Field of Invention

The invention relates to modulators capable of facilitating ion flow through sodium leak channel (NALCN) and their use in therapy.

Background

Most patients with cancer die as a result of metastasis ¹ -the process by which cancer cells spread from the primary tumour to other organs in the body. Cancers cells can spread throughout the body via various mechanisms and some of them are able to form new tumors in other parts of the body. Metastatic cancer cells can also remain inactive at a distant site for long periods of time before they begin to grow again, if at all. Blocking metastasis could markedly improve the survival of patients with cancer; but how this process is triggered within the complex cascade of tumourigenesis remains unclear.

As such, there is a need to develop therapeutics which can prevent and inhibit metastasis.

Summary of the Invention

The present inventors have identified a single ion channel, NALCN, as a key regulator of both malignant and non-malignant cell metastasis, providing important insights to the metastatic process and a novel target for anti-metastatic therapies. Among 10,022 human cancers, NALCN loss-of-function mutations were selectively enriched in advanced gastric and colorectal cancers. Deletion of Nalcn from mice susceptible to developing gastric, intestinal or pancreatic adenocarcinoma did not alter the incidence of these tumours, but markedly increased levels of circulating tumour cells (CTCs) and seeding of peritoneal, kidney, liver and lung metastases. Treatment of these mice with gadolinium-a Nalcn channel blocker-similarly increased CTCs and metastasis. Remarkably, deletion of Nalcn from mice that lacked oncogenic mutations and never developed cancer, caused similar shedding of cells into the peripheral blood at levels equivalent to those seen in tumour-bearing animals. These cells trafficked to distant organs where they formed apparently normal structures, including kidney glomeruli and tubules, rather than tumours. The transcriptomes of these circulating cells in tumour and non-tumour-bearing mice were indistinguishable and closely related to those of human CTCs. Thus, NALCN regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumourigenesis and unmasking a potential new target for anti-metastatic therapies.

As such the present invention relates to an ion channel modulator for use in the treatment of cancer and/or metastasis, wherein the ion channel modulator is capable of facilitating ion flow through sodium leak channel (NALCN). In an aspect the invention relates to a pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator is capable of facilitating ion flow through NALCN.

In an aspect the invention relates to a method of treating or preventing cancer, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator is capable of facilitating ion flow through NALCN.

In an aspect the invention relates to a method of treating or preventing metastasis, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator is capable of facilitating ion flow through NALCN.

In an aspect the invention relates to a method of reducing the level of circulating tumour cells comprising contacting a cell with a, ion channel modulator, wherein said ion channel modulator is capable of facilitating ion flow through NALCN.

In an aspect the invention relates to a method of modulating the level of solid tissue cell shedding comprising contacting a cell with a, ion channel modulator, wherein said ion channel modulator is capable of facilitating ion flow through NALCN.

In an aspect the invention relates to an ion channel modulator for use in the treatment of a fibrotic disease wherein the ion channel modulator is capable of facilitating ion flow through NALCN.

In an aspect the invention relates to a method of treating or preventing a fibrotic disease, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator is capable of facilitating ion flow through NALCN.

Figures

Figure 1. NALCN loss-of-function characterises aggressive intestinal cancer, (a) Volcano plot of differential gene expression between Prom1 ⁺ cells isolated from mouse stomach epithelium and P7 ^KP -GACs: down-regulated ion channels highlighted, (b) t-Distributed Stochastic Neighbor Embedding plot of 10,022 samples from 32 human cancer types: NALCN- mutant samples and cancer-types enriched for NALCN mutations highlighted (p-value, dN/dS shown), (c) Mutant residues significantly enriched in pore turret and voltage sensing domains of NALCN. (d) Impact of 196 different NALCN mutations on pore radius determined by HOLE analysis, (e) NALCN pore closure caused by mutations in different stages of cancer (*=p<0.05, Mann-Whitney). Figure 2. NALCN loss-of-function increases tumour metastasis, (a) Unsupervised hierarchical clustering of 77 primary and metastatic P7 ^KP -GAC, V7 ^KP -IAC, Pdx1 ^KP -PAC tumours as well as four P1 ;Pten ^FIX/FIX ;Tp53 ^FIX/FIX (PI™*) primary hepatocellular carcinomas. Heatmap below reports enrichment of the indicated primary tumour transcriptome in each metastatic tumour, (b) Macroscopic images of exemplar ZsGreen ⁺ (ZSG) metastatic tumours ([met] outlined). (c) Photomicrographs (left haematoxylin and eosin, right immunohistochemistry/fluorescence) of the metastases in (b). Scale bar 50pm. (d) Left in each, cumulative total number of metastases per autopsied mouse of the indicated genotype at the indicated time post tamoxifen induction (age for Pdx1 ^KP mice; p=Mann-Whitney for total tumour burden in /Va/cn-deleted versus wild-type mice, see also Supplementary Tables 5 and 6). Right in each, organ heat maps of total metastases per mouse of the indicated genotype. Numbers of male:female (M:F) and P3:P60 induced mice are shown, (e) Cumulative metastatic burden and organ metastases heatmap plots of V7 ^KP -IAC in gadolinium or control treated Nalcn ^+/+ mice. (*=p<0.05, **=p<0.005, Mann-Whitney).

Figure 3. NALCN loss-of-function increases shedding of circulating tumour cells, (a) Scatter plot of CZCs (expressed as % of total peripheral blood cells) identified in peripheral blood of the indicated mice that were, or were not treated with gadolinium (ns=not significant, *=p<0.05, **=p<0.005, Mann-Whitney), (b) Uniform Manifold Approximation and Projection (UMAP) of single cell RNA sequencing profiles of CZCs and mouse peripheral blood mononuclear cells, (c) Heatmap reporting geneset enrichment analysis in the UMAP clusters identified in (b). Test Genesets were derived from 2,086 different tissue and cell types including bulk RNAseq of mouse normal tissues and tumours, huCTC signatures, and normal PBMCs (Extended Data Fig. 7; Methods), (d) Exemplar co-immunofluorescence of CZCs and PBMCs in peripheral blood smears of P1 ^KP (top) and V1 ^KP (bottom) mice (ZsGreen [ZSG], scale bar=10pm). (e) Top left, exemplar macroscopic direct green fluorescence imaging of a whole mouse lung showing Pdx1 ^KP -PAC CZC metastases in a recipient immunocompromised mouse. Other images show exemplar haematoxylin and eosin or co-immunofluorescence of metastases of P7 ^KP -GAC or V7 ^KP -IAC CZC metastases in immunocompromised recipient mice (scale bar=10pm). (f) Organ heat maps of total metastases per mouse identified in recipient mice injected with the indicating CZCs.

Figure 4. NALCN loss-of-function increases shedding of circulating non-tumour cells, (a) Scatter plot of CZCs (expressed as % of total cells) identified in peripheral blood of the indicated non-tumour bearing mice (***=p<0.0005, Mann-Whitney), (b) Uniform Manifold Approximation and Projection (UMAP) of 201 ,183 single cell RNA sequencing profiles (SCS) of PBMCs and tumour bearing (t) and non-tumour bearing (nt) CZCs as well as cells derived from the indicated normal and malignant mouse tissues, (c) Exemplar co-immunofluorescence of CZCs and PBMCs in peripheral blood smears of P1 ^R Nalcn ^FIX/FIX mice (ZsGreen [ZSG], scale bar=10pm). (d) Organ heat maps of total numbers of CZC cell clusters per mouse identified in organs of recipient mice injected with the indicated P1 ^R Nalcn ^FIX/FIX CZCs. (e) Exemplar co- immunofluorescence of P1 ^R Nalcn ^FIX/FIX CZCs (arrows) incorporated into the kidnesy of recipient mice (arrows indicated ZSG ⁺ cells, scale bar=50pm). (f) Confocal laser scanning microscope image of P1 ^R Nalcn ^FIX/FIX CZCs incorporated into the renal cortex of recipient mice (scale bar=100pm).

Figure 5. Fibrosis-free survival following conditional deletion of NALCN at P3 in P1 ^R mice. Survival curve for all organs (a) or the indicated organs (b-i). P value reports the log-rank statistic. The numbers of animals of each genotype are shown.

Detailed Description

The embodiments of the invention will now be further described. In the following passages, different embodiments are described. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary.

Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012).

In a first aspect the invention relates to an ion channel modulator for use in the treatment of cancer and/or metastasis, wherein the ion channel modulator is capable of facilitating ion flow through sodium leak channel NALCN, referred to as NALCN herein. Ion channels are crucial components of cellular excitability and are involved in many diseases. The present inventors have herein demonstrated that NALCN plays a crucial role in both malignant and non-malignant cell metastasis. NALCN is a nonselective monovalent cation channel which is a sole member of a distinct branch of voltage-gated sodium and calcium channels that regulates the resting membrane potential and excitability of neurons. NALCN is expressed most abundantly in the nervous system and conducts a persistent sodium leak current that contributes to tonic neuronal excitability. The sequence of NALCN is known and may comprise the sequence provided in ENSG00000102452 (ensemble), 259232 (NCBI Entrez Gene), 19082 (HGNC), Q8IZF0 (UniProtKB/Swiss-Prot), or 611549 (OMIM). In one embodiment the sequence of NALCN comprises SEQ ID NO.1. There are multiple splice variants of NALCN and the present invention extends to these variants. NALCN forms a polypeptide chain of 24 transmembrane helices (TM) that form four homologous functional repeats, also referred to as a-subunits, connected by intracellular linkers. Each functional repeat comprises voltage sensing domain, pore helices and ion selectivity filter.

It has been shown herein that loss of function of NALCN contributes to an increase in circulating tumour cell (CTC) levels and seeding of metastases. As such by modulating the activity of NALCN it may be possible to regulate metastasis. The ion channel modulator of the present invention modulates NALCN such the ion flow through NALCN is facilitated. Ion channel modulators may be categorised as ion channel blockers or ion channel openers. Ion channel blockers are generally antagonistic compounds which act to prevent the response that is normally provided by the opening of the channel. Ion channel openers are generally agonistic compounds which act to facilitate the response that is normally provided by the opening of the channel. In a preferred embodiment the ion channel modulator is an ion channel opener, or a compound that is agonistic to NALCN.

Where the ion channel modulator is referred to as a “compound” this encompasses an ion channel modulator which is a small molecule, an antibody or fragment thereof, a peptide, a synthetic peptide, a transcriptional modulator such as triplex forming oligonucleotides, synthetic polyamides, zinc finger proteins, or post-transcriptional modulator such as RNAi, siRNA, miRNA.

As such the ion channel modulator may modulate the activity of NALCN by directly acting on the protein NALCN. For example, as a small molecule, an antibody a peptide, or a synthetic peptide. The ion channel modulator may modulate the activity of NALCN by altering the expression level of the NALCN gene or the NALCN protein. For example, as a transcriptional or post- transcriptional modulator.

Where the ion channel modulator acts on the NALCN protein. The modulation may be achieved by binding one or more of the a-subunits of NALCN. The modulation may be achieved by the modulator disrupting the protein-protein interactions between a-subunit and auxiliary subunits of NALCN or disrupting interactions between NALCN and protein(s) associated with NALCN. The modulation may be achieved by the modulator interfering with or enhancing the movement of the voltage sensor or other elements of the gating machinery.

In an aspect the invention relates to a pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator is capable of facilitating ion flow through NALCN.

In an aspect the invention relates to a method of treating or preventing cancer and /or metastasis, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising an ion channel modulator, wherein the ion channel modulator is capable of facilitating ion flow through NALCN. In an aspect the invention also relates to the use of an ion channel modulatorforthe manufacture of a medicament for the treatment or prevention of cancer and/or metastasis.

As discussed above the ion channel modulator facilitates ion flow through the NALCN. The ion channel modulator may achieve the ion flow through NALCN by targeting NALCN directly. The ion channel modulator may achieve the ion flow through NALCN by targeting one of the proteins associated with NALCN. NALCN protein forms a channelosome complex within the cell membrane. The channelosome includes various proteins associated with NALCN including G- protein-coupled receptors, UNC-79, UNC-80, SLO2.1 , FAM155A, FAM155B, NCA localization factor-1 , and src family tyrosine kinases. As such one of these proteins that is associated with NALCN may be targeted by the ion channel modulator. The proteins associated with NALCN which may be targeted by the ion channel modulator are selected from: M3 muscarinic receptor (M3R), UNC80, UNC79, FAM155A, Fam155B, SLO2.1 , NCA localization factor-1 , src family tyrosine kinases.

In embodiments, the ion channel modulator may comprise, for example, gadolinium chloride, verapamil, high levels of exogenous Ca2+, 2-aminoethoxydiphenyl borate, nifedipine, nimodipine, flunarizine, ethoxzolamide, N-benzhydryl quinuclidine compounds, and/or L703606.

The NALCN protein comprises multiple domains, as such the ion channel modulator may target one or more of these domains in order to facilitate ion flow through NALCN. The ion channel modulator may target one of more of the pore turret domains, voltage sensing domains or linker domains of NALCN. The linker domains may be linker domains that extend either extracellularly or intracellularly. The ion channel modulator may target one or more of the domains comprising any one of the amino acid sequences set out in SEQ ID NO. 2 to 23. The domains of NALCN and their sequences are set out in the following table:

Table 1

The ion channel modulator may also interact with specific residues within NALCN. For example the ion channel modulator may interact with one or more of the amino acid residues of NALCN selected from; L588M, P573, R855, K1213, T71 , P225, D1527, D416, C1348, R297, V1386, A1091 , V1229, D134, T272, R43, A1157, V1036, M520, R1500, V320, V53, W1085, E1458,

N1274, V1542, Y1300, R1174, H1523, F332, Q549, L999, F540, A1421 , R1384, H569, A1435.M55, R1495, C245, F110, V510, C970, E454, V273, R1556, S174, S1068, V385, S384, A401 , S902, R1495, A276, R1540, L517, R295, R382, H876, F300, R164, E257, R995, G1526, D291 , V1239, E1552, N1475, M55, L1553, Y1349, E323, A1044, T1281 , V1007, L253, L564, F1427, V949, Q279, T539, R159, K452, R1127, V1490, G555, E62, L1461 , L942, R166, P65,

D952, I322, F154, K1163, L305, R152, W1085, R143, A1444, R989, R143, R1193, D1466, M520, V1285, S52, 151 , E1518, E532, L1279, V1329, T57, A1378, S121 , K498, R1094, V120, A88, A401 , L1548, G1303, M150, D1277, E432, L1442, P1082, T1 165, G1316, R1273, E128, E906, F1311 , R1481 , T204, T552, F389, D1527, P908, A1166, I577, G954, G1013, P65, E1016, N1070, S980, A1217, V1503, T1320, A223, A310, R1127, D1504, D1277, E128, K1491 , Q553, V511 , F1250, S1374, D211 , T1 149, D1099, M1425, M1003, P467, R43, L222, V400, M1244, A424, F1410, G193, H39, W219, F1018, R1193, K1069, V50, R1498, K1230, S403, S1264, R995, Q238, 11433, P66, L428, D1171 , A1107, S1033, 11017, K1259, M986. It has been demonstrated herein that mutations at each of these positions can result in the closure of the NALCN pore. Without wishing to be bound by theory it is hypothesised that these amino acid residues may be involved in regulating the opening of the NALCN pore. As such by targeting one or more of these residues it may be possible to facilitate ion flow through NALCN.

The ion channel modulator or the pharmaceutical composition may be administered by any suitable route for example any parenteral or enteral route. For example, any suitable route, includes but is not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably administration may be orally, sublingually, buccally, intravenously, intramuscularly, subcutaneously, rectally, or intranasally. Various oral dosage forms can be used, including such solid forms as tablets, capsules, liquids, granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple- compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents.

The ion channel modulator or the pharmaceutical composition may take the form of one or more dosage units.

In an embodiment the pharmaceutical composition of the invention may further comprise one or more additional active agents, pharmaceutically acceptable carrier, diluent or adjuvant. The pharmaceutically acceptable carrier, diluent or adjuvant may vary depending on the dosage form used. For example, various oral dosage forms can be used, including such solid forms as tablets, capsules, liquids, granules and bulk powders. Tablets can be compressed, tablet triturates, enteric-coated, sugar-coated, film-coated, or multiple-compressed, containing suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent preparations reconstituted from effervescent granules, containing suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, melting agents, coloring agents and flavoring agents, can be in the form of a liquid, e.g., a solution, emulsion or suspension. Liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides, polyethylene glycols, glycerin, or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The ion channel modulator or the pharmaceutical composition can be enclosed in an ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.

An intravenous formulation of the ion channel modulator or the pharmaceutical composition may be in the form of a sterile injectable aqueous or non-aqueous (e.g. oleaginous) solution or suspension. The sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, a solution in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, phosphate buffer solution, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of the intravenous formulation of the invention.

The ion channel modulator or the pharmaceutical composition can be prepared using methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining with water so as to form a solution. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension.

In an embodiment the subject to be treated is a mammal, preferably a human.

In an embodiment the ion channel modulator or the pharmaceutical composition is for use in the treatment of cancer, wherein said cancer is selected from gastric cancer, gastric adenocarcinoma, colorectal cancer, lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bone cancer, pancreatic cancer, colon cancer, colorectal cancer, skin cancer, cancer of the head or neck, head and neck squamous cell carcinoma, melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, hepatocellular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, thymoma, urothelial carcinoma leukemia, prostate cancer, prostatic adenocarcinoma mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, and multiple myelomas. In an embodiment the cancer is selected from gastric, intestinal or pancreatic cancer.

In an embodiment the ion channel modulator is for use in the treatment or prevention of metastasis. Metastasis is the process by which cancer cells spread from a primary tumour to other organs in the body. Metastasis may be malignant or non-malignant. The ion channel modulator may be for use in the treatment or prevention of metastasis which occurs at any site in the body, for example the site of metastasis may include lungs, liver, brain, peritoneal, lymph nodes, bones, bone marrow, breast, ovary, adrenal glands, pancreas, skin and soft tissue, kidney, stomach, colon, lacteal gland, prostate gland, bladder, bronchus, or nerve. In an embodiment the ion channel modulator may be for use in the treatment or prevention of peritoneal, kidney, liver or lung metastases. The ion modulator may be for use in the treatment of a subject who has been identified as at a high risk of metastasis.

Wherein the ion channel modulator is used in the prevention of a disease for example metastasis the modulator may be administered prophylactically.

The ion channel modulator or pharmaceutical composition may be used combination with a further anti-cancer treatment and/or anti-metastatic treatment. The further anti-cancer and/or metastatic treatment may be selected from chemotherapy, hormone therapy, immunotherapy, radiation therapy, stem cell therapy, surgery or targeted therapies such as small molecule therapy, antibody therapy, checkpoint inhibitors or CAR-T therapy.

The term "combination" as used herein, is meant to encompass the administration of the ion channel modulator simultaneously, separately or sequentially with administration of the further anti-cancer treatment.

In an aspect the invention also relates to a method of reducing the level of circulating tumour cells comprising contacting a cell with a, ion channel modulator, wherein said ion channel opener is capable of facilitating ion flow through NALCN.

It has been shown herein that loss of function of NALCN markedly increases the levels of circulating tumour cells. Therefore, by facilitating ion flow through NALCN the level of circulating tumour cells can be reduced. In an aspect the invention also relates to a method of modulating the level of solid tissue cell shedding comprising contacting a cell with a, ion channel modulator, wherein said ion channel modulator is capable of facilitating ion flow through NALCN. Modulating the level of cell shedding from solid tissue may comprise increasing the level or decreasing the level.

In an embodiment the cell is contacted in vitro, ex vivo or in vivo.

In an aspect the invention also relates to an ion channel modulator for use in the treatment of a fibrotic disease wherein the ion channel modulator is capable of facilitating ion flow through NALCN.

In an aspect the invention also relates to a method of treating or preventing a fibrotic disease, comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention.

Fibrosis, or the accumulation of extracellular matrix molecules that make up scar tissue, is a common feature of chronic tissue injury. A “fibrotic disease” refers to a disease where fibrosis occurs. Fibrotic diseases include gadolinium-induced systemic fibrosis, renal fibrosis, pulmonary fibrosis, hepatic cirrhosis. Gadolinium-induced fibrosis may also be referred to as nephrogenic systemic fibrosis. It has been shown herein that exposure to gadolinium, an NALCN blocker increased the level of CTCs and metastasis. Gadolinium-induced fibrosis can manifest as severe cutaneous and systemic fibrosis following the administration of gadolinium-based contrast agents. The present inventors have shown that loss-of-function of NALCN (no exposure to gadolinium) in animal models lead to the formation of multi-organ Gadolinium-induced fibrosis demonstrating NALCN is the likely underlying genetic cause for this syndrome. This type of fibrosis possess features similar to many fibrosis diseases which go through common mechanisms of action to result in fibrosis. Therefore, NALCN may regulate the process of fibrosis. As such the compositions of the invention have therapeutic utility in fibrotic disease.

The terms “treat”, “treatment” or “treating,” as used herein refer to administering a compound to a subject for prophylactic and/or therapeutic purposes. As explained herein, the ion channel modulator can be used to treat cancer and/or metastasis. Thus in one embodiment, the ion channel modulator is being used to prevent the onset of cancer and/or metastasis and is administered prophylactically. As such the term “prevention” refers to preventing the onset of cancer and/or metastasis, in particular the ion channel modulator may be used to prevent the onset of metastasis. For example the ion channel modulator may be used to treat a subject identified as being at a high risk of metastasis. The term “prevention” can refer to a reduction in the risk of contracting a cancer, metastasis or fibrotic disease. The term “prophylactic or preventative treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” refers to administering treatment to a subject already suffering from a disease or condition.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The term “comprising” or “comprises” where used herein means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of’ or “consists essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components and the like.

The term “consisting of’ or “consists of’ means including the components specified but excluding other components.

Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists essentially of’ or “consisting essentially of’, and also may also be taken to include the meaning “consists of’ or “consisting of’.

The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention, as set out herein are also applicable to all other aspects or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each aspect or exemplary embodiment of the invention as interchangeable and combinable between different aspects and exemplary embodiments.

The invention is further illustrated in the following non-limiting examples.

Examples

Intestinal cancers, including those of the stomach, are thought to arise from stem cells ^7-9 ; but how oncogenic mutations transform intestinal stem cells to produce invasive cancer remains unclear. The inventors have shown previously that Promininl (Prom1) marks basal stem cells in gastric antral glands, and that their lineage forms adenocarcinomas in Prom1 ^CreERT2/LacZ ;Kras ^G12D ;Trp53 ^FIX/FIX (P1 ^KP ) mice following expression of mutant-Kras ^G72D and deletion of Trp53 ⁷ . Prom1 ⁺ , but not PromT, cells isolated from P1 ^KP gastric adenocarcinomas (P7 ^KP -GAC) propagated these tumours readily as allografts in immunocompromised mice; suggesting that Prom1 ⁺ R7 ^KP -GAC cells are the malignant counterparts of antral gland basal stem cells.

Example 1. Nalcn loss-of-function is a feature of advanced cancer

To understand better how antral gland basal stem cells are corrupted during transformation, we compared their transcriptomes with those of Prom1 ⁺ R7 ^KP -GAC cells. Ion channels and solute carriers were enriched among genes downregulated in Prom1 ⁺ P7 ^KP -GAC cells (adjusted p- value=1 ,7e ^-3 ; Fig. 1a;). Review of 10,022 human cancers within The Cancer Genome Atlas showed that non-synonymous mutations in NALCN are enriched among gastric (n=43/422; dN/dS ratio, p=0.007) and colorectal adenocarcinomas (n=45/528, p=0.04; Fig. 1 b) ⁵⁶ . Mapping these mutated residues on the cryo-electron microscope structure of NALCN, embedded and relaxed within a 575-POPC lipid bilayer in silico ³ ' ⁰ " , revealed significant spatial-clustering within the pore turret and voltage sensing domains that regulate channel opening (p=0.03; Fig. 1c). HOLE analysis ¹² -that estimates ion channel pore radius size-performed on the end frame of an equilibrium molecular dynamics simulation of membrane embedded NALCN, predicts that 76% (n=224/295) of these mutations occlude the NALCN selectivity filter, and so will close the channel ²³ (Fig. 1d;). Among 221 patients for whom both disease stage and NALCN mutation status were available ^{13 14} , NALCN mutations predicted to cause the greatest pore closure were enriched in the most advanced cancers (Fig. 1 e;). Further, human GACs in which NALCN was mutated, upregulated genes expressed during epithelial-mesenchymal transition (EMT, p- value=1 .26e ^-9 )-a feature of invasive cancer ¹⁵ .

As a first step to test if Nalcn regulates cancer progression, we altered its function in P7 ^KP -GAC cells using genetic (/Va/cn-shRNA and /VALC/V-cDNA lentiviral transduction) or chemical (Nalcn channel blocker, gadolinium chloride [GdCh] ⁴ ) approaches. Whole-cell voltage-clamp analysis of P7 ^KP -GAC cells showed a linear GdCh sensitive current to voltage steps in the ±80 mV range as previously reported ⁴ . This current was eliminated in /Va/cn ^shRNA transduced P7 ^KP -GAC cells. Decreasing Nalcn function in P7 ^KP -GAC cells increased their proliferation in vitro and conferred an EMT morphology and transcriptome (adjusted p-value=5.29e ^-6 ) on orthotopic tumour allografts of these cells ¹⁶ . Conversely, increasing P7 ^KP -GAC cell NALCN expression, increased the GdCh-sensitive current, decreased proliferation, and produced a striking hyper-epithelialized morphology in allografts.

Example 2. Loss of Nalcn promotes cancer metastasis

To study how Nalcn loss-of-function impacts cancer initiation and progression in intact tissues, we generated mice harboring a conditional Nalcn allele in which exons 5 and 6 of the gene were flanked by loxP sites Nalcn™ . These mice were bred with P1 ^KP , Villinl- Cre ^ERT2 ;Kras ^G12D ;Trp53 ^FIX/FIX (V1 ^KP ) or Pdx1-Cre;Kras ^G12D ;Trp53 ^FIX/+ (Pdx1 ^KP ) mice to produce equivalent numbers of male and female mice that were either /Va/cn-wild-type (Nalcn ^+/+ ), Nalcn ^+/Flx or Nalcn ^FIX/FIX (total n=551 ;). All mice carried the Rosa26-ZsGreen (Rosa26 ^ZSG ) lineage tracing allele. Cancers in V1 ^KP and Pdx1 ^KP mice are restricted by Cre expression to the intestine ^{17 18} and pancreas ¹⁹²⁰ , respectively. prom1 ^CreERT2/LacZ is expressed by a variety of stem/progenitor cells and induces tumours of the small intestine, liver, lung, salivary glands, prostate, uterus, skin, and stomach in P1 ^KP mice ⁷⁸ . Since tissues can display age-dependent susceptibility to transformation ⁷ we activated Cre-recombination in P1 ^KP and V1 ^KP mice using tamoxifen at postnatal day (P) 3 or P60. Mice displaying signs of tumour development were euthanised and subject to whole-body macro- and microscopic autopsy. As expected, V1 ^KP (n=127/141) and Pdx1 ^KP (n=55/55) mice developed intestinal and pancreatic tumours, respectively. P1 ^KP mice developed tumours in the stomach (n=49/269), small intestine (n=59/269) and other sites (n=1 O8/269) ^{7 18} ’ ²⁰ : 99% (n=212/214) of P1 ^KP mice developed a single primary cancer . Neither age of induction, sex or Nalcn status altered significantly the site, type, size or incidence of primary tumours, or tumour-free survival in these mouse models. Thus, Nalcn function does not appear to impact the capacity of Kras and Trp53 oncogenic mutations to transform tissues.

However, hetero- or homozygous deletion of Nalcn dramatically increased tumour metastasis to the peritoneum, retroperitoneum, liver, lymph nodes, lungs and/or kidneys in P1 ^KP , V1 ^KP and Pdx1 ^KP mice (Fig. 2a-c). Metastatic and primary tumours were readily distinguished from one another by: expert pathologist, blinded histology review; co-segregation of 'matched' primary and secondary tumour transcriptomes by unsupervised hierarchical clustering; and highly- selective enrichment within metastatic tumour transcriptomes of histology-predicted primary tumour genesets (Fig. 2a and c). Intestinal adenocarcinomas (lACs) in V1 ^KP Nalcn ^+/+ mice (n=27 mice) and pancreatic adenocarcinomas (PACs) in Pdx1 ^KP Nalcn ^+/+ mice (n=19 mice), produced 2.8+4.9SE and 5.5+4.0SE metastases/mouse, respectively (Fig. 2d;). In stark contrast, these same tumours in V1 ^KP Nalcn ^+/Flx (n=51), V1 ^KP Nalcn ^FIX/FIX (n=26), Pdx1 ^KP Nalcn ^+/Flx (n=23), and Pdx1 ^KP Nalcn ^FIX/FIX (n=13) mice produced 16.2+5.7SE (Mann-Whitney, p=0.03 relative to Nalcn ^+/+ ), 26.0+10.18SE (p=0.0009), 15.0+3.62SE (p=0.007), and 13.5+5.01 SE (p=0.02) metastases/mouse, respectively. Nalcn deletion from V7 ^KP -IACs increased metastasis in particular to the peritoneum, kidneys and liver, while Nalcn deletion from Pdx1 ^KP -PACs increased metastasis to the peritoneum and lungs (Fig. 2d). Similar patterns of IAC and GAC metastatic burden were observed among 80 P1 ^KP mice that developed these, but not other, cancers: P1 ^KP Nalcn ^+/+ (11.6+3.45SE metastases/mice), P1 ^KP Nalcn ^+/Flx (42.2+11 .23SE metastases/mice) and P1 ^KP Nalcn ^FIX/FIX (40.24.0+15.51 SE metastases/mice): Nalcn loss significantly increased metastasis to the lungs and peritoneum in these mice (Fig. 2d;).

To validate further /Va/cn-loss-of-function as a driver of cancer metastasis, we treated additional cohorts of V1 ^KP Nalcn ^+/+ (n=37), V1 ^KP Nalcn ^FIX/+ (n= 17) and V1 ^KP Nalcn ^FIX/FIX (n=8) mice with the Nalcn channel blocker gadolinium chloride (2pg/kg/week up to 30 weeks). lACs developing in gadolinium treated V1 ^KP Nalcn ^+/+ mice (n=28) produced 18.3+5.94SE metastases/mice relative to 2.8+4.9SE in controls (p=0.02; Fig. 2e;). Notably, gadolinium did not increase IAC metastasis in either V1 ^KP Nalcn ^FIX/+ or V1 ^KP Nalcn ^FIX/FIX mice, confirming that the agent induced metastasis by blocking Nalcn-mediated currents.

Example 3. Loss of Nalcn increases the number of circulating tumour cells

Since loss of Nalcn function increased metastasis and enriched primary tumour transcriptomes with genesets expressed by human circulating tumour cells (CTCs; Extended Data Fig. 4i), we reasoned that loss of Nalcn function might increase the release of CTCs from primary tumours into the peripheral blood: CTCs are shed from tumours as precursors of metastatic disease ²¹ . Nucleated circulating ZSG ⁺ cells (CZCs) were quantified by fluorescence-activated cell sorting (FACS) from the peripheral blood of p _rO m1 ^CreERT2/LacZ (n=337 mice), Villin-1 ^CreER (n=121 mice), or Pdx1 ^Cre (n=40 mice) mice carrying the ROSA ^ZSG allele and various combinations of oncogenic and Nalcn ^FIX alleles. Following blood sampling, all mice underwent whole body autopsy. An average of 4.5e ³ +1 .1 SE CZCs/ml of blood (0.078%+0.02SE total cells) were isolated from all mice after an average of 296+9.8SE days following Cre-recombination . Across all three Cre- lines, the number of CZCs was highly correlated with both the presence of a primary tumour and the total number of metastases (multiple linear regression, T=10.43, p<0.0001 ;), independent of mouse sex or age of induction. Nalcn deletion, or gadolinium treatment, increased significantly the level of CZCs in tumour bearing P1 ^KP , V1 ^KP and Pdx1 ^KP mice (Fig. 3a). Since neither Prom1 ^CreERT2/LacZ , Villin-1 ^CreERT2 , or Pdx1 ^Cre recombine haematopoeitic cells in the bone marrow, then these data suggest strongly that CZCs are CTCs shed from primary tumours through a process regulated by Nalcn.

To better understand the origin of CZCs, we generated single cell RNA sequence (SCS) profiles of CZCs isolated from mice with P7 ^KP -GAC (n=1 ,701 cells) or V7 ^KP -IAC (n=119), as well as peripheral blood mononuclear cells (PBMCs, n=559), and compared these with published SCS profiles of human breast, lung, pancreatic and prostate CTCs (n=360) and PBMCs (n=500) ^22-27 . Human CTCs comprised three overlapping clusters, that were readily resolved from PBMCs : 'huCTCI ' (enriched with epithelial [adjusted p-value=1 .Oe ^-26 ] and dendritic cell [adjusted p- value=0.003] genesets); huCTC3 (CD71 ⁺ erythroid cell enriched [adjusted p-value=1 ,9e- ⁴³ ]); and huCTC2 (sharing profiles of huCTCI and 3). huCTC1-3 expressed p-globin (/7BB)-a survival factor for human CTCs ²⁴ -as well as HBA1, HBA2, and HBD. Mouse CZCs formed seven clusters whose transcriptomes closely matched huCTCI (mCZC2-5), huCTC2 (mCZC2-7) and huCTC3 (mCZC6 and 7), and included orthologues of HBA1, HBA2 (Hba-a1, Hba-a2), HBB (Hbb-bs, Hbb-bf), ANXA2 and LGALS3, as well as genes expressed in normal and malignant stomach and small intestine (Fig. 3c;). Co-immunofluorescence of peripheral blood smears taken from mice with V7 ^KP -IAC and P1 ^KP -GAC confirmed CZC expression of Hba-a2, Lgals3, and epithelial cell markers (Krt80, Cdh1) and Cdx2 that marks intestinal epithelium (Fig. 3d). PBMCs did not express these markers but did express markers of PBMCs e.g., Cd45.

To test directly if CZCs are CTCs, we injected separate aliquots of 25,000 CZCs isolated from mice with Pdx1 ^KR -PAC, P1 ^KP -GAC or V7 ^KP -IAC into the tail veins of eight immunocompromised mice. Within 75 days, all mice developed respiratory distress and contained numerous ZSG ⁺ metastases in the lungs, liver, kidneys and peritoneum (Fig. 3e and f). Thus, CZCs include CTCs that recapitulate the transcriptome of human CTCs and are shed into the peripheral blood through a process regulated by Nalcn.

Example 4. Nalcn regulates solid tissue cell-shedding independent of cancer

Preventing CTC shedding into the peripheral blood could stop metastasis; but disentangling this process from the complex cascade of tumourigenesis has proved challenging. To test if Nalcn regulates cell shedding from solid tissues independent of tumourigenesis, we looked for CZCs in the peripheral blood of Prom1 ^CreERT2/LacZ Rosa26 ^ZSG Nalcn ^+/+ (P1 ^R Nalcn^n=87), P1 ^R Nalcn ^+/FIX (n=48) and P1 ^R Nalcn ^FIX/FIX (n=37) mice that lacked oncogenic alleles and never developed tumours. Remarkably, CZCs were readily isolated from the peripheral blood of these mice, and deletion of Nalcn increased the numbers of these cells significantly-to a degree similar to that seen in tumour bearing animals (Figs. 3a and 4a). SCS profiles of CZCs isolated from non- tumour-bearing (ntCZCs) mice co-clustered with CZCs from tumour bearing animals (tCZCs) and with IAC and GAC metastases SCSs (Fig. 4b). The great majority of tCZCs and ntCZCs SCSs did not cluster with profiles generated from primary lACs, GACs, or normal lung, liver, small intestine, stomach, kidney, uterus or epididymis cells (Fig. 4b). Similar to human CTCs ¹ , the SCS profiles of tCZCs and ntCZCs were highly-enriched for genesets expressed by gastric and small intestinal stem/progenitor cells (tCZC1 nt/tCZC1-4), huCTC-1 (tCZC1 , nt/tCZC8 and 9), huCTC-2 (nt/tCZC4-9) and huCTC-3 (nt/tCZC8 and 9;). Co-immunofluorescence of blood smears confirmed that both ntCZCs and tCZCs share markers of huCTCs, including Hba-a1 (Figs. 3d and 4c).

To understand the fate of CZCs in non-tumour bearing mice, we injected separate aliquots of 25,000 CZCs isolated from P1 ^R Nalcn ^FIX/FIX mice into the tail veins of six immunocompromised mice. All recipient mice remained clinically well after an average of 100 days but contained numerous ZSG ⁺ /CdhT7lcam1 ⁺ donor-cell clusters within their lungs, liver, kidneys and peritoneum at a frequency similar to metastatic tumours formed by tail vein injections of tCZCs (Fig. 3f, 4d-f). ntCZCs appeared to incorporate into, and/or form component parts of, apparently normal recipient organs-the most extreme example being their incorporation into glomeruli, vessels and/or tubules of the kidney (Fig. 4d-f). Thus, Nalcn regulates cell shedding from solid tissues independent of cancer, divorcing this process from tumourigenesis and unmasking an oncogene-independent metastatic pathway.

Example 5. Nalcn-blockade causes gadolinium-induced systemic fibrosis

While P1 ^R Nalcn ^+/FIX (n=1 18) and P1 ^R Nalcn ^FIX/FIX (n=112) mice did not develop tumours, whole body autopsy of these mice revealed increasing fibrosis of the kidneys and skin-that are sites of prom1 ^CreERT2/LacZ driven recombination ⁷ -relative to P1 ^R Nalcn ^+/+ (n=65) mice. Nalcn deletion did not increase fibrosis of the liver, lungs, pancreas, stomach or intestines. This pathology arose after >400 days and replicated that of gadolinium-induced systemic fibrosis (GISF, previously called nephrogenic systemic fibrosis)-a debilitating condition manifested by the development of severe cutaneous and systemic fibrosis following the administration of gadolinium-based contrast agents (GBCA) ²⁸ . Thus, our data directly implicate gadolinium-blockade of NALCN as the mechanism underpinning GISF.

Discussion

Most patients with cancer die as a result of metastasis-the process by which cancer cells spread from the primary tumour to other organs in the body. Current understanding of metastasis is predicated on the idea that oncogenic mutations drive a cascade of events in which stem-cell like cancer cells leave the primary tumour, enter the blood stream, and travel to distant sites where they form new malignant growths ^{1 29} . If correct, this model requires the presence of a primary tumour at some stage in the disease history, and assumes that the process is abnormal and unique to malignancy. By demonstrating that a single ion channel, NALCN, regulates cell trafficking from both non-malignant and malignant tissues to distant organs, we provide important new insights to the metastatic process and possible explanations for long-standing enigmatic observations.

Developing anti-metastatic therapies has proven difficult since potential therapeutic targets in primary tumours that drive metastases e.g., mutant oncoproteins, have proved hard to find ¹ . By divorcing the process of CTC shedding from 'upstream' tumourigenesis, our data unmask Nalcn function, and thereby the manipulation (depolarization) of resting membrane potential, as a promising new approach to block metastasis. Gadolinium-blockade of Nalcn increased the abundance of tCZCs in our mice; therefore, drugs capable of re-opening the channel might be effective anti-metastatic drugs. Precedent for this approach is provided by drugs that open the chloride-ion channel mutated in the disease cystic fibrosis ³⁰ .

A model in which metastases always descend from a primary tumour is hard to reconcile with the observation that metastases can emerge many years after removal of a localised cancer ³¹ and that up to 5% of patients with metastases lack an apparent primary tumour ³² . Loss of Nalcn function in our mice caused an abundant and persistent shedding of cells that embed in distant organs, even in the absence of a primary tumour. Since human epithelial tissues contain fields of phenotypically normal cells that harbour oncogenic mutations ³³³⁴ , then loss of NALCN function in these cells could provide a source of CTCs that form metastases in the absence of a primary tumour, or long after a primary tumour has been removed from within the field of mutant cells. It is likely that such cells would need to acquire additional mutations to form tumours at the metastatic site, compatible with the relative rarity of these phenomena. Our data may also explain why CTCs have been found in the bone marrow of patients who lack metastases. While these cells could represent 'dormant' CTCs as previously suggested ²⁹ , equivalent to ntCZCs in our mice, they may be shed from non-transformed epithelia that have lost NALCN function, but not gained the ability to form metastatic tumours.

Our observations also raise important questions: 'How does loss of Nalcn function promote cell shedding?' And, since we observed CZCs in P1 ^R Nalcn ^+/+ mice, albeit at lower levels than in Nalcn deleted animals, 'Is epithelial cell trafficking a normal phenomenon that is corrupted in cancer?' Since Nalcn loss-of-function promoted an EMT phenotype and transcriptome in tumours and CTCs in our mice, Nalcn may regulate gene transcription in a manner similar to that of calcium-ion channels ³⁵ : the calcium pump PMCA4 was reported to regulate an EMT transcriptome in gastric cancer cells ³⁶ . Further work will uncover the role of epithelial cell trafficking in normal tissue maintenance or other disease states. Our observation that deletion of Nalcn replicated GISF in the kidneys and skin of aged animals pinpoint Nalcn-channel blockade as the likely mechanism underpinning this debilitating condition. Since P1 ^KP mice succumbed to cancer well before the onset of organ fibrosis in P1 ^R mice, and Nalcn deletion in P1 ^R mice did not induce stomach, intestine, pancreas, lung or liver fibrosis-principal sites of primary and metastatic tumours in P1 ^KP mice-then fibrosis is unlikely to contribute to metastasis in /Va/cn-deleted mice. However, since limited exposure to gadolinium can induce GISF in humans, it is a note of concern that gadolinium-contrast imaging of cancer patients could accelerate metastasis.

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