FIELD OF THE INVENTION The present invention relates to the field of oncology, and more particularly relates to combinations, pharmaceutical compositions, or kit-of-parts, and their use for preventing and/or treating cancer diseases, and in particular, chemoresistant or potentially chemoresistant cancer diseases. BACKGROUND OF THE INVENTION

Cancer refers a group of diseases characterized by the development of abnormal cells that divide uncontrollably and can infiltrate and destroy normal body tissue. Cancer is the second-leading cause of death in the world. Numerous therapies, in particular chemotherapies, have been developed to treat the various cancer diseases. However, sometimes, cancer cells can overcome the efficacy of the anticancer agents and may become chemoresistant.

Mounting evidences indicate that lysosomes are involved in resistance to anticancer agents. One proposed mechanism is the sequestration of the anticancer agent into the lysosome which results in reduction of the accessibility of the drug to its cellular targets as well as of its effectiveness. This effect ultimately has been suggested to contribute to resistance to chemotherapy (Gong Y et al., J Pharmacol Exp Ther 2006; 316: 242-247; Herlevsen et al., Mol Cancer Ther 2007; 6: 1804-1813; Smith et al., Cancer Res 1992; 52: 4000^008; Zhitomirsky et al., Oncotarget 2015; 6: 1143-1156; Kroemer et al., Oncotarget 2017; 8: 112168-112169).

G-quadruplexes (G4s) are four-stranded non-canonical nucleic acids secondary structures which form in guanine-rich DNA and RNA sequences. Many G-quadruplex forming sequences are found to be associated with cancer. Some compounds, named G-quadruplex interactive ligands (or G-quadruplex ligands, or G4 ligands, abbreviated as G4L) have been shown to strongly interact with G-quadruplex DNA and to inhibit cancer proliferation. Those G- quadruplex ligands were proposed as anticancer agents based on their antiproliferative and chemosensitizing effects against tumor models both in vitro and in vivo (Li Q et al., Current Pharmaceutical Design 2012; 18, 1973-1983; Asamitsu S etai, Molecules 2019; 254:429).

Sun et al., (J Inorg Biochem, 2015) report a study characterizing the binding properties of enantiomers of complexes of polypyridyl and ruthenium (A-Ru and A-Ru) to G- quadruplex DNA. In this report, cellular uptake of ruthenium-based complexes was studied with a combination of A-Ru complex and chloroquine.

However, the accumulation of some DNA ligands in lysosomes have been described (Kang et al. Integr Biol Quant Biosci Nano Macro 2013; 5: 1217-1228; Shivalingam A et al. Nat Commun 2015; 6: 8178). For example, it has been shown that BMVC (3,6-bis(1- methyl-4-vinylpyridinium) carbazole diiodide) is particularly localized in the lysosomes of cancer cells resistant to chemotherapy and that the induction of lysosomal membrane permeabilization (LMP) with LLOMe (L-Leucyl-L-Leucine methyl ester) promotes its nuclear relocation (Kang et al. Integr Biol Quant Biosci Nano Macro 2013; 5: 1217-1228).

Therefore, there is a need to prevent the accumulation of anticancer G-quadruplex ligands (G4L) in the lysosomes of cancer cells to improve their efficiency. There is also a need to be able to liberate anticancer G-quadruplex ligands which could have been sequestrated into lysosomes of cancer cells.

There is a need to increase the efficacy, in particular to synergistically increase the efficacy, of anticancer G-quadruplex ligands.

There is a need to increase the efficacy of anticancer G-quadruplex ligands towards the target cancer cells.

There is a need to increase, and in particular to synergistically increase, the apoptotic activity of anticancer G-quadruplex ligands towards the cancer target cells.

There is a need to provide new combinations of anticancer active agents with increased efficacy, in particular with synergistic efficacy, towards the target cancer cells.

There is a need to provide new combinations of active agents with a lysosomotropic effect. There is a need to provide new combinations of active agents able to efficiently trigger lysosomal membrane permeabilization in target cancer cells.

There is a need to provide new combinations of active agents with increased efficacy, in particular with synergistic efficacy, towards lysosomal membrane permeabilization- induced cancer cells death.

There is a need to increase the induction of lysosomal dependent non-apoptotic cell death.

Lysosomal dependent non-apoptotic cell death is of tremendous interest in cancer therapy as cancer cells often display defective apoptotic machinery rendering them resistant to therapy.

The present invention has for purpose to satisfy all or part of those needs.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a combination comprising at least one anticancer G-quadruplex ligand and at least one lysosomotropic agent; or a pharmaceutically acceptable salt thereof.

The present invention lies on the discovery that chemoresistance, in particular lysosomal-induced chemoresistance, of cancer cells to anticancer G-quadruplex ligands can be removed or strongly reduced, or prevented, by a treatment combining at least one anticancer G-quadruplex ligand (anticancer G4L) with at least one lysosomotropic agent. It has been surprisingly observed that such a combination exerts, with an unexpected synergic effect, an apoptotic effect on cancer cells and chemoresistant cancer cells. More particularly, it has been surprisingly observed that such a combination can induce lysosomal membrane permeabilization into cancer cells, and trigger cancer cells death.

The inventors have unexpectedly found that the combination of an anticancer G4L, such as 20A or quarfloxin, and a lysosomotropic agent, such as chloroquine or Lys05, provokes a significant lysosomal membrane permeability and triggers a massive cell death in various cancer cell lines. The inventors have unexpectedly observed that an anticancer G4L, such as 20A or quarfloxin, and a lysosomotropic agent, such as chloroquine or Lys05, can act synergistically to provoke a significant lysosomal membrane permeability.

The inventors have unexpectedly observed that an anticancer G4L, such as 20A or quarfloxin, and a lysosomotropic agent, such as chloroquine or Lys05, can act synergistically to induce massive cancer cells death.

In particular, the inventors have unexpectedly observed that combining an anticancer G4L, such as 20A or quarfloxin, and a lysosomotropic agent, such as chloroquine or Lys05, triggers a massive cell death in cervical cancer cells, such as HeLa, and in lung adeno-carcinoma cells, such as the A549 cell line, in bone osteosarcoma epithelial cells, such as U20S,and the PDX cells from lung cancer patients, while a separate treatment with each compound leads to limited cell death.

In particular, the inventors have observed that combining an anticancer G4L, such as 20A or quarfloxin, and a lysosomotropic agent, such as chloroquine or Lys05, each at a sub-efficient dose, can induce a level of cancer cell death greater than the sum of cancer cell deaths observed with each drug used separately at the same dose.

The inventors have observed that combining an anticancer G4L, such as 20A or quarfloxin, and a lysosomotropic agent, such as chloroquine or Lys05, can synergistically act to induce cell death in cancer cells, such as cervical cancer cells or in lung adeno-carcinoma cells.

In one embodiment, in a combination of the invention, an anticancer G4L may be selected in the group consisting in triarylpyridines; fluoroquinolones and derivatives thereof; 2,6-diamidoanthraquinone and derivatives thereof; IZNP1 ; TH3; IZCZ-3; benzofuran and derivatives thereof; Tz1 ; furopyridazinone and derivatives thereof; GTC365; acridine and acridinium derivatives thereof; berberine and epiberberine; naphtalene diimides (NDI); quindoline; indoloquinolines; quinazolone derivatives; telomestatin; L1 H1 -70TD; cyanine derivatives; topotecan; porphyrin and porphyrazine derivatives thereof, such as N-methyl MesoPorphyrin IX (NMM) or TMPyP4; isaindigotone and derivatives thereof; SYUIQ-FM05; pyridostatin; bisquinolinium derivatives such as 360A or PhenDC3; carbazole derivatives; bleomycin; epigallocatechin gallate; theaflavin-3,3'-digallate; or a pharmaceutically acceptable salt thereof.

In one embodiment, in a combination of the invention, an anticancer G4L may be selected among triarylpyridines; fluoroquinolones and derivatives thereof; or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention relates to a combination comprising at least one anticancer G-quadruplex ligand selected among triarylpyridines and fluoroquinolones and at least one lysosomotropic agent; or a pharmaceutically acceptable salt thereof.

In one embodiment, the present invention relates to a combination comprising at least one anticancer G-quadruplex ligand selected among triarylpyridines and at least one lysosomotropic agent; or a pharmaceutically acceptable salt thereof.

A triarylpyridine suitable for the invention may be compound 20A as disclosed in Smith et al., Org Biomol Chem. 2011 ; 9(17) :6154-62).

A fluoroquinolone or a derivative thereof suitable for the invention maybe quarfloxin or CX-5461 .

In one embodiment, in a combination of the invention, an anticancer G4L may be triarylpyridine 20A; quarfloxin; or a pharmaceutically acceptable salt thereof.

In one embodiment, in a combination of the invention, a lysosomotropic agent may be selected in the group consisting in chloroquine and derivatives thereof, such as oxychloroquine or hydroxychloroquine; nanaomycin; siomycin A; helenalin; Lys05; siramesine; GNS561 ; or a pharmaceutically acceptable salt thereof.

In one embodiment, in a combination of the invention, a lysosomotropic agent may be selected in the group consisting in chloroquine; oxychloroquine; hydroxychloroquine; Lys05; siramesine; GNS561 ; and in particular is chloroquine or Lys05; or a pharmaceutically acceptable salt thereof; and more particularly is chloroquine diphosphate or Lys05. In a combination of the invention, a lysosomotropic agent may be chloroquine; hydroxychloroquine; or a pharmaceutically acceptable salt thereof such as chloroquine or hydroxychloroquine diphosphate.

In a combination of the invention, a lysosomotropic agent may be chloroquine; or a pharmaceutically acceptable salt thereof, such as chloroquine diphosphate.

In a combination of the invention, a lysosomotropic agent may be Lys05.

In one embodiment, in a combination of the invention, an anticancer G4L may be a triarylpyridine or fluoroquinolone, or a derivative thereof, and a lysosomotropic agent may be chloroquine, hydroxychloroquine, or Lys05, or a pharmaceutically acceptable salt thereof.

In one embodiment, in a combination of the invention, an anticancer G4L may be a triarylpyridine; a fluoroquinolone or a derivative thereof; or a pharmaceutically acceptable salt thereo, and a lysosomotropic agent may be chloroquine; Lys05; or a pharmaceutically acceptable salt thereof.

In one combination of the invention, an anticancer G4L may be 20A; quarfloxin; or a pharmaceutically acceptable salt thereof, and a lysosomotropic agent may be chloroquine; Lys05; or a pharmaceutically acceptable salt thereof.

According to another aspect, the present invention is directed to a pharmaceutical composition comprising a combination as disclosed herein and a pharmaceutically acceptable excipient or carrier.

According to another aspect, the present invention relates to a kit-of-parts comprising at least a first and a second containers, the first container is containing a first composition comprising at least one anticancer G-quadruplex ligand according to the invention, and the second container is containing a second composition comprising at least one lysosomotropic agent according to the invention.

According to another aspect, the invention relates to a combination according to the invention, or a pharmaceutical composition according to the invention, or a kit-of-parts according to the invention, for use in prevention and/or treatment of a cancer disease. According to one embodiment, a combination or a pharmaceutical composition or a kit-of-parts according to the invention may be for use in a synergistic prevention and/or treatment of a cancer disease.

According to one embodiment, a combination or a pharmaceutical composition or a kit-of-parts according to the invention may be for use in prevention and/or treatment of a chemoresistant cancer disease.

In a combination or a pharmaceutical composition or a kit-of-parts according to the invention, the anticancer G4L and the lysosomotropic agent may be for simultaneous, separate or sequential use.

According to one embodiment, a combination or a pharmaceutical composition or kit-of-parts according to the invention may be for use in prevention and/or treatment of cancer disease selected from breast cancer; colon cancer; rectal cancer; endometrial cancer; gastric carcinoma (including gastrointestinal carcinoid tumors and gastrointestinal stromal tumors); glioblastoma; hepatocellular carcinoma; cervical carcinoma; lung adeno-carcinoma (including small cell lung cancer and non-small cell lung cancer (NSCLC)); melanoma, including uveal melanoma; medulloblastoma; ovarian carcinoma; osteosarcoma; pancreatic cancer; prostate cancer; acute myelogenous leukemia (AML); chronic myelogenous leukemia (CML); non- Hodgkin's lymphoma; thyroid carcinoma; and pediatric tumors, such as leukemia, brain tumors, nephroblastoma, neuroblastoma, lymphoma, embryonic tumors, and rhabdosarcoma.

In embodiments, the invention also relates a method of synergistically preventing and/or treating a cancer disease in a subject in need thereof, said method includes administering to the subject a synergistically therapeutic effective amount of at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, thereby treating a cancer disease in said subject. The method includes observing a prevention or a treatment, such as a relieving, of the cancer disease.

In embodiments, the invention also relates to a method of preventing and/or treating a cancer disease in a subject in need thereof, said method includes administering to the subject a therapeutically effective amount of at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, thereby treating a cancer disease in said subject.

In embodiments, the invention also relates to a method for the prevention and/or treatment of a chemoresistant and/or potentially chemoresistant cancer disease in a subject in need thereof, said method includes administering to the subject a therapeutic effective amount of at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, thereby treating a chemoresistant cancer disease in said subject. The method includes observing a prevention or a treatment, such as a relieving, of the chemoresistant and/or potentially chemoresistant cancer disease. In embodiments, the invention also relates to a method for the synergistic prevention and/or treatment of a chemoresistant and/or potentially chemoresistant cancer disease in a subject in need thereof, said method includes administering to the subject a synergistically therapeutic effective amount of at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, thereby treating a chemoresistant cancer disease in said subject. The method includes observing a synergistic prevention or a synergistic treatment, such as a relieving, of the cancer disease.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that 20A and chloroquine act in concert to trigger cell death. A) HeLa cells and A549 cells were treated with the indicated concentration of 20A with (CQ - grey bars) or without (control - black bars) 25mM of chloroquine for 24h. Cell death was assessed by evaluating plasma membrane permeability using propidium iodide dye following flow cytometer analysis. The data represents the mean ± SD of 6 values obtained from three independent experiments each performed in duplicate. ^** p-value < 0.01 using Mann-Whitney test. B) Autophagy-proficient sg NT 1 (cells transduced with control plasmid NTC1 - white bars) and -deficient HeLa cells sg ATG5 and sg ATG7 (ATG5 and ATG7 KO cells - respectively light and dark grey bars) were treated without (DMSO) with 5mM 20A either in the presence or absence 25mM of chloroquine (CQ) for 24h, or with chloroquine alone. Cell death was assessed by evaluating plasma membrane permeability using propidium iodide dye following flow cytometer analysis. The data represents the mean ± SD of 9 values obtained from three independent experiments each performed in triplicate p-value ^** < 0.01 and ^*** < 0.001 , ns = non-significant using Mann-Whitney test. Figure 2 shows that 20A and chloroquine act in concert to trigger LMP in the cancer cells U20S expressing Galectin3-mCherry cells. A) The percentage of cells displaying Galectin 3 punctae are scored (0 punctae - light grey; 1 or 2 punctae - dark grey; 3+ punctae - medium grey). Data are presented as mean ± SD of 15 values obtained from 5 randomly chosen fields in each of the three independent experiments. B) The number of Galectin 3 punctae per cell in absence (control) and presence of chloroquine (CQ), and with (light grey bars) or without (black bars) A20 was scored and results are expressed as percentage of cells with indicated Gal-3 puncta. Data are presented as the mean percentage of at least 1000 cells analyzed from 5 randomly chosen field in each of the three independent experiments. ^**** p- value < 0.0001 using Mann-Whitney test Figure 3 shows that apoptosis is involved in cell death induced by the combination of chloroquine and 20A. A) HeLa cells (left panel) and A549 (right panel) cells were treated with 5 mM and 3.5 mM 20A respectively in either the presence or absence of 25 mM chloroquine for 24h. Apoptotic cell death was assessed by immunoblot analysis of cleaved forms of either Caspase 3 or PARP1 . Actin-b was detected as a loading control. B) HeLa cells (left panel) and A549 (right panel) cells were treated with (+) or without (-) 20 mM QVD-OPH (QVD) two hours prior to the addition of 0 (-), or 5 mM 20A (+) and 3.5 mM 20A (+) for HeLa cells and A549 cells, respectively. Where indicated, cells are also exposed (+) to 25 mM chloroquine (CQ) for 24h. Apoptotic cell death was assessed by measurement of loss of mitochondrial transmembrane potential (DYGTI). The data represents the mean ± SD of 6 values obtained from three independent experiments each performed in duplicate. ^** p-value < 0.01 using Mann-Whitney test.

Figure 4 shows that 20A and Lys05 (Ly05) act in concert to trigger cell death. U20S cells were treated with the indicated concentration of 20A (0, 1 , 2, 3, 4 and 5 mM) with (Ly05 1 or 5 mM - respectively light or medium grey bars) or without (no Ly05 - black bars) Lys05 for 24h. Cell death was assessed by evaluating plasma membrane permeability using DAPI dye staining following flow cytometer analysis. The data represents the mean ± SD of 6 values obtained from three independent experiments each performed in duplicate. ^** p-value < 0.01 using Mann-Whitney test.

Figure 5 shows that quarfloxin (CX) and Chloroquine (Chloro) act in concert to trigger cell death. U20S cells were treated with the indicated concentration of quarfloxin (CX) (0, 0.5, 1 , and 2 mM) with (Chloro 20 mM or 25 mM - respectively light or medium grey bars) or without (No Chloro - black bars) Chloroquine for 24h. Cell death was assessed by evaluating plasma membrane permeability using DAPI dye staining following flow cytometer analysis. The data represents the mean ± SD of 6 values obtained from three independent experiments each performed in duplicate. ^** p-value < 0.01 using Mann-Whitney test.

DETAILED DESCRIPTION OF THE INVENTION While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.

The abbreviations used herein have their conventional meaning within the chemical and biological arts. It is understood that aspects and embodiments of the present disclosure described herein include “having,” “comprising,” “consisting of,” and “consisting essentially of” aspects and embodiments. The words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of the stated element(s) (such as a composition of matter or a method step) but not the exclusion of any other elements. The term “consisting of” implies the inclusion of the stated element(s), to the exclusion of any additional elements. The term “consisting essentially of” implies the inclusion of the stated elements, and possibly other element(s) where the other element(s) do not materially affect the basic and novel characteristic(s) of the invention. It is understood that the different embodiments of the disclosure using the term “comprising” or equivalent cover the embodiments where this term is replaced with “consisting of” or “consisting essentially of”.

Anticancer G-quadruplex ligands

One of the active agents considered within the invention is an anticancer G- quadruplex ligand (anticancer G4L).

G-quadruplexes (G4s) are non-canonical nucleic acids secondary structures that form within guanine-rich strands of regulatory genomic regions. G4s have been extensively described in the human genome and have been found as having implications for various pharmacological and biological events, including cancers. Ligands (or agent) interacting with G4s have attracted great attention as potential anticancer therapies.

As used herein, “ligand” refers to a molecule able to reversibly bind to a targeted macromolecule such as a protein or a nucleic acid and having a functional role, such as structural stabilization, catalysis, enzyme activity modulation, or signal transmission.

An “anticancer agent”, and preferably an “anticancer G-quadruplex ligand” as used herein refers to a molecule used to treat cancer through prevention, destruction or inhibition of cancer cells or tissues. Anticancer agents may be selective for certain cancers or certain tissues. An anticancer G4L suitable for the invention is able to bind and stabilize G- quadruplex structures, hereby inducing cancer cell death and then anticancer activity. Not all G4Ls have anticancer activity. But G4Ls with anticancer activity are known to the skilled person (Asamitsu et al., Molecules 2019; 254:429; Sengupta et al., Molecules 2019; 254:582 ; Shivalingam et al. Nat Commun 2015; 6: 8178; Smith et al., Org Biomol Chem. 2011 ; 9(17):6154-62; Li et al., Current Pharmaceutical Design 2012; 18, 1973-1983).

Several methods have been developed to identify G4Ls with anticancer activity which can be suitable for the invention (Li et al. Current Pharmaceutical Design 2012; 18, 1973-1983). As screening methods usable to identify G4Ls, one may mention virtual screening, NMR methods, fluorometric-based methods, competition dialysis, or electrospray ionization mass spectrometry. All those methods are then completed with in vitro or in vivo analysis, such as xenograft cancer models, to show anticancer activity of the selected compounds.

Among the anticancer G4Ls that may be useful for the invention, one may mention the following compounds and their derivatives.

Within the invention, “derivative(s)” with respect to a particular compound or a particular family of compounds is intended to mean a compound or a set of compounds presenting substantially the same chemical structure for substantially the same biological or pharmacological activity. A derivative of a compound or of a family of compound is a bioisostere. A bioisostere is a structural analogue of a compound (parent compound) obtained by substitution of an atom or a group of atoms in the parent compound for another with similar electronic and steric characteristics and which retains the biological or pharmacological activity of the parent compound. For example, a chlorine -Cl group may be replaced by a trifluoromethyl -CF ₃ group, or by a cyano -CºN group. In another example a phenyl -C ₆ H ₅ ring can be replaced by a different aromatic ring such as thiophene or naphthalene. Accordingly, the chemical structure of a derivative according to the invention may differ from the chemical structure of a reference compound (i) by the absence of one or more substituents and/or (ii) the presence of one or more supplemental substituents and/or (iii) the substitution of one or more substituents by another having substantially the same physico-chemical properties, for instance a fluorine by a chlorine or a methyl by an ethyl, in such a manner that the derivative has substantially the same biological or pharmacological activity than the reference compound.

Within the disclosure, the term “substantially” used in conjunction with a feature of the disclosure intends to define a set of embodiments related to this feature which are largely but not wholly similar to this feature in such a manner that the difference does not materially affect the nature of the feature.

- Triarylpyridines having an anticancer G4L activity, such as the compounds described in Smith et al. ( Org Biomol Chem. 2011 ; 9(17):6154-6220A), in particular 20A (compound 3 in Smith et al.) which has been proposed against cervical carcinoma, lung adeno carcinoma, and osteosarcoma;

- Fluoroquinolones and derivatives thereof having a G4L activity such as quarfloxin (also known as CX-3543) and the related non-fluorinated compound pidnarulex CX-5461.

CX-5461 and CX-3543 have a specific toxicity against breast cancer cells. Furthermore, quarfloxin has completed Phase II trials as a candidate therapeutic agent against several tumors, including neuroendocrine tumors, carcinoid tumors, and lymphoma.

- 2,6-diamidoanthraquinone and derivatives thereof;

- IZNP1 (2-(4-(4,5-bis(4-(4-methylpiperazin-1 -yl)phenyl)-1 H-imidazol-2-yl)phenyl)- 6-(4-methylpiperazin-1 -yl)-1 H-benzo[de]isoquinoline-1 ,3(2H)-dione), proposed against squamous cell carcinoma;

- TH3 (thiazole peptide TH3 as disclosed in Dutta et al., Nucleic Acids Res.

2018;46(11):5355-5365), proposed against lung cancer, cervical cancer; IZCZ-3 (9-ethyl-3-[1 -(4-methoxyphenyl)-4,5-bis[4-(4-methylpiperazin-1 - yl)phenyl]imidazol-2-yl]carbazole) proposed against squamous cell carcinoma, cervical cancer, liver cancer, malignant melanoma;

- benzofuran and derivatives thereof, proposed against myeloma; - Tz1 (triazole derivative as disclosed in Panda et ai., Nat Commun. 2017;8:18103)

T¾ 1 proposed against colorectal carcinoma;

- Furopyridazinone and derivatives thereof proposed against human acute T cell leukemia; - GTC365 (2-[3-[[4-[(3-Nitroacridin-9-yl)amino]phenyl]sulfamoyl]propy l]guanidine hydrochloride), proposed against breast cancer (adenocarcinoma);

- acridine and acridinium derivatives thereof;

- berberine and epiberberine;

- naphtalene diimides (NDI); - quindoline, proposed against colorectal cancer, cervical cancer, liver cancer, multiple myeloma, lung cancer;

- indoloquinolines;

- quinazolone derivatives;

- telomestatin, a macrocycle naturally occurring in Streptomyces annulatus ;

- L1 H 1 -70TD (a telomestatin derivative as disclosed in WO 2009/157505 A1 )

- cyanine derivatives;

- topotecan;

- porphyrin and porphyrazine derivatives thereof, such as N-methyl Meso Porphyrin IX (NMM) or TMPyP4, a cationic porphyrin proposed against mammary tumors and human prostate carcinomas;

- isaindigotone and derivatives thereof;

- SYUIQ-FM05 (N’-(7-Fluoro-5-N-methyl-10H-indolo[3,2-b]quinolin-5-ium)- N,N- dimethylpropane-1 ,3-diamine iodide); - Pyridostatin;

- bisquinolinium derivatives, such as 360A (2-N,6-N-bis(1-methylquinolin-1-ium-3- yl)pyridine-2, 6-dicarboxamide) or PhenDC3 (2-N,9-N-bis(1-methylquinolin-1-ium-3-yl)-1 ,10- phenanthroline-2,9-dicarboxamide;trifluoromethanesulfonate);

- carbazole derivatives; - bleomycin;

- epigallocatechin gallate and theaflavin-3,3'-digallate (TFDG) from green tea and black tea;

- or a pharmaceutically acceptable salt thereof.

In one embodiment, a G-quadruplex ligand is not a metal-based complex. In another embodiment, a G-quadruplex ligand is not a ruthenium-based complex. As exemplary embodiment, it is not a polypyridyl chiral ruthenium (II) complex. In one embodiment, it is not a A-[Ru(phen) ₂ (p-DMNP)] ²⁺ or a A-[Ru(phen) ₂ (p-DMNP)] ²⁺ as described in Sun et al., J Inorg Biochem, 2015.

In one embodiment, a combination as disclosed herein does not comprise, or does consist of, a combination of a ruthenium-based complex and of chloroquine.

In one embodiment, a combination as disclosed herein does not comprise, or does consist of, a combination of A-[Ru(phen) ₂ (p-DMNP)] ²⁺ or a A-[Ru(phen) ₂ (p-DMNP)] ²⁺ and of chloroquine.

In one embodiment, a combination as disclosed herein does not comprise, or does consist of, a combination of A-[Ru(phen) ₂ (p-DMNP)] ²⁺ and of chloroquine.

In embodiments, pharmaceutically acceptable salts of an anticancer G4L may also be used.

As used herein, a "pharmaceutically acceptable salt" of an agent in accordance with the invention means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent agent. It is understood that the pharmaceutically acceptable salts are non-toxic. As used herein, the term “salt” refers to acid or base salts of agents used in the present invention. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts. The neutral forms of the agents are preferably regenerated by contacting the salt with a base or acid and isolating the parent agent in the conventional manner. The parent form of the agent differs from the various salt forms in certain physical properties, such as solubility in polar solvents.

According to one embodiment, an anticancer G4L useful for the invention may be preferably selected in the group consisting in triarylpyridines; fluoroquinolones and derivatives thereof; acridine and acridinium derivatives; berberine and epiberberine; naphtalene diimides (NDI); quindoline; indoloquinolines; quinazolone derivatives; telomestatin; L1 H1 -70TD; cyanine derivatives; topotecan ; porphyrin and porphyrazine derivatives, such as N-methyl MesoPorphyrin IX (NMM) or TMPyP4; SYUIQ-FM05; pyridostatin; bisquinolinium derivatives such as 360A or PhenDC3; or a pharmaceutically acceptable salt thereof.

According to another embodiment, an anticancer G4L useful for the invention may be selected in the group consisting in triarylpyridines; fluoroquinolones; acridines; berberine and epiberberine; naphtalene diimides (NDI); quindoline; indoloquinolines; quinazolones; telomestatin; L1 H1-70TD; cyanines; topotecan ; porphyrin and porphyrazines, such as N- methyl MesoPorphyrin IX (NMM) or TMPyP4; SYUIQ-FM05; pyridostatin; bisquinoliniums, such as 360A or PhenDC3; or a pharmaceutically acceptable salt thereof.

According to one embodiment, an anticancer G4L useful for the invention may be preferably selected in the group consisting in triarylpyridines; fluoroquinolones and derivatives thereof; or a pharmaceutically acceptable salt thereof.

According to one preferred embodiment, an anticancer G4L useful for the invention may be a triarylpyridine or a pharmaceutically acceptable salt thereof, and preferably may be the triarylpyridine 20A, or a pharmaceutically acceptable salt thereof, and more preferably is the triarylpyridine 20A.

Other triarylpyridine compounds suitable for the invention are described in the publication of Smith et al. Org Biomol Chem. 2011 ; 9(17):6154-62. 20A is the triarylpyridine compound n°3 of the publication of Smith etal. Org Biomol Chem. 2011 ; 9(17):6154-62.

According to one embodiment, an anticancer G4L useful for the invention may be a fluoroquinolone, a derivative thereof, or a pharmaceutically acceptable salt thereof.

In one preferred embodiment, a fluoroquinolone suitable for the invention may be quarfloxin (CX-3543), or a pharmaceutically acceptable salt thereof, and preferably is quarfloxin.

In one preferred embodiment, a fluoroquinolone derivative suitable for the invention may be CX-5461 (pidnarulex), or a or a pharmaceutically acceptable salt thereof, and preferably is CX-5461 . In one preferred embodiment, an anticancer G4L useful for the invention may be triarylpyridine 20A, quarfloxin or CX-5461 , or a or a pharmaceutically acceptable salt thereof, and preferably is triarylpyridine 20A or quarfloxin; or a pharmaceutically acceptable salt thereof, and preferably is triarylpyridine 20A or quarfloxin.

An anticancer G4L is used in a therapeutically effective amount.

An “effective amount” or a “therapeutically effective amount” as used herein is an amount sufficient for an agent or a combination of agents, as considered here, to contribute to the treatment or prevention of a cancer disease or of a symptom of a cancer disease. The exact amounts will depend on the various factors, such as the purpose of the treatment, the age, weight, or sex of the patient, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

Effective amounts, or dosages, may be varied depending upon the requirements of the patient and the anticancer G4L being employed. The dose administered to a patient, in the context of the present invention, should be sufficient to induce a beneficial cancer treatment or prevention in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects. Determination of the proper dosage for a particular situation is within the skill of the practitioner, and will vary depending on the compounds, the combinations, the disease state and its severity, the bioavailability characteristics of the compounds, the age of the patient to be treated, and the like.

A suitable therapeutically effective amount for an anticancer G4L in accordance with the invention, for example 20A or quarfloxin, may be, for example, within the range of 5 to 25, preferably 10 to 20, more preferably about 15 mg/kg. This dosage may be particularly useful for triarylpyridine 20A or for fluoroquinolone quarfloxin.

The duration of time for which an anticancer G4L in accordance with the invention can be administered will vary depending on various factors such as the cancer, the patient, the compound, and can be ascertained using known techniques in the art. An anticancer G4L in accordance with the invention, such as in particular triarylpyridine 20A or fluoroquinolone quarfloxin, maybe administered for several days, weeks or months, and for example for 2 weeks, or more preferably for one week. According to one amendment, an anticancer G4L in accordance with the invention maybe administered for 5 consecutive days over 7 days, for two weeks.

Lysosomotropic agent

One active agent considered within the invention is a lysosomotropic agent. Lysosomotropic agent are compounds, lipophilic or amphiphilic with a basic moiety, able to be protonated and trapped within lysosomes (Giralod et al., Biochem Soc Trans, 2014; Kuzu eta!., Pharmacological Research, 2017).

Lysosomes are advanced organelles involved in many cellular processes and are considered crucial regulators of cell homoeostasis. The interior is acidic with a pH of <5 and contains over 50 hydrolases, able to degrade all constituents of the cell. Lysosomes are limited by a single 7-10-nm phospholipid bilayer that functions as an interface to regulate communication between the lysosomal lumen and the cytosol. Owing to their high hydrolase content, leakage of lysosomal content to the cytosol is potentially harmful to the cell. Partial permeabilization of the membrane induces apoptosis, whereas massive lysosomal rupture induces necrosis. Lysosomal membrane permeabilization (LMP) is characterized as any damage to the lysosomal membrane that results in the release of the lysosomal contents into the cytosol

A lysosomotropic agent suitable for the invention is a compound able to relocate all or part of the lysosomal content to the cytosol. A lysosomotropic agent suitable for the invention is an active agent which accumulate inside acidic compartments.

As examples of methods suitable to identify lysosomotropic agent suitable for the invention, one may mention the imaging method described by Nadanaciva et al. (Toxicology in Vitro. 2011 , 25(3), 715-723) or by Seo et al., ( Biochemical and Biophysical Research Communications, 2014, 450(1), 189-194).

Among the lysosomotropic agents that may be useful for the invention, one may mention:

- chloroquine and derivatives thereof, such as oxychloroquine or hydroxychloroquine. Chloroquine has already been proposed for the treatment of some cancer diseases;

- Lys05 (N-(7-chloroquinolin-4-yl)-N'-[2-[(7-chloroquinolin-4-yl)ami no]ethyl]-N'- methylethane-1 ,2-diamine trihydrochloride);

- Siramesine;

- GNS561 (as disclosed in Brun et al., Invest New Drugs 37, 1135-1145 (2019);

- nanaomycin;

- siomycin A;

- helenalin;

- or a pharmaceutically acceptable salt thereof.

“Derivative” has the definition as previously provided.

In embodiments, pharmaceutically acceptable salts of a lysosomotropic agent may also be used. A "pharmaceutically acceptable salt" has the definition as previously provided.

According to one embodiment, a lysosomotropic agent may be chloroquine; oxychloroquine; hydroxychloroquine; Lys05; siramesine; GNS561 ; or a pharmaceutically acceptable salt thereof.

According to one embodiment, a lysosomotropic agent may be chloroquine; oxychloroquine; hydroxychloroquine; or a pharmaceutically acceptable salt thereof.

According to one embodiment, a lysosomotropic agent may be chloroquine or a pharmaceutically acceptable salt thereof, and preferably is chloroquine diphosphate.

According to one embodiment, a lysosomotropic agent may be Lys05 or a pharmaceutically acceptable salt thereof, and preferably is Lys05. According to one embodiment, a lysosomotropic agent may be selected among the group consisting in chloroquine; chloroquine diphosphate; hydroxychloroquine, and Lys05.

According to one embodiment, a lysosomotropic agent may be selected among the group consisting in chloroquine; Lys05; and a pharmaceutically acceptable salt thereof. According to one embodiment, a lysosomotropic agent may be selected among the group consisting in chloroquine diphosphate and Lys05.

A lysosomal membrane permeabilization inducing agent is used in a therapeutically effective amount. “Effective amount” or a “therapeutically effective amount” have the definition as previously provided. Dosages may be varied depending upon the requirements of the patient and the lysosomal membrane permeabilization inducing agents being employed.

A lysosomotropic agent, such as chloroquine or hydroxychloroquine, or a pharmaceutically acceptable thereof, may be administered at a therapeutically effective amount ranging from 10 to 1000 mg/per dose, from 20 to 800 mg/dose, from 50 to 400 mg/dose, from 100 to 300 mg/dose.

For instance, a dosage useful for chloroquine may be in a range from 100 to 1000 mg/dose, from 150 to 800 mg/dose, or from 200 to 500 mg/dose. Combinations, Pharmaceutical compositions, and Kit-of-parts

According to one embodiment, the present invention is directed to a combination comprising at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof.

It is to be understood that the term a "combination" as used herein envisages the simultaneous, sequential or separate administration of the components of the combination. In one aspect of the invention, a combination envisages simultaneous administration of the anticancer G4L and the lysosomotropic agent. In a further aspect of the invention, a combination envisages sequential administration of those agents. In another aspect of the invention, a combination envisages separate administration of those agents. Where the administration of those agents is sequential or separate, the delay in administering the second component should not be such as to lose the benefit of the effect of the combination therapy, in particular the synergistic effect. The combination according to the invention comprises therapeutically active amount of the active agents. Accordingly, a combination in accordance with the invention is therapeutically active.

A combination according to the invention is able to induce lysosomal membrane permeabilization or LMP, and to trigger cancer cells death. The active agents of the combination of the invention, an anticancer G4L and a lysosomotropic agent, are able to synergistically induce LMP and to synergistically trigger cancer cells death.

The active agents of a combination of the invention, that is an anticancer G4L and a lysosomotropic agent, may not induce LMP when used alone. However, when they are used in a combination of the invention, the resulting combination may induce LMP and may trigger cancer cells death.

The high content of hydrolytic enzymes in lysosomes makes them potentially harmful to the cell. If the lysosomal membrane is damaged (e.g. by membrane permeabilization), lysosomes release their contents into the cytosol, setting off indiscriminate degradation of cellular components. The distinctive sign of LMP is the translocation of soluble lysosomal components, such as cathepsins and other hydrolases, from the lysosomal lumen to the cytosol. Accordingly, LMP can be measured by a variety of simple techniques. Immunofluorescence techniques using antibodies against cathepsins such as CB and CD can reveal the redistribution of these proteases from lysosomes to the cytosol.

Several methods have been developed to detect LMP which can be useful to identify combinations suitable for the invention.

One may mention methods measuring the release into the cytosol of lysosomal enzymes such as cathepsins. These proteins can be visualized by fluorescence microscopy. Cathepsins confined within intact lysosomes are visualized as a punctate pattern of intense fluorescence and can be co-stained with antibodies against LAMP-1 or LAMP-2. By contrast, cathepsins released during LMP produce a diffuse fluorescence pattern throughout the cell. Cytosolic release of cathepsins can also be detected by Western blot of the cytosolic fractions after cell fractionation. This method can be used to estimate the extent of LMP by simultaneously determining the levels of several cathepsins of different sizes over time. Alternatively, cathepsins in cytosolic extracts can be detected using cathepsin-specific substrates (eg, Magic Red) and a fluorescence plate reader. Fluorescence microscopy can also be used to count cathepsin-positive cells in tissue sections. Those methods, and others, are well-known to the skilled person.

According to another embodiment, the invention relates to a pharmaceutical composition comprising a combination comprising at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier.

Further, according to another embodiment, the invention is directed to a kit-of-parts comprising at least a first and a second containers, said first container containing a first composition comprising at least one anticancer G4L, and said second container containing a second composition comprising at least one lysosomotropic agent.

In embodiments, the compositions of the kit-of-parts are pharmaceutical compositions.

The anticancer G4L and lysosomotropic agent are as defined herein.

In one embodiment, the anticancer G4L is as defined herein 20A or quarfloxin.

In one embodiment, the lysosomotropic agent is as defined herein chloroquine, or a pharmaceutically acceptable salt thereof, such as chloroquine diphosphate, or is Lys05.

Compositions or combinations provided herein comprise the active agents in a therapeutically effective amount, i.e., in an amount effective to achieve its intended purpose. As exposed above, the actual effective amount for a particular application will depend, inter alia, on the condition being treated and various other factors well-known in the art such as the age, the weight, the sex of the patient, the presence of other potential aggravating conditions, or the diet. Determination of a therapeutically effective amount of a compound of the invention is well within the capabilities of those skilled in the art.

The pharmaceutical compositions or combinations described herein can be prepared according to techniques known to the skilled person by using a combination of at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable excipient or carrier.

These compositions may comprise one or more pharmaceutically acceptable excipients or carriers. Suitable carriers and excipients and their formulations are described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). By pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained.

The compositions can be in any form deemed appropriate by the skilled person, such as solid, semi-solid, liquid, granular, inhalation or aerosol inhalation.

The liquid forms may be appropriate forms for oral or systemic administration.

Compositions suitable for oral administration can be capsules, tablets, pills, powders, granules, solutions or suspensions in aqueous or non-aqueous liquids, foam or beaten edible, liquid oil in water emulsions or liquid water in oil emulsions.

For example, for oral administration in capsule or tablet form, the active agents mentioned herein may be combined with pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and similar. There may also be present flavourings, preservative, colouring coating and/or dispersant agents.

Compositions suitable for parenteral administration may include sterile aqueous or non-aqueous solution for injection which may contain antioxidants, buffers, bacteriostatic and solutes which render the solution isotonic with the blood of the intended recipient, and aqueous or non-aqueous sterile suspensions which may include suspending and thickening agents. These compositions may be sterilized by conventional, well known sterilization techniques. A parenteral composition may include a solution or suspension of the compounds in a vehicle such as sterile water or a parenterally acceptable oil. Alternatively, the solution can be lyophilized. The lyophilized parenteral pharmaceutical composition can be reconstituted with a suitable solvent just prior to administration. The compositions may be presented in single dose or multi-dose containers, for example, sealed ampoules or vials, and may be stored in lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from powders, granules, lyophilized and sterile compresses. In the case of parenteral administration, the composition may also be provided with the active ingredients in separate containers that can be suitably admixed according to the desired dosage taking into account the weight, age, gender and health status of the patient in need thereof.

The anticancer G4L and the lysosomotropic agent, or a pharmaceutically acceptable salt thereof, may also be presented in separate compositions, packaged in separate containers, as a kit-of parts. The separate compositions may then be admixed before administration for a simultaneous administration, or they may be administered separately or sequentially. Cancer diseases, Therapeutic uses & Methods of treatment

In embodiments, the combination or the pharmaceutical composition or a kit-of- parts according to the invention are for use in prevention and/or treatment of a cancer disease.

In embodiments, the invention relates to the use of a combination according to the invention for the manufacture of a medicament for the prevention and/or treatment of a cancer disease.

In embodiments, the invention relates to the use of a combination according to the invention for the manufacture of a kit-of-parts for the prevention and/or treatment of a cancer disease, preferably a chemoresistant cancer disease. “Treating” or “treatment” as used herein, and as well-understood in the art, includes any approach for obtaining beneficial or desired results in a subject’s cancer condition. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more cancer symptoms or conditions, diminishment or reduction of the extent of a cancer disease or of a cancer symptom, stabilizing, i.e., not worsening, the state of a cancer disease or of a cancer symptom, prevention of a cancer disease or of a cancer symptom’s spread, delay or slowing of cancer disease or cancer symptom progression, amelioration or palliation of the cancer disease state, diminishment of the reoccurrence of cancer disease, and remission, whether partial or total and whether detectable or undetectable. In other words, "treatment" as used herein includes any cure, amelioration, or reduction of a cancer disease or symptom. A “reduction” of a symptom or a disease means decreasing of the severity or frequency of the disease or symptom, or elimination of the disease or symptom.

“Prevent” or “preventing” as herein refers to a decrease in the risk of occurrence of a cancer disease or symptom in a patient. As indicated above, the prevention may be complete, i.e. no detectable symptoms or disease, or partial, such that fewer symptoms or less severity of the disease are observed than would likely occur absent treatment.

In other embodiments, the combination or the pharmaceutical composition or the kit-of-parts according to the invention are for use in synergistic prevention and/or treatment of a cancer disease.

In other embodiments, the combination or the pharmaceutical composition or the kit-of-parts according to the invention are for use in prevention and/or treatment of a chemoresistant cancer disease.

In other embodiments, the combination or the pharmaceutical composition or the kit-of-parts according to the invention are for use in a synergistic prevention and/or treatment of a chemoresistant cancer disease. In embodiments the manufactured medicament or kit-of-parts described herein are for a synergistic prevention and/or synergistic treatment of a cancer disease, preferably a chemoresistant cancer disease.

Within the present description, "synergy" or "therapeutic synergy" are used when the combination of two products at given doses is more efficacious than the best of the two products alone considering the same doses.

Because of the synergistic effect of a combination according to the invention, each of the active agent, i.e. the anticancer G4L and the lysosomotropic agent, composing the combination may be present in an amount below their usual prescribed effective amount or therapeutically effective amount as active agent.

According to one embodiment, the anticancer G-quadruplex ligand and the lysosomal membrane permeabilization inducing agent may be each present in a combination according to the invention in a synergistic amount. A synergistic amount is, with respect to a given active agent, a fraction of the usually prescribed effective amount or therapeutically effective amount.

As used herein, a "usually prescribed effective amount or therapeutically effective amount of an active agent" refers to the amount or dose of a therapeutic agent typically used as a unit dose in the standard medical protocols or indicated in the accompanying documentation of the packages of the therapeutic agents known to the skilled person. This value, recognizable by the skilled person, varies from drug to drug and cannot be defined in a way applicable to all the commonly used therapeutic agents. The definition, applied to any active agent, indicates exactly the dosage commonly prescribed and specified in the medical and pharmacological protocols for therapy for each active agent known to the skilled person. In one embodiment, a synergistically therapeutic efficient amount of the anticancer

G4L may be about 0.1 , 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 70.0, 80.0, 90.0, 95.0, or 99.0% of the therapeutically efficient amount of the anticancer G4L provided herein when used separately from the lysosomotropic agent. In one embodiment, a synergistically therapeutic efficient amount of the lysosomotropic agent may be about 0.1 , 1.0, 2.0, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 70.0, 80.0, 90.0, 95.0, or 99.0% of the therapeutically efficient amount of the lysosomotropic agent provided herein when used separately from the anticancer G4L.

In other embodiments, a combination or a pharmaceutical composition or a kit-of- parts according to the invention comprises at least one anticancer G4L in a synergistically therapeutic efficient amount and at least one lysosomotropic agent in a synergistically therapeutic efficient amount.

In embodiments, the invention also relates to a method of preventing and/or treating a cancer disease in a subject in need thereof, said method includes administering to the subject a therapeutically effective amount of at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, thereby treating a cancer disease in said subject.

“Patient” or “subject in need thereof” as used herein refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of a composition according to the invention. Non-limiting examples include mammals: human, bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, or non-mammalian animals. In some embodiments, a subject or a patient is a mammal, and preferably is human.

In embodiments, the invention also relates a method of synergistically preventing and/or treating a cancer disease in a subject in need thereof, said method includes administering to the subject a synergistically therapeutic effective amount of at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, thereby treating a cancer disease in said subject. The method includes observing a prevention or a treatment, such as a relieving, of the cancer disease.

In embodiments, the invention also relates to a method for the prevention and/or treatment of a chemoresistant and/or potentially chemoresistant cancer disease in a subject in need thereof, said method includes administering to the subject a therapeutic effective amount of at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, thereby treating a chemoresistant cancer disease in said subject. The method includes observing a prevention or a treatment, such as a relieving, of the chemoresistant and/or potentially chemoresistant cancer disease. In embodiments, the invention also relates to a method for the synergistic prevention and/or treatment of a chemoresistant and/or potentially chemoresistant cancer disease in a subject in need thereof, said method includes administering to the subject a synergistically therapeutic effective amount of at least one anticancer G4L and at least one lysosomotropic agent, or a pharmaceutically acceptable salt thereof, thereby treating a chemoresistant cancer disease in said subject. The method includes observing a synergistic prevention or a synergistic treatment, such as a relieving, of the cancer disease.

In embodiments, in the methods described herein, the at least one anticancer G4L and at least one lysosomotropic agent, or pharmaceutically acceptable salts thereof, are administered as a combination. In embodiments, in the methods described herein, the at least one anticancer G4L and at least one lysosomotropic agent, or pharmaceutically acceptable salts thereof, are administered as a pharmaceutical composition.

According to one embodiment, the invention is directed to a combination or a pharmaceutical composition or a kit-of-parts or a method employing such combination, pharmaceutical composition, or a kit-of-parts, as described here, wherein the anticancer G4L and the lysosomotropic agent are for simultaneous, separate or sequential use.

The anticancer G4L and the lysosomotropic agent may be administered in combination either simultaneously ( e.g ., as a mixture), separately but simultaneously (e.g., via separate intravenous lines) or sequentially (e.g., one agent is administered first followed by administration of the second agent). Thus, the term combination is used to refer to concomitant, simultaneous or sequential administration of the anticancer G4L and the lysosomotropic agent. As used herein, "administering" or "administered" means administration by any route, such as oral administration, administration as a suppository, topical contact, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, subcutaneous or transmucosal ( e.g ., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal) administration, or the implantation of a slow-release device, e.g., a mini-osmotic pump, to a subject, including parenteral. Parenteral administration includes intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.

Those terms are also meant to include modes of administration of the selected active agents, as considered in the present invention, to a single patient and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. Administration can include delivery to a patient of the active agents, e.g. an anticancer G4L and a lysosomotropic agent, simultaneously in the form of a single entity or dosage. Administration can also include that the active agents are both administered to a patient as separate entities either simultaneously, concurrently, or sequentially with no specific time limits, such that the administration provides therapeutically effective levels of the combination of active agents in the body of the patient.

In one embodiment, the anticancer G4L and the lysosomotropic agent are administered simultaneously or separately.

In another embodiment, the anticancer G4L and the lysosomotropic agent are administered sequentially.

In some embodiments, during the course of a treatment, the G4L and the lysosomotropic agent may at times be administered sequentially and at other times be administered simultaneously or separately.

In embodiments, where the anticancer G4L and the lysosomotropic agent are administered sequentially, the anticancer G4L is administered at a first time point and the lysosomotropic agent is administered at a second time point, wherein the first time point precedes the second time point. Alternatively, in embodiments, where the anticancer G4L and the lysosomotropic agent are administered sequentially, the lysosomotropic agent is administered at a first time point and the anticancer G4L is administered at a second time point, wherein the first time point precedes the second time point.

In instances where the anticancer G4L and the lysosomotropic agent are administered simultaneously, the active agents are admixed prior to administration. In embodiments, uses or methods in accordance with the invention preferably implement 20A or quarfloxin as the anticancer G4L, and chloroquine or Lys05, as the lysosomotropic agent, or a pharmaceutically acceptable salt thereof.

The course of a treatment with a combination of the invention is best determined on an individual basis, depending on the particular characteristics of the subject and the type of treatment selected. The treatment, such as those disclosed herein, can be administered to the subject on a daily, twice daily, bi-weekly, monthly or any applicable basis that is therapeutically effective. The treatment can be administered alone or in association with any other treatment disclosed herein or known in the art. The additional treatment can be administered simultaneously with the first treatment, at a different time, or on an entirely different therapeutic schedule ( e.g ., the first treatment can be daily, while the additional treatment is weekly).

In some embodiments, the combination or the pharmaceutical composition as described herein can be administered simultaneously, separately, or sequentially, daily for a period of time extending from at least one day up to several days, for instance up to 30 days, or several months, for instance up to 3 months. The administration may be repeated for several period of times separated by a period of time without administration. For instance, the administration may be carried out over two period of one month separated by a period of one month without administration. Such sequence may be repeated at least once or several times.

In some embodiments, the combination or the pharmaceutical composition as described herein can be administered once, twice, three time or 4 time a day, or once a day over a period of time ranging from a few minutes, e.g. one minutes, up to several minutes, e.g. 40, 50 or 60 minutes or up to several hours, one, two, three or up to 6 or 8 hours. The exact dosage, frequency and length of administration will depend on the particular combination of anticancer G4L and lysosomotropic agent used, the particular condition to be treated, the severity of the condition to be treated, age, weight and the overall physical condition of the particular patient as well as on other medications that the patient is taking, as is well known to experts in the field. It is also clear that this quantity can be effectively lowered or increased depending on the responses of the treated patient and/or according to the evaluation of the physician prescribing the compounds of the present invention. The effective doses given here are therefore only indicative. A cancer, in particular a chemoresistant cancer, which may be considered within the invention may be selected from breast cancer; colon cancer; rectal cancer; endometrial cancer; gastric carcinoma (including gastrointestinal carcinoid tumors and gastrointestinal stromal tumors); glioblastoma; hepatocellular carcinoma; cervical carcinoma; lung adeno carcinoma (including small cell lung cancer and non-small cell lung cancer (NSCLC)); melanoma, including uveal melanoma; medulloblastoma; ovarian carcinoma; osteosarcoma; pancreatic cancer; prostate cancer; acute myelogenous leukemia (AML); chronic myelogenous leukemia (CML); non-Hodgkin's lymphoma; thyroid carcinoma; and pediatric tumors, such as leukemia, brain tumors, nephroblastoma, neuroblastoma, lymphoma, embryonic tumors, and rhabdosarcoma. In a more preferred embodiment, the cancer disease considered within the invention is a cervical carcinoma, lung adeno-carcinoma, and osteosarcoma. The lung cancer may be in particular small cell lung cancer or non-small cell lung cancer (NSCLC).

The cancer, in particular the lung adeno-carcinoma, the cervical cancer or the osteosarcoma, may be a chemoresistant cancer. Within the present description, the term “chemoresistance" or “chemoresistant cancer” refers to a reduction or a lack of responsivity of a cancer cell to a particular anticancer agent, used alone or in combination with other drugs commonly used in anticancer therapies. More particularly, a chemoresistant tumor or a chemoresistant cancer refers to a cancer that does not undergo cell death in response to a chemotherapeutic or anticancer agent.

As used herein "composition effective in the treatment of chemoresistant and/or potentially chemoresistant cancer" means composition which have the effect of inhibiting from the beginning the development of chemoresistant cells or to revert chemoresistance of cells treated with these substances and to restore the apoptotic pathway. A combination of the invention is a such composition.

REFERENCES

Amaravadi R.K, Winkler J.D., Lys05 - A new lysosomal autophagy inhibitor. Autophagy. 2012 Sep 1 ; 8(9): 1383-1384

Ambrogio C, Gomez-Lopez G, Falcone M, Vidal A, Nadal E, Crosetto N et at. Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS-driven lung adenocarcinoma. Nat /Wed 2016; 22: 270-277.

Aits S, Kricker J, Liu B, Ellegaard A-M, Hamalisto S, Tvingsholm S etal. Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay. Autophagy 2015; 11 : 1408-1424.

Asamitsu S, Obata S, Yu Z, Bando T, and Sugiyama H. Recent Progress of Targeted G-Quadruplex-Preferred Ligands Toward Cancer Therapy. Molecules 2019; 254:429 Brun, S., Bassissi, F., Serdjebi, C. et at. GNS561 , a new lysosomotropic small molecule, for the treatment of intrahepatic cholangiocarcinoma. Invest New Drugs 37, 1135- 1145 (2019). https://doi.org/10.1007/s10637-019-00741 -3

Dutta D, Debnath M, Muller D, et al. Cell penetrating thiazole peptides inhibit c- MYC expression via site-specific targeting of c-MYC G-quadruplex. Nucleic Acids Res. 2018;46( 11 ) :5355-5365. doi : 10.1093/nar/gky385

Fitch T.R., Northfelt D.W., Griffin P.P., Goldston M., Lim J.K., Padgett C.S., Von Hoff D.D.and Papadopoulos K.P. Phase I clinical trial of quarfloxin administered weeklyx3 of a four week cycle. Journal of Clinical Oncology 26, no. 15_suppl (May 20, 2008) 14667-14667 Giraldo A.V.M., Appelqvist H., Ederth T. and Ollinger K., Lysosomotropic agents: impact on lysosomal membrane permeabilization and cell death. Biochem. Soc. Trans. 2014; 42, 1460-1464

Goldman SDB, Funk RS, Rajewski RA and Krise JP. Mechanisms of amine accumulation in, and egress from, lysosomes. Bioanalysis 2009; 1 : 1445-1459.

Gong Y, Duvvuri M, Duncan MB, Liu J and Krise JP. Niemann-Pick C1 protein facilitates the efflux of the anticancer drug daunorubicin from cells according to a novel vesicle- mediated pathway. J Pharmacol Exp Ther 2006; 316: 242-247. Herlevsen M, Oxford G, Owens OR, Conaway M, and Theodorescu D. Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes. Mol Cancer Ther 2007; 6: 1804-1813.

Homewood CA, Warhurst DC, Peters W, and Baggaley VC. Lysosomes, pH and the Anti-malarial Action of Chloroquine. Nature 1972; 235: 50-52.

Kang C-C, Huang W-C, Kouh C-W, Wang Z-F, Cho C-C, Chang C-C et al. Chemical principles for the design of a novel fluorescent probe with high cancer-targeting selectivity and sensitivity. Integr Biol Quant Biosci Nano Macro 2013; 5: 1217-1228.

Kazmi F, Hensley T, Pope C, Funk RS, Loewen GJ, Buckley DB etal. Lysosomal Sequestration (Trapping) of Lipophilic Amine (Cationic Amphiphilic) Drugs in Immortalized Human Hepatocytes (Fa2N-4 Cells). Drug Metab Dispos 2013; 41 : 897-905.

Kroemer G and Galluzzi L. Lysosome-targeting agents in cancer therapy. Oncotarget 2017; 8: 112168-112169.

Kuzu O.F., Toprak M. Noory A.M. and Robertson G.P., Effect of lysosomotropic molecules on cellular homeostasis. Pharmacological Research, 2017, 117, 177-184

Li O, Xiang JF, Zhang H and Tang YL. Searching Drug-Like Anti-cancer Compound(s) Based on G-Ouadruplex Ligands. Current Pharmaceutical Design 2012; 18, 1973-1983.

Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999)

Martinez R., Burrage A.M., Wiethoff C.M., and Wodrich H., High temporal resolution imaging reveals endosomal membrane penetration and escape of adenoviruses in real-time. Methods Mol Biol. 2013; 1064: 211-226.

Montespan C, Marvin SA, Austin S, Burrage AM, Roger B, Rayne F et al. Multi- layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid. PLoS Pathog 2017; 13: e1006217. Nadanaciva S., Lu S., Gebhard D.F., Jessen B.A., Pennie W.D. and Will Y. A high content screening assay for identifying lysosomotropic compounds. Toxicology in Vitro. 2011 , (25)3, 715-723

Panda D, Saha P, Das T, Dash J. Target guided synthesis using DNA nano- templates for selectively assembling a G-quadrupiex binding c-MYC inhibitor. Nat Common. 2017;8:161G3. Published 2017 Jul 14. doi:10.1038/ncomms16103

O’Prey J, Sakamaki J, Baudot AD, New M, Van Acker T, Tooze SA et al. Application of CRISPR/Cas9 to Autophagy Research. Methods Enzymol 2017; 588: 79-108.

Piao S, Amaravadi RK. Targeting the lysosome in cancer. Ann N Y Acad Sci 2016; 1371 : 45-54.

Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins.

Remington: The Science and Practice of Pharmacy, 21st Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Seo I., Jha B.K., Lim JG., Suh SI., Suh MH., and Baek WK., Identification of lysosomotropic compounds based on the distribution and size of lysosomes, Biochemical and Biophysical Research Communications, 2014, 450(1), 189-194

Sengupta A, Ganguly A, and Chowdhury S. Promise of G-Quadruplex Structure Binding Ligands as Epigenetic Modifiers with Anti-Cancer Effects. Molecules 2019; 254:582 Shivalingam A, Izquierdo MA, Le Marois A, Vysniauskas A, Suhling K, Kuimova

MK et al. The interactions between a small molecule and G-quadruplexes are visualized by fluorescence lifetime imaging microscopy. Nat Commun 2015; 6: 8178.

Schneider, C. A.; Rasband, W. S. & Eliceiri, K. W. "NIH Image to ImageJ: 25 years of image analysis", Nature methods, 2012, 9(7): 671-675 Smith NM, Labrunie G, Corry B, Tran PL, Norret M, Djavaheri-Mergny M, Raston

CL, and Mergny JL. Unraveling the relationship between structure and stabilization of triarylpyridines as G-quadruplex binding ligands. Org Biomol Chem. 2011 ; 9(17):6154-62. Smith PJ, Sykes HR, Fox ME, and Furlong IJ. Subcellular distribution of the anticancer drug mitoxantrone in human and drug-resistant murine cells analyzed by flow cytometry and confocal microscopy and its relationship to the induction of DNA damage. Cancer Res 1992; 52: 4000^008. Sun D, Liu Y, Yu Q, Liu D, Zhou Y, Liu J. Selective nuclei accumulation of ruthenium (II) complex enantiomers that target G-quadruplex DNA. J Inorg Biochem. 2015;150:90-99. doi:10.1016/j.jinorgbio.2015.04.003

Zhitomirsky B and Assaraf YG. Lysosomal sequestration of hydrophobic weak base chemotherapeutics triggers lysosomal biogenesis and lysosome-dependent cancer multidrug resistance. Oncotarget 2015; 6: 1143-1156.

WO 2009/157505 A1 - Telomerase inhibitor

EXAMPLES

Example I: Materials and methods

Cell culture

The human cervical cancer cell line HeLa was purchased from the American Type Culture Collection (ATCC) and the human lung carcinoma A549 cell line is a generous gift of Prof. Jean-Frangois Riou. The autophagy-deficient (ATG5 and ATG7 depleted) HeLa cell lines were generated as described by O’Prey et at. (Methods Enzymol 2017; 588: 79-108). The U20S Galectin3-mCherry cell line was as described in Montespan etal. (PLoS Pathog 2017; 13: e1006217) or in Martinez etal. (Methods Mol Biol. 2013; 1064: 211-226). U20S cell lines are cultivated in DMEM, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 pg/ml streptomycin (Gibco-Life technologies, 1540-122). The lung adenocarcinoma PDX cell lines (PDX2) and (PDX3) are generous gifts from Dr. D. Santamaria (Ambrogio etal., Nat /We 2016; 22: 270-277).

HeLa cells were grown in RPMI 1640 culture media supplemented with 2 mM glutamine (Gibco-Life technologies), 10% fetal bovine serum (Gibco-Life technologies), 100 units/ml penicillin, and 100 pg/ml streptomycin (Gibco-Life technologies, 1540-122), while A549, U20S and PDX cell lines were grown in DMEM, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 pg/ml streptomycin (Gibco-Life technologies, 1540- 122). For U20S expressing Galectin3-mCherry 200 pg/ml hygromycin B (#10687010, Invitrogen) was added for cell culture, and removed from media before the experiments. All cell lines used in this study were cultivated at 37 ‘C in a humidified atmosphere with 5% C0 ₂ .

Reagents and antibodies

Antibodies against the following proteins were used: Cleaved-Caspase3 (#9664), LAMP-I (#9091) from Cell Signaling Technology, PARP-1 (C-2-10) (#BML-SA249-0050) from Enzo life sciences, b-Actin (#NB600-501) from Novus Biologicals, HRP conjugated rabbit (#111 -035-003) and HRP conjugated mouse (#115-035-174) from Jackson ImmunoResearch, Alexa488 conjugated anti-rabbit (#A11008) from Invitrogen. The Hoechst 33258 (#14530), Propidium iodide (#P4864), Chloroquine diphosphate salt (#C6628), QVD-OPH (#SML0063) were purchased from Sigma-Aldrich. Tetramethylrhodamine methyl-ester, TMRM (T-668) was purchased from Molecular Probes, Fluoromount G (#00-4958-02) was purchased from Invitrogen. 20A was synthesized as previously described (Smith et a!., Org Biomol Chem. 2011 ; 9(17):6154-62 - compound n °3). Quarfloxin, a fluoroquinolone derivative with antineoplastic activity (also know as CX-3543), was obtained from Selleckchem. Lys05 was obtained from Sigma (Ref: SML2097).

Mitochondrial and lysosomal staining and evaluation of 20A localization Cells were grown on glass coverslips on a 6-well plate (6 c 10 ⁴ cells/well), treated with or without 5 mM 20A for 6h. Cell were stained with 50nM Mitotracker Green (#M7514, Molecular probes) and 50nM Lysotracker Red DN-99 (#L7528, Molecular probes) 30 min before the end of the incubation time of 20A. Cells were placed in a perfusion chamber with DMEM without red Phenol containing 15nM Lysotracker Red DN-99. Image acquisition was made on a Zeiss LSM 510 METAconfocal microscope, with an Apoplan x63 objective. Identical exposures were used for each channel throughout individual experiments, and no images were altered after capture. Co-localization of blue fluorescence (20A) with the lysosome cellular compartment was analyzed by superposing fluorescence profile of the different cellular compartments.

Evaluation of cell death

To evaluate cell death, plasma membrane permeability is scored after labeling cells with specific fluorescent probes (propidium iodide (P4864, Sigma) (Figure 1) or Hoechst 33342 (B2261 , Sigma) uptake (Figures 4 and 5). Briefly, supernatant and attached cells were collected, pelleted at 1800rpm for 5min and loaded with either 1 pg/ml PI or Hoechst 33345 (10 mrLhI) during 15 min at room temperature. Cells were then analyzed by flow cytometer (FACSCalibur, Becton Dickinson) and the FlowJo software v10. Apoptotic cell death was also determined by measuring the loss of mitochondrial transmembrane potential (DYGP) by using tetramethyl rhodamin methyl ester perchlorate dye (TMRM). Briefly, supernatant and attached cells were collected, pelleted at 1800rpm for 5min and loaded with 200nM TMRM for 30min at 37 °C in PBS supplemented with 20mM verapamil. Cells were then analyzed by flow cytometer (FACSCalibur, Becton Dickinson). Apoptosis was also evaluated by Western blotting analyses of cleaved forms of either PARP 1 or caspase 3. Where indicated, the caspase inhibitor, QVD- OPH (Sigma, SML0063) was added to cells to evaluate the possible implication of caspases in cell demise.

Lysosomal membrane permeabilization (LMP) analysis

Quantification of Galectin 3 puncta

Lysosomal membrane damage was assessed thanks to U20S expressing Galectin3-mCherry cells. Cells were plated on 12-well plates (7.5 c 10 ⁴ cells/well) treated with or without 20A in either the presence or absence of 25mM chloroquine for 24h. Cells were then washed with PBS, fixed with 4% (v/v) paraformaldehyde for 10 min at room temperature and then nuclei were counterstained for 15 min with Hoechst 33258. Image acquisition was made with a Leica DMI8 epifluorescence microscope, with a x20 objective (NA 0.40) equipped with a digital CMOS camera (Hamamatsu photonics) and filter bloc for detection of phase contrast and blue (exc: 325-375nm / em: 435-485nm) and red (exc: 541 -551 nm / em: 565-605nm) fluorescence. Each images of cells was collected with identical exposure times and scaled equally. Five images were acquired on each condition and then analyzed thanks to ImageJ software (Schneider etal., 2012, 9(7): 671-675) in order to count manually the number of cells, and automatically the number of Galectin3 punctae within each cell by using the “Find Maxima” tool.

Evaluation of Galectin 3 and LAMP1 co-localization

To assess for lysosomal membrane permeabilization (LMP) co-localization study of Galectin3 puncta and LAMP1 was carried out by immunofluorescence study. Cells grown on glass coverslips were fixed in ice cold methanol at -20 °C for 15 min then blocked/permeabilized with 3% BSA containing (Sigma, CAS Number 9048-46-8 ) 0.3% Triton X-100 (Sigma 9002-93-1) for 1 h. Then cells were incubated with primary antibody overnight at 4 ^q C. After washing, cells were incubated with secondary antibody (anti-rabbit Alexa Fluor 488 antibody) for 1 h at room temperature in a dark chamber and stained with 2pg/ml of Hoechst 33258 (H3569, Promega) for 10min. After two PBS wash, cells were mounted with Fluoromount (F4680, Sigma-Aldrich) and images were acquired using a Zeiss LSM510 Meta confocal microscope (Zeiss, Gottingen, Germany) with an oil immersion Apoplan 63x objective (NA 0.75). z-stack series were made with 0.6pm interval and the corresponding images represent a z-projection of maximal intensity. Identical exposures were used for each channel throughout individual experiment. No images were altered after capture. Processing of images is performed thanks to ImageJ software.

Immunoblot assay Cell extracts were prepared in 10mM Tris, pH 7.4, 1% SDS, 1mM sodium vanadate, 2mM PMSF (93482, Sigma-Aldrich), 1% Protease Inhibitor Cocktail (P8849, Sigma- Aldrich), and 1% Halt Phosphatase Inhibitor Cocktail (1862495, Thermo Fisher Scientific) and treated with Benzonase Endonuclease (71205, Merck Millipore) for 5min at room temperature, and boiled for 5min. Fractions (30-50 pg) of cellular extract proteins were subjected to SDS- PAGE using a Tris/glycine buffer system based on the method of Laemmli. After electrophoresis, proteins were transferred to a nitrocellulose membrane (10600003 Amersham Protrant Premium 0.45 NC, GE Healthcare Life Sciences). Protein loading was assessed by Ponceau red staining of membranes. Blots were then incubated with primary antibodies using the manufacturer's protocol followed by the appropriate horseradish peroxidase-conjugated secondary antibody. Mouse anti-Actin antibody was used to assess equal loading of the samples. Immunostained proteins were visualized on a chemiluminescence detector equipped with a camera (FUSION FX7, Fisher Bioblock Scientific) using the enhanced chemiluminescence (ECL) detection system. Statistical analysis.

Each experiment was performed three times. Results were expressed as the mean value ± standard deviation (SD). Unless otherwise stated, statistical analysis was performed by Mann-Withney unpaired statistical test p < 0.05 was considered statistically significant. The software used was GraphPad PRISM.

Example II: 20A accumulates at the lysosomal compartment and causes enlargement of the lysosomes

Because 20A causes enrichment of genes involved in the lysosomal pathway, first it was examined whether this compound affects the lysosomal compartment in HeLa cells with the lysosomal marker Lysotracker Red.

The obtained results showed that 20A accumulates in the lysosomes and causes lysosomal enlargement. HeLa cells were with treated or without 5mM 20A for 6h and then stained with Lysotracker Red (LTR) and Mitotracker Green (MTG) to visualize lysosomes and mitochondria, respectively. Magnified views of confocal images of control cells and 20A treated cells showed that 20A fluorescence co-localize with lysotracker. It was noted the presence of enlarged lysosomes in cells subjected to 20A treatment.

Example III: 20A and chloroquine act in concert to trigger LMP and cell death.

The presence of 20A within the lysosome resembles to a mechanism referred as lysosomal drug sequestration that has been observed in response to some cancer therapy (Piao et a!., Ann N Y Acad Sci 2016; 1371 : 45-54). Growing evidence revealed that lysosomotropic agents such as chloroquine can prevent lysosomal drug sequestration and thus sensitize cells towards cancer therapies (Zhitomirsky et a!., Oncotarget 2015; 6: 1143— 1156; Goldman etal., Bioanalysis 2009; 1 : 1445-1459; Kazmi etal. Drug Metab Dispos 2013; 41 : 897-905). To test this hypothesis, the effect of combination of 20A and chloroquine on cell death in both HeLa and A549 cancer cell lines was investigated. In order to properly evaluate the effect of the combination of the two drugs (20A and chloroquine), it was decided to use drug doses that trigger a low level of cell death (<10%) when used alone (5 or 6 mM and 3.5 or 4 mM for HeLa cells and A549 cells respectively, knowing that the IC ₅ o of 20A is 6.3 mM and

5mM for HeLa and A549 cells, respectively). For all experiments, a sublethal dose of chloroquine (25mM - CQ) was also used.

As shown in Figure 1A, the combination of chloroquine (25 mM) and 20A (5 mM) promoted a robust activation of cell death process (50% cell death) in HeLa cells as compared to 20A or chloroquine alone (less than 5% cell death). A similar effect is observed when A549 cells are treated with the combination of 20A (3.5 mM) and chloroquine (25 mM) indicating that chloroquine greatly potentiates 20A-induced cell death.

Those results show that chloroquine and a triarylpyridine compound, 20A, acted synergistically to induce cell death.

Next, it was examined the lysosome size and number in response to the combined 20A/chloroquine treatment. The obtained image data revealed that 20A and chloroquine both caused the lysosomal enlargement as evidenced by Lampl staining but more interestingly is that this phenotype is markedly increased when the two drugs are combined.

Next, it was explored whether the accumulation of autophagosomes by chloroquine is responsible for the robust induction of cell death observed under the combination of 20A and chloroquine. To this purpose, we compared cell death induced by the combination of 20A and chloroquine in autophagy-proficient cells (control sg NTC1 cells) and autophagy-deficient cells (cell line with complete autophagy disruption by deletion of two key autophagy genes, sg ATG5 and sg ATG7). As shown in Figure 1 B, the autophagy-deficient cells (in which the formation of autophagosome is completely inhibited) manifest a comparable rate of cell death in response to the combination of 20A and chloroquine cells than the autophagy-proficient cells, suggesting that chloroquine sensitizes cells to 20A treatment through an autophagy-independent mechanism.

One mechanism through which chloroquine can promote cell death relies on the induction of lysosomal membrane permeabilization (Homewood et al. Nature 1972; 235: 50- 52). Recently, the detection of Galectin 3 puncta at damaged endo-lysosome membranes was shown to be a highly sensitive method to evaluate LMP (Aits etal. Autophagy 2015; 11 : 1408- 1424). To explore this possibility, a cell line expressing mCherry-tagged Galectin 3 was used and the cells with one or more Galectin 3 puncta were counted as “Gal3 positive cells”.

U20S cells expressing Galectin3-mcherry were treated or not with 3 mM 20A either in the presence or absence of 25mM chloroquine for 24h and then immunostained for LAMP1 . The obtained image data, obtained by z-projection of merge confocal images of nuclei (blue signal) and LAMP1 (green signal) revealed that 20A and chloroquine both causes the lysosomal enlargement as evidenced by Lamp1 staining but more interestingly is that this phenotype is markedly increased when the two drugs are combined.

As shown in the Figure 2A the percentage of Gal3 positive cells is nearly equal in cells subjected to a sublethal dose of 20A and untreated cells (respectively 3% and 2%) but increases to 8 % when cells are exposed to chloroquine. More importantly, the percentage of Gal3 positive cells increased massively (39%) when chloroquine was associated with 20A treatment. It is worth noting that under the latter condition, 26% of Gal3 positive cells harbor one or two Gal3 puncta and the rest (13%) displays either 3 or more Gal3 punctae (Figure 2B) suggesting the presence of several endo-lysosomal membrane damages under this condition.

Next, it was analyzed if the Gal3 puncta are situated at the lysosomal membrane by co-staining cells with LAMP1 , a specific marker of the lysosome. U20S cells expressing Galectin3-mcherry were treated or not with 3mM 20A either in the presence or absence of 25mM chloroquine for 24h and then immunostained against LAMP1 . Results from z-projection of confocal images of Galectin3 (red signal), nuclei (Hoechst - blue signal) and LAMP1 (green signal) showed a colocalization of Lamp1 (green signal) and galectin 3 (red signal) indicating lysosomal membrane rupture. As shown by the images, a large overlap in colocalization was observed when cells were simultaneously exposed to both drugs confirming that Gal3 positive cells represent cells harboring leaky lysosomes. It was also noticed an important increase in the size of the lysosome (as evidenced by LAMP1 staining) in cells treated with chloroquine either alone or with 20A which mirrored the lysosomal swelling process that has been observed under certain conditions in which LMP is induced.

Altogether, these results clearly showed that the combination of 20A and chloroquine caused the lysosomal enlargement associated with an important damage to the lysosomal membrane. Therefore, 20A and chloroquine acted in concert to trigger LMP in cancer cells.

Example IV: Apoptosis is involved in cell death induced by chloroquine/20A

It was investigated if apoptosis is implicated in the massive cell death induced by chloroquine/20A treatment. As presented in Figure 3A, the combination of chloroquine and 20A treatment promoted a robust cleavage of PARP-1 and caspase 3 in both HeLa and A549 cells while no significant cleavage was observed when cells are treated solely with sublethal dose of each compound.

To further evaluate the role of caspase in the occurrence of cell death, a pan inhibitor of caspase (QVD-OPH) was used and a feature of cell death, namely loss of mitochondrial transmembrane potential, was evaluated on HeLa and A549 cells. As shown in the Figure 3B, this feature of cell death was significantly reduced when cells were treated with QVD-OPH prior the addition of Chloroquine (CQ)/20A, suggesting that caspases play a key role in the execution of cell death.

Example V: Combined treatment with chloroquine and 20A significantly activates cell death in patient-derived xenograft cell lines To validate the results in a clinically relevant setting, the effect of chloroquine and 20A was evaluated in patient-derived xenograft cell lines from lung cancer (A549, PDX2 and PDX3). A549 lung cancer cell lines and two PDX from lung cancer PDX2 and PDX3 were treated with 3.5 mM 20A, in either the presence or absence of 25 mM chloroquine for 24h. Cell death was assessed by scoring the percentage of propidium iodide positive cells by flow cytometer analysis. The obtained results showed that both PDX cell lines are highly sensitive to the combination treatment, suggesting the relevance of this combination for further studies in in vivo lung cancer models.

Example VI: 20A combined with Lys05 significantly activates cell death

To evaluate whether the synergistic induced cell death of cancer cells could be observed with another lysosomotropic agent, the combination of 20A with Lys05 was evaluated. Lys05 is a dimeric chloroquine which accumulates in the lysosome and blocks autophagy (Amaravadi R.K, Winkler J.D., Autophagy. 2012 Sep 1 ; 8(9): 1383-1384).

As shown in Figure 4, the combination of Lys05 (Ly05: 0, 1 mM or 5 mM) with increasing amount of 20A (0, 1 mM, 2 mM, 3 mM, 4 mM and 5 mM) promoted a robust activation of cell death process (up to 80% cell death) in U20S cells as compared to 20A or Lys05 alone (less than 10% cell death).

Those results show that Lys05, a lysosomotropic agent, and a triarylpyridine compound, 20A (anticancer G4L), acted synergistically to induce cell death.

Example VII: Quarfloxin combined with chloroquine significantly activates cell death

To evaluate whether the synergistic induced cell death of cancer cells could be observed with another anticancer G4L, the combination of quarfloxine (CX) with chloroquine (Chloro) was evaluated. Quarfloxin is a fluoroquinolone derivative with antineoplastic activity which has been evaluated in clinical trial (Fitch T.R., Northfelt D.W., Griffin P.P., Goldston M., Lim J.K., Padgett C.S., Von Hoff D.D., Papadopoulos K.P. Journal of Clinical Oncology 26, no. 15, suppl (May 20, 2008) 14667-14667).

As shown in Figure 5, the combination of chloroquine (Chloro) (0, 20 mM, or 25 mM) with increasing amount of quarfloxin (CX) (0, 0.5 mM, 1 mM or 2 mM) promoted a robust activation of cell death process (up to 60% cell death) in U20S cells as compared to quarfloxin or chloroquine alone (less than 10% cell death).

Those results show that quarfloxin, a fluoroquinolone derivative with anticancer G4L activity, with chloroquine, a lysosomal membrane permeabilization inducing agent, acted synergistically to induce cell death.