Inhibition of autolysosomes by repurposing drugs as a promising therapeutic strategy for the treatment of cancers

Figures & data

Table 1. Dysfunctional autophagy is associated with different human diseases.

Cancers	Breast	Haplo insufficiency of Beclin 1 (Liang et al. 1999), mutation and deletion of EI24/PIG8 (Zhao et al. 2005)
	Ovarian	Haplo-insufficiency of Beclin 1 (Cai et al. 2014)
	Endometrial	ATG4 C, ULK4 and FIP200 along with MTOR-activating mutations (Devis-Jauregui et al. 2021)
	Prostate	Haplo-insufficiency of Beclin 1 (Levy et al. 2017)
	Brain	Mutation in PARK2 (Veeriah et al. 2010)
	Lung	Mutation in PARK2 (Xiong et al. 2015)
	Gastric/colorectal	Mutation in UVRAG and PARK2 (Kim et al. 2008; Poulogiannis et al. 2010; Park et al. 2014)
	Carcinoma (nasopharyngeal/ hepatocellular/ esophageal squamous cell carcinoma)	MicroRNA 106a-5p upregulation (Zhu et al. 2021), LncRNA NBR2 downregulation (Sheng et al. 2021); Genetic polymorphisms of ATG5 (Yang et al. 2017)
	Sarcoma	Overexpression of Bcl-2 and Bcl-XL; Mutation in p53 (Min et al. 2017)
Bone	Paget’s disease	Mutation in p62 (Usategui-Martin et al. 2020)
Lung	Asthma	Genetic polymorphisms of ATG5 (Martin et al. 2012)
	Cystic fibrosis	Mutation in CFTR (Groman et al. 2002); Sequestration of Beclin 1 in perinuclear aggregates (Luciani et al. 2011; Maiuri et al. 2019)
	Lymphangioleiomyomatosis	Mutations in TSC1/2 (Muzykewicz et al. 2009)
Heart/kidney	Ischemia/ reperfusion injury	Overexpression of Beclin 1 (Hamacher-Brady et al. 2006; Li et al. 2020)
Central nervous system	Alzheimer’s disease	Mutation in PSEN1 (Janssen et al. 2003; Tran and Reddy 2020)
	Parkinson’s disease	Mutations in PINK1 and PARK2 (Pickrell and Youle 2015; Barazzuol et al. 2020)
	Amyotrophic lateral sclerosis (ALS)	Mutation in p62; SOD1; OPTN; dynactin and dynein (Lee et al. 2015)
	SENDA	Mutation in WDR45 (WIPI4) (Mollereau and Walter 2019)
	Hungtington’s disease	Upregulation of mRNA expression of LC3A, ULK2, and LAMP2 and downregulation of PINK1, WDFY3 or ALFY, and FKBP1A (Martin et al. 2015)
	Hereditary spastic paraparesis	Mutations in TECPR2; SPG15 or KIF5A (Oz-Levi et al. 2012; Renvoise et al. 2014; Lopez et al. 2015)
Intestine	Crohn’s disease	T300A mutation in Atg16L1 (Ma et al. 2021); polymorphisms and mutations in NOD2 (Yamamoto and Ma 2009) and IRGM (Mehto et al. 2019)
	Ulcerative colitis	Downregulation of ATF4 and VDR (Shao et al. 2021); SNPs in REDD1/SMURF1 (Franke et al. 2010; Angelidou et al. 2018)
Polycystic kidney disease		Mutation in Pkd1/2 (Nowak and Edelstein 2020); BCL-2 deficiency (Veis et al. 1993)
Vacuolar myopathies in skeletal muscle		Accumulation of LC3-II (Margeta 2020), mutations in LAMP2, VMA21, and CLN3 (Weihl et al. 2015)
Liver	α1-Antitrypsin deficiency	Z mutation (SERPINA1) (Marciniak and Lomas 2010)
	NAFLD	Down-regulation of ATG7 (Khambu et al. 2018)
Immune diseases	Vici syndrome	Recessive mutation in EPG5 (Byrne et al. 2016)
	Lupus (SLE)	Polymorphisms in ATG5 and PRDM1 (Zhou et al. 2011)
	RA	Overexpression of Beclin 1, ATG5 and LC3 (Zhu et al. 2017)
Microbial infection	Mycobacterium tuberculosis	Polymorphism in IRGM (Xie et al. 2017)
	Mycobacterium leprae	Polymorphism in NOD2 (Leturiondo et al. 2020)
Metabolic Dysfunction	Type II diabetes	Loss of function of ATG7 and ER stress (Yao et al. 2021)
	Metabolic syndrome	Downregulation of ATG7 and ATG5; insulin sensitivity (Menikdiwela et al. 2020)
	Obesity (obesogenesis)	Deletion in ATG7, LAMP2 or TFEB (Zhang et al. 2018)
Aging		Overexpression of ATG5 in mice increases lifespan (Aman et al. 2021)
Lysosomal Disease	Danon disease	Mutation in LAMP2 (Lieberman et al. 2012; Majer et al. 2012)
	Pompe disease	Acid α-glucosidase (GAA) deficiency (Lieberman et al. 2012)
	Gaucher disease	Mutation in glucocerebrosidase (V394L) (Lieberman et al. 2012)

ATF4 (activating transcription factor 4), ATG16L1 (autophagy-related 16 like 1), CFTR (cystic fibrosis transmembrane conductance regulator), DRAM1 (DNA damage-regulated autophagy modulator protein 1), EPG5 (ectopic P-granules autophagy protein 5 homolog), FIP200 (RB1 inducible coiled-coil 1), FKBP1A (FK506-binding protein 1A), IRGM (immunity-related GTPase family M), KIF5A (kinesin heavy-chain), LncRNA NBR2 (long noncoding RNA neighbor of BRCA1 gene 2), NAFLD (Nonalcoholic fatty liver disease), NOD2 (nucleotide binding oligomerization domain containing 2), OPTN (optineurin), PAMPs (pathogen-associated molecular patterns), PARK2 (Parkin RBR E3 ubiquitin protein ligase), PINK1 (PTEN induced putative kinase 1), Pkd1/2 (polycystin 1/2), PSEN1 (presenilin 1), RA (rheumatoid arthritis), SENDA (Static encephalophagy of childhood with neurodegeneration in adulthood), SLE (systemic lupus erythematosus), SMURF1 (SMAD specific E3 ubiquitin protein ligase 1), SNPs (Single-nucleotide polymorphisms), SOD1 (superoxide disumutase 1), TSC1/2 (tuberous sclerosis complex 1 or 2), UVRAG (UV radiation resistance associated), VDR (vitamin D receptor), WDR45 (WD repeat domain 45).

Figure 1. Different classes of autophagy process. The autophagy is classified as (A) macroautophagy where cytosolic constituents are submerged and delivered to the lysosome; (B) microautophagy where cytosolic wastage products are straightly submerged and co-opted by lysosome via redisposition of its membrane and (C) chaperone-mediated autophagy (CMA) where KFERQ motif holding cellular proteins documented by the cytosolic HSP70 chaperone and translocated to the lysosomal membrane via interacting with LAMP2 receptor leading to their degradation. Macroautophagy (simply autophagy) proceeds the isolation membrane (IM) formation. Cellular stresses culminate in AMPK stimulation and mTORC1 suppression that in turn makes the ULK1 complex dissociated with mTORC1 and initiates autophagy. Through a series of interchain responses, this IM confiscates its cargos into an autophagosome (AP, a double-membrane vesicle) that in turn kisses and fuses with the lysosome to make autolysosome (AL). The AL cargos are degraded by an acidic niche containing hydrolytic enzymes in lysosomal lumen. The degrading products are used for energy production or biosynthesis of macromolecules. TFEB coordinates the stress responses like starvation via lysosome and controls countless cellular processes including autophagy and lysosomal biogenesis (Al-Bari and Xu 2020).

Figure 2. The central position of lysosomes at the ALP. Activated lysosomes and autolysosomes are associated with terminal degradation stage through endocytic and autophagic pathways. Exogenous products are endocytosed by endosomes and autolysosomes act as an integral part of the autophagy for degradation. The membrane of lysosome plays as a podium for the gathering of mTORC1 complex, which plays a main role for nutrient sensing signals of lysosomes to control energy levels through downstream mediators including TFEB. Interior pHs of intracellular vesicles steadily decrease laterally from early endosomes to late endosomes to lysosomes, the mostly acidic organelles. Lysosomes create and preserve their pH homeostasis by applying v-ATPase carrier. The positive charged ions such as K⁺ can be dispatched by K⁺ ion channel or by exchanged by Cl⁻ influx carrier via ClC-7, a Cl⁻/H⁺ antiporter.

Figure 3. Control of ALP by mTORC1-dependent and independent signal transductions. Amino acid (AA) signal, growth factors like insulin and cellular ATP regulate autophagy via mTORC1 mTORC1-dependent and independent pathways. Insulin stimulates both mTORC1 and mTORC2 via the traditional PI3K-AKT-TSC1/2 (tuberous sclerosis complex 1/2)-RHEB pathway. In Ras-MAPK (ERK) signal, ERK and RSK suppress TSC2 to induce mTORC1. Low nutrition status induces AMPK and TSC complex, suppressing RHEB and mTORC1. Carriage of AAs controls mTORC1 action via AA transporters like SLC3A2 in cell membrane as well as SLC38A9 in lysosomal membrane. The RAGULATOR complex ties up the RAGs to the surface of lysosome. V-ATPase is essential for the induction of mTORC1 on lysosomal surface. RAG A/B is controlled by the GATOR1 complex and GATOR2. KICSTOR complex acts as an essential action in tethering GATOR1 to the lysosomal surface, thus suppressing the activity of RAG A/B and mTORC1. Lysosomal membrane-oriented arginine (Arg) transporter, SLC38A9 binds with the RAGs and RAGULATOR to stimulate mTORC1. DNA damage inhibits mTORC1 activity by activating p53 in an AMPK- and TSC1/2-conditional way. Activated mTORC1 controls TFEB intracellular position via its phosphorylation (at Ser²¹¹) and subsequent confiscation with 14-3-3 protein. mTORC2 regulates autophagy via FOXO3 pathway. Phosphorylated FOXO3 interacts with 14-3-3 protein in cytoplasm and prevents induction of gene transcription. mTOR-independent cell signaling controlling autophagy comprises cAMP-Epac-PLCϵ-IP3, Ca²⁺-calpains–G_sα and inositol (Ins) signaling pathways. Intracytosolic Ca²⁺ status can be amplified by multiple Ca²⁺ channels including the TRP channel. An upsurge in cytosolic Ca²⁺ levels stimulates calpains and then Gsα/AC/cAMP/Epac/Rap2B/PLCϵ signal which regulates autophagy by endorsing IP3 via its receptor, IP3R activation. IP3 binds to IP3 receptor (IP3R) on ER to release stored Ca²⁺ that impairs AP maturation by blocking AP-lysosome fusion. Ca²⁺ efflux through lysosomal NAADP triggered TRP channel can be extra augmented by Ca²⁺ secretion from IP3R causing activation of the CaMKKβ–AMPK signal and AP formation. However, Ca²⁺ secretion from lysosomes can influence lysosomal pH and repeal fusion of lysosomes with APs. On the other hand, an upsurge Ca²⁺ concentration in cytosol next stimulates CAMKK-β, tracked by AMPK-mediated autophagy induction. In normal cases, phosphorylated TFEB by mTORC1 is sequestered with 14-3-3 protein in cytoplasm. Under fasting, lysosomal mTORC1 is inactive form and upsurge Ca²⁺ is secreted from lysosome via TRPML1 channel. Then Ca²⁺ stimulates CaN (calcineurin) that interacts and dephosphorylates TFEB. This form of TFEB is incapable to interact with 14-3-3 protein and easily moves to the nucleus (Al-Bari and Xu 2020).

Figure 4. The Janus-faced contradictory actions of autophagy in cancer cells. Autophagy acts as a Janus-faced play or the Yin-Yang faced function on cancer development. In healthy cells, it functions as a tumor inhibitory mechanism by ensuring optimal energy supply, preserving genomic integrity, promoting the degradation of damaged organelle and cellular oncogenes, and endorsing first-line immunity against microbial infection. In advanced tumors, however, autophagy actions can be reinstated. Autophagy functions as an oncogenic phenomenon by endorsing cancer development via declining stresses, the preservation of cancer stemness potential and metastasis; executing cells resistant to anoikis; helping the persistence of cancer cells in a dormant state and supporting the senescent cell state.

Table 2. Selected examples of ALP inhibitors with therapeutic benefits.

Agent	Derivative	Target of actions	Stage of autophagy	Indications	Remarks	Affected cancer cell type tested
Lysosomal lumen alkalizers: chloroquine analogues
Chloroquine	Aminoquinolines	Impaired lysosomal acidity	Fusion and degradation	Permitted for malaria	Nonselective suppression of ALP	Different cell types (McAfee et al. 2012; Selvakumaran et al. 2013; Levy et al. 2017; Rebecca et al. 2017)
Hydroxy-chloroquine	Aminoquinolines	Impaired lysosomal acidity	Fusion and degradation	Permitted for malaria, SLE and RA	Nonselective suppression of ALP	Different cell types (McAfee et al. 2012; Rangwala et al. 2014; Rebecca et al. 2017)
Quinacrine	9-aminoacridine	Impaired lysosomal acidity	Degradation	Putative not classic female sterility	Nonselective suppression of ALP	Different cell types (Goodall et al. 2014)
Mefloquine	Quinoline	Impaired lysosomal acidity	Degradation	Permitted for Malaria	Nonselective suppression of ALP	Different cell types (Sharma et al. 2012; Goodall et al. 2014)
Quinine	Quinoline	Impaired lysosomal acidity	Degradation	Permitted for malaria	Nonselective suppression of ALP	Different cell types (Liu et al. 2016)
Spautin-1	MBCQ	Suppression of USP10 & USP13	Nucleation	Preclinical cancers	Exception of CQ analogue	Different cell types (Liu et al. 2011)
Lys05	Aminoquinolines	Impaired lysosomal acidity	Fusion and degradation	Preclinical, cancer	Nonselective suppression of ALP	Different cell types (Amaravadi and Winkler 2012; McAfee et al. 2012)
ARN16090	ARN5187 analogue	Impaired lysosomal acidity	Fusion and degradation	Preclinical cancer	Suppression of NR1D2/REV-ERBβ	Colon cancer HCT116 (De Mei et al. 2015; Torrente et al. 2015)
SAR405	–	Inhibition of Vps34	Nucleation	Preclinical cancer	Kinase selectivity profile	Renal tumor cells (Pasquier 2015)
EAD1	Substituted triazole	accumulation of LC3-II	Nucleation	Experimental	More potent than CQ and HCQ	Human lung cancer cells (Nordstrom et al. 2015; Sironi et al. 2019)
Verteporfin	Benzoporphyrin	Suppression of LC3 lipidation	Elongation	Permitted as PDT	Exception of CQ analogue	Different cell types (Lu et al. 2009; Donohue et al. 2011)
VATG-027	1,2,3,4 tetrahydroacridine	Impaired lysosomal acidity	Fusion	Preclinical trials	Potent suppression than CQ	Mutant BRAF melanomas (Goodall et al. 2014)
Clioquinol ionophore	8-hydroxyquinoline	Impaired lysosomal acidity	Degradation	Permitted urinary infections	Autophagy initiation by interruption of mTOR signaling	Different cell types (Cao et al. 2014)
Nitazoxanide	2-AN-5NB	Impaired lysosomal acidity	Degradation	Antiprotozoal drug	Broad-spectrum anti-infection agent	Bladder cancer cells (Wang et al. 2018)
Carvedilol	methoxyphenoxy- 2-propanol	Impaired lysosomal acidity	Degradation	Synthetic antihypertensive	Nonselective β-adrenoceptor blocker	Different cell types (Meng et al. 2018)
ML-9	Naphthalene sulphonamide	Impaired lysosomal acidity	Diffusion and degradation	Inhibitor of Akt	Nonspecific inhibition of kinase	Prostate cancer cells (Kondratskyi et al. 2014)
L-asparagine	Nonessential amino acid	Inhibition of efflux pathway of lysosome	Fusion	Preclinical	Increase catalytic activity of L-asparaginase	Breast cancer cells (Knott et al. 2018)
Azithromycin	Macrolide antibiotic	Prevention of autophagosomal acidification	Fusion and degradation	Chronic infection	Failure to kill intracellular mycobacteria	Myeloma cells (Renna et al. 2011; Moriya et al. 2013)
Clarithromycin	Macrolide antibiotic	Prevention of autophagosomal acidification	Fusion and degradation	Infection	Failure to kill intracellular mycobacteria	Myeloma cells (Nakamura et al. 2010; Moriya et al. 2013)
Ciprofloxacin and norfloxacin	Quinolone Antibiotics	LMP induction	Diffusion and degradation	Infection	Caspase-independent cell death with MMP	Cervical carcinoma (Groth-Pedersen and Jaattela 2013)
Nanomycin and siomycin A	Quinone antibiotics	LMP induction Cathepsin D	Diffusion and degradation	Infection	Selectively inhibits DNMT3B	Colon carcinoma (Groth-Pedersen and Jaattela 2013)
Liensinine	Isoquinoline alkaloid	Impairment of RAB7 recruitment to lysosomes	Fusion and degradation	Preclinical	Unaffected lysosomal pH	Breast cancer cells (Zhou et al. 2015)
Matrine	Quinolizidine alkaloid	Impaired endosomal/lysosomal acidification	Diffusion and degradation	Preclinical	Induction of autophagy	Different cell types (Wang et al. 2013; Zhang et al. 2020)
MHY1485	Morpholino-triazine	Impaired lysosomal acidity	fusion	In vitro studies	mTOR inducer and autophagy inhibitor	Experimentally used (Yang et al. 2017)
Vacuolar H^+-ATPase inhibitors
Bafilomycin A1	Antibiotic	v-ATPase suppression	Fusion and degradation	Clinical trial	Global v-ATPase suppressor	Experimentally used (Li et al. 2016)
Doxorubicin Adriamycin	Anthracycline antibiotic	v-ATPase inhibition	Fusion and degradation	Permitted for leukemia	DNA-damaging agent	MEFs (Aits and Jaattela 2013; Li et al. 2016)
Manzamine A	Alkaloid	v-ATPase suppression	Fusion and degradation	Preclinical	Global v-ATPase suppressor	Cervical cancers (Kallifatidis et al. 2013; Karan et al. 2020)
Cleistanthin-A	Glycoside	v-ATPase suppression	Fusion and degradation	In vitro works	Global v-ATPase suppressor	Different cell types (Zhao et al. 2015; Liu et al. 2020)
Archazolid	Macrocyclic lactone	v-ATPase suppression	Fusion and degradation	In vitro works	Decrease of cathepsin B activity	Mammary epithelial cells (Kubisch et al. 2014; Merk et al. 2017)
E64d	Fungal metabolite	Protease suppression	Incomplete proteolysis	Not recorded for Leishmania	Nonspecific suppressor	Experimentally used in MCF-7 cells (Schurigt et al. 2010)
Lysosomal hydrolase inhibitors
Omeprazole	Pyramidal racemate	V-ATPase inhibitors	V-ATPase inhibitors, cathepsin B/L	Acid reflux, ulcers	Risk of gastric cancer for long-term use	Leukemia, lymphoma (Groth-Pedersen and Jaattela 2013; Cheung and Leung 2019)
Elaiophylin	Macrodiolide antibiotic	Abolition of maturation of cathepsin B and D.	Degradation	Antimicrobial and antihelminthic actions	Raise of autophagosome accrual	Ovarian cancer cells (Zhao et al. 2015)
Pepstatin A	Hexapeptide metabolite	Inhibition of cathepsin D	Partial lysosomal proteolysis	Not recorded	Reversible nonspecific suppressor	Experimentally used (Maynadier et al. 2013)
Vildagliptin	Cyanopyrrolidine	Cathepsin K Inhibitor	Partial Lysosomal proteolysis	Not recorded	Selective DPP-4 inhibitor	Colorectal cancers, lung cancer cells (Jang et al. 2015; Davidson and Vander Heiden 2017; Jang et al. 2019)
Acid sphingomyelinase (ASM) modulators
Siramesine	Sigma (σ) receptor agonist	ASM modulator	Modulation of LMP	treatment of anxiety	Lysosomotropic agent	Breast carcinoma (Groth-Pedersen and Jaattela 2013; Petersen et al. 2013)
HPS70 inhibitors
PES	Pifithrin-μ	ASM modulator	Modulation of LMP	HPS70 inhibitor	Lysosomotropic agent	NSCLC cells (Granato et al. 2013; Zhou et al. 2017)
Tetrandrine	bisbenzylisoquinoline alkaloid	Inhibition of lysosomal acidification	Diffusion and degradation	Preclinical	Activation of autophagy in HCC	Different cell types (Qiu et al. 2014; Wong et al. 2017)
Thapsigargin	Sesquiterpene lactone	Inhibitor of SERCA; elevation of cytoplasmic Ca^2+ levels	Nucleation, Fusion and degradation	Experimental agent	Induction of autophagy caused by ER stress	Different cell types (Ganley et al. 2011)
Paclitaxel (taxol)	Taxane	Inhibition of Vps34 and autophagosome trafficking	Initiation, degradation	Approved for breast, ovarian and lung cancers	Inhibition of autophagy reverses taxol resistance	Different cell types (Wang et al. 2020)
Geldanamycin	Natural product	Inhibition of cathepsin D	Initiation, degradation	Hsp90	Sustaining proliferative Signalling	Different cell types (Davidson and Vander Heiden 2017)

DNMT (DNA methyltransferases), DPP-4 (dipeptidyl peptidase-4), LMP/MMP (lysosomal/ mitochondrial membrane permeabilization), MBCQ (4- [[3,4-(Methylenedioxy) benzyl] amino]-6-chloroquinazoline), MEFs (mouse embryonic fibroblasts), NAC (N-acetyl-L-cysteine), NSCLC (non-small cell lung cancer), PES (2-phenylethyenesulfonamide).

Table 3. Selected examples of therapeutic benefits from ALP inhibition in animal models.

Agent	Host	Tumor type	Therapy	Comments
In vivo efficacy studies of CQ analogue as monotherapy
CQ	nu/nu mice	U373-EGFRwt (H)		ITR to epithelial tumor (Jutten et al. 2013; Galluzzi et al. 2017; Verbaanderd et al. 2017; Mainz and Rosenfeldt 2018)
	Athymic BALB/c nu/nu mice	CD133^+ and CD133^- cells (H)	–	Sensitive to LCSCs in TME
	Nude mice	HepG2-GFP (H)	–	Sensitive to liver cancer
	NOD-SCID mice	SKMel23 cells (H)	–	Selectively promotes apoptosis in melanoma cells
	nu/nu mice	B16-F10 cells (M)	–	ITR to melanoma model (Maes et al. 2014)
	Nude nu/nu mice	MCF7 and MDAMB231 cells	–	Potential response to TNBC
	NMRI nu/nu mice	HCT116 and HT29 cells	–	Limitations in efficacy of CQ in TME (Pellegrini et al. 2014)
CQ (Lys05)	nu/nu mice	1205Lu cells, C8161 cells, HT29 cells (H)	–	Standalone therapeutic effect in melanoma and CRC (McAfee et al. 2012)
HCQ	nu/nu mice	Pancreatic cancer (H)	–	ITR to pancreatic cancer
In vivo efficacy studies of autophagy inhibitor as combination therapy
CQ	Athymic nu/nu mice (4-6 wk)	U87MG glioma cells (H)	TMZ	ITR to glioblastoma
	C.B.17 SCID mice (8-10 wk)	HT29 colon carcinoma cells (H)	Bevacizumab, oxaliplatin	ITR to treatment in CRC (Selvakumaran et al. 2013)
	Swiss nu/nu mice (6 wk)	MDA-MB-231 breast cells (H)	CyP and/or doxorubicin	ITR to breast cancers
	NOD/SCID mice	HT29 cells (H) PROM1/CD133^+ CCSC	PDT	ITR to CRC, restoration of sensitivity to PDT
	BALB/c nude mice (5-6wk)	FaDu hypopharyngeal cells (H)	Cisplatin	ITR to HSCC model
	Wistar rats (8 wk)	C6 brain cells	TMZ (+curcumin)	ITR to brain tumor
	BALB/c nu/nu mice (4-6 wk)	EC109/CDDP oesophageal cells (H)	Cisplatin	ITR of cisplatin
	BALB/c mice (8 wk)	SGC7901 gastric cancer cells (H)	Cisplatin	Chemosensitivity to cisplatin
	NMRI nude mice (8 wk)	MDA-MB-231 and MCF-7 breast cancer cells	Rapamycin	Tumor growth reduction
	BALB/c nu/nu and BALB/c mice (4-6 wk)	CaR-1, HT-29, colon26 colon cancer cells	Temsirolimus (TEM)	Suppressed tumor growth and triggered enhanced apoptosis
	BALB/c nude mice (6 wk)	PC-9/wt and PC-9/gefB4 lung cancers	Gefitinib	Suppressed tumor growth
	BALB/c nude mice	HCC-827 lung cancers	Gefitinib (+ Akt inhibitor)	Significantly inhibited tumor growth
	Swiss albino mice	Ehrlich ascites carcinoma cells	Sunitinib	Significantly induced apoptosis and reduced tumor growth
	CD-1 nude mice (6-7 wk)	H3122CR-1 lung cancer cells	Crizotinib	Sensitization of drug resistant lung cancer cells to crizotinib
	NOD-SCID mice (6 wk)	Karpas-299 lymphoma cells	Crizotinib	Significantly suppressed tumor growth and increased apoptosis
	BALB/c nude mice (6-8 wk)	U251 glioblastoma cells	Vandetanib	Increased apoptosis and reduced tumor growth
	Athymic nude mice (4-5 wk)	JIMT-1 breast cancer cells	Trastuzumab	Significantly suppressed tumor growth
	New Zealand White rabbits	VX2 liver tumors	TACE	Significantly reduced tumor growth
HCQ	Rag2^−/− mice	MDA-MB-231 cells (H)	Epirubicin	Enhanced response to breast cancer model
	Nude NCr Nu-M mice (6 wk)	UACC903 melanoma cells	Temsirolimus	Promotes apoptosis and tumor suppression
	Athymic nude mice (5-6 wk)	H358 or H460 human NSCLC cells	Erlotinib	Significant sensitization to erlotinib therapy
	Athymic BALB/c nude mice (5-6 wk)	SPC-A1 lung cancer cells (H)	Crizotinib	Increased apoptosis and reduced tumor growth

5-FU (5-fluorouracil), ATM (ataxia telangiectasia mutated), CCSC (colorectal cancer stem cells), CQ (chloroquine), CRC (colorectal carcinoma), EGFR (epidermal growth factor receptor), H (human), HCC (hepatocellular carcinoma), HCQ (hydroxy-chloroquine), HDIL-2 (high-dose interleukin-2), HSCC (hypopharyngeal squamous cell carcinoma), ITR (improved therapeutic response), LCSCs (liver cancer stem cells), M (mouse), NMU (N-methyl-N-nitrosourea), NOD (non-obese diabetic), PDAC (pancreatic ductal adenocarcinoma), PDT (photodynamic therapy), SCID (severe combined immunodeficiency mice), TACE (transcatheter arterial chemoembolisation), TME (tumor microenvironment), TMZ (Temozolomide), TNBC (triple-negative breast cancers), wk (week-old), wt (wild-type).

Table 4. Ongoing and completed clinical trials with ALP inhibitors.

Treatment strategy
Inhibitor	Other agents	Disease	Trial phase	Identifier	Sponsor
CQ	–	SCLC	1/T	NCT01575782	Maastricht Radiation Oncology
	–	SCLC	1/T	NCT00969306	Maastricht Radiation Oncology
	TMZ	Gliosarcoma	1/NR	NCT04397679	Barbara Ann Karmanos Cancer Institute
	Trametinib	Metastatic N-RAS Melanoma	R	NCT03979651	Hospices Civils de Lyon
HCQ	Radiation	COVID-19, cancer	2/R	NCT04381988	Memorial Sloan Kettering Cancer Center
	Trametinib	Biliary cancer	2/NR	NCT04566133	National Cancer Institute (NCI)
	Ulixertinib	GI neoplasms	1/R	NCT04145297	University of Utah
	Trametinib	Pancreatic cancer	1/R	NCT03825289	University of Utah
	Letrozole, Palbociclib	Breast cancer AJCC v8	1/2/R	NCT03774472	M.D. Anderson Cancer Center
	Azithromycin	Cancer, COVID 19	2/R	NCT04341207	Gustave Roussy, Cancer Campus, Grand Paris
	LY3214996	Pancreatic cancer	2/R	NCT04386057	Kimberly Perez
	Cobimetinib, Atezolizumab	GI cancer	1/2/R	NCT04214418	Columbia University
	Binimetinib	Metastatic pancreatic adenocarcinoma	1/R	NCT04132505	M.D. Anderson Cancer Center
	Everolimus	Breast cancer stage IIB	2/R	NCT03032406	University of Pennsylvania
	Binimetinib	NSCLC	2/NR	NCT04735068	University of Pennsylvania
	Abemaciclib	Breast cancer	2/NR	NCT04523857	University of Pennsylvania
	Sorafenib	Hepatocellular Cancer	2/R	NCT03037437	The University of Texas Health Science Center at San Antonio
	–	Prostate cancer recurrent	2/R	NCT04011410	Andrew C. James, MD
	SUBA-itraconazole	Prostate cancer	1/2/R	NCT03513211	St Vincent's Hospital, Sydney
	TMZ	Glioblastoma	2/3/R	NCT03008148	Johnpro Biotech, Inc.
	CPI-613	Myelodysplastic syndromes	1/2/NR	NCT03929211	Wake Forest University Health Sciences
	Trametinib	Metastatic N-RAS melanoma	R	NCT03979651	Hospices Civils de Lyon
	Dabrafenib, Trametinib	Glioma	1/2/R	NCT04201457	Pediatric Brain Tumor Consortium
	Nivolumab, Ipilimumab	Melanoma	1/2/R	NCT04464759	Ravi Amaravadi, MD
	Docetaxel, Gemcitabine	Recurrent Osteosarcoma	1/2/R	NCT03598595	M.D. Anderson Cancer Center
	Carfilzomib, Dex	Multiple Myeloma	1/R	NCT04163107	Norwegian University of Science and Technology
	CPI-613	Sarcoma, Clear Cell	1/2/R	NCT04593758	Rafael Pharmaceuticals Inc.
	ABC294640	Cholangiocarcinoma	2/R	NCT03377179	RedHill Biopharma Limited
	–	2019 Novel Coronavirus	1/2/NR	NCT04731051	King Hussein Cancer Center
	Radiation	COVID-19, Cancer	2/R	NCT04381988	Memorial Sloan Kettering Cancer Center
	Trametinib	Biliary Cancer	2/NR	NCT04566133	National Cancer Institute (NCI)
Verteporfin	SpectraCure P18 System	Recurrent Prostate Cancer	1/R	NCT03067051	SpectraCure AB
Doxo-rubicin	Cisplatin	Ovarian cancer	1/R	NCT02475772	Ruhr University of Bochum
	Trastuzumab	Breast cancer	1/R	NCT02562378	Roche Pharma AG
	–	Cutaneous T cell lymphoma	1/R	NCT02192021	University of Pittsburgh
	Ixazomib, gemcitabine	Bladder cancer	1 /2/R	NCT02420847	M.D. Anderson Cancer Center
	–	Glioblastoma	2/R	NCT02758366	Meyer Children's Hospital
	–	Liver cancer	1/R	NCT02753881	Yale University
	Pembrolizumab	Sarcoma	1/2/R	NCT02888665	Fred Hutchinson Cancer Center
Clarithro-mycin	Lansoprazole, amoxicillin, metronidazole	Gastric diffuse large B-cell lymphoma	2/R	NCT02388581	National Health Research Institutes, Taiwan
	Selinexor, Dex, lenalidomide	Multiple myeloma	1/2/R	NCT02343042	Karyopharm Therapeutics Inc
	Antimicrobial agents	Neoplasms	2/R	NCT02366884	Dr. Frank Arguello Cancer Clinic
	Lenalidomide, Dex	Multiple myeloma	3/R	NCT02575144	PETHEMA Foundation
	Lenalidomide, Dex	Multiple myeloma	3/R	NCT04287660	The First Affiliated Hospital of Soochow University
	Triple therapy	Gastric cancer	4/R	NCT02047994	International Agency for Research on Cancer
	Daratumumab, Pomalidomide, Dex	Multiple myeloma	2/NR	NCT04302324	Weill Medical College of Cornell University
	Lenalidomide, Dex	Multiple myeloma in Relapse	2/R	NCT04063189	The First Hospital of Jilin University
Cipro-floxacin	Metronidazole, neomycin	Rectal cancer	2/NR	NCT02682485	University of Chicago
	Etoposide	Leukemia	1/2/R	NCT02773732	University of Florida
	Bile Acid Binder	Colon adenocarcinoma	R	NCT04003181	University of Aarhus
	–	Pancreatic ductal adenocarcinoma	1/R	NCT04523987	National University Hospital, Singapore
	Antibiotic	Acute lymphoblastic leukaemia	NR	NCT04678869	University College, London
Paclitaxel	Enzalutamide + carboplatin	Endometrial cancer	2/R	NCT02684227	M.D. Anderson Cancer Center
	fruquintinib	Advanced gastric cancer	3/R	NCT03223376	Sun Yat-sen University
	PU-H71	Metastatic breast cancer	1/R	NCT03166085	Memorial Sloan Kettering Cancer Center
	Gemcitabine	Small cell lung cancer	2/R	NCT02769832	Celgene Corporation
	Gemcitabine	Pancreatic adenocarcinoma	4/R	NCT03401827	Seoul National University Hospital
	Gemcitabine	Adenocarcinoma pancreas	4/ R	NCT02812992	AIO-Studien-GmbH

ANT (active, not recruiting), C (completed), Dex (dexamethasone), IELSG (International Extranodal Lymphoma Study Group), NCI (National Cancer Institute), NR (not yet recruiting), NSCLC (Non-Small Cell Lung Cancer), R (recruitment), S (suspended), SCLC (small cell lung cancer), T (terminated), TMZ (temozolomide).

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Data availability statement

Data sharing does not apply to this article as no new data were created or analyzed in this study.