline, 4,4 -bis(diethy1ami- associate with growth indolency and enhanced chemosensitivity of α β noethoxy) , -diethyldiphenylethane induce lipidosis, which is mani- these tumors (Altinoz et al., 2007). Hence, pimozide may act similar to fested by accumulation of membranous lysosomes and enhanced levels antidepressants and progesterone in induction of lipidosis and asso- of polar lipids in tissues indicating disturbed phospholipid metabolism ciated inhibition of glial tumor growth via its CAD structure. Indeed, 29 (Pappu and Hauser, 1981). For instance, in liver slices, CADs block the years after pioneering studies of Pappu and Hauser which showed synthesis of triacylglycerol and phosphatidylcholine and produced a

98 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109

Fig. 4. Pimozide binding and activation of PPAR-γ Pathway. (From: Jack Vanden Heuvel, PennState College of Agricultural Sciences, http://nrresource.org/nuclear_ receptors/peroxisome-proliferator-activated-receptor-gamma-pparg-nr1c3/) NRR Science. pimozide actions as a cationic amphiphilic drug, Wiklund et al. de- witnessed a remission of measurable disease (Taub and Baker, 1979). A monstrated that tumoricidal actions of pimozide may associate with its 60-year-old woman had a pigmented melanotic mole removed from her CAD structure and perturbation of cholesterol homeostasis, which will right leg and recurrent metastases were resected from lymph-nodes in be discussed below (Wiklund et al., 2010). the right groin 12, 17, and 23 months later. After 27 months, the patient Penfluridol (16 μM), pimozide (20 μM) and trifluoperazine (66 μM) became systemically ill and multiple liver and lung metastases were were shown to bind the mitochondrial inner membrane with high af- seen on liver scan and chest X-ray (Taub and Baker, 1979). Dacarbazine finity and to inhibit ADP-stimulated respiration in isolated rat liver 850 mg/m2 was given as a single intravenous dose, but 3 weeks later a mitochondria (Ruben and Rasmussen, 1981). These drugs did not exert new lung lesion had appeared and the other metastatic lung lesions had any effect on either uncoupler- or Ca2+ stimulated respiration; and the increased in size (Taub and Baker, 1979). Thereafter, oral pimozide was inhibition of ADP-stimulated respiration seemed to result from started 4 mg daily for 1 week, 8 mg/day for the second week, and 12 mg blockage of the oligomycin sensitive ATPase independent from inhibi- daily after that (Taub and Baker, 1979). After 4 weeks of pimozide (7 tion of calmodulin (Ruben and Rasmussen, 1981). It has been long weeks after the dose of dacarbazine) lung lesions were smaller by believed that cancer cells mostly rely on glycolysis for gaining energy 1–2 mm. 1 week later, lung lesions were smaller by 50%, and serum- due to the Warburg effect, yet recent studies revealed that cancer cells alkaline-phosphatase had fallen from 194 to 98 i.u./I (normal range also rely heavily on mitochondrial oxidative phosphorylation (OX- 30–85). After 8 weeks of pimozide therapy (11 weeks after dacarba- PHOS) for growth and survival (Deng and Haynes, 2017). Glycolysis zine) lung lesions were no longer visible and a liver scan showed greater alone may not be sufficient for highly increased energetic requirements than 50% reduction in the size of lesions (Taub and Baker, 1979). Side- of malignant cells; and hence pimozide inhibition of mitochondrial effects included lethargy, xerostomia, and xerophthalmia. Pimozide respiration may also help to limit cancerous growth. Indeed, a relatively dosage has been tapered off and stopped, and the patient remained in recent gene set enrichment analysis (GSEA) revealed that cancer cell remission 20 weeks after starting pimozide (Taub and Baker, 1979). sensitive to pimozide-induced growth inhibition exhibit over expression Melanin synthesis starts with the binding of melanocyte stimulating of tricarboxylic acid cycle (TCA)-related discriminating genes, IDH3A, hormone (MSH) to a cell surface receptor with subsequent release of c- IDH1, IDH3G, ACO2 and ACLY (Covell, 2012). The precise lethal dose of AMP which triggers the hydroxylation of phenylalanine to tyrosine, pimozide in humans is unknown, the oral LD50 is 228 mg/kg in mice, which is again hydroxylated to form L-dopa (Krummel et al., 1982). L- 5120 mg/ kg in rats, 188 mg/kg in guinea pigs, and 40 mg/kg in dogs dopa may then undergo decarboxylation to form dopamine or may (Chen et al., 2017). This indicates a very high safety window for pi- undergo a series of redox reactions to form an indole-quinone ring mozide, if its lethal dose is similar to dogs, 2.8 g might cause lethality in which polymerizes to form melanin (Krummel et al., 1982). This humans; on the other hand, a relatively very lower dosage (12 mg) pathway appears to be unique to melanocytes and melanomas and some caused regression of metastatic melanoma, which will be mentioned observations indicated that manipulation of this pathway may change below. the growth of some melanomas. Previous studies indicated that pharmacologic levels of melanin intermediates (L-dopa, dopamine, and their analogues) may block macromolecular synthesis in some melanoma cell 3. Pimozide. First studies showing its anticancer activity in cultures and enhance survival in tumor-bearing mice (Krummel et al., melanoma and neuroblastoma 1982). On this background, a potent antidopamine drug, pimozide was analyzed in murine melanomas and its antiproliferative effects were In 1979, Taub and Baker employed pimozide to treat a post- correlated with macromolecular synthesis using assays for competitive menopausal woman with metastatic pigmented melanoma and

99 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109 dopamine binding activity (receptors) and melanin synthesis (Krummel diagnosed with pancreatic cancer, ∼85% harbour pancreatic ductal et al., 1982). Three murine melanomas (Fl, F10, B16) were in- adenocarcinoma (PDAC); and unfortunately, most of them succumb to vestigated. The amelanotic B16 cell line showed no inhibition by dopa, their disease within 6 months after diagnosis. Jandaghi et al. performed dopamine, or pimozide when assayed for 14C-Leu or 3H-TdR in- microarray analysis to analyze gene expression profiles of 195 PDAC corporation (Krummel et al., 1982). Using a competitive binding assay, and 41 non-tumor pancreatic tissue samples (Jandaghi et al., 2016). only low levels of dopamine binding were present. The very melanotic They also employed an extensive analysis of PDAC transcriptome by F1 cell line showed no inhibition by dopa or dopamine, but pimozide superimposing interaction networks of proteins encoded by aberrantly inhibited 14C-Leu and 3H-TdR (indicative of S-phase/DNA synthesis/ expressed genes over signaling pathways associated with PDAC. In an cell proliferation) incorporation in a dose-response manner; 50% in- analysis of 38,276 human genes and loci, they defined 1676 genes − hibition was witnessed at 10 9 M concentration with no loss in cell which expressions were significantly increased and 1166 genes which viability or changes in cell numbers (Krummel et al., 1982). Competi- expressions were significantly decreased in PDAC, in comparison to tive dopamine binding was present (19 pmoles per g of wet tissue) with non-tumor pancreatic tissues (Jandaghi et al., 2016). Notably, one gene a Kd of 0.2 nM, figures approximating those seen in dog caudate nu- that was upregulated and linked to diverse signaling pathways that are cleus controls. The F10 line, with melanin production between the B16 dysregulated in PDAC was G protein Subunit alpha i2 (GNAI2). GNAI2 and F1, was intermediate in terms of inhibition of 14C-Leu and 3H-TdR mediates the effects of dopamine receptor D2 (DRD2) on cAMP-sig- incorporation and dopamine binding (Krummel et al., 1982). Purified naling; PDAC tissues had a slight but significant increase in DRD2 melanin did not bind and, thus, does not appear to explain the obtained mRNA. However, tissue microarray analysis of DRD2 across 40 normal binding results. These findings indicate that pimozide inhibits 14C-Leu pancreatic tissues, 63 PDAC sections and 49 samples of chronic pan- and 3H-TdR incorporation in murine melanoma cells and that compe- creatitis (CP, associated with high risk of PDAC) illuminated that DRD2 titive dopamine binding (receptor) correlates with this inhibition protein levels were significantly increased in PDAC and CP in com- (Krummel et al., 1982). Neifeld administered pimozide to previously parison to benign tissue samples (> 3-fold), with the highest level de- treated metastatic melanoma patients (Neifeld et al., 1983). Among the tected in PDAC (Jandaghi et al., 2016). Most importantly, RNAi 24 evaluable patients, two exerted complete response, two exerted knockdown of DRD2 or blockage with pharmacologic antagonists (pi- partial response, and two had disease stabilization. Responses were mozide and haloperidol) decreased proliferation of pancreatic cancer witnessed in soft tissue, lymph nodes, liver, and lung and toxic effects cells, triggered endoplasmic reticulum stress and apoptosis, and de- included extrapyramidal manifestations in nine patients and malaise in creased cell migration (Jandaghi et al., 2016). Notably, exposure to seven (Neifeld et al., 1983). pimozide caused a dose-dependent inhibitory effect on cell proliferation Neuroblastomas origin from neural crest cells and are characterized in all by the production of catecholamines, especially dopamine and nora- examined pancreas cancer cell lines (in the range of 5–20 μM, in drenaline. In some studies, dopamine and dopamine analogs (6-hy- some pancreas cancer cells 20 μM of pimozide killed all cells). Pimozide droxydopamine and L-dopa methylester) were shown to demonstrate effects were more prominent in MiaPaCa-2 and BxPC-3 cells, which had tumoricidal activity in neuroblastoma cells (McGrath and Neifeld, higher expression levels of DRD2 among the cell lines, and weaker on a 1985). C-1300 neuroblastoma cells origin from a spontaneously oc- primary normal fibroblast cell line used as a control (Jandaghi et al., curing tumor in albino A/J mice and harbour the catecholamine bio- 2016).The authors have underlined that schizophrenic patients re- synthetic pathway (McGrath and Neifeld, 1984). McGrath and Neifeld ceiving DRD2 antagonist neuroleptics have declined incidence rates of tempted to manipulate this pathway for modifying neuroblastoma cell various cancers, including those of rectum, colon and prostate, and also growth and survival in neuroblastoma-bearing mice, and to correlate that lower cancer incidence rates were reported in patients with Par- these findings with membrane dopamine-binding activity (McGrath and kinson’s disease in which the dopaminergic signaling is hampered Neifeld, 1984). The dopamine antagonists domperidone, pimozide, and (Jandaghi et al., 2016). spiroperidol blocked macromolecular synthesis in vitro as shown by reduced 3H-TdR and 14C-Leu incorporation in a dose-response manner. 5. Pimozide inhibition of breast cancer growth and induction of − Inhibitions of 56, 49, and 43% percent were noted at 10 6 M con- radiotherapy sensitization. Calmodulin, σ-Receptors and t-type centration of each drug, respectively, with no cell death (McGrath and calcium channels Neifeld, 1984). Dopamine agonists exerted no significant inhibition. Scatchard analysis of dopamine binding was consistent with a single Calmodulin (Fig. 5) is a heat stable, calcium-binding protein in- class of receptor sites with a mean concentration of 13.2 ± 2.0 pmole/ volved in versatile biological processes including cellular proliferation g wet weight of tissue and mean dissociation constant (Kd) = 0.69 ± and disassembly of the mitotic apparatus and is increased in rapidly 0.38 nM. Amice injected with 1 × 106 tumor cells and treated with growing liver tumors and transformed fibroblasts (Lee and Hait, 1985). daily pimozide or domperidone had lived significantly longer in com- Pimozide is one of the most potent calmodulin antagonist among the parison to controls (15 versus 8.5 days, p < 0.001) as well as a sig- neuroleptic drugs currently approved for clinical use (Strobl et al., nificant prolongation in overall survival (35 versus 25 days, 1990). Pimozide, thioridazine and calmoduline antagonists W-13, and p < 0.001) (McGrath and Neifeld, 1984). One year later, the same W-12 were analyzed whether these agents are capable to block growth group reported their findings about the actions of domperidone, pi- of MCF-7 human breast cancer cells. All compounds inhibited growth of mozide, and spiroperidol in four human neuroblastoma cell lines MCF-7 cells in vitro (Strobl et al., 1990). The order of potency for (McGrath and Neifeld, 1985). All dopamine antagonists inhibited DNA proliferation blockage in the presence of 2% stripped calf serum was 3 14 synthesis ( H-TdR assay) and macromolecule synthesis ( C-Leu in- pimozide (Ki 2 μM) > thioridazine (Ki 5 μM) > W-13 (Ki 15 μM) > W- corporation) in neuroblastoma cells in a dose-response dependent 12 (Ki 39 μM). Similar concentrations of these compounds also inhibited manner. At least 50% inhibition was found at 1 μM concentration of estradiol-induced growth of MCF-7 cells, but estrogen receptor (ER) each drug (McGrath and Neifeld, 1985). interactions did not seem to be involved (Strobl et al., 1990). Pimozide and thioridazine did not influence the estradiol binding of the MCF-7 4. Dopamine receptor D2 overexpression in pancreas cancer and ER, nor did pimozide interact with the induction of progesterone re- its inhibition with pimozide ceptors by estradiol (Strobl et al., 1990). Furthermore, pimozide also inhibited incorporation of 3H-thymi- Overall five-year survival rate of all cancer patients is 63%, while it dine into MCF-7 cells induced by growth-stimulating protein hormones is about 5% for pancreatic cancer – which remained mostly unchanged in serum-free medium. The Ki for pimozide in serum-free medium for the last three decades (Jandaghi et al., 2016). Among patients alone, 0.46 μM,was similar to that defined in the presence of insulin

100 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109

Fig. 5. Molecular Structure of Calmodulin. (From: //open- i.nlm.nih.gov/) (A) Ribbon structure of free CaM (blue) with four bound Ca2+ ions (yellow). (B) A 2.4 Å ribbon structure of CaM bound to a peptide (green) corresponding to the CaM binding domain of CaMKII (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article).

(0.42 μM), insulin-like growth factor I (0.54 μM) and epidermal growth factor (0.43 μM) (Strobl et al., 1990). The antiproliferative effects of pimozide on breast cancer cells were not limited to the MCF-7 cell line. Pimozide also inhibited proliferation of the ER-positive T47D and ZR75-1B human breast cancer cells and the ER-negative human breast cancer cell MDA-MB-231 (Strobl et al., 1990). The authors have underlined that calmodulin inhibitory and antiproliferative concentrations of pimozide and thioridazine were similar; and also that calmodulin-antagonists W-13 and W-12 blocked growth of breast cancer cells. Hence, they deduced that pimozide and thioridazine efficacy to inhibit breast cancer growth may occur via antagonizing calmodulin dependent pathways (Strobl et al., 1990). Lastly, and maybe most importantly, concentrations of pimozide and thioridazine that inhibit breast cancer cell growth in vitro are achievable in patients receiving these drugs (Strobl et al., 1990). Antipsychotic/neuroleptic drugs bind to sigma receptors (Fig. 6) (both σ-receptor-1 and σ-receptor-2) with high affinity and pimozide binds to σ-receptor with a Ki of 139 nM (Vilner and Bowen, 1993). Blood levels of pimozide range between 2 to 40 nM in healthy volun- teers treated with therapeutic doses of pimozide (Strobl et al., 1998). Human kidney, colon and brain tumors as well as cell lines obtained from melanomas, breast and lung carcinomas express high levels of both σ-receptor-1 and σ-receptor-2; therefore, even low doses of pi- Fig. 6. Schematic Structure of Sigma (σ)-Receptors. mozide may modify σ-receptor-associated pathways selectively in malignancies in comparison to benign tissues (Strobl et al., 1998). More- dose of γ-radiation. The radiation dose required for 1% survival was over, in murine cancer cells, high affinity σ-receptor-2 expression was decreased by a factor of 2, while treatment of WI-38 human embryonic found to be selectively induced in proliferative phase in comparison to lung cells with pimozide did not increase their sensitivity to γ-radiation quiescent G-1 arrested cells, indicating that σ-receptor-2 may act even (Strobl et al., 1998). Pimozide (2.5 μM) induced early apoptotic more potent on rapidly dividing malignant cells. changes in MCF-7 cells and 10 μM pimozide activated a complete Strobl et al. studied whether neuroleptic drugs that bind σ-receptors apoptotic process resulting in the killing of > 90% of the cells within inhibit growth and sensitize to radiation therapy in MCF-7 human 24 h. MCF-7 cells exposed to γ-radiation alone (8 Gy) exerted giant cell breast cancer cells (Strobl et al., 1998). Blockage of growth by ap- formation, mitotic arrest, and a limited degree of apoptosis and ne- proximately 50% occurred in cells treated with pimozide (0.6 μM), crosis. Within 50 h of treatment with a combination of γ-radiation and haloperidol (10 μM), and the σ-receptor ligand DTG (1,3-di(2-tolyl) pimozide, cell numbers were robustly reduced in comparison to cul- guanidine, 20 μM), but no growth inhibition happened in cells treated tures exposed to either γ-radiation or pimozide alone (Strobl et al., with clozapine, a neuroleptic lacking σ-receptor binding activity, or 1998). The authors have proposed that pimozide augmented the ra- dextromethorphan, a selective σ-receptor-1 ligand (Strobl et al., 1998). diation sensitivity of MCF-7 cells by a mechanism not shared by halo- Pimozide (2.5 μM), but not haloperidol (3.6 μM), enhanced the sensi- peridol, and the concentration of pimozide in MCF-7 cells as a result of tivity of MCF-7 cells to γ-radiation in clonogenic survival assays. Pi- an enrichment of σ-receptor-2 sites might achieve the mozide significantly lowered clonogenic survival following a 5 or 8 Gy

101 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109 radiosensitization. Indeed, some studies showed that MCF-7 breast concentrations that fully saturate σ-receptors (Wiklund et al., 2010). cancer cells σ-receptor-2 but not σ-receptor-1 (Strobl et al., 1998). Indeed, haloperidol, a prominently more potent σ-receptor antagonist It has been also shown that MCF-7 cells which express T-type Ca+2 than pimozide, was much less cytocidal. Overall, the investigators channels are more sensitive to the growth inhibitory effects of pimozide proposed that tumoricidal activity of neuroleptics was not mediated by and mibefradil, a more selective T-type Ca+2 channel-inhibitor (an old the neuroreceptor systems involved in their psychotropic effects drug which was used in treatment of chronic angina pectoris) (Bertolesi (Wiklund et al., 2010). As suggested above, qRT-PCR confirmed that et al., 2002). The proliferation of Y79 and WERI-Rb1 retinoblastoma essential regulatory genes for cholesterol metabolism (HMGCR – HMG and MCF7 breast cancer cells, all of which express T-channel current Coenzyme A Reductase, INSIG1 – Insulin Induced Gene-1 Protein and and mRNA for T-channel subunits, is blocked by pimozide and mibe- LDLR – Low Density Lipoprotein Receptor) were upregulated by pi- fradil with IC50 values between 0.6 and 1.5 μM. Although a strong mozide and olanzapine (Wiklund et al., 2010). Dysregulation of cho- correlation between T-channel expression and antiproliferative activity lesterol trafficking activates the sterol regulatory elementbinding pro- was witnessed, the following observations indicate that these two drugs tein (SREBP) transcription factors which involve in the uptake and may inhibit cell growth also via diverging mechanisms: 1) pimozide and synthesis of cholesterol and genes defined to be upregulated by anti- mibefradil acted additive on inhibiting T-channel currents, yet sy- psychotics (HMGCR, INSIG1 and LDLR) are all SREBP target genes nergistically on cell proliferation; 2) an increase in apoptosis and de- (Wiklund et al., 2010). Whether it is the cell’s perceived depletion of crease of the anti-apoptotic bcl-2 levels in Y79 and MCF7 cells were cholesterol (induced by its perturbed trafficking) or accumulation of observed only in pimozide-treated cells, whereas the toxicity is pri- excess intracellular cholesterol that causes cytocidal actions is not il- marily necrotic in mibefradil treatment; and 3) antiproliferative effi- luminated. Some studies revealed that perturbations in cholesterol le- cacy of mibefradil is reduced in Y79 cells transfected with T-channel vels influence the AKT signaling and that depletion of cholesterol hin- antisense and in differentiated Y79 cells (with lowered T-channel ex- ders cell division though the inability to synthesize new cell membranes pression), but growth inhibition by pimozide is affected to a lesser (Wiklund et al., 2010). The authors have also underlined that calmo- extent (Bertolesi et al., 2002). These results suggest that pimozide and dulin antagonism may be related to amphiphilic structure and both mibefradil inhibit cell proliferation via diverging mechanisms and that amphiphilic and anti-calmodulin actions overlap on regulatory genes, in the case of pimozide, other antitumoral mechanisms shall accompany second messenger systems, such as the PI3-Kinase-AKT or additive ef- the T-channel inhibition. fects on cholesterol homeostasis (Wiklund et al., 2010). Wiklund’s findings are in close aggreement with very early findings that pimozide 6. Pimozide inhibition of cholesterol homeostasis which associate significantly alters brain phospholipids via its CAD structure (Pappu with its tumoricidal activity on a Broad range of malignancies and Hauser, 1981).

Wiklund et al. have hypothesized that the reported decline in ma- lignancy risk in patients with schizophrenia may be because neuroleptic 7. Pimozide, fatty acid binding Protein-4, PPAR-γ and cancer agents have anticancer potential (Wiklund et al., 2010). Therefore, they cachexia analyzed the effects of 6 antipsychotic agents with diverse structural and pharmacological properties (reserpine, chlorpromazine, haloper- Sleeman et al. investigated the appetite-stimulating (orexigenic) idol, pimozide, risperidone and olanzapine), on the growth of cell lines efficacy of the peripheral dopamine receptor antagonist domperidone derived from lymphoma, neuroblastoma, non-small cell lung cancer and the central dopamine antagonist pimozide in tumor-bearing anor- and breast carcinoma (Wiklund et al., 2010). With the exception of exic rats (Sleeman et al., 1989). Dopamine antagonists were employed risperidone, all neuroleptics exerted selective inhibition of the viability intraperitoneally Days 7–15 after the subcutaneous implantation of the of cancer cells in comparison to benign cells. Among these drugs, the Walker 256 carcinosarcoma. The dose of pimozide given was 0.1 mg/kg most potent antitumoral agent was pimozide (Wiklund et al., 2010). By daily. While all doses of dopamine antagonists caused an initial decline employing Affymetrix microarrays and qPCR, they revealed that for the in body weight and food intake, the body weight of pimozide-injected antipsychotic drugs, olanzapine and pimozide, tumoricidal actions oc- animals was not decreased significantly (in the early stages of drug cured via effects on cholesterol homeostasis (Wiklund et al., 2010). treatment) as it was observed with the various doses of domperidone Supporting these findings, cholesterol synthesis inhibitor, mevastatin (Sleeman et al., 1989). There was significantly more food in the sto- increased tumoricidal actions of olanzapine and pimozide. Pimozide machs of domperidone and pimozide-treated animals in comparison to and olanzapine exerted increasing cytotoxicity from 12 to 48 h in time those of the vehicle-treated, tumor-implanted animals. 16 years later, course studies, in parallel to the time-dependent onset of cytotoxicity elegant studies conducted by Wang and colleagues demonstrated that induced by the amphiphile, U18666 A (Wiklund et al., 2010). Hence, pimozide selectively inhibits Fatty Acid Binding Protein-4 (FABP-4), the authors have proposed Class II cationic amphiphilic properties of subsequently activates PPAR-γ and induces weight gain (Wang et al., neuroleptic agents mediated their tumoricidal actions by changing 2015). Fatty acid-binding proteins (FABPs) is a cluster of ∼15 kDa cholesterol homeostasis and the biophysical properties of cellular cytoplasmic proteins which transport endogenous fatty acids for me- membranes (Wiklund et al., 2010). tabolic process or storage. FABP4 (aP2) is highly expressed in adipo- They argued against the proposal that tumoricidal action of neu- cytes and its gene contains peroxisome proliferator response elements roleptics is primarily mediated via the dopaminergic system due to 2 (PPREs) and therefore can be regulated by PPARγ; FABP enhances li- reasons. First, the tumoricidal levels of neuroleptics were orders of polysis and reduces insulin sensitivity (Fig. 7). With molecular docking magnitude greater than those required to saturate dopaminergic re- studies, it was shown that pimozide selectively binds and inhibits FABP- ceptor system. For instance, the IC50 for pimozide (a powerful D2R 4, which leads to activation of PPAR-γ and enhancement in weight gain antagonist) for neuroblastoma cells (6 μM) is 600-fold greater than the (Wang et al., 2015). These findings are of essential importance, as very estimated cerebral levels associated with clinically effective D2R oc- recent studies showed that insulin resistance and PPAR-associated cupancy (in the order of 10 nM) in patients with schizophrenia (esti- pathways involve in cancer cachexia. Indeed, the PPAR-γ-agonist an- mation based on a therapeutic plasma level of 6 nM and a plasma-to- tidiabetic pioglitazone, L-carnitine and a plant extract from Chry- brain drug accumulation ratio of 1.6) (Wiklund et al., 2010). For σ- santhemum morifolium were shown to alleviate cancer cachexia via sti- receptors, they revealed that first generation neuroleptics (which are σ- mulating PPAR-γ (Beluzi et al., 2015; Jiang et al., 2015; Ohsawa et al., receptor antagonists) acted as more potent anticancer agents than 2016). second generation agents (which are not σ-receptor antagonists). On the other hand, tumoricidal neuroleptic levels are well in excess of

102 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109

including genes involved in cell diﬀerentiation, growth and apoptosis resistance (Nelson et al., 2011). Although STATs may be essential for tumor growth, they are dispensable for physiologic cellular function, likely because of redundancies in normal signaling. For instance, aberrant activation of STAT3 (Fig. 9) is a frequent event in a range of human tumors, but in the hyper-IgE syndrome, a dominant inhibitory form of STAT3 is present since conception which is compatible with normal human development (Nelson et al., 2011). Similarly, targeted knock-out of STAT-5 in mice results in relatively healthy animals with minimal defects in hematopoiesis, including fewer CD19+ cells and a mild impairment in erythropoiesis. Hence, targeting STATs harbours the potential for a very high therapeutic index in treating malignancies driven by constitutive activation of these proteins (Nelson et al., 2011). Zhou et al. demonstrated that pimozide blocked prostate cancer cell proliferation in a dose- and time-dependent manner by inducing G1 phase cell cycle arrest, decreased colony formation and sphere forming, as well as inhibited cell migration in both DU145 and LNCaP cells (Zhou et al., 2016). Moreover, pimozide reduced the basal phosphorylation of STAT3 at tyrosine 705 and reversed the expression of phosphorylation of STAT3 induced by IL-6 addition, indicating that pimozide can block cellular STAT3 activation (Zhou et al., 2016).

10. Pimozide inhibition of stem cell features, STAT-3 and Wnt/β- Catenin signaling in liver cancer

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies globally and is a prominent cause of cancer-related deaths, its 5-year overall survival rate in the United States is less than 20% (Fako et al., 2016). The multi-kinase inhibitor sorafenib is the only FDA-approved targeted treatment drug for the treatment of HCC, which only provides a small survival advantage Fig. 7. Pimozide binding to FABP-4 and FABP-4 Stimulation of Lipolysis and (∼2.8 months) in comparison to supportive care for patients with ad- Reduction of Insulin Sensivity and Cardiac Contraction. (Adapted From: vanced stage HCC (Fako et al., 2016). HCC patients typically have a Pasterkamp G. Lipid-immunity cross-talk: a role for adipocyte fatty acid binding relatively poor prognosis partly because HCC cells are often refractory protein? Arterioscler Thromb Vasc Biol. 2012; 32:2043-4. doi:10.1161/ to standard chemotherapy and radiotherapy and tumor-initiating cells ATVBAHA.112.254797). or cancer stem cells are held responsible for this resistance (Chen et al., 2017). Chen et al. have revealed that pimozide blocked cell prolifera- 8. Pimozide as an inhibitor of lung cancer tion and sphere formation in HCC cells (Chen et al., 2017). Noteworthy, pimozide also blocked the maintenance and tumorigenicity of HCC Fortney et al. used computational biological approaches and mainly tumor stem cells, especially the side population (SP) or CD133-positive employed the Connectivity Map (CMap), which catalogues the tran- cells, as assessed by colony formation, sphere formation and transwell scriptional responses to drug treatment in human cell lines for over a migration assays with concomitant declines in stemness genes Bmi-1, c- thousand small molecules, to determine novel active molecules which Myc and Nanog (Chen et al., 2017). Moreover, pimozide inhibited can interfere with lung cancer signaling (Fortney et al., 2015). They STAT3 activity in HCC cells by attenuating STAT3- dependent luciferase found that pimozide interferes with major molecular driving forces of activity and down-regulating downstream genes of STAT3 signaling lung cancer (Fortney et al., 2015). When they tested pimozide activity (Chen et al., 2017). As an oncogene, STAT3 is constitutively activated in A549, H460, HCC4006, and H1437 lung cancer cell lines, they re- by the phosphorylation of Tyr705 in various cancers providing signals vealed that pimozide exerted robust antitumor activity in all tested cell for cell proliferation, vascularization and chemoresistance (Fig. 8). lines (Fortney et al., 2015). This activity seemed to be independent of STAT3 also contributes to oncogenesis by preventing apoptosis, inva- calmodulin inhibition and showed no synergism with cisplatin (Fortney sion, and metastasis as well as suppressing antitumor immune responses et al., 2015). (Fig. 8)(Zhou et al., 2016). Phosphorylated STAT3 dimerizes and translocates into the nucleus to induce the transcription of downstream genes, such as BCL-xL, MCL1, and c-Myc (Chen et al., 2017). STAT3 9. Pimozide inhibition of STAT-3 and stemness in prostate cancer activation is associated with poor prognosis in HCC and inhibition of STAT3 is shown to reduce proliferation and chemoresistance in HCC Activating mutations of tyrosine kinases are frequent events in cells, which can be achieved with sorafenib, a ﬁrst-line therapy in cancer initiation and progression and mutant kinases induce versatile treating patients with advanced liver cancer. One of the important ac- signaling cascades that culminate in the activation of genes that drive tivators of STAT-3 and stem cell features in HCC is the cytokine IL-6 and the malignant behavior (Nelson et al., 2011). The deﬁnition of tran- very importantly pimozide was found to block both basal and IL-6 sti- scription factors that mediate the actions of aberrantly activated kinases mulated STAT-3 activation and downstream target genes including is an attractive target for cancer therapy. One family of transcription BCL-xL, MCL1, and c-Myc (Chen et al., 2017). factors activated by tyrosine kinases is the signal transducer and acti- One of the important molecular cascades providing stemness fea- vator of transcription (STAT) family, which is constituted by latent tures in HCC is mediated by wnt/β-catenin signaling and subsequent proteins residing in the cytoplasm that are activated by phosphoryla- induction of EpCAM (Fako et al., 2016). Cytoplasmic stabilization of β- tion on critical tyrosine residues (Nelson et al., 2011). After translo- catenin and its subsequent translocation into the nucleus and tran- cating to the nucleus, STATs induce transcription of their target genes, scriptional activation of down-stream target genes, is regulated by

103 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109

Fig. 8. Pimozide inhibition of STAT-3 and versatile Tumorigenic Roles of STAT-3 Signaling Pathway (From: Pan Y. Role of Stat3 in nasopharyngeal carcinoma. OA Cancer 2013; 1(2):17). phosphorylations. Several phosphorylated residues, including Ser191/ EpCam levels in Hep3B and HepG2 HCC cells, both of which harbour a Ser605 by JNK2, Ser552 by AKT, and Ser675 by protein kinase A (PKA), homogenous population of stem cells that are positive for the stem-cell are important for β-catenin stabilization and nuclear localization. Fako marker EpCAM (Fako et al., 2016). As a control, they also investigated et al. studied eﬀects of pimozide on stemness, wnt/β-catenin and the actions of pimozide on the proliferation of EpCAM(-) Sk-Hep-1

Fig. 9. Pimozide (PMZ)-Inhibition of STAT5 in Leukemia (From: Turhan AG. STAT5 as a CML target: STATinib therapies? Blood. 2011;117: 3252-3. doi: 10.1182/ blood-2011-01-332569).

104 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109 hepatoma cell line. Hep3B cells contain non-mutated β-catenin protein tyrosine phosphorylation of STAT5 and induced signiﬁcant apoptosis of with proper wnt/β-catenin signaling. Pimozide blocked cell growth in malignant myeloproliferative cells (Bar-Natan et al., 2012). all HCC cells in a dose-dependent manner following 48-hour treatment, STAT5 also drives the oncogenesis of acute myelogenous leukemia but with higher potency in the EpCAM(+) cell lines. They selected to (AML) harbouring the FLT3 internal tandem duplication (ITD) mutation employ 10 μM concentration of pimozide for subsequent studies, as (Nelson et al., 2012). FLT3 ITD is a constitutively active tyrosine kinase

10 μM treatment occurred at ∼IC60-IC70 concentration in both EpCAM that triggers activation of STAT5, providing the proliferation and sur- (+) cell lines, but only the ∼IC25 in Sk-Hep-1 cells (Fako et al., 2016). vival of AML cells (Nelson et al., 2012). Pimozide blocked the tyrosine Exposure of HCC cells to pimozide blocked cell growth, viability, phosphorylation of STAT5, leading to the death of AML cells via colony, and spheroid formation, while stimulating apoptosis, in both apoptosis. Pimozide also exerted an additional effect with the tyrosine Hep3B and HepG2 cells (Fako et al., 2016). Pimozide also decreased kinase inhibitors midostaurin (FLT3 inhibitor) and sunitinib in the gene expression, protein expression and the transcriptional activity of blockage of STAT5 tyrosine phosphorylation and the triggering of β-catenin and levels of EpCAM (Fako et al., 2016). apoptosis. Most importantly, pimozide also lowered the tumor burden in a mouse model of FLT3-driven AML (Nelson et al., 2012). PI3K in- 11. Pimozide as an inhibitor of STAT-3 and STAT-5 in leukemias hibitor GDC-0941 or the Akt inhibitor MK-2206 triggered apoptosis through the mitochondria-mediated intrinsic pathway more efficiently In chronic myelogenous leukemia (CML) the BCR/ABL fusion kinase in hematopoietic 32D cells driven by FLT3-TKD (32D/TKD) than FLT3- drives main pathways of cell growth and drug resistance and the de- ITD (32D/ITD), which prominently activated STAT-5 (Nogami et al., velopment of the BCR/ABL inhibitor imatinib mesylate caused a para- 2015). The resistance to GDC-0941 and MK-2206 was achieved through digm shift in the treatment of CML and also started a new era of tar- the constitutively activated STAT-5 mutant STAT-5-A1*6 in 32D/TKD geted drugs in treatment of cancer (Nelson et al., 2011). Unfortunately, cells, while it could be blocked by pimozide both in 32D/ITD cells or CML cells can develop resistance to imatinib via point mutations in FLT3-ITD-expressing human leukemia MV4–11 cells (Nogami et al., BCR/ABL that alleviate the binding of imatinib to the active site of the 2015). Furthermore, GDC-0941 or MK-2206 induced depho- kinase. One such mutation, T315I, renders CML cells completely re- sphorylation of 4EBP1 stronger in 32D/TKD than in 32D/ITD, which sistant not only to imatinib but also to the second-generation BCR/ABL was increased by pimozide. Lastly, in primary AML cells with FLT3-ITD, inhibitors nilotinib and dasatinib (Nelson et al., 2011). In CML, the pimozide augmented 4EBP1 dephosphorylation and Mcl-1 down- BCR/ABL fusion kinase leads the constitutive activation of STAT-5, regulation (Nogami et al., 2015). But pimozide inhibition of AML may thereby stimulating the expression of genes providing cell survival. also occur via additional mechanisms. Inhibitor of DNA binding 1 (ID1) Hence, Nelson et al. employed a cell-based screen to determine drugs transcription factor is required for the growth and progression of di- that inhibit STAT-dependent gene expression and identified pimozide verse malignancies, including leukemia (Mistry et al., 2013). ID1 is as a STAT-5 inhibitor (Fig. 9)(Nelson et al., 2011). Pimozide selectively normally polyubiquitinated and degraded by the proteasome and USP1, attenuated STAT-5 tyrosine phosphorylation, although it did not block a ubiquitin-specific protease, deubiquitinates ID1 and saves it from BCR/ABL or other tyrosine kinases and inhibited the expression of proteasome degradation; hence, blockage of USP1 function may be a STAT5 target genes and triggered cell cycle arrest and apoptosis in CML plausible novel strategy of cancer and leukemia treatment (Mistry et al., cells (Nelson et al., 2011). Moreover, pimozide produced similar effects 2013). Importantly, pimozide lowered USP1 levels, triggered ID1 de- in the presence of the T315I BCR/ABL mutation that renders CML cells gradation and inhibited K562 cell growth in vitro and in vivo (Mistry resistant to available BCR/ABL kinase inhibitors. Furthermore, si- et al., 2013). Various somatic ribosome defects were defined in ma- multaneously inhibiting STAT-5 with pimozide and the kinase in- lignancies, yet their oncogenic mechanisms are not well illuminated. hibitors imatinib or nilotinib exerted increased effects in reducing Recurrent R98S mutation in ribosomal protein L10 (RPL10-R98S) was STAT-5 phosphorylation and in inducing programmed cell death discovered in T cell ALL; and in engineered RPL10-R98S mouse lym- (Fig. 9)(Nelson et al., 2011). phoid cells, proteomic screening defined increased expression of var- Myeloproliferative neoplasms (MPNs) are a group of clonal dis- ious Jak-Stat signaling proteins (Girardi et al., 2017). These findings orders that originate from the malignant dedifferentiation of hemato- were similar in hematopoietic cells from transgenic Rpl10-R98S mice poietic stem cells. A single point mutation in the Janus kinase 2 (Jak2) and T-ALL xenograft samples. RPL10-R98S expressing cells exerted gene was defined in the majority of patients with Philadelphia chro- JAK-STAT pathway hyper-activation upon cytokine stimulation; and mosome (Ph)-negative MPN, which may function as a selective target importantly, they exhibited enhanced sensitivity to pimozide (Girardi for Jak2 inhibition (Bar-Natan et al., 2012). Small molecule Jak2 in- et al., 2017). hibitors are currently tested in clinical trials, yet their efficacy is not well illuminated. In addition to the inadequate blockage of mutated 12. Pimozide as a cancer chemosensitizing agent against DNA Jak2 in vivo or resistance development through alternative pathways, damaging agents and inhibition of ubiquitine specific protease, many MPNs contain further mutational events (for instance, mutation USP-1 in exon12 of Mpl, or the KIT D618 V mutation in patients with systemic mastocytosis), and thus are insensitive to Jak inhibitors (Bar-Natan Kennedy et al. investigated whether calmodulin antagonists, pimo- et al., 2012). STAT-5 constitutes a convergence point of these oncogenic zide and trifluoperazine influence bleomycin induced cytotoxicity and pathways, which stimulates the expression of genes involved in cell DNA damage in L1210 murine leukemia cells (Kennedy et al., 1986). proliferation and survival (Fig. 9). As STAT5 is a dominant mediator of The combined cytotoxicity of bleomycin and calmodulin antagonists the effects of Jak2 V617 F, the discovery of agents that block this exerted true pharmacological synergism and was observed with the transcription factor is a plausible approach for the management of MPN exposure to the calmodulin antagonists both during or after bleomycin (Bar-Natan et al., 2012). Hypothetically, the combined inhibition of exposure (Kennedy et al., 1986). Furthermore, the DNA damage of both STAT5 and Jak2 may provide even higher therapeutic efficacy. bleomycin and the bleomycin-like drugs, talisomycin S10b or peplo- Hence, Bar-Natan et al. investigated the efficacies of pimozide alone mycin, was also augmented by calmodulin antagonists (Kennedy et al., and combined with a Jak2 inhibitor on the myeloproliferative cells 1986). The synergism seen with these drug combinations is reflected by including Ba/F3 cells that was transfected with erythropoietin receptor changes in DNA integrity and their obtained data indicated that the and the mutant form of Jak2 (BAFEJ), as well as the HEL and SET2 inhibition of DNA repair may be responsible for the increased cyto- human leukemia cells that endogenously express Jak2 V617 F (Bar- toxicity and DNA damage when calmodulin antagonists are added to Natan et al., 2012). Exposure of each cell line to pimozide alone and in bleomycin (Kennedy et al., 1986). Lazo et al. confirmed thart the in- synergy with the Jak2 inhibitor Jak inhibitor-1 led to a decrease in creased cytotoxicity occurred without a change in (a) cellular

105 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109 bleomycin content, (b) drug efflux, or (c) bleomycin metabolism (Lazo crosslinking agents, including mitomycin C, diepoxybutane, and cis- et al., 1986). The amount of DNA damage after coincubation of bleo- platin (Chen et al., 2011). Eight FA proteins form a multisubunit mycin and a calmodulin antagonist was more intense in intact LI210 complex that ubiquitinates FANCD2, which is essential for the repair of cells than with bleomycin alone, yet no increase in DNA breakage was DNA interstrand crosslinks. Since USP1 involves in the two important observed in vitro with plasmid DNA suggesting that calmodulin an- DNA damage response pathways, it is a promising candidate to discover tagonists acted via inhibition of DNA repair (Lazo et al., 1986). Lazo small molecule inhibitors to block cancer cells’ repair of DNA lesions et al. also tested bleomycin-pimozide combination on SK-OV cells iso- and to increase their killing with DNA-damaging chemotherapeutics lated from a human ovarian adenocarcinoma, A-2S3 cells isolated from (Chen et al., 2011). USP1 belongs to a class of DUBs that require a a human squamous cell carcinoma originated in the submaxillary gland partner protein for enzymatic function and forms a stable complex with and on human bone marrow cells (Lazo et al., 1986). They showed that UAF1, which increases its catalytic activity. Since many USPs exist and (a) there is enhanced cytotoxicity to human cancer cells when cal- function as complexes in vivo, it is compulsory to discover inhibitors modulin antagonists are added to bleomycin; (b) the effect is dose de- against the corresponding USP complexes (Chen et al., 2011). Chen pendent for both type of drugs; (c) the maximum effect was observed et al. subjected the human USP1/UAF1 complex to a quantitative high- when cells are treated concurrently with the calmodulin antagonist and throughput screening (hHTS) and defined potent and selective in- bleomycin; (d) calmodulin antagonists can overcome inherent bleo- hibitors of USP1/UAF1. The top inhibitors, pimozide and GW7647, mycin resistance of malignant cells; (e) bone marrow cells are also blocked USP1/UAF1 noncompetitively with a Ki of 0.5 and 0.7 μM, sensitive to this combination. In the presence of 5 μM bleomycin, 95% respectively, and displayed selectivity against a number of deubiquiti- of SK-OV cells were killed with 5 μM of pimozide (Lazo et al., 1986). nases, deSUMOylase, and cysteine proteases (Chen et al., 2011). Now, we will discuss that pimozide is capable to block ubiquitin-spe- Moreover, the USP1/UAF1 inhibitors exerted synergism with cisplatin cific protease-associated DNA repair mechanisms. in blocking cisplatin-resistant non-small cell lung cancer (NSCLC) cell Ubiquitin is a 76 amino acid long peptide that can be covalently proliferation (Chen et al., 2011). Hence, pimozide is an important attached to proteins to modulate their stability or localization and was candidate of cancer chemosensitization, because it could both block the first discovered as a “destruction tag” leading to the degradation of the translesion synthesis and Fanconi anemia pathways essential for DNA modified protein in the proteasome (García-Santisteban et al., 2013). damage repair. On the other hand, García-Santisteban et al. studied 7 But ubiquitin and ubiquitin-like proteins also involve in versatile cel- human lung cancer cell lines and have shown that pimozide-cisplatin lular processes, including membrane trafficking, transcription regula- activity was synergistic in one cell line, additive in 3 cell lines and tion and DNA damage response (Chen et al., 2011). Deubiquitinases antagonistic in 3 (García-Santisteban et al., 2013). Hence, further (DUBs) are enzymes that cleave the isopeptide bond formed between careful studies are necessary before employing pimozide-cisplatin the C-terminal carboxylate of ubiquitin and a lysine residue amino combination in a clinical setting. group on the target protein (Chen et al., 2011). By antagonizing the activity of E3 ligases, DUBs can rescue a ubiquitinated protein from 13. Pimozide activity in glioblastoma. Role of calmodulin, σ- proteasomal degradation, or alter its fate in a more subtle way by receptors, autophagy, serotonin receptor 5-HT7 and USP-1 editing the length and topology of its ubiquitin tag (García-Santisteban et al., 2013). DUB activity modulates the ubiquitination of diverse Lee and Hait analyzed the effect of several classes of calmodulin cellular proteins, and thus regulates their levels, activity and localiza- inhibitors - including pimozide - on the activity of calmodulin prepared tion. Besides these functions, several DUBs also play a housekeeping from C6 astrocytoma cells and also investigated whether these drugs function in the maintenance of the cellular free ubiquitin pool (García- inhibit cell proliferation in C6 astrocytoma (Lee and Hait, 1985). They Santisteban et al., 2013). The activity of diverse signaling pathways observed antiproliferative effects of these drugs on C6 astrocytoma cells regulating malignant behavior, such as those mediated by p53, EGFR, which exerted a good correlation between the inhibitory activity of NFκB, TGFβ or WNT, is, at least in part, controlled by DUBs; and hence, these drugs on calmodulin. Pimozide inhibition of calmodulin had an drugs which interfere with the functioning of DUBs are accepted to be IC50 value of 6 μM and inhibition of C6 glioblastoma growth had an IC50 potential candidates for cancer treatment (García-Santisteban et al., value of 10 μM(Lee and Hait, 1985). Vilner and Bowen suggested that a 2013). higher concentration of pimozide acted cytotoxic on C6 glioblastoma The human genome encodes close to 100 DUBs which can be clas- cells and proposed that cytotoxicity of neuroleptics on glioma cells sified into five families and ubiquitin-specific proteases (USPs) con- correlated with their affinity to σ-receptors (Vilner and Bowen, 1993). stitute the largest DUB family. Among the human USPs, USP1 exerts One prominent feature of σ-receptors is their affinity for haloperidol special importance as it is implicated in DNA damage response (Chen and several other neuroleptic drugs which are dopamine D2 receptor et al., 2011). Disruption of USP1 in chicken DT40 cells enhanced the antagonists. After exchange of standard culture medium for medium sensitivity to DNA crosslinkers and knockout of the murine USP1 gene containing 100 μM of pimozide, significant rounding of cells and loss of in mice resulted in increased mitomycin-C sensitivity (Chen et al., viability was observed after 18 to 24 h and the calculated Ki of pimozide 2011). Analysis of the COSMIC mutation database revealed 23 non- for σ-receptor was 139 nM (Vilner and Bowen, 1993). synonymous mutations of the USP1 gene in diverse tumor types, thir- Autophagy is a cellular catabolic process providing the turnover of teen of these found in lung cancer samples (García-Santisteban et al., intracellular organelles and proteins through a lysosome-dependent but 2013). A survey of the Oncomine Research Edition database uncovers proteasome-independent degradative pathway (Zhang et al., 2007). An that USP1 mRNA expression is significantly changed (Fold change > autophagosome encapsulates cytoplasmic organelles including mi- 1.5; p < 0.05) in several tumor types. Aberrant overexpression is the tochondria, endoplasmic reticulum, and ribosomes, by forming a most common change, which is particularly frequent in cervical and double-membrane vesicle. The outer membrane of the autophagosome gastric cancer, melanoma and sarcoma (García-Santisteban et al., fuses with the lysosome delivering the sequestered content to the ly- 2013). USP1 deubiquitinates PCNA and FANCD2 in cells. In eukaryotes, sosomal lumen for degradation (Zhang et al., 2007). Autophagy is es- PCNA ubiquitination is essential for the normal DNA damage response sential for the survival of cells under starvation conditions because it (Chen et al., 2011). Monoubiquitination of PCNA is required for provides recycling of the intracellular material for producing essential translesion synthesis that promotes lesion-bypass synthesis across the energy and macromolecules. On the other hand, excess autophagy is damaged base. FANCD2 protein is an important constituent of the detrimental for cells and agents which robustly trigger autophagic human Fanconi anemia (FA) pathway. Genetic deficiency of FA proteins cascades exert anticancer actions (Levy et al., 2017). 17 genes are re- leads to chromosomal instability and carcinogenesis; while, on the quired for autophagy in yeast (referred to as the ATG genes) (Zhang other hand, it also increases sensitivity of cancer cells to DNA et al., 2007). In mammalian cells, mTOR kinase, the target of

106 I. Elmaci, M.A. Altinoz Critical Reviews in Oncology / Hematology 128 (2018) 96–109 rapamycin, provides the major blocking signal that inhibits autophagy pathways (Kast, 2010). under nutrient-rich conditions. In opposite, mammalian type III PI3- Lee et al. analyzed the expression of ubiquitination-specific pro- kinase, the homolog of yeast VPS34 and inhibitable by 3-methyladenine teases by transcriptome analysis and revealed that USP1 is highly ex- (3-MA) (a nonspecific inhibitor of PI3-kinase), triggers the onset of pressed in GBM (Lee et al., 2016). Employing the patient GBM-derived autophagy (Zhang et al., 2007). To illuminate the mechanism of au- primary tumor cells, they blocked USP1 by shRNA-mediated knock- tophagy and determine small molecules that stimulate it, Zhang et al. down or pimozide and investigated stem cell marker expression, pro- performed a high-throughput image-based screen by employing light liferation, and clonogenic tumor growth (Lee et al., 2016). USP1 was chain 3 (LC3) coupled to green fluorescent protein (GFP) (Zhang et al., highly expressed and more so in glioblastoma stem cell enrichment 2007). Mammalian LC3, the ortholog of yeast ATG8, specifically bea- marker (CD133 or CD15) positive cells. USP1 enhanced protein stability cons the autophagosome membrane; indeed, LC3-GFP-positive autop- of the ID1 and CHEK1, important regulators of DNA damage response hagosomes per cell were very low under normal growth but were ra- and stem cell maintenance (Lee et al., 2016). Targeting USP1 both by pidly increased under serum starvation or exposure to rapamycin. RNA interference or pimozide decreased clonogenic growth and sur- Agents that enhance the levels of LC3-GFP, are not necessarily able to vival of glioblastoma stem cells and increased radiation sensitivity of increase the degradative activities of autophagy, rather they may in- glioblastoma cells. Lastly and most importantly, USP1 blockage with crease LC3-GFP due to cytocidal effects, lysosomal perturbation and pimozide alone or with radiation increased the survival of glio- blockage of autophagy (Zhang et al., 2007). Hence, more sophisticated blastoma-bearing mice, which occured with dramatic prolongation methods are necessary to uncouple agents which selectively induce when both modalities were combined (Lee et al., 2016). autophagic protein degradation from those inducing toxic autophagic effects. Zhang et al. performed a novel series of image-based screens 14. Another possible target of pimozide. Translationally and assay criteria to truly define agents which selectively induce au- controlled tumor protein (TCTP)? tophagic protein degradation and using these screens, they analyzed 480 compounds (at dosages between 3 to 12 μM) in the ICCB known Above, we had indicated that both pimozide and thioridazine may bioactive library (BIOMOL catalog 2840; www.biomol.com)(Zhang block tumor growth via antagonizing calmodulin (Strobl et al., 1990). et al., 2007). They performed their analysis on a human glioblastoma There also exists findings indicating that certain antihistaminic com- H4 cell line stably expressing human microtubule-associated protein pounds (hydroxyzine and promethazine) and pharmaceutical molecules (MAP) LC3-GFP and discovered eight compounds that can induce au- with a similar structure (eg. thioridazine and sertraline) induces cancer tophagy and promote long-lived protein degradation, which include cytotoxicity and reverses the malignant phenotype by downregulating fluspirilene, pimozide, trifluoperazine, niguldipine, nicardipine, amio- TCTP (Translationally Controlled Tumor Protein) (Tuynder et al. 2004). darone, loperamide, and penitrem A (Zhang et al., 2007). Very note- As demonstrated in Table 1, pimozide also inhibits histamine H1 re- worthy, 3 of these agents were neuroleptics and 2 of these are diphe- ceptor with Ki value about 700 nM; such a concentration would not be nylbutylpiperidines (fluspirilene and pimozide) (Zhang et al., 2007). possible with its current clinical application, yet it is also possible that it The remaining compounds consist of five agents, including three would accumulate within the malignant tissues. Above, we also in- FDA-approved drugs for cardiovascular indications, niguldipine, ni- dicated strong anti-melanoma activity of pimozide (Taub and Baker, cardipine and amiodarone, that inhibit intracellular Ca2+ currents, 1979; Krummel et al., 1982; Neifeld et al., 1983); and noteworthy, loperamide, a FDA-approved drug for diarrhea, and penitrem A, a ihibition of TCTP induces strong melanoma cell cytotoxicity, blocks fungal neurotoxin which can not be developed as a clinical drug (Zhang clonogenic growth, migration, and in vivotumor growth (Boia-Ferreira et al., 2007). The authors have performed these studies to discover et al., 2017) which may associate with pimozide’s anti-melanoma po- agents which would alleviate diseases with aberrant protein folding and tential. Lastly, we shall mention that TCTP blocks MDM2 auto-ubiqui- accumulation such as Huntington’s Disease and underlined that 2 of tination and stimulates MDM2-mediated ubiquitination and degrada- these 8 compounds, trifluoperazine and pimozide were proposed to tion of P53 (Amson et al., 2011).Very noteworthy, TCTP alleviate Huntington symptoms (Zhang et al., 2007). Under basal con- haploinsufficient mice are more vulnerable to P53-dependent apoptosis ditions, pimozide may protect neurons which accumulate high levels of and expression of TCTP very highly correlates with worse survival in toxic proteins, yet complete shut-down of autophagic mechanisms and breast cancer (p < 0.0005) and its silencing reduces stemness of breast protein turnover with higher doses of pimozide may trigger death of cancer cells (Amson et al., 2011). Hence, many of the pimozide’s ver- cancer cells alone and presumably in synergy with chemo-radiotherapy. satile chemosensitizing and radiosensitizing actions in cancer cells may Indeed, suppression of mTOR-upregulated resistance to autophagy is occur via downregulating TCTP and subsequent elevation of P53, as suggested to be a critical strategy to block glioblastoma stem cells wild-type, non-mutant P53 induces chemo-radiosensitivity in cancer (Ryskalin et al., 2017). (Chung and Irwin, 2010). Among the 500 similar seven-transmenbrane non-olfactory receptors, about a dozen are serotonin (5-HT) receptors (Kast, 2010). 15. Conclusions and future directions Each 5-HT receptor has its unique molecular structure, intracellular second messengers engaged, unique distributions within brain regions Novel utilization of non-antineoplastic drugs for cancer treatment is and on neurones and glia. Among these, 5-HT7 (Fig. 10 depicts its a highly logical approach, which would allow to define new cancer pathway) is of interest to neuro-oncology, since it was shown to be anticancer drugs from the known small molecules that have already expressed in malignant glioma cells and since three psychiatrical drugs undergone rigorous tests for human safety. Such an approach would potently inhibit it, including risperidone, paliperidone and pimozide. fast-track the development and approval of novel treatment strategies,

Pimozide has the highest affinity at 5-HT7 of any marketed drug (Kast, while reducing life-threatening risks. As suggested above, comprehen- 2010). 5-HT7 activation induces erk1/2 kinase pathways and the sive in vivo screening of about 300 central nervous system acting synthesis of IL-6 in glioblastoma cells (Kast, 2010). Resected glio- compounds revealed that psychoactive agents were 18-fold more likely blastoma tissue is robustly positive for IL-6 by immunohistochemistry to exert anticancer potential when compared with a random population and ELISA. High amplification of IL-6 gene was revealed in half of of molecules. More importantly, due to their robust capability to tra- surgical glioblastoma specimens, and that half has significantly shorter verse the blood-brain barrier, it is even more plausible to test their survival than did the non-amplified half. IL-6 is responsible for the in- efficacies in brain malignancies. Pimozide exerts pleiotropic effects on duction of STAT-3 and the synthesis of the angiogenic protein VEGF various psychoactive receptors, versatile actions on cell membranes and (Kast, 2010). Taken together, pimozide was proposed to block growth mitochondrial functioning, fatty acid metabolism in likely association of glioblastoma via interfering with the 5-HT7-receptor associated with cancer cachexia, prooncogenic signaling molecules such as STATs

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Fig. 10. Pimozide inhibition of 5-HT7 Receptor and Signaling Pathway of 5-HT7 Receptor (From: Guseva D, Wirth A, Ponimaskin E. Cellular mechanisms of the 5- HT7 receptor-mediated signaling. Front Behav Neurosci. 2014; 8:306. doi: 10.3389/fnbeh.2014.00306). and Wnt/β-catenin and on USP-1 related DNA repair mechanisms and Med. 18 (1), 91–99. tumoral chemosensitivity. Therefore, we strongly believe that pimozide Awouters, F.H., Lewi, P.J., 2007. Forty years of antipsychotic drug research—from haloperidol to paliperidone–with Dr. Paul janssen. Arzneimittelforschung 57 (10), deserves to be studied in further cell culture and animal model of ma- 625–632. lignancies. Such studies would reveal whether antineoplastic effects of Bar-Natan, M., Nelson, E.A., Walker, S.R., Kuang, Y., Distel, R.J., Frank, D.A., 2012. Dual pimozide are also relevant in diverse cancer cell lines and models, inhibition of Jak2 and STAT5 enhances killing of myeloproliferative neoplasia cells. Leukemia 26 (6), 1407–1410. which would also pave to develop proper Phase-I trials for potential Beluzi, M., Peres, S.B., Henriques, F.S., Sertié, R.A., Franco, F.O., Santos, K.B., Knobl, P., application in clinical management of patients suffering from cancers Andreotti, S., Shida, C.S., Neves, R.X., Farmer, S.R., Seelaender, M., Lima, F.B., and glial brain tumors. Batista Jr, M.L., 2015. Pioglitazone treatment increases survival and prevents body weight loss in tumor-bearing animals: possible anti-cachectic effect. PLoS One 10 (3), e0122660. http://dx.doi.org/10.1371/journal.pone.0122660. Funding Bertolesi, G.E., Shi, C., Elbaum, L., Jollimore, C., Rozenberg, G., Barnes, S., Kelly, M.E., 2002. The Ca(2+) channel antagonists mibefradil and pimozide inhibit cell growth ff – No funding was received for this review manuscript. via di erent cytotoxic mechanisms. Mol. Pharmacol. 62 (2), 210 219. Boia-Ferreira, M., Basílio, A.B., Hamasaki, A.E., Matsubara, F.H., Appel, M.H., Da Costa, C.R.V., Amson, R., Telerman, A., Chaim, O.M., Veiga, S.S., Senff-Ribeiro, A., 2017. Ethical approval TCTP as a therapeutic target in melanoma treatment. Br J. Cancer 117 (5), 656–665. Chen, J., Dexheimer, T.S., Ai, Y., Liang, Q., Villamil, M.A., Inglese, J., Maloney, D.J., Jadhav, A., Simeonov, A., Zhuang, Z., 2011. Selective and cell-active inhibitors of the This is a review manuscript, which does not involve any studies on USP1/UAF1 deubiquitinase complex reverse cisplatin resistance in non-small cell human subjects or animal experiments. Hence, no ethical approval was lung cancer cells. Chem. Biol. 18 (11), 1390–1400. needed. Chen, J.J., Cai, N., Chen, G.Z., Jia, C.C., Qiu, D.B., Du, C., Liu, W., Yang, Y., Long, Z.J., Zhang, Q., 2017. The neuroleptic drug pimozide inhibits stem-like cell maintenance and tumorigenicity in hepatocellular carcinoma. Oncotarget. 8 (11), 17593–17609. 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