Interleukin-1 receptor type 1 inhibitors for use in the treatment of cancer - Patent Application 20070122997

Abstract

The present invention provides inhibitors for use in the treatment of KRAS mutant cancers, uses and methods for treating KRAS mutant cancers, methods for selecting subjects predicted to be therapeutically responsive to a treatment comprising an inhibitor. The present invention also provides kits.

Description

[Technical field]

[0001] The present invention provides inhibitors for use in the treatment of KRAS mutant cancers, uses and methods for treating KRAS mutant cancers, and methods for selecting subjects predicted to be therapeutically responsive to treatments comprising inhibitors. The present invention also provides kits. [Background technology]

[0002] Cancer is a leading cause of death worldwide, and its treatment and outcome have been dramatically revolutionized by targeted therapies. As the most frequently mutated oncogene, Kirsten rat sarcoma viral oncogene homolog (KRAS) has attracted considerable attention.

[0003] Many clinical studies have shown that targeted therapy significantly extends progression-free survival and is less toxic than standard chemotherapy. For example, targeted therapy in patients with epidermal growth factor receptor (EGFR)-sensitive mutations or anaplastic lymphoma kinase (ALK) gene fusions has significantly improved survival. Unfortunately, despite 40 years of unique drug efforts, there is still no effective strategy targeting KRAS mutations, except for sotorasib, which has just been approved to target mutated KRAS (KRAS G12C). Due to the unique characteristics of the KRAS protein, KRAS is considered a challenging therapeutic target and even deemed "undruggable." Thus, many efforts have focused on targeting KRAS indirectly by targeting its downstream signaling effectors using telomerase inhibitors and epigenetic approaches such as RNA interference. However, most of these strategies have failed due to lack of activity or selectivity. In addition, patients with KRAS mutations usually have poor responses to current standard therapies. Thus, there is an urgent and unmet need to target KRAS-driven cancers. Summary of the Invention

[0004] Against this background, the present inventors have surprisingly found that KRAS mutant cancers activate NF-κB in tumor-associated myeloid cells to induce IL-1β, which is necessary for their sustained growth. As shown in the accompanying examples, IL-1β is induced from macrophages via versican (VCAN).

[0005] In a first aspect, the present invention provides an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling for use in treating cancer in a subject, wherein the cancer comprises a KRAS mutation and elevated levels and / or activity of veriscan (VCAN).

[0006] This aspect of the invention also includes the use of an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling in the manufacture of a medicament for treating cancer in a subject, wherein the cancer comprises a KRAS mutation and elevated levels and / or activity of veriscan (VCAN).

[0007] This aspect of the invention also includes a method of treating cancer in a subject, wherein the cancer comprises a KRAS mutation and elevated levels and / or activity of veriscan (VCAN), the method comprising administering to the subject an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

[0008] Interleukin-1 receptor type 1 (IL-1R1), also called IL-1RI, is a membrane-bound protein that regulates inflammatory responses through agonistic and antagonistic regulation of cytokine activity. Interleukin-1 alpha (IL-1α) and beta (IL-1β) are the prototypic members of the IL-1 family of cytokines. Within the complex regulatory network of the IL-1 pathway, the cytokines IL-1α and IL-1β interact with the extracellular domain of IL-1R1, triggering recruitment of an accessory receptor, IL-1RAcP, resulting in a functional receptor complex that initiates the IL-1R1 signaling cascade. In addition to the agonists IL-1α and IL-1β, IL-1R1 also binds to the antagonist IL-1Ra, which is unable to trigger IL-1R1 association with IL-1RAcP, thereby competitively blocking IL-1 signaling via IL-1R1 binding.

[0009] By "inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling" we include the meaning of any molecule that can downregulate, antagonize, suppress, reduce, prevent, decrease, block, and / or reverse the biological activity and / or effect of IL-1R1. For the avoidance of doubt, we also include any molecule that blocks or reduces the expression of an IL-1R1 agonist (such as IL-1β) and / or increases the degradation of an IL-1R1 agonist (such as IL-1β) and / or increases the expression of an IL-1R1 antagonist (such as IL-1RA).

[0010] Methods for evaluating IL-1RI inhibition are known in the art. For example, it is possible to assay the activity of IL-1 agonist cytokines (such as IL-1α and IL-1β). Some exemplary assays for IL-1 activity are described in Boraschi et al. (2006) and include T cell proliferation assays, IL-6 and IL-8 production assays, and calcium influx inhibition. In one exemplary assay, the ability of an inhibitor can be evaluated for its ability to inhibit IL-1β-stimulated release of IL-6 from human fibroblasts. Inhibition of IL-1β-stimulated cytokine release in MRC5 cells correlates with the ability of the inhibitor to inhibit IL-1-mediated activity in vivo. Details of the assay are described in Dinarello et al., Current Protocols in Immunology, Ch.6.2.1-6.2.7, John Wiley and Sons Inc., 2000. Briefly, human MRC5 human fibroblasts (ATCC number CCL-171, Manassas VA, USA) are grown to confluency in multi-well plates. The cells are treated with titrated doses of inhibitors and controls. The cells are then contacted with 100 pg / ml of IL-1β in the presence of the titrated inhibitors and / or controls. Negative control cells are not stimulated with IL-1β. The amount of IL-6 released in each group of treated cells is measured using an IL-6 ELISA kit (e.g., BD Pharmingen, Franklin Lakes, NJ, USA). Controls that can be used include buffer alone, IL-1Ra, and antibodies against IL-1β. The effectiveness of inhibitors can also be evaluated in vivo. For an exemplary assay, see Economides et al., Nature Med., 9:47-52 (2003). Briefly, mice are injected intraperitoneally with titrated doses of inhibitors and controls. 24 hours after injection, mice are subcutaneously injected with recombinant human IL-1β at a dose of 1 μg / kg. Two hours after injection of IL-1β (time of peak IL-6 response), mice are sacrificed and blood is collected and processed for serum.

[0011] IL-1 induces the release of inflammatory mediators such as IL-6 via IL-1R1. Serum IL-6 levels can be assayed by ELISA. Percent inhibition can be calculated based on the ratio of IL-6 detected in experimental animal serum to that detected in control. Other exemplary assays for IL-1R1 activity in vivo are described in Boraschi et al. and include anorexia, hypoglycemia, and neutrophilia assays.

[0012] A further example of assaying IL-1R1 activity is described in Example 3 of WO2012 / 016203 (incorporated by reference).

[0013] We include the meaning that IL-1R1-mediated signaling is reduced in the presence of an inhibitor compared to IL-1R1-mediated signaling in the absence of the inhibitor. Inhibition is not limited to complete inhibition or prevention of IL-1R1 signaling. It may be that in a given application, some low level of IL-1R1 signaling that does not adversely affect patient outcome can be tolerated. In an embodiment, the inhibitor reduces IL-1R1 signaling by at least 10%, e.g., at least 20%, 30%, 40%, or 50%, compared to IL-1R1-mediated signaling in the absence of the inhibitor. In an embodiment, the inhibitor reduces IL-1R1 signaling by at least 50%, e.g., at least 60%, 70%, 80%, or 90%, e.g., 95%, compared to IL-1R1-mediated signaling in the absence of the inhibitor.

[0014] An inhibitor may inhibit IL-1R1 signaling with an IC50 of less than 100, 50, 20, 10, or 5 nM.

[0015] There are two general mechanisms of inhibiting IL-1R1 signaling: binding to the IL-1 receptor (e.g., Isunakinra) or directly binding to an agonistic IL-1 cytokine (e.g., rilonacept and canakinumab). By binding to the receptor, the inhibitors can block downstream signaling, for example, by reducing or preventing the recruitment of IL-1RAcP. By directly binding to an agonistic IL-1 cytokine, the inhibitors effectively sequester IL-1α and / or IL-1β, thus preventing them from binding to IL-1R1.

[0016] In embodiments, the inhibitor may be an inhibitor that selectively inhibits IL-1R1 signaling. For example, the inhibitor may inhibit and / or reduce the biological activity of IL-1R1 to a greater extent than it inhibits the biological activity of an unrelated cell surface receptor. Preferably, the agent inhibits the biological activity of IL-1R1 at least 5-fold, or at least 10-fold, or at least 50-fold more than it inhibits the biological activity of another unrelated cell surface receptor. More preferably, the agent inhibits the biological activity of IL-1R1 at least 100-fold, or at least 1,000-fold, or at least 10,000-fold more than it inhibits the biological activity of another unrelated cell surface receptor.

[0017] By "biological activity of IL-1R1", we include the biological effects of IL-1R1, which refers to any function(s) exhibited or performed by the naturally occurring and / or wild-type forms of IL-1R1 measured or observed in vivo (i.e., in the protein's natural physiological environment) or in vitro (i.e., under laboratory conditions). Such activators include induction of inflammatory mediators, such as IL-6. Assays for measuring induction of IL-6 are described herein. Biological activity of IL-1R1 can also be tested using the NF-κB reporter gene assay described herein.

[0018] In an embodiment, the inhibitor is an inhibitor that binds to IL-1R1 to inhibit the biological activity of IL-1R1. In an embodiment, the inhibitor is an inhibitor that selectively binds to IL-1R1.

[0019] By an inhibitor that "selectively binds" to IL-1R1, we include the meaning that the inhibitor binds to IL-1R1 with a higher affinity than to an unrelated cell surface receptor. In embodiments, the inhibitor binds to IL-1R1 with an affinity that is at least 5-fold, or at least 10-fold, or at least 50-fold higher than to an unrelated cell surface receptor. In embodiments, the agent binds to IL-1R1 with an affinity that is at least 100-fold, or at least 1,000-fold, or at least 10,000-fold higher than to an unrelated cell surface receptor. Binding to IL-1R1 may be determined by methods well known in the art, including ELISA and surface plasma resonance (SPR) (such as those described in Example 4 of WO2012 / 016203, which is incorporated by reference).

[0020] It will be understood that inhibition of IL-1R1 signaling following binding of an inhibitor to IL-1R1 may be referred to as "direct inhibition". For example, an inhibitor may occupy the binding pocket of IL-1R1, thus denying agonist cytokines such as IL-1α and IL-1β the opportunity to bind. Such binding inhibition can be determined using assays and methods well known in the art, for example, using a BIAcore chip with immobilized IL-1R1 and incubating with soluble IL-1β with or without the inhibitor to be tested. Such binding inhibition can also be determined using flow cytometry or ELISA.

[0021] ELISA assays typically involve the use of enzymes that give a colored reaction product, usually in a solid-phase assay. Enzymes such as horseradish peroxidase and phosphatase have been widely used. A method to amplify the phosphatase reaction is to use NADP as a substrate to generate NAD, which now acts as a coenzyme for a second enzyme system. Pyrophosphatase from Escherichia coli provides a good conjugate, as the enzyme is not present in tissues, is stable, and gives a good reaction color. Chemiluminescence systems based on enzymes such as luciferase can also be used.

[0022] ELISA methods are well known in the art, see, e.g., The ELISA Guidebook (Methods in Molecular Biology), 2000, Crowther, Humana Press, ISBN-13:978-0896037281, the disclosure of which is incorporated herein by reference.

[0023] In embodiments, the inhibitor does not bind to IL-1R1 to inhibit IL-1R1 signaling. It will be understood that this may be referred to as "indirect inhibition". For example, the inhibitor may bind to the agonist cytokines IL-1β and / or IL-1α, thus interfering with the ability of IL-1β and / or IL-1α to bind to the cognate receptor IL-1RI, thus inhibiting IL-1R1 signaling. Alternatively, the molecule may be able to sequester naturally occurring IL-1α and / or IL-1β, such that IL-1α and / or IL-1β cannot bind to IL-1R1. Alternatively, the inhibitor may be able to block the recruitment of the accessory receptor IL-1RAcP to IL01R1, resulting in no signaling.

[0024] Preferably, the inhibitor is selected from the group comprising a peptide, a polypeptide, a nucleic acid molecule (such as an aptamer), a small molecule, an antibody, a peptidomimetic, a natural product, a monobody, or a carbohydrate.

[0025] By "nucleic acid", also referred to as "oligonucleotide", "nucleic acid sequence", "nucleic acid molecule" and "polynucleotide", we include DNA sequences or analogs thereof, or RNA sequences or analogs thereof.

[0026] In an embodiment, the nucleic acid inhibitor may be an aptamer. Aptamers can be considered as chemical antibodies with the properties of nucleotide-based therapeutics. Aptamers are small nucleic acid molecules that specifically bind to molecular targets such as proteins. Unlike nucleic acid therapeutics, which act by hybridizing to another nucleic acid target, aptamers form a three-dimensional shape that allows them to specifically bind to enzymes, growth factors, receptors, viral proteins, and immunoglobulins. Nucleic acid aptamers generally include a primary nucleotide sequence that allows the aptamer to form a secondary structure (e.g., by forming a stem-loop structure) that allows the aptamer to bind to its target. In the context of the present invention, aptamers can include DNA, RNA, nucleic acid analogs (e.g., peptide nucleic acids), locked nucleic acids, chemically modified nucleic acids, or combinations thereof. Aptamers can be designed for a given ligand by a variety of procedures known in the art (see Buglak et al., Int J Mol Sci. 2020 Nov 10; 21(22): 8420).

[0027] In embodiments, the inhibitors are small molecules, including but not limited to small synthetic organic molecules capable of inhibiting IL-1R1 signaling. Their molecular weight is usually less than 800 Da and they have properties including good solubility, bioavailability, PK / PD, metabolism, etc.

[0028] By "peptide" we include short chains of amino acid monomers linked by peptide (amide) bonds. By "polypeptide" we include long, continuous, unbranched peptide chains.

[0029] In an embodiment, the inhibitor is an antibody. Antibodies are characterized in that they contain an immunoglobulin domain and therefore they are members of the immunoglobulin superfamily of proteins. By "antibody" we include the meaning of fragments thereof. An antibody fragment is a portion of an intact full-length antibody, such as an antigen-binding fragment or variable region(s) of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), multispecific antibody fragments such as bispecific, trispecific, and multispecific antibodies (e.g., diabodies, triabodies, tetrabodies), minibodies, chelating recombinant antibodies, tribodies or bibodies, intrabodies, nanobodies, small modular immunopharmaceuticals (SMIPs), binding domain immunoglobulin fusion proteins, camelized antibodies, VHH-containing antibodies, and any other polypeptides formed from antibody fragments.

[0030] By "antibody" and "antibodies", we include the meaning of polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies, single chain Fv antibody fragments (scFv), single variable domains (VhH), Fab fragments, and F(ab)2 fragments. Polyclonal antibodies are heterogeneous populations of antibody molecules specific for a particular antigen that can be obtained from the sera of immunized animals. Polyclonal antibodies are produced using well known methods. Monoclonal antibodies, which are homogeneous populations of antibodies against a particular epitope contained within an antigen, can be prepared using standard hybridoma technology. In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by a series of cell lines in culture, such as that described in Kohler, G. et al. Nature. 1975, 256:495, the human B-cell hybridoma technique (Kosbor et al. Immunology Today, 1983, 4:72; Cole et al. Proc. Natl. Acad. Sci. USA. 1983, 80:2026), and the EBV-hybridoma technique (Cole et al. "Monoclonal Antibodies and Cancer Therapy", Alan R. Liss, Inc., 1983, pp. 77-96). Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. Hybridomas producing monoclonal antibodies can be cultured in vitro or in vivo. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Chimeric antibodies can be produced by standard techniques.

[0031] By "antibody" we also include antibody mimetics. By "antibody mimetics" we include organic compounds that are not structurally related to antibodies but can bind to targets in a manner similar to that of antigen-antibody interactions. They are usually artificial peptides or proteins with a molar mass of about 3-20 kDa. Affibodies are a type of antibody mimic, and their protein scaffold is derived from the B domain of Staphylococcus aureus protein A.

[0032] By "carbohydrates" we include macromolecules composed of carbon, hydrogen, and oxygen. They are organic compounds organized in the form of aldehydes or ketones with multiple hydroxyl groups pendant from the carbon chain.

[0033] By "monobody" we include the meaning of a synthetic binding protein constructed using the fibronectin type III domain (FN3) as a molecular scaffold.

[0034] By "subject", we include the meaning of a patient or individual in need of treatment and / or prevention of a disease or condition described herein. The subject may be a vertebrate, such as a vertebrate mammal. In embodiments, the subject is selected from the group including primates (e.g., humans, monkeys, apes), rodents (e.g., mice, rats, hamsters, guinea pigs, gerbils, rabbits), canines (e.g., dogs), felines (e.g., cats), equines (e.g., horses), bovines (e.g., cows), and / or porcines (e.g., pigs). Preferably, the subject is a human.

[0035] By "cancer comprises a KRAS mutation," we include the meaning of cancers that have KRAS mutations, such as increased copy number (amplification) of the KRAS gene, KRAS gene fusions, KRAS rearrangements, and / or missense or nonsense gene mutations. It will be understood that KRAS mutations are oncogenic mutations, such as those that drive tumor initiation and maintenance. Non-limiting examples of such KRAS gene mutations include missense or nonsense mutations at codon 5, codon 12, codon 13, codon 14, codon 18, codon 19, codon 23, codon 31, codon 38, codon 59, codon 61, codon 62, codon 97, codon 117, codon 118, codon 119, codon 121, codon 140, codon 143, codon 145, codon 146, codon 151, codon 153, codon 168, codon 171, codon 180, codon 185, codon 187, and codon 188 of the KRAS gene.More specific examples of these include p.E3K, p.K5E, p.Y5N, p.G10dup, p.A11_G12dup, p.G12A, p.G12C, p.G12D, p.G12R, p.G12S, p.G12V, p.G13C, p.G13D, p.G13R, p.G13V, p.V14I, p.S17T, p.A18N, p.L19F, p.I21R, p .Q22K, p.L23R, p.I24N, p.Q31*, p.D33E, p.P34L, p.I36M, p.D38N, p.D38Y, p.T58K, p.A59G, p.A59 E, p.A59T, p.Q61E, p.Q61R, p.Q61H, p.Q61P, p.Q61K, p.Q61L, p.E62Y, p.E62K, p.E63K, p.R68M, p.R 68S, p.R97I, p.Y71C, p.K88*, p.D92Y, p.E98*, p.P110S, p.K117N, p.M118V, p.D119N, p.P121H, p. R123*, p.A130V, p.R135T, p.P140H, p.D143G, p.S145L, p.A146T, p.A146P, p.A146V, p.G151A, p.D Examples of the KRAS mutations include, but are not limited to, 153V, p.A155D, p.T158A, p.E168fs, p.R164Q, p.I171M, p.I171Nfs*14, p.K176Q, p.K180del, p.K185fs, p.I187V, p.M188V, p.X2_splice, p.KRAS-LMNTD1, p.KRAS-SLC2A14, etc. More specific examples of KRAS mutations in codon 12 include, but are not limited to, p.G12A, p.G12C, p.G12D, p.G12R, p.G12S, p.G12V, etc.

[0036] Determining the mutation status, including the KRAS gene mutation status, can be performed by in vitro methods using methods well known in the art, for example, the step of determining the gene mutation status in the patient includes taking a sample from the patient and then determining the gene mutation status of the sample.The sample can include, for example, at least one of serum, whole fresh blood, peripheral blood mononuclear cells, frozen whole blood, fresh plasma, frozen plasma, urine, saliva, skin, hair follicle, bone marrow, tumor tissue, tumor biopsy, or archived paraffin-embedded tumor tissue.

[0037] The KRAS mutation status can be, for example, at the level of the genomic DNA, protein and / or mRNA transcript of the KRAS gene.

[0038] The determination of KRAS mutation can be carried out using a method selected from the group including: (a) PCR, (b) RT-PCR, (c) FISH, (d) IHC, (e) immunodetection method, (f) Western blot, (g) ELISA, (h) radioimmunoassay, (i) immunoprecipitation, (j) FACS, (k) HPLC, (l) surface plasmon resonance, (m) optical spectroscopy, and (n) mass spectrometry. The presence of KRAS gene mutation(s) can be further detected by any sequencing method, including dideoxy sequencing, pyrosequencing, PYROMARK®, KRAS assay, and allele-specific PCR assay. The determination of KRAS gene can be detected using a kit known in the art, for example, RASKET kit (trade name, MEBGEN), therascreen KRAS RGQ PCR Kit (Qiagen).

[0039] Versican (VCAN) is an extracellular matrix proteoglycan. VCAN is encoded by a single gene and is located on chromosome 5q12-14 in the human genome. The human VCAN gene is divided into 15 exons spanning 90-100 kb. By "Versican (VCAN)", we include the meaning of all splice variants of VCAN, including the known splice variants V0, V1, V2, V3 and V4.

[0040] By "elevated levels and / or activity of Veriscan (VCAN)", we include the meaning of increased levels and / or activity of VCAN in the cancer of a subject compared to the level and / or activity of VCAN in a reference subject(s). This elevated level and / or activity may be present in the cancer cells themselves, but because VCAN is secreted from cancer cells, elevated levels and / or activity may be present in the tumor microenvironment, such as surrounding blood vessels, immune cells, fibroblasts, signaling molecules and / or the extracellular matrix. By "cancer cells", we include the meaning of cells with uncontrolled cell proliferation, such as tumor cells.

[0041] In one embodiment, the "reference subject" is the same subject. Thus, if the level and / or activity of VCAN in a cancer is increased compared to the level and / or activity of VCAN in a non-cancerous sample or tissue from a subject, the cancer may have an elevated level and / or activity of VCAN. Alternatively, if the level and / or activity of VCAN in a cancer is increased compared to the level and / or activity of VCAN in tissue containing the cancer at an earlier time point (e.g., before the onset of malignancy, before the start of treatment, or during treatment), the cancer may have an elevated level and / or activity of VCAN.

[0042] In alternative embodiments, the "reference subject(s)" are one or more healthy subjects, such as subjects who do not have cancer or who do not otherwise have the same type of cancer (e.g., one affecting the same tissue). The healthy subjects may be in the same age group, and optionally of the same sex, as the subjects with cancer.

[0043] In certain embodiments, the level and / or activity of VCAN is at least 5%, e.g., at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 41%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 102%, 104%, 105%, 106%, 107%, 108%, 109%, 109%, 109%, 110 An increase of 42%, 43%, 44%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, ±91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, ±150%, 175%, 200%, 225%, 250%, 275%, 300%, 350%, 400%, 500% or at least 1000% is considered elevated.

[0044] In certain embodiments, the level and / or activity of VCAN is considered elevated if it is increased by at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, or at least 500% in the subject's cancer compared to the level and / or activity of VCAN in a reference subject(s).

[0045] In certain embodiments, the level and / or activity of VCAN is considered elevated if it is increased by at least 25%, at least 50%, at least 75%, at least 100%, or at least 200% in the subject's cancer compared to the level and / or activity of VCAN in a reference subject(s).

[0046] In certain embodiments, the level and / or activity of VCAN is considered to be elevated if it is increased by at least 50% in the cancer of the subject compared to the level and / or activity of VCAN in the reference subject(s). In certain embodiments, the level and / or activity of VCAN is considered to be elevated if it is increased by at least 100% in the cancer of the subject compared to the level and / or activity of VCAN in the reference subject(s). In certain embodiments, the level and / or activity of VCAN is considered to be elevated if it is increased by at least 200% in the cancer of the subject compared to the level and / or activity of VCAN in the reference subject(s). In one embodiment, the level and / or activity of VCAN is considered to be elevated if it is increased by at least 1.5-fold or 2-fold in the cancer of the subject compared to the level and / or activity of VCAN in the reference subject(s).

[0047] In some embodiments, the level and / or activity of VCAN in a subject is compared to a normal level and / or activity of VCAN. By "normal level and / or activity of VCAN, we include the average level and / or activity of VCAN in a reference subject(s).

[0048] It should be understood that when assessing whether VCAN level and / or activity is elevated, it may be desirable to normalize the VCAN level and / or activity in a sample and compare the normalized VCAN level and / or activity to the normalized VCAN level and / or activity in a sample from a healthy subject. For example, the level and / or activity may be normalized to control for differences in sample volume or content (e.g., cell number). For example, the VCAN level and / or activity may be normalized to the level and / or activity of another product in the cell, such as beta-actin.

[0049] The level and / or activity of VCAN may be elevated due to an alteration in VCAN. By "alteration" of VCAN, we include mutations, copy number alterations, rearrangements and / or fusions of the gene encoding VCAN. Alterations including missense mutations include those listed in The Cancer Genome Atlas (TCGA) pan-cancer dataset.

[0050] The level and / or activity of VCAN can be measured by any method known in the art or described herein. The level and / or activity of VCAN can be determined directly, i.e., by measuring the protein level of VCAN or by measuring the mRNA of VCAN. In one embodiment, the level of VCAN is the protein level of VCAN. In one embodiment, the level of VCAN is the mRNA level of VCAN.

[0051] For example, the level of VCAN, such as in a tissue sample, can be determined by assessing (e.g., quantifying) the transcript RNA of VCAN in the sample, for example, using Northern blotting, PCR analysis, real-time PCR analysis, or any other technique known in the art or described herein. In one embodiment, the level of VCAN, such as in a tissue sample, can be determined by assessing (e.g., quantifying) the mRNA of VCAN in the sample. The level of VCAN, such as in a tissue sample, can also be determined by assessing (e.g., quantifying) the protein level of VCAN in the sample, for example, using immunohistochemistry analysis, Western blotting, ELISA, immunoprecipitation, flow cytometry analysis, or any other technique known in the art or described herein.

[0052] In one embodiment, the level of VCAN, such as in a tissue sample, is determined by assessing (e.g., quantifying) protein expression of VCAN in the sample using immunohistochemistry. As shown in the accompanying examples, VCAN protein expression was significantly increased in lung adenocarcinoma (LUAD) compared to adjacent lung tissue (Figure 4N). Antibodies for use in assays to measure levels of VCAN in a sample (e.g., tissue samples (e.g., lung, kidney, skin, thymus, breast, pancreas, thyroid, bladder, liver, cervix, endometrium, colon, rectum and / or stomach samples) are known in the art or can be readily developed using approaches known to the skilled artisan. Exemplary antibodies that can be used in assays to measure levels of VCAN in a sample include rabbit anti-versican (E-AB-36300; Elabscience, Wuhan, China), and anti-VCAN (ab19345; Abcam, London, UK; RRID:AB_444865).

[0053] In one embodiment, the level of VCAN, such as in a tissue sample, is determined by assessing (e.g., quantifying) the mRNA expression of VCAN in the sample using qPCR. As shown in the accompanying examples, the expression of VCAN mRNA is significantly increased in human MPE compared to benign pleural effusion (BPE) (Figure 4O). Primers that can be used to determine the mRNA expression of VCAN are included in Table 4.

[0054] The level and / or activity of VCAN can be determined indirectly, i.e., by measuring the level and / or activity of a molecule (e.g., protein or mRNA) known to be affected by the level and / or activity of VCAN. For example, the activity of NF-κB can be measured to determine whether the level and / or activity of VCAN is increased in cancer.

[0055] The activity of VCAN can be measured by any assay known in the art, including, but not limited to, reporter gene assays (e.g., containing a VCAN-responsive reporter gene construct), measuring the induction of IKKβ, or any other biological activity assay. Exemplary assays that can be used to measure the activity of VCAN are described herein.

[0056] In an embodiment, NF-κB reporter gene assays can be used to assess VCAN activity. Specifically, the NF-κB reporter Raw 264.7 cell line (containing NFκB.GFP.luciferase (NGL)) is designed to monitor the nuclear factor kappa B (NF-κB) signaling pathway. It contains a firefly luciferase gene driven by four copies of the NF-κB response element located upstream of a minimal TATA promoter. After activation by proinflammatory cytokines or lymphokine receptor stimulants, endogenous NF-κB transcription factors bind to the DNA response element and induce transcription of the luciferase reporter gene. As shown in the accompanying examples, VCAN potently activates macrophage NF-κB-driven transcription (Figure 4F, G). Furthermore, shRNA-mediated Vcan silencing reduced their ability to induce NF-κB activation in reporter gene assays (Figure 4I-M).

[0057] In an embodiment, the activity of VCAN is determined by measuring the induction of IKKβ by immunoblotting and / or microarray. As shown in the accompanying examples, VCAN induced IKKβ in primary mouse bone marrow-derived macrophages (BMDMs) (Figure 4H).

[0058] VCAN levels and / or activity can be assessed in any tissue sample obtained from a subject according to the methods described herein. In certain embodiments, VCAN levels and / or activity are assessed in samples obtained from the lung, kidney, skin, thymus, breast, pancreas, thyroid, bladder, liver, cervix, endometrium, colon, rectum and / or stomach of a subject according to the methods described herein.

[0059] By "treat", "treatment", and "treating", we include the meaning of a reduction or amelioration of the progression, severity, and / or duration of a disorder, e.g., cancer, or an improvement in one or more symptoms, preferably one or more identifiable symptoms, of a disorder resulting from the administration of one or more therapies. In certain embodiments, the terms "treat", "treatment", and "treating" refer to an improvement in at least one measurable physical parameter of a cancer, such as tumor growth, that is not necessarily identifiable by the patient. In other embodiments, the terms "treat", "treatment", and "treating" refer to an inhibition of the progression of a cancer, either physically, e.g., by stabilization of an identifiable symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat", "treatment", and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number. Using lung cancer as an example, the term treatment can refer to at least one of the following: alleviating one or more symptoms of lung cancer, delaying the progression of lung cancer, reducing tumor size in a lung cancer patient, inhibiting the growth of lung cancer tumors, increasing overall survival, increasing progression-free survival, preventing or delaying lung cancer tumor metastasis, reducing (e.g., eradicating) existing lung cancer tumor metastasis, reducing the incidence or burden of existing lung cancer tumor metastasis, or preventing recurrence of lung cancer.

[0060] In embodiments, the cancer is determined to be a cancer that contains a KRAS mutation.

[0061] In embodiments, the cancer comprises elevated levels of VCAN mRNA and / or VCAN protein.

[0062] In embodiments, an elevated level and / or activity of VCAN is a level and / or activity of VCAN that is elevated in a test sample from a subject relative to the level and / or activity of VCAN in a reference sample.

[0063] In embodiments, the reference sample is taken from a reference subject as described herein.

[0064] It will be understood by those skilled in the art that in addition to measuring the activity of VCAN in the subject's cancer, the method of the present invention may also include measuring the same activity of VCAN in one or more reference samples.

[0065] It will be understood by those skilled in the art that in addition to measuring the level of VCAN in the cancer of the subject, the method of the present invention can also include measuring the level of VCAN in one or more reference samples from corresponding tissue.For example, when VCAN level is measured in the test sample that comprises lung tissue, it is preferred that VCAN level is measured in the reference sample that comprises lung tissue.

[0066] By "sample to be tested", "test sample" or "reference sample", we include tissue or body fluid samples taken or derived from an individual, the sample comprising endogenous proteins and / or nucleic acid molecules and / or carbohydrate moieties. The sample may be a cell sample, a tissue sample, a blood sample, a serum sample, or a sample of pleural or peritoneal fluid. Preferably, the test sample comprises cancerous cells. The sample may be a biopsy sample taken from a subject, e.g., one suspected or known to contain cancer cells. The sample may be archived paraffin-embedded tumor tissue.

[0067] In embodiments, the cancer involves elevated levels and / or activity of IL-1β.

[0068] By "elevated levels and / or activity of IL-1β" we include the meaning of increased levels and / or activity of IL-1β in the cancer of a subject compared to the level and / or activity of IL-1β in a reference subject(s). This elevated level and / or activity may be present in the cancer cells themselves, but since IL-1β is secreted from myeloid cells, elevated levels and / or activity may be present in the tumor microenvironment, such as surrounding blood vessels, immune cells (such as macrophages), fibroblasts, signaling molecules and / or the extracellular matrix.

[0069] In one embodiment, the "reference subject" is the same subject. Thus, a cancer may have elevated levels and / or activity of IL-1β if the level and / or activity of IL-1β in the cancer is increased compared to the level and / or activity of IL-1β in a non-cancerous sample or tissue from the subject. Alternatively, a cancer may have elevated levels and / or activity of IL-1β if the level and / or activity of IL-1β in the cancer is increased compared to the level and / or activity of IL-1β in tissue containing the cancer at an earlier time point (e.g., before the onset of malignancy, before the start of treatment, or during treatment).

[0070] In alternative embodiments, the "reference subject(s)" are one or more healthy subjects, such as subjects who do not have cancer or who do not otherwise have the same type of cancer (e.g., one affecting the same tissue). The healthy subjects may be in the same age group, and optionally of the same sex, as the subjects with cancer.

[0071] In certain embodiments, the level and / or activity of IL-1β is at least 5%, e.g., at least 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 41%. , 42%, 43%, 44%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, ±91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 125%, ±150%, 175%, 200%, 225%, 250%, 275%, 300%, 350%, 400%, 500% or at least 1000% increase.

[0072] In certain embodiments, the level and / or activity of IL-1β is considered to be elevated if it is increased by at least 10%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 75%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, or at least 500% in the cancer of the subject compared to the level and / or activity of IL-1β in the reference subject(s). In certain embodiments, the level and / or activity of IL-1β is considered to be elevated if it is increased by at least 25%, at least 50%, at least 75%, at least 100%, or at least 200% in the cancer of the subject compared to the level and / or activity of IL-1β in the reference subject(s).

[0073] In certain embodiments, the level and / or activity of IL-1β is considered to be elevated if it is increased by at least 50% in the cancer of the subject compared to the level and / or activity of IL-1β in the reference subject(s). In certain embodiments, the level and / or activity of IL-1β is considered to be elevated if it is increased by at least 100% in the cancer of the subject compared to the level and / or activity of IL-1β in the reference subject(s). In certain embodiments, the level and / or activity of IL-1β is considered to be elevated if it is increased by at least 200% in the cancer of the subject compared to the level and / or activity of IL-1β in the reference subject(s). In one embodiment, the level and / or activity of IL-1β is considered to be elevated if it is increased by at least 1.5-fold or 2-fold in the cancer of the subject compared to the level and / or activity of IL-1β in the reference subject(s).

[0074] In some embodiments, the level and / or activity of IL-1β in the subject is compared to a normal level and / or activity of IL-1β. By "normal level and / or activity of IL-1β, we include the average level and / or activity of IL-1β in a reference subject(s).

[0075] It should be understood that when assessing whether IL-1β levels and / or activity are elevated, it may be desirable to normalize the level and / or activity of IL-1β in a sample and compare the normalized level and / or activity of IL-1β to the normalized level and / or activity of IL-1β in a sample from a healthy subject. For example, the level and / or activity may be normalized to control for differences in sample volume or content (e.g., cell number). For example, the level and / or activity of IL-1β may be normalized to the level and / or activity of another product in the cell, such as beta-actin.

[0076] The level and / or activity of IL-1β may be increased due to an alteration in IL-1β. By "alteration" of IL-1β, we include mutations, copy number alterations, rearrangements and / or fusions in the gene encoding IL-1β. Alterations, including missense mutations, include those listed in The Cancer Genome Atlas (TCGA) pan-cancer dataset.

[0077] The level and / or activity of IL-1β may be measured by any method known in the art or described herein. The level and / or activity of IL-1β may be determined directly, i.e., by measuring IL-1β protein levels, or by measuring IL-1β mRNA. In one embodiment, the level of IL-1β is the protein level of IL-1β. In one embodiment, the level of IL-1β is the mRNA level of IL-1β.

[0078] For example, the level of IL-1β, such as in a tissue sample, can be determined by assessing (e.g., quantifying) the transcript RNA of IL-1β in the sample, for example, using Northern blotting, PCR analysis, real-time PCR analysis, or any other technique known in the art or described herein. In one embodiment, the level of IL-1β, such as in a tissue sample, can be determined by assessing (e.g., quantifying) the mRNA of IL-1β in the sample. The level of IL-1β, such as in a tissue sample, can also be determined by assessing (e.g., quantifying) the level of IL-1β protein in the sample, for example, using immunohistochemical analysis, Western blotting, ELISA, immunoprecipitation, flow cytometry analysis, or any other technique known in the art or described herein.

[0079] In one embodiment, the level of IL-1β, such as in a tissue sample, is determined by assessing (e.g., quantifying) the protein expression of IL-1β in the sample using ELISA. As shown in the accompanying examples, the inventors have shown that KRAS mutation status correlates with IL-1β expression and protein levels (see FIG. 3L). Antibodies and reagents for use in assays to measure the level of IL-1β in a sample (e.g., tissue samples, such as lung, kidney, skin, thymus, breast cancer, pancreas, thyroid, bladder, liver, cervix, endometrium, colon, rectum and / or stomach samples) are known in the art or can be readily developed using approaches known to those of skill in the art. Examples of reagents that can be used in assays to measure the level of IL-1β in a sample include the IL-1β ELISA (catalog number 900-K47) from Peprotech (London, UK) and the R&D Systems High Sensitivity IL-lb ELISA kit.

[0080] In one embodiment, the level of IL-1β, such as in a tissue sample, is determined by assessing (e.g., quantifying) IL-1β mRNA expression in the sample using qPCR. As shown in the accompanying examples, IL-1β mRNA expression was increased in KRAS mutant cancers ( FIG. 3K ). Primers that can be used to determine VCAN mRNA expression are included in Table 4.

[0081] IL-1β levels and / or activity can be determined indirectly, i.e., by measuring the levels and / or activity of molecules (e.g., proteins or mRNAs) known to be affected by IL-1β levels and / or activity. For example, to determine whether IL-1β levels and / or activity are increased in cancer, IL-1β-stimulated release of IL-6 from human fibroblasts can be measured as described herein.

[0082] The activity of IL-1β can be measured by any assay known in the art, including, but not limited to, IL-1β-stimulated release of IL-6 from human fibroblasts or any other biological activity assay. Exemplary assays that can be used to measure the activity of IL-1β are described herein.

[0083] IL-1β levels and / or activity can be assessed in any tissue sample obtained from a subject according to the methods described herein. In certain embodiments, IL-1β levels and / or activity are assessed in samples obtained from the lung, kidney, skin, thymus, breast, pancreas, thyroid, bladder, liver, cervix, endometrium, colon, rectum, and / or stomach of a subject according to the methods described herein.

[0084] In embodiments, the elevated level and / or activity of IL-1β is an elevated level and / or activity of IL-1β compared to the level and / or activity of IL-1β in a reference sample.

[0085] In an embodiment, the cancer comprises an inhibitor of elevated levels and / or activity of NF-κB kinase (IKK)β. As shown in the accompanying examples, KRAS mutant tumors cause IKKβ activation and IL-1β release via secreted versican (see FIG. 4H), and the VCAN-IKKβ axis is necessary for the sustained growth of KRAS mutant tumors. Methods for determining whether a cancer comprises elevated levels and / or activity of IKKβ are described herein and include the same methods described for VCAN and IL-1β. By "elevated levels and / or activity of IKKβ", we include the meaning of an increased level and / or activity of IKKβ in the cancer of the subject compared to the level and / or activity of IKKβ in the reference subject(s). "Elevated" levels and / or activity are as defined in relation to VCAN and / or IL-1β. This elevated level and / or activity may be present in the cancer cells themselves, but because IKKβ is expressed in myeloid cells, elevated levels and / or activity may be present in the tumor microenvironment, such as surrounding blood vessels, immune cells (such as macrophages), fibroblasts, signaling molecules, and / or the extracellular matrix. As shown in the accompanying examples, IKKβ signaling in primary macrophages is required for differentiation and expression of key pro-inflammatory genes, including Il1b.

[0086] The level and / or activity of IKKβ may be elevated due to an alteration in IKKβ. By "alteration" of IKKβ, we include mutations, copy number alterations, rearrangements and / or fusions in the gene encoding IKKβ. Alterations including missense mutations include those listed in The Cancer Genome Atlas (TCGA) pan-cancer dataset.

[0087] In an embodiment, the reference sample is a non-cancerous sample.

[0088] In embodiments, a non-cancerous sample is a sample comprising non-cancerous cells or tissue from a subject.

[0089] In embodiments, the non-cancerous samples are from the same subject or different subjects.

[0090] In embodiments, determining whether a cancer contains elevated levels and / or activity of veriscan (VCAN), elevated levels and / or activity of IL-1-1β, and / or elevated levels and / or activity of IKKβ is performed in vitro. In other words, determining whether a cancer contains elevated levels and / or activity of veriscan (VCAN), elevated levels and / or activity of IL-1-1β, and / or elevated levels and / or activity of IKKβ is performed on a sample already obtained from a subject.

[0091] In embodiments, the cancer is selected from the group including lung cancer (such as non-small cell lung cancer (NSCLC)), kidney cancer, skin cancer, thymic cancer, breast cancer, pancreatic cancer, thyroid cancer, bladder cancer, liver cancer, cervical cancer, endometrial cancer, colorectal cancer, and gastric cancer.

[0092] In an embodiment, the cancer is a cancer that causes malignant pleural effusion (MPE). By "malignant pleural effusion (MPE)", we mean the accumulation of fluid and cancer cells that collect between the chest wall and the lung. Some types of cancer are more likely to cause pleural effusion. For example, about 40% of people with lung cancer will develop pleural effusion at some point during the course of their cancer. Cancers that cause MPE include breast cancer, lung cancer, lymphoma, mesothelioma, and ovarian cancer.

[0093] In embodiments, the cancer is selected from the group comprising lung adenocarcinoma (LUAD), colon adenocarcinoma (COAD), rectal adenocarcinoma (READ), and uterine endometrial carcinoma (UCEC).

[0094] In embodiments, the inhibitor is selected from the group including peptide IL-1 receptor antagonists, nucleotide IL-1 receptor antagonists, peptide fragments of IL-1R1, anti-IL-1 antibodies, anti-IL-1R1 antibodies, decoy IL-1 receptor (optionally a soluble IL-1 receptor, or IL-1 TRAP).

[0095] In embodiments, the inhibitor is a peptide that acts as an IL-1 receptor antagonist, such as an antagonistic cytokine, which binds to IL-1R1 but does not allow the accessory receptor to form the necessary trimeric complex, thus inhibiting IL-1 signaling. Naturally occurring and modified forms of IL-1RA may be useful in inhibiting IL-1R1 signaling.

[0096] IL-1 receptor antagonist (IL-1RA; also called IRAP for IL-1 receptor antagonist protein) acts as a natural antagonist of IL-1α and IL-1β by binding to the IL-1 receptor, but does not transduce intracellular signals or biological responses. IL-1RA is a competitive ligand because it does not interact with the accessory receptor, IL-1RAcP, and therefore cannot signal. A gene encoding this antagonist of IL-1 has been described (see Hannum et al. (1990) Nature 343:336-340, Eisenberg et al. (1990) Nature 343:341-346, and Carter et al. (1990) Nature 344:633-638). Peptide IL-1RA inhibits the biological activity of IL-1 both in vitro and in vivo and has been shown to be effective in animal models of septic shock, rheumatoid arthritis, graft-versus-host disease, stroke, and cardiac ischemia. Normal animals, including humans, can be infused intravenously with high doses of this protein without altering physiological or metabolic parameters. For example, human volunteers infused with 133 mg / h IL-1RA for 72 hours showed no changes in clinical or laboratory values. See Dinarello et al. (1993) J. Amer. Med. Assoc. 269:1829-1835. Thus, in an embodiment, the inhibitor is a naturally occurring IL-1RA, such as human IL-1RA.

[0097] In an embodiment, the inhibitor is a modified form of IL-1RA. In an embodiment, the inhibitor is r-metHuIL-1ra (also known as Anakinra, Kineret®), a recombinant form of the naturally occurring IL-1 receptor antagonist. Anakinra is a non-glycosylated form of human IL-1RA that competitively inhibits IL-1α and IL-1β binding to the IL-1 receptor type 1. This polypeptide is 153 amino acids in length, has a molecular weight of 17.3 kDa, and is identical to the naturally occurring non-glycosylated form of human IL-1RA, except for the addition of an N-terminal methionine.

[0098] In embodiments, the present disclosure provides peptide inhibitors that comprise an amino acid sequence that is at least 80, 82, 85, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or 100% identical to a sequence disclosed herein, e.g., a sequence listed in Table 6 or the Examples, and / or WO2012 / 016203, which are incorporated by reference in their entireties. [Table 1]

[0099] P01.P01 contains three segments from IL-1Ra (SEQ ID NO:6) corresponding to amino acids Ala12-Val48, Ile60-Val83, and Asp95-Tyr147 of SEQ ID NO:6, and the remaining four segments from IL-1β (SEQ ID NO:7). Overall, P01 has 74 of the 153 amino acids from IL-1β (about 48% identity) and 119 amino acids from IL-1Ra (about 77% identity). Several amino acids in P01 and other exemplary proteins disclosed herein are conserved between IL-1β and IL-1Ra and therefore contribute to the identity percentages to both IL-1β and IL-1Ra, so that these percentages add up to more than 100%.

[0100] The amino acid sequence of IL-1Ra (human) referred to herein is: RPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQED (SEQ ID NO: 6).

[0101] The amino acid sequence of IL-1β (human) referred to herein is: APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQFVSS (SEQ ID NO: 7).

[0102] P02. P02 contains three segments from IL-1Ra corresponding to amino acids Ala12-Val48, Ile60-Val83, and Ser110-Tyr147 of SEQ ID NO: 6, and the remaining four segments from IL-1β. Overall, P02 has 85 of the 153 amino acids from IL-1β (about 55% identity) and 108 amino acids from IL-1Ra (about 70% identity).

[0103] P03. P03 contains two segments from IL-1Ra corresponding to amino acids Ala12-Lys45 and Phe100-Lys145 of SEQ ID NO: 6, and the remaining three segments from IL-1β. Overall, P03 has 94 of the 153 amino acids from IL-1β (about 61% identity) and 91 amino acids from IL-1Ra (about 64% identity).

[0104] P04. P04 contains two segments from IL-1Ra corresponding to amino acids Ala12-Lys45 and Ala114-Lys145 of SEQ ID NO: 6, and the remaining three segments from IL-1β. Overall, P04 has 104 of the 153 amino acids from IL-1β (about 68% identity) and 89 amino acids from IL-1Ra (about 58% identity).

[0105] P05. P05 contains two segments from IL-1Ra corresponding to amino acids Arg14-Lys45 and Phe120-Tyr147 of SEQ ID NO: 6, and the remaining three segments from IL-1β. Overall, P05 has 108 of the 153 amino acids from IL-1β (about 70% identity) and 85 amino acids from IL-1Ra (about 55% identity).

[0106] Peptide inhibitors can include a range of residues that differ from IL-1β and IL-1Ra, as exemplified in Table 7 below. In embodiments, peptide inhibitors can have 48-70% of the residues from IL-1β and 55-78% of the residues from IL-1Ra. In embodiments, peptide inhibitors can have 48-72% of the residues from IL-1β and 55-78% of the residues from IL-1Ra. (Because some amino acid residues are conserved between the two proteins, the sum of the identity percentages for IL-1β and IL-1Ra can exceed 100%). [Table 2]

[0107] In embodiments, the inhibitor is 45-72% identical to IL-1β and 53-80% identical to IL-1Ra, or 50-72% identical to IL-1β and 53-71% identical to IL-1Ra, or 60-72% identical to IL-1β and 53-68% identical to IL-1Ra, or 65-72% identical to IL-1β and 54-60% identical to IL-1Ra, or 68-72% identical to IL-1β and 54-57% identical to IL-1Ra.

[0108] The peptide inhibitor may include a methionine at the N-terminus relative to the amino acid sequence of P01, P02, P03, P04, or P05, and / or the peptide inhibitor may include the amino acid sequence of P01, P02, P03, P04, or P05 without an alanine at the N-terminus.

[0109] A peptide inhibitor may include a tag, such as a hexahistidine sequence, such as GSHHHHHH. The tag may be N-terminal or C-terminal relative to the inhibitor sequence. In an embodiment, the hexahistidine tag is C-terminal to the inhibitor sequence.

[0110] In embodiments, the inhibitor is at least 80% identical to a sequence selected from any one of SEQ ID NO:1 (P01), SEQ ID NO:2 (P02), SEQ ID NO:3 (P03), SEQ ID NO:4 (P04), and SEQ ID NO:5 (P05).

[0111] Calculations of "homology" or "sequence identity" between two sequences (these terms are used interchangeably herein) can be performed as follows: The sequences are aligned according to the alignment provided herein, or, in the absence of a suitable alignment, according to an optimal alignment determined as the best score, e.g., using the Needleman and Wunsch algorithm as implemented in the Needle algorithm of the EMBOSS package using the Blosum 62 scoring matrix with a gap penalty of 10, and a gap extension penalty of 1. See Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453; Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44; Addison Wesley and the European Bioinformatics Institute (Cambridge UK) EMBOSS: The European Molecular Biology Open Software Suite (2000); Rice, P. et al., A., Trends in Genetics 16, (6) pp. 276--277, and online at https: / / www.ebi.ac.uk / Tools / psa / . The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.To determine the collective identity of a sequence of interest to a group of reference sequences, positions are considered to be identical if they are identical to at least one amino acid at the corresponding position in any one or more of the group of reference sequences. For lists of segments, features, or regions, identity can be calculated collectively for all members of such lists to arrive at an overall identity percentage.

[0112] As used herein, the term "corresponding to" is used to indicate the position of an amino acid residue in a polypeptide of interest relative to a reference polypeptide. Generally, the position is as shown by an alignment such as Figure 4 of WO2012 / 016203, which is incorporated by reference in its entirety.

[0113] In embodiments, the inhibitor is at least 90% identical to a sequence selected from any one of SEQ ID NO:1 (P01), SEQ ID NO:2 (P02), SEQ ID NO:3 (P03), SEQ ID NO:4 (P04), and SEQ ID NO:5 (P05).

[0114] In embodiments, the inhibitor is at least 95% identical to P01, P02, P03, P04, or P05. In embodiments, the inhibitor is identical to P01. In embodiments, the inhibitor is identical to P02. In embodiments, the inhibitor is identical to P03. In embodiments, the inhibitor is identical to P04. In embodiments, the inhibitor is identical to P05.

[0115] In certain embodiments, the inhibitor is isnakinra.

[0116] By "isunakinra" we include the meaning of an IL-1β and IL-1 receptor antagonist protein chimera comprising P05 (SEQ ID NO:5), as described in WO2012 / 016203, which is incorporated by reference.

[0117] A preferred formulation of Isunakinra is described in WO2014 / 160371, which is incorporated by reference in its entirety.

[0118] In addition to antagonist peptide cytokines (such as isakinra and anakinra), it is possible to use small peptides that bind to IL-1R1 and act as antagonists. One such peptide, AF10847, was crystallized with IL-1RI to determine its mechanism of antagonism, showing that this peptide binding site A of IL-1RI induced a conformational change in the receptor that precluded cytokine binding (see Vigers GP, Dripps DJ, Edwards CK, 3rd, Brandhuber BJ. X-ray crystal structure of a small antagonist peptide bound to interleukin-1 receptor type 1. J Biol Chem. (2000) 275:36927-33).

[0119] In embodiments, the inhibitor is a nucleotide that acts as an IL-1 receptor antagonist, such as an RNA or DNA aptamer. An aptamer is an oligonucleotide fragment that can bind to a protein target. The DNA aptamer SL1067 binds to IL-1α and disrupts its ability to bind to its cognate receptor IL-1RI (see Ren X, Gelinas AD, Von Carlowitz I, Janjic N, Pyle AM. Structural basis for IL-1alpha recognition by a modified DNA aptamer that specifically inhibits IL-1alpha signaling. Nat Commun. (2017) 8:810).

[0120] In an embodiment, the inhibitor is a decoy IL-1 receptor. By "decoy IL-1 receptor" we include the meaning of a synthetic or naturally occurring IL-1 receptor that is capable of binding IL-1 cytokine but lacks the cytoplasmic domain required for signaling. The active receptor complex consists of the type I receptor (IL-1RI) and IL1RAcP (for IL-1 receptor accessory protein). IL-1RI is responsible for binding the three naturally occurring ligands (IL-1α, IL-1β and IL-1RA) and can do so in the absence of IL1RAcP. However, signaling requires the interaction of IL-1α or IL-1β with IL1RAcP, so that when IL-1β or IL-1α binds to IL-1RI, IL-1RAcP is recruited to initiate signaling. Thus, such a decoy receptor may act by sequestering IL-1α or IL-1β from the active IL-1R1. Such decoy receptors would not be bound to the plasma membrane and would therefore be soluble, which would allow them to sequester free cytokines, thus preventing them from binding to cell surface expressed IL-1R1.

[0121] Such decoy receptors can sequester accessory proteins (IL-1RAcP) and thus prevent them from participating in IL-1 signaling. IL-1 type II receptor (IL-1RII) can bind IL-1 agonists (IL-1α and IL-1β) and then recruit IL-1RAcP for the creation of the IL-1 ternary complex, but due to the lack of an intracellular TIR domain, signaling cannot occur. IL-1RII is a decoy receptor, either as an antagonist of its membrane form or a cleaved secreted form, and can inhibit IL-1 activity. For an overview, see Dinarello (1996) Blood 87:2095-2147.

[0122] By "IL-1 TRAP" we include the meaning of a recombinant nucleic acid molecule encoding a fusion polypeptide that forms a multimer capable of binding interleukin-1 (IL-1) to form a non-functional complex. IL-1 TRAP is essentially as described in WO2004039951A2. IL-1 TRAP is a decoy receptor that can bind to circulating IL-1α and IL-1β molecules, effectively blocking the engagement of IL-1β to IL-1R1 and inhibiting downstream activation of downstream signaling pathways. IL-1 TRAP incorporates the extracellular domains of both receptor components required for IL-1 signaling, the IL-1 type I receptor (IL-1RI) and the IL-1 receptor accessory protein (AcP), in a single molecule. Because it contains both receptor components, IL-1 TRAP binds IL-1β and IL-1α with picomolar affinity, while IL-1RI binds only in the absence of AcP with an affinity constant of about 1 nanomolar. IL-1 TRAP was generated by fusing sequences encoding the extracellular domains of AcP, IL-1RI, and Fc inline, without any intervening linker sequence. An expression construct encoding the fusion protein is transfected into Chinese Hamster Ovary (CHO) cells, and a high-producing strain is isolated that secretes IL-1 TRAP into the medium. IL-1 TRAP is a dimeric glycoprotein with a protein molecular weight of 201 kDa, including glycosylation, and has a total molecular weight of approximately 252 kDa. The dimers are covalently linked by disulfide bonds in the Fc region. Such IL-1 TRAPS can neutralize IL-1 receptor signaling by acting as a decoy receptor. An example is Rilonacept (trade name Arcalyst, Regeneron Pharmaceuticals).

[0123] In embodiments, the inhibitor is an IL-1 antibody, such as an anti-IL-1β antibody and / or an anti-IL-1α antibody. Antibodies with specific binding affinity to IL-1β can be generated by standard methods. Alternatively, the antibodies can be commercially available, for example, from R&D Systems, Inc., Minneapolis, MN. For example, Canakinumab (Ilaris®) is an anti-IL-1β neutralizing monoclonal antibody that blocks binding to the IL-1 receptor. Gevokizumab is a fully human monoclonal anti-human IL-1 beta antibody of IgG2 isotype. LY 2189102 is a humanized interleukin-1 beta (IL-1β) monoclonal antibody. It will be understood that the IL-1β antibody and / or anti-IL-1α antibody or fragment thereof neutralizes anti-IL-1α associated with the biological activity of IL-1β and / or the signaling function of IL-1RI. IL-1β antibodies can neutralize the biological activity of IL-1β by binding to IL-1β and preventing the binding of bound IL-1β to IL-1RI. Binding of IL-1β to IL-1RI can be determined by immobilizing an IL-1β-binding antibody, contacting IL-1β with the immobilized antibody and determining whether IL-1β is bound to the antibody, and contacting a soluble form of IL-1RI with the bound IL-1β / antibody complex and determining whether soluble IL-1RI is bound to the complex. The same applies to IL-1α antibodies.

[0124] IL-1β binding antibodies can neutralize the biological activity of IL-1β by binding to IL-1β without substantially preventing the binding of bound IL-1β to IL-1RI. This means that they can bind to and neutralize IL-1β while still allowing IL-1β to bind to IL-1RI. This can result in an effective reduction of IL-1α and IL-1β biological activity since there are fewer unbound IL-1RI sites for IL-1α to bind to, thereby resulting in an overall reduction in IL-1RI signaling. The same applies to IL-1α antibodies.

[0125] In embodiments, the inhibitor is an anti-IL-1R1 antibody. AMG108 is a fully human IgG2 monoclonal antibody that binds to IL-1R type 1 and non-selectively inhibits the activity of both forms of IL-1 (IL-1α and IL-1β).

[0126] Antibody fragments having specific binding affinity for IL-1β, IL-1α or IL-1R1 can be produced by known techniques. For example, such fragments include, but are not limited to, F(ab')2 fragments, which can be produced by pepsin digestion of an antibody molecule, and Fab fragments, which can be produced by reducing disulfide bridges of F(ab') fragments. Alternatively, Fab expression libraries can be constructed. See, e.g., Huse et al. 1989, Science, 246:1275. Single-chain Fv antibody fragments are formed by linking heavy and light chain fragments of the Fv region via an amino acid bridge (e.g., 15-18 amino acids), resulting in a single-chain polypeptide. Single-chain Fv antibody fragments can be produced by standard techniques. See, e.g., U.S. Pat. No. 4,946,778.

[0127] The inhibitors of the present invention or formulations thereof can be administered by any conventional method, including parenteral (eg, subcutaneous or intravenous) injection.

[0128] The inhibitors disclosed herein or used as described herein may be administered orally, topically, parenterally, by inhalation or aerosol, sublingually, via implants, including ophthalmic implants, transdermally, via buccal administration, rectally, as an ophthalmic solution, by injection, including ophthalmic injection, intravenously, intra-aortically, intracranially, subdermal, intraperitoneally, subcutaneously, nasally, sublingually, intrathecally, or rectally, or by other means, in dosage unit formulations containing conventional pharma- ceutically acceptable carriers.

[0129] The pharmaceutical composition may be formulated in any pharma- ceutical useful form, for example, as an aerosol, cream, gel, gelcap, pill, microparticle, nanoparticle, injection or infusion solution, capsule, tablet, syrup, transdermal patch, subcutaneous patch, dry powder, inhalation formulation, medical device, suppository, buccal or sublingual formulation, parenteral formulation, or ophthalmic solution or suspension. Some dosage forms, for example, tablets and capsules, are subdivided into appropriately sized unit doses containing an appropriate amount of active ingredient, for example, an effective amount to achieve a desired purpose. An effective amount of the inhibitor described herein may be incorporated into nanoparticles, for example, for convenience of delivery and / or sustained release.

[0130] Pharmaceutical compositions suitable for pulmonary administration can be delivered by a wide range of passive respiratory and active power-driven single / multiple dose dry powder inhalers (DPIs).The most commonly used devices for respiratory delivery include nebulizers, metered dose inhalers, and dry powder inhalers.Several types of nebulizers are available, including jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers.The selection of an appropriate pulmonary delivery device depends on parameters such as the nature of the drug and its formulation, the site of action, and the pathophysiology of the lungs.

[0131] Although the compound of the present invention can be administered alone, it is preferable to present it as a pharmaceutical composition together with one or more acceptable excipients, diluents, and / or carriers. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the present invention and not harmful to the recipient thereof. Typically, the carrier is sterile, pyrogen-free water or saline. The pharmaceutical compositions contemplated herein may optionally include a carrier. The carrier must have sufficiently high purity and sufficiently low toxicity to be suitable for administration to the patient to be treated. The carrier may be inert or may have pharmaceutical benefits of its own. The amount of carrier utilized in conjunction with the compound is sufficient to provide a practical amount of material for administration per unit dose of the compound. Classes of carriers include, but are not limited to, binders, buffers, colorants, diluents, disintegrants, emulsifiers, fillers, flavorings, glidants, lubricants, pH modifiers, preservatives, stabilizers, surfactants, solubilizers, tableting agents, and wetting agents. Some carriers may be listed in more than one class, for example, vegetable oils may be used as lubricants in some formulations and as diluents in others. Exemplary pharmaceutically acceptable carriers include sugar, starch, cellulose, powdered tragacanth, malt, gelatin, talc, and vegetable oils. Examples of other matrix materials, fillers, or diluents include lactose, mannitol, xylitol, microcrystalline cellulose, calcium diphosphate, and starch. Examples of surfactants include sodium lauryl sulfate and polysorbate 80. Examples of drug complexing or solubilizing agents include polyethylene glycol, caffeine, xanthene, gentisic acid, and cylodextrin. Examples of disintegrants include sodium starch gycolate, sodium alginate, sodium carboxymethylcellulose, methylcellulose, colloidal silicon dioxide, and croscarmellose sodium. Examples of binders include methylcellulose, microcrystalline cellulose, starch, and gums such as guar gum and tragacanth.Examples of lubricants include magnesium stearate and calcium stearate. Examples of pH modifiers include acids such as citric acid, acetic acid, ascorbic acid, lactic acid, aspartic acid, succinic acid, phosphoric acid, and the like; bases such as sodium acetate, potassium acetate, calcium oxide, magnesium oxide, trisodium phosphate, sodium hydroxide, calcium hydroxide, aluminum hydroxide, and buffers that generally contain a mixture of an acid and its salt. Optional other active agents may be included in the pharmaceutical composition that do not substantially interfere with the activity of the compound of the present invention.

[0132] Preferably, the inhibitor or a pharmaceutical composition comprising the inhibitor is administered to a subject in a therapeutically effective amount. By "therapeutically effective amount," we include the meaning of the amount of the inhibitor or a pharmaceutical composition comprising the inhibitor to produce a desired pharmacological effect in a subject, such as treating the subject as described herein.

[0133] The pharmaceutical compositions may be provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions, containing a suitable amount of the compound or its pharma- ceutically acceptable derivatives. The inhibitors may be formulated and administered in unit dosage forms or multiple dosage forms. Unit dosage forms, as used herein, refer to physically discrete units suitable for human and animal subjects and individually packaged, as known in the art. Each unit dosage contains a predetermined amount of the inhibitor sufficient to produce the desired therapeutic effect, in association with the necessary pharmaceutical carrier, vehicle, or diluent. Examples of unit dosage forms include ampoules and syringes, as well as individually packaged tablets or capsules. A unit dosage form may be administered in fractions or multiples thereof. A multiple dosage form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dosage forms. Examples of multiple dosage forms include vials, bottles of tablets or capsules, or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit doses that are not segregated within a package.

[0134] The inhibitor and / or composition may be formulated for administration in a single dosage. To formulate the composition, a weight fraction of the compound is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier at an effective concentration such that the condition being treated is alleviated, prevented, or one or more symptoms are ameliorated.

[0135] The inhibitors provided herein can be administered at once or divided into several smaller doses administered at time intervals. It is understood that the exact dosage and duration of treatment is a function of the disease being treated and can be empirically determined using known testing protocols or by extrapolation from in vivo or in vitro test data. It is noted that concentration and dosage values ​​can also vary with the severity of the condition being alleviated. It is further understood that the specific dosage regimen for any particular subject can be adjusted over time according to individual needs and the professional judgment of the person administering or supervising the administration of the composition, and the concentration ranges described herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions.

[0136] The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease and / or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0137] In some embodiments, the inhibitor may be administered via multiple routes of administration, either simultaneously with or subsequent to other doses of the same or different inhibitor.

[0138] For oral and parenteral administration to human patients, the daily dosage levels of the compounds of the invention are typically about 0.015-25 mg / kg, e.g., about 5-20 mg / Kg, administered in single or divided doses. In some embodiments, the dosage administered to a patient is typically 0.1 mg / kg-100 mg / kg of patient body weight. In some embodiments, the dosage administered to a patient is about 1 mg / kg to about 75 mg / kg of patient body weight. Preferably, the dosage administered to a patient is 1 mg / kg-20 mg / kg of patient body weight, more preferably 1 mg / kg-5 mg / kg of patient body weight. However, smaller dosages and less frequent administration are possible.

[0139] The inhibitors of the present invention may also be administered parenterally, for example, intravenously, intraarterially, intraperitoneally, intramedullary, intraventricular, intrasternal, intracranially, intramuscularly, or subcutaneously, or by infusion techniques. Injectables may be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins.

[0140] The composition can be formulated according to the usual procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If necessary, the composition can also contain a solubilizing agent and a local anesthetic, such as lignocamne, to ease pain at the injection site. However, such compositions can be administered by routes other than intravenous.

[0141] Formulations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready to be combined with a solvent immediately prior to use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle immediately prior to use, and sterile emulsions. Solutions can be either aqueous or non-aqueous.

[0142] The concentration of the pharma- ceutically active compound is adjusted so that injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight, and condition of the patient or animal, as is known in the art.

[0143] In embodiments, the subject is also administered an inhibitor of VCAN.

[0144] By "inhibitor of VCAN" we include the meaning of any molecule that inhibits, reduces, blocks and / or antagonizes the biological activity of VCAN. For the avoidance of doubt, we also include any molecule that prevents or reduces the expression of VCAN and / or increases its degradation.

[0145] Inhibitors of VCAN may include TLR1 / 2 receptor antagonists, VCAN siRNA, anti-VCAN antibodies, soluble TLR1 / 2, monobodies and aptamers.

[0146] By "biological activity of VCAN", we include the biological effects of VCAN, which refers to any function(s) exhibited or performed by the naturally occurring and / or wild-type forms of VCAN measured or observed in vivo (i.e., in the protein's natural physiological environment) or in vitro (i.e., under laboratory conditions). Such active agents include those known in the art or described herein. Methods for assessing VCAN inhibition are known in the art and described herein.

[0147] We include the meaning that the activity of VCAN is reduced in the presence of an inhibitor compared to the activity of VCAN in the absence of the inhibitor. Inhibition is not limited to complete inhibition of VCAN activity. In a given application, some low level of VCAN activity that does not adversely affect patient outcome may be acceptable. In an embodiment, an inhibitor of VCAN reduces VCAN activity by at least 10%, for example, at least 20%, 30%, 40%, or 50%, compared to the activity of VCAN in the absence of the inhibitor. In an embodiment, an inhibitor of VCAN reduces VCAN activity by at least 50%, for example, at least 60%, 70%, 80%, or 90%, for example, 95%, compared to the activity of VCAN in the absence of the inhibitor.

[0148] In embodiments, the VCAN inhibitor may be an inhibitor that selectively inhibits VCAN. For example, the VCAN inhibitor may inhibit and / or reduce the biological activity of VCAN to a greater extent than inhibiting the biological activity of an unrelated protein. Preferably, the agent inhibits the biological activity of VCAN at least 5 times, or at least 10 times, or at least 50 times more than inhibiting the biological activity of another unrelated protein. More preferably, the agent inhibits the biological activity of VCAN at least 100 times, or at least 1,000 times, or at least 10,000 times more than inhibiting the biological activity of another unrelated protein.

[0149] Preferably, the VCAN inhibitor is selected from the group comprising a small molecule, a peptide, a polypeptide, a nucleic acid molecule (such as an aptamer), an antibody, a peptidomimetic, a natural product, a monobody, or a carbohydrate.

[0150] In an embodiment, the inhibitor of VCAN is a toll-like receptor 1 / 2 (TLR1 / 2) inhibitor. As shown in the accompanying examples, the toll-like receptor 1 / 2 (TLR1 / 2) inhibitor Cu-CPT22 (3,4,6-trihydroxy-2-methoxy-5-oxo-5H-benzocycloheptene-8-carboxylic acid hexyl ester) blocks versican (VCAN)-induced bone marrow NF-κB activation and MPE of Kras mutant cancer cells in vivo. In an embodiment, the TLR1 / 2 inhibitor is Cu-CPT22. Other known inhibitors of TLR1 / 2 are known in the art, including NCI35676 (a natural product derived from gallnut and oak bark named purpurogallin). It will be understood that the best strategy for targeting versican is to focus on regions that are not isoform-specific.

[0151] The subject may be administered a combination of an inhibitor of IL-1R1 signaling and a VCAN inhibitor. The term "in combination" is understood to mean that two or more drugs are administered sequentially or simultaneously, in other words, they do not necessarily have to be co-formulated. Alternatively, the term "in combination" is understood to mean that two or more drugs are administered in a manner that is expected to overlap the effective therapeutic concentrations of the drugs for a majority of the time period in the patient's body. The inhibitor of IL-1R1 signaling and one or more combination partners (e.g., another drug, also referred to as a "therapeutic agent" or "adjunct") may be administered simultaneously or separately independently within a time interval, especially when these time intervals allow the combination partners to exhibit a coordinated, e.g., synergistic effect. As used herein, terms such as "co-administration" or "combined administration" are meant to encompass administration of selected combination partners to a single subject (e.g., patient) in need thereof, and are intended to include therapeutic regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The drugs may be administered to a patient as separate entities simultaneously, in parallel, or sequentially without any specific time limit, such administration providing therapeutically effective levels of the two compounds in the patient's body, and the treatment regimen providing the beneficial effect of the drug combination in treating the conditions or disorders described herein. The latter also applies to cocktail therapy, e.g., administration of three or more active ingredients.

[0152] In embodiments, the subject also receives one or more chemotherapeutic agents.

[0153] It will be understood that the subject may also be administered a chemotherapeutic agent that is the standard of care for the particular cancer.

[0154] Cancer chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, topoisomerase inhibitors, mitotic inhibitors, antitumor antibiotics, immunotherapeutics including checkpoint inhibitors, receptor tyrosine kinase inhibitors, and various agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin, anthracenediones (anthraquinones or dioxoanthracenes) such as mitoxantrone and anthracyclines, substituted ureas such as hydroxyurea, methylhydrazine derivatives such as procarbazine (N-methylhydrazine, MIH), and adrenal cortical suppressants such as mitotane (o,p'-DDD) and aminoglutethimide, taxol and analogs / derivatives, and hormone agonists / antagonists such as flutamide and tamoxifen.

[0155] In another aspect, the invention provides a method for selecting a subject having cancer, the cancer being predicted to be therapeutically responsive to a treatment comprising an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling, the method comprising: a) determining whether the cancer contains a KRAS mutation; b) determining whether the cancer has elevated levels and / or activity of veriscan (VCAN); The method further comprises selecting a subject as having a cancer that is predicted to be therapeutically responsive to a treatment comprising an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling if the cancer from the subject comprises a KRAS mutation and has elevated levels and / or activity of VCAN.

[0156] It will be understood that determining whether a cancer contains a KRAS mutation and determining whether a cancer has elevated levels and / or activity of veriscan (VCAN) can be performed using any of the methods described herein. Preferences for KRAS mutations and elevated levels and / or activity of veriscan (VCAN) include those described herein, as well as preferences for inhibitors of interleukin-1 receptor type 1 (IL-1R1) signaling.

[0157] In embodiments, determining whether the cancer has elevated levels and / or activity of VCAN includes determining whether the level and / or activity of VCAN in the cancer is elevated relative to the level and / or activity of VCAN in a reference sample.

[0158] In an embodiment, the method comprises: and c) determining whether the cancer has elevated levels and / or activity of IL-1β, wherein if the cancer from the subject comprises a KRAS mutation, elevated levels and / or activity of VCAN, and has elevated levels and / or activity of IL-1β, the subject is selected as having a cancer that is predicted to respond therapeutically to a treatment comprising an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

[0159] It will be understood that determining whether a cancer has elevated levels and / or activity of IL-1β can be carried out using any of the methods described herein, including those described herein, where preference for elevated levels and / or activity of IL-1β.

[0160] In embodiments, determining whether the cancer has elevated levels and / or activity of IL-1β includes determining whether the level and / or activity of IL-1β in the cancer is elevated relative to the level and / or activity of IL-1β in a reference sample.

[0161] In embodiments, any of steps (a), (b) and (c) are performed in vitro and / or on a sample provided by the subject.

[0162] Preferences for the sample include those described above. For example, the sample may be a cell sample, a tissue sample, a blood sample, a serum sample, or a pleural or peritoneal fluid sample. Preferably, the test sample contains cancerous cells. The sample may be a biopsy sample taken from a subject, e.g., one that contains suspected or known cancer cells. The sample may be archived paraffin-embedded tumor tissue. The sample may be a tissue sample (e.g., a lung, kidney, skin, thymus, breast, pancreas, thyroid, bladder, liver, cervix, endometrium, colon, rectum, and / or stomach sample).

[0163] In embodiments, the method further includes treating a subject selected as having a cancer predicted to be therapeutically responsive to a treatment comprising an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling with an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

[0164] In another aspect, the invention provides the use of veriscan (VCAN) as a biomarker to determine whether a subject having a cancer that contains a KRAS mutation is suitable for treatment with an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

[0165] A preference is given to inhibitors of interleukin-1 receptor type 1 (IL-1R1) signaling herein.

[0166] As described in the accompanying examples, VCAN is overexpressed in human KRAS mutant cancers and can function as a diagnostic and prognostic biomarker.

[0167] By "biomarker" we include the meaning of a naturally occurring biological molecule, or a component or fragment thereof, the measurement of which can provide information useful for the prognosis and / or diagnosis of cancers suitable for treatment with inhibitors of interleukin-1 receptor type 1 (IL-1R1) signaling. For example, a biomarker can be a naturally occurring protein or carbohydrate moiety, or an antigenic component or fragment thereof.

[0168] In embodiments, this use includes determining whether a subject having a cancer that includes a KRAS mutation includes elevated levels and / or activity of VCAN as described herein.Thus, it will be appreciated that VCAN can be useful as a biomarker for identifying subjects amenable to treatment with an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

[0169] This use may include measuring the expression of a nucleic acid molecule encoding VCAN. Measuring the expression of VCAN may be performed using a method selected from the group consisting of Southern hybridization, Northern hybridization, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative real-time PCR (qRT-PCR), nanoarray, microarray, macroarray, autoradiography, and in situ hybridization. Alternatively or additionally, the nucleic acid molecule is a ctDNA molecule, a cDNA molecule, or an mRNA molecule.

[0170] This use may include measuring the expression of a protein molecule encoding VCAN. Measuring the expression of VCAN can be performed using any suitable method known in the art, such as ELISA, Western blotting, immunofluorescence, immunohistochemistry, etc.

[0171] In embodiments, IKKβ is also used as a biomarker to determine whether a subject having a cancer that contains a KRAS mutation is suitable for treatment with an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling. In embodiments, this use involves determining whether a subject having a cancer that contains a KRAS mutation contains elevated levels and / or activity of IKKβ as described herein.

[0172] The present invention provides kits that can be used in any of the above methods.

[0173] In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: (i) a reagent for detecting the presence of a KRAS mutation; and (ii) a reagent for determining VCAN level and / or activity.

[0174] It will be appreciated that such kits may be used to identify subjects suitable for treatment with inhibitors of interleukin-1 receptor type 1 (IL-1R1) signaling in the methods described herein.

[0175] By "reagent for detecting the presence of KRAS mutation", we mean any suitable reagent that can be used to detect the presence of KRAS mutation in a subject or a sample taken therefrom. Methods for detecting KRAS mutation are described herein. For example, the reagent can be a mutation-specific primer that can be used in PCR, such as QPCR.

[0176] By "reagent for determining the level and / or activity of VCAN" we include the meaning of any suitable reagent that can be used to detect the level and / or activity of VCAN in a subject or a sample taken therefrom. The reagent for determining the level and / or activity of VCAN may comprise or consist of a nucleic acid, such as a primer for use in PCR, or a probe for use in immunohistochemistry. The reagent for determining the level and / or activity of VCAN may comprise or consist of an antibody or an antigen-binding fragment thereof, or a variant thereof. In alternative or additional embodiments, the reagent(s) is / are immobilized on a surface (e.g., a multi-well plate or array). Alternatively or additionally, the reagent may be directly or indirectly labeled with a detectable moiety. Suitable detectable moieties are well known in the art. For example, an antibody may be conjugated to a detectable moiety, such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody may be conjugated to a detectable moiety). Reagents for determining the level and / or activity of VCAN may comprise or consist of an antibody or antigen-binding fragment thereof, or a variant thereof, for use in an ELISA to detect VCAN.

[0177] The detectable moiety can be a fluorescent and / or luminescent and / or chemiluminescent moiety that can be detected when exposed to certain conditions. For example, a fluorescent moiety may need to be exposed to radiation (i.e., light) at a particular wavelength and intensity to cause excitation of the fluorescent moiety, thereby enabling it to emit detectable fluorescence at a particular wavelength that can be detected.

[0178] Alternatively, the detectable moiety may be an enzyme capable of converting a (preferably undetectable) substrate into a detectable product that can be visualized and / or detected. Examples of suitable enzymes are discussed in more detail below, e.g., with respect to ELISA assays.

[0179] Alternatively, the detectable moiety can be a radioactive atom that is useful for imaging. Suitable radioactive atoms include 99mTc and 123I for scintigraphic studies. Other easily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI), such as 123I, 131I, 111In, 19F, 13C, 15N, 17O, gadolinium, manganese, or iron. Obviously, the agent to be detected (such as KRAS, VCAN, IL-1β, and / or IKKβ) must have sufficient suitable atomic isotopes so that the detectable moiety is easily detectable in the test sample and / or control sample described herein, and / or antibody molecule for use in detecting the selected protein).

[0180] Radioactive or other labels can be incorporated into the agent of the invention (i.e., the protein present in the sample of the method of the invention and / or the binding agent of the invention) in known manner. For example, if the binding moiety is a polypeptide, it can be biosynthesized or synthesized by chemical amino acid synthesis using suitable amino acid precursors, for example, containing fluorine-19 instead of hydrogen. Labels such as 99mTc, 123I, 186Rh, 188Rh, and 111In can be attached, for example, via a cysteine ​​residue in the binding site. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Comm. 80, 49-57) can be used to incorporate 123I. Reference ("Monoclonal Antibodies in Immunoscintigraphy", JF Chatal, CRC Press, 1989) describes other methods in detail. Methods for conjugating other detectable moieties (such as enzymatic, fluorescent, luminescent, chemiluminescent, or radioactive moieties) to proteins are well known in the art.

[0181] The reagent for determining the level and / or activity of VCAN may be the NF-κB reporter Raw264.7 cell line described herein.

[0182] In embodiments, the kit comprises: (iii) further comprising a reagent for determining the level and / or activity of IL-1β.

[0183] By "reagent for determining IL-1β level and / or activity" we include the meaning of any suitable reagent that can be used to detect IL-1β level and / or activity in a subject or a sample taken therefrom. Reagents for determining IL-1β level and / or activity may comprise or consist of nucleic acids such as primers for use in PCR or probes for use in immunohistochemistry. Reagents for determining IL-1β level and / or activity may comprise or consist of antibodies or antigen-binding fragments thereof, or variants thereof. In alternative or additional embodiments, the reagent(s) are immobilized on a surface (e.g., a multi-well plate or array). Alternatively or additionally, the reagents may be directly or indirectly labeled with a detectable moiety. Suitable detectable moieties are well known in the art and described herein. Reagents for determining IL-1β level and / or activity may comprise or consist of antibodies or antigen-binding fragments thereof, or variants thereof, for use in ELISA to detect IL-1β.

[0184] In an embodiment, the kit comprises a reagent for determining the level and / or activity of IKKβ. By "reagent for determining the level and / or activity of IKKβ", we include the meaning of any suitable reagent that can be used to detect the level and / or activity of IKKβ in a subject or a sample taken therefrom. The reagent for determining the level and / or activity of IKKβ may comprise or consist of a nucleic acid, such as a primer for use in PCR, or a probe for use in immunohistochemistry. The reagent for determining the level and / or activity of IKKβ may comprise or consist of an antibody or an antigen-binding fragment thereof, or a variant thereof. Such reagents include any such reagents described in the methods for determining the level and / or activity of IKKβ described herein.

[0185] The invention also provides an inhibitor for use, a use, a method or a kit substantially as herein described with reference to the accompanying description and / or drawings.

[0186] Preferred, non-limiting examples embodying certain aspects of the present invention will now be described with reference to the following tables and drawings. [Brief description of the drawings]

[0187] [Figure 1]Non-oncogene addiction of KRAS mutant cancers to interleukin (IL)-1β. (A, B) TP53, KRAS, EGFR, and BRAF mutation frequencies in the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) and The Cancer Genome Atlas (TCGA) lung adenocarcinoma (LUAD) patients. Data from (2, 12), https: / / www.cbioportal.org / , and https: / / bit.ly / 3uSO0D4. Shown are patient and mutation numbers (n) and percentages (%), as well as probability (P), χ2 test (A) or hypergeometric test (B). (C) Data summary of KRAS alterations versus IL1B mRNA expression in the Cancer Genome Atlas (TCGA) pan-cancer dataset from the United States (n=10,967 specimens from 10,953 patients with 31 different cancers). Data from https: / / www.cbioportal.org / and https: / / bit.ly / 3c1KOyl. RSEM, RNA-Seq by Expectation Maximization. Note elevated IL1B mRNA expression in KRAS mutant cancers. (D) Data summary (left) and representative image (right; inlay: isotype control) of KRAS alterations on IL-1β protein expression in lung adenocarcinomas (LUAD) and adjacent lung tissue from n=36 resected patients from Munich, Germany. Note elevated IL-1β protein expression in KRAS mutant LUAD. (E) Data summary of subcutaneous (sc) tumor and malignant pleural effusion (MPE) volumes in C57BL / 6 mice competent (WT) or diploinsufficient (Il1b- / -) in the Il1b allele at the indicated time points following sc or intrapleural injection of 5 or 2 x 105 tumor cells, respectively, with (left; n=10, 10, 30, 30, 10, 10, 20, and 20 from left to right) or without (right; n=10 / group) Kras mutation. Note the requirement of Kras mutant tumors for host IL-1β. Shown are raw data (circles), rotated kernel density distributions (violins), medians (dashed lines), quartiles (dotted lines), and P, probability, Kolmogorov-Smirnov or Kruskal-Wallis tests (A), two-way ANOVA (B, above graphs), and Bonferroni post-hoc tests (B, within graphs), or unpaired tests (C).*** and ****: P<0.001 and P<0.0001, respectively, compared with diploid patients, Dunn's post-hoc test. [Diagram 2] Kras mutant tumors activate NF-κB in tumor-infiltrating macrophages. (A) Color-coded cancer cell lines used along with tissue of origin and KRAS mutation status. (B-E) Bioluminescence images with pseudocolor scale (B,C) and data summary (D,E) from WT and HIV-LTR. luciferase (HLL: B and D), and NF-κB.GFP.luciferase (NGL; C and E) NF-κB reporter mice at 14 days (B,D) or at consecutive time points (C,E) after pleural injection of tumor cells. Note that in these models, bioluminescence is exclusively emitted by the host and not by tumor cells. (B,C) Dashed area indicates the thorax. (F) Photo / biofluorescence image overlay with pseudocolor scale of NGL mouse lung explants after 14 days of pleural LLC cells shows NF-κB reporter GFP signal (κB.eGFP) across the pleural tumor (contour; n=10). (G) GFP immunoreactivity in pleural tumor sections colocalizes with the macrophage marker CD68 (arrows; n=10). (H,I) Flow cytometry contour plots (H) and data summaries (I) of pleural tumor cells from NGL mice obtained 14 days after pleural injection stained for myeloid marker CD11b and κB.LUC reporter. Percentages in (H) are for CD11b+LUC+ cells. Data in (D,E,I) are given as raw data (circles), medians (dashed lines), quartiles (dotted lines), and kernel density distributions (violin plots) color-coded as in (A). Sample size (n) = 5–10 / group; P, probability, one-way ANOVA or two-way ANOVA; *, **, ***, and ****, P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively, compared with mice injected with RAW264.7, PANO2, or B16F10 cells at the same time points, Bonferroni post-hoc test. [Diagram 3]Tumor-secreted factors drive IKKβ activation, differentiation, and IL-1β secretion in macrophages. (A) Color-coded cancer cells with Kras mutation status. (B-E) Bioluminescence images with pseudocolor scale (B,E), immunoblots (C), and data summaries (D,E) from exposure of RAW264.7 macrophages stably expressing pNGL (B-D) and mouse bone marrow-derived macrophages (BMDM) obtained from NGL mice 1 week after 100 ng / mL M-CSF exposure (E) to cell-free tumor-conditioned medium or DMEM (white box) with or without bortezomib pretreatment (1 h, 1 μg / mL to 3 μM). (D,E) n=5 / group; P, probability, one-way ANOVA or two-way ANOVA; **** and ####, P<0.0001 compared with other groups or saline-treated cells, respectively, Bonferroni post-hoc test. (F) Differentiation marker expression assessed by flow cytometry in mouse bone marrow cells before (day 0) and after (day 7) 1 week of M-CSF exposure. (G) Microarray strategy and top 5 differentially expressed genes (ΔGE) in mouse BMDM compared to cancer cells (ΔGE1) and tumor-adapted BMDM compared to naive BMDM (ΔGE2). n=5 / group; P, probability, one-way ANOVA. (H) Bioluminescence images and data summary of NGL, NGL;Tnf- / -, and NGL;Il1b- / - mice 14 days after pleural injection of LLC cells. n=13 / group; P, probability, one-way ANOVA; ** and ***, P<0.01 and P<0.001, respectively, compared to NGL mice, Bonferroni post-hoc test. (I-L) Histograms of naive or tumor-conditioned BMDM for macrophage differentiation markers (I, J), Tnf and Il1b mRNA (K), and IL-1β protein (L) expression (I). Data summary (J-L). n=5-10 / group, P, probability, one-way ANOVA; *** and ****, P<0.001 and P<0.0001, respectively, compared with DMEM and B16F10 conditioned medium, Bonferroni post-hoc test. Data are presented as raw data (circles), median (dashed line), quartiles (dotted line), and kernel density distribution (violin plot), color-coded as in (A). [Figure 4]Tumor-secreted versican, macrophage IKKβ, drives metastasis and is a cancer biomarker. (A-E) Kras mutant and wild-type cancer cell RNA and supernatants were subjected to microarray and LC-MSMS analysis, respectively. Shown are the experimental design (A), the top 20 over-represented transcripts (B) and secreted proteins (C), and Vcan / VCAN mRNA / protein expression (D, E). n=2-3 / group, P, probability, two-way ANOVA; bold, false discovery rate (FDR) q<0.05 compared to Kras wild-type cells, Benjamini, Hochberg, and Yekutieli two-step linear ascending procedure. (F, G) Representative bioluminescence images (F) and data summary (G) from pNGL RAW264.7 cells exposed to lipopolysaccharide (LPS; 1 μg / mL) or recombinant protein (1-2 nM). n=5 / group, P, probability, two-way ANOVA; ****, P<0.0001 compared with other groups, Bonferroni post-hoc test. (H) Immunoblots of mouse BMDMs exposed to VCAN. n=5 / group. (I-M) LLC cells stably expressing control (shC) and validated anti-Vcan (shVcan) shRNA were injected intrapleurally into NGL mice. Shown are immunoblots (I), Vcan mRNA expression (J), and data summaries (K,L) obtained 14 days after tumor cells, as well as representative photographs and bioluminescence images (M). (I,J) n=5 / group, (K-M) n=16 / group, P, probability, unpaired Student's t-test. (N) Images and data summaries of VCAN expression for n=41 tumor / normal tissue pairs from patients with resected lung adenocarcinoma. (O) VCAN mRNA expression in 10 benign and 15 malignant pleural effusions. (P) Data summary and representative images of bioluminescence of pNGL RAW264.7 cells following exposure to benign (n=6; top triplicate) and malignant (n=11; bottom triplicate) pleural effusion and tumor-conditioned media (each triplicate row is from one patient). (N-P) P, probability, unpaired Student's t-test. (B-D, G, J-L, N-P) Shown are raw data (circles), kernel density distributions (violins), and medians / quartiles (dashed / dotted lines). [Diagram 5]IKKβ mediates pro-tumor NF-κB activity, differentiation, and IL-1β secretion in macrophages. (A, B) Bioluminescence images with pseudocolor scale (A) and data summary (B) of pNGL RAW264.7 cells 72 h after infection with control (shC), anti-GFP (shGFP), or anti-inhibitor of NF-κB kinase (shChuk, shIkbkb, shIkbke, or shTbk1) specific shRNA. n = 8 independent experiments / group, P, probability, one-way ANOVA; ****, P < 0.0001 compared to shC, Bonferroni post-hoc test. (C-F) Bone marrow-derived macrophages (BMDMs) were derived from mT / mG;Lyz2.Cre, Chukf / f;Lyz2.Cre, and Ikbkbf / f;Lyz2.Cre mice using 1-week exposure to 100 ng / mL M-CSF. Shown are images and mean ± SD % green cells (C) of bone marrow cells from mT / mG;Lyz2.Cre mice during / after weekly treatment with M-CSF, flow cytometry histograms (left) and data summary (right) of marker expression (D), top differentially expressed genes by microarray (E), and interleukin (IL)-1β secretion by ELISA (F). (C,D) n=5 independent experiments / group, P, probability, Fisher's exact test or one-way ANOVA, **, P<0.01 compared to other groups, Bonferroni post-hoc test. (E) n=1 pooled triplicate / group, P, prob, 2-way ANOVA. (F) n=10 independent experiments, P, prob, 1-way ANOVA; ** and ***, P<0.01 and P<0.001, respectively, compared to control, Bonferroni post-hoc test. (G) Chukf / f;Lyz2.Cre and Ikbkbf / f;Lyz2.Cre mice received intrapleural LLC or MC38 cells and were evaluated for malignant pleural effusion (MPE) 14 days later. Data summary for n=40, 15, and 21 single transgenic control, Chukf / f;Lyz2.Cre, and Ikbkbf / f;Lyz2.Cre mice injected with LLC cells, respectively, and n=40, 15, and 25 mice injected with MC38 cells, respectively.P, probability, two-way ANOVA; *, **, and ****, P<0.05, P<0.01, and P<0.0001, respectively, compared with control, Bonferroni post-hoc test. (H) Schematic of the proposed mechanism for non-oncogene addiction of KRAS mutant cancers to IL-1β. To this end, KRAS mutant cancers secrete VCAN to co-select IKKβ in macrophages within the metastatic niche, which drives IL-1β secretion by macrophages to promote tumor progression. [Figure 6]Pharmacological abrogation of non-oncogene addiction of Kras mutant tumors to IL-1β. (A-D) The IL-1 receptor antagonist isunakinra limits nuclear factor (NF)-κB activation and malignant pleural effusion (MPE) from Kras mutant cancer cells. (A) FVB (n=10) and C57BL / 6 (n=45) mice received subcutaneous injections of 5x106 FULA1 (FVB mice) or LLC, MC38, B16F10, or PANO2 (C57BL / 6 mice) cells harboring G12C, G13R, Q61R, or wild-type (WT) Kras alleles. Mice were allowed to take tumors for 10-17 days and treated with daily intraperitoneal phosphate buffered saline (PBS) or 20 mg / Kg (LLC cells) or 50 mg / Kg (all other cells) Isunakinra until control tumor volumes reached 1 cm3 (PANO2 cells) or 2 cm3 (all other cell lines). Shown are treatment (Tx) start, number of mice (n), tumor volumes as mean (circles) and SD (bars), two-way ANOVA probability (P) for treatment efficacy, and mean Isunakinra efficacy (%) at the last time point. ** and ***: P<0.01 and P<0.001, respectively, Bonferroni post-hoc test. (B-D) C57BL / 6 mice (n=48) received intrapleural 2x105 LLC cells stably expressing the κB.LUC reporter (NGL), were allowed for tumor ingestion for 5 days, and received daily intraperitoneal PBS or 20 mg / Kg isunakinra. After 1 mg intravenous D-luciferin and bioluminescence imaging, mice were sacrificed when diseased (n=17 / treatment) or 14 days after LLC cells (n=7 / treatment). Shown are representative thoracic (dotted) bioluminescence images with pseudocolor scale (B), Kaplan-Meier survival estimates (curves) with log-rank probability (P) and hazard ratios (HR) (C), and data summaries of thoracic bioluminescence and MPE volumes shown as raw data points (circles), medians (dashed), quartiles (dotted), kernel density distributions (violins), and probabilities (P), unpaired Student's t-tests (H). (E–H) The toll-like receptor 1 / 2 (TLR1 / 2) inhibitor Cu-CPT22 blocks versican (VCAN)-induced myeloid NF-κB activation and MPE in Kras mutant cancer cells in vivo.(E,F) Representative bioluminescence images with pseudocolor scale (E) and summary of results (F) of RAW264.7 macrophages stably expressing NGL pretreated with 1% DMSO or increasing concentrations of Cu-CPT22 in 1% DMSO and exposed to 10 nM lipopolysaccharide (LPS) or 1 nM recombinant VCAN (1 h latency). Cells were assessed for bioluminescence 24 h after LPS / VCAN treatment and for MTT reduction 72 h after LPS / VCAN treatment. n=3 and n=6 independent experiments / groups for κB.LUC and MTT, respectively, are shown as 50% inhibitory / lethal concentrations (IC50 / LC50), mean (circles), and SD (bars). (G,H) Representative images (G) and data summaries (H) of thoracic bioluminescence and MPE volumes of κB.luc mice 14 days after pleural injection of 2×105 LLC cells, followed by daily intraperitoneal injections of 100 μl corn oil 10% DMSO (n=10) or 20 mg / kg Cu-CPT22 diluted in 100 μl corn oil 10% DMSO (n=10) starting 5 days after LLC cells. Shown are raw data points (circles), medians (dashed lines), quartiles (dotted lines), kernel density distributions (violins), and probabilities (P), unpaired Student's t-test. [Figure 7](Figure S1). Seven mouse cell lines with different Kras alleles and transcriptional regulation of interleukin (IL)-1β by nuclear factor (NF)-κB. (A, B) Sanger sequencing traces for Kras codons 12, 13, and 61 of all cell lines used. Arrows point to Kras mutations. MC38 cells have the wild-type (Wt / WT; A) Kras allele, LLC and AE17 cells G13R, FULA1 cells G12C, (B) as well as all other cell lines. Note that all mutant Kras alleles are heterozygous to the Wt allele. (C) IL1B as an NF-κB target gene in a ChIPseq dataset from the CHEA transcription factor target dataset (https: / / maayanlab.cloud / Harmonizome / dataset / CHEA+Transcription+Factor+Targets). We downloaded RELA (red) and RELB (blue) binding sequence motifs from the ENCODE portal (https: / / www.encodeproject.org / ) using the identifiers: ENCFF507YCV (CHIP-seq on HuH-7.5 cells) and ENCFF615HZF (CHIP-seq on 8988T cells), respectively. E-values ​​represent the statistical significance of the motif in terms of the probability of being found in a set of random sequences of similar size. TSS: transcription start site. [Figure 8] (Figure S2). Expression of IL1B correlates with KRAS and macrophage marker ADGRE1 in human tumors. Gene expression data from The Cancer Genome Atlas (TCGA) pan-cancer dataset (n=10,071 patients). Correlation of IL1B with KRAS (A) and macrophage marker ADGRE1 (encoding F4 / 80) (B) mRNA levels. Shown are raw data points (circles) colored by KRAS alteration status, regression line and equation (line), as well as Spearman and Pearson correlation coefficients with probability (P) and squared correlation coefficient (R2). Data from https: / / www.cbioportal.org / . [Figure 9](Figure S3). Expression of IL1B correlates with lineage-specific markers in human tumors. Gene expression data from The Cancer Genome Atlas (TCGA) pan-cancer dataset (n=10,071 patients). Correlation between IL1B mRNA levels and neutrophil marker ELANE (top left; encodes neutrophil elastase), pan-lymphocyte marker CD3D (top center; encodes cluster of differentiation 3), mast cell marker KIT (top right; encodes c-KIT), cancer cell marker KRT18 (bottom left; encodes cytokeratin 18), fibroblast marker ACTA2 (bottom center; encodes alpha-smooth muscle actin), and endothelial marker F8 (bottom right; encodes factor VIII). Shown are raw data points (circles) colored by KRAS mutation status, regression line and equation (line), and Spearman and Pearson correlation coefficients with probability (P) and squared correlation coefficient (R2). Data from https: / / www.cbioportal.org / . [Figure 10](Figure S4). NF-κB activation in pleural metastases of NGL mice. (A, B) Representative photographic / bioluminescence images with pseudocolor scale (A) and data summary (B) from wild-type (WT / WT), heterozygous (WT / NGL), and homozygous (NGL / NGL) NF-κB.eGFP.LUC reporter mice 5 min after retro-orbital injection of 1 mg D-luciferin. Sample size (n) = 10 / group, P, probability, one-way ANOVA; ****, P < 0.0001 compared to WT / WT mice, Bonferroni post-hoc test. (C, D) Representative photographic / biofluorescence (C) images with pseudocolor scale and data summary (D) of κB.eGFP reporter signal (green) in lung explants of NGL mice 14 days after pleural injection of 2 × 105 Lewis lung carcinoma (LLC) cells. Note the NF-κB reporter signal (κB.eGFP) on the pleural tumor (outline). n=10 / group, P, probability, unpaired Student's t-test. (E,F) Representative photographic / bioluminescence image overlays with pseudocolor scale of thoracic bioluminescence (E) and summary of results (F) of NGL mice bearing metastatic malignant pleural effusions 14 days after pleural injection of 2×105 B16F10 cutaneous melanoma, MC38 colon adenocarcinoma, or LLC cells before (undrained) and after (drained) pleural catheter insertion, and after fluid removal. n=5 / group, P, probability, repeated measures two-way ANOVA; ns and *, P>0.05 and P<0.05 for pre-post drainage comparisons, respectively. Data in (B,D,F) are given as raw data (circles), medians and quartiles (lines), and kernel density distributions (violin plots). [Figure 11] (Fig. S5). NF-κB activation in pleural metastases of NGL mice. Pleural metastases from mice treated as in Fig. 10C,D (Fig. S4C,D) show endogenous κB. eGFP reporter signal colocalizing with the pan-hemocyte proliferation marker CD45 (A) and macrophage marker CD68 (B) in tumor-infiltrating myeloid cells and macrophages (arrows). Nuclear Hoechst 33258 counterstain. [Figure 12](Figure S6). NF-κB activation in metastasis-associated macrophages. NGL NF-κB reporter mice received pleural PBS or DMEM (controls), or 2 × 105 B16F10 cutaneous melanoma, MC38 colon adenocarcinoma, or Lewis lung carcinoma (LLC) cells 14 days before sacrifice. Pleural fluid and pleural tumor cells were stained with antibodies against hematopoietic marker CD45, myeloid markers CD11b and Ly6c, and endogenous κB.LUC reporter. Representative flow cytometry dot plots showing sequential gating strategies for pleural fluid (A) and tumor (B) macrophages, as well as histograms (C) and data summaries (D) of κB.LUC reporter signal in pleural macrophages. Data in (D) are given as raw data (circles), medians and quartiles (lines), and kernel density distributions (violin plots). Sample size (n) = 5 / group; P, probability, one-way ANOVA; ****, P < 0.0001 compared with mice injected with B16F10 cells, Bonferroni post-hoc test. MFI, mean fluorescence intensity. [Figure 13] (Figure S7). NF-κB activation in metastasis-associated macrophages. NGL NF-κB reporter mice received 2 × 105 pleural B16F10 cutaneous melanoma, MC38 colon adenocarcinoma, or Lewis lung carcinoma (LLC) cells and were sacrificed 14 days later. Pleural tumors were assessed for κB.eGFP reporter signal by fluorescence microscopy after nuclear counterstaining with Hoechst 33258. Data summary (A) and representative fused microscopy images (B–D) of κB.eGFP+ pleural tumor cells. Data in (A) are given as raw data (circles), medians and quartiles (lines), and kernel density distributions (violin plots). Sample size (n) = 10 / group, P, probability, one-way ANOVA; ****, P < 0.0001 compared with mice injected with B16F10 cells, Bonferroni post-hoc test. hpf, high power fields. [Figure 14](Figure S8). There was no effect of mast cells on the host NF-κB response to pleural metastasis, and the intensity of the host NF-κB response to ectopic tumor growth was reduced. (A-B) Representative photographic / bioluminescence image overlays with pseudocolor scale (A) and summary of results (B) from NGL mice additionally carrying none (NGL) or one (NGL;Cpa3.Cre) Cpa3.Cre allele, rendering them mast cell competent or deficient, respectively. Images and data were obtained 14 days after pleural injection of 2 × 105 Lewis lung carcinoma (LLC) cells and 5 min after retro-orbital injection of 1 mg D-luciferin. Sample size (n) = 8 / group, P, probability, unpaired Student's t test. (C-D) Representative photographic / bioluminescence image overlays with pseudocolor scale (C) and a summary of results (D) from NGL mice 21 days after subcutaneous injection of 5x105 B16F10 skin melanoma, MC38 colon adenocarcinoma, or LLC cells and 5 min after retro-orbital injection of 1 mg D-luciferin. n=12 / group, P, probability, one-way ANOVA; ****, P<0.0001 compared to B16F10 cells, Bonferroni post-hoc test. Data in (B,D) are given as raw data (circles), medians and quartiles (lines), and kernel density distributions (violin plots). [Figure 15](Figure S9). Requirement of mutant Kras signaling for host NF-κB activation during pleural metastasis. (A) Tumor cells harboring Kras mutations (MC38 and LLC cells) were engineered to stably express shRNAs encoding random control (shC) or anti-Kras specific (shKras) sequences. Kras-wild-type RAW264.7 myelomonocytic leukemia cells were used as controls. Shown are the color-coded tumor cell lines used along with shRNA expression and Kras mutation status. (B, C) Tumor cells were injected into the pleural cavity of NGL mice (5 × 105 / mouse). (D, E) RAW264.7 macrophages expressing pNGL plasmid were pretreated with saline or the proteasome and NF-κB inhibitor bortezomib (1 μg / mL equivalent to 3 μM) and then incubated with tumor-conditioned medium (1:1 dilution in DMEM). Shown are representative photographs / bioluminescence images with pseudocolor scale (B, D) and a summary of the results of breast bioluminescence of NGL mice at 14 days after tumor cell (C) and a summary of the results of cell bioluminescence of pNGL RAW264.7 macrophages at 4 hours after tumor conditioned medium (E). (C) n=10 / group; (E) n=5 independent experiments / group; P, probability, two-way ANOVA; ** and ***, P<0.01 and P<0.001, respectively, compared to shC; #, P<0.05, respectively, compared to saline pretreated cells. Data in (C, E) are given as raw data (circles), medians and quartiles (lines), and kernel density distributions (violin plots). [Figure 16](Figure S10). Adoptive bone marrow transplantation determines host NF-κB response to pleural metastasis. NF-κB.eGFP.LUC reporter (NGL) and wild-type (WT) recipients received total body irradiation (1100 Rad) followed by adoptive bone marrow replacement (BMT) from WT and NGL donors. After 1 month required for bone marrow chimerism, 500 μg of liposomal clodronate was administered intrapleurally. After another month required for replacement of pleural bone marrow cells with transplanted bone marrow cells, mice received 2 × 105 intrapleural LLC cells and were imaged for bioluminescence 14 days later. Shown are experimental schematics (each box represents 1 month after birth) (A, B), color-coded table with experimental groups and specimen size (n) (C), summary of results (D), and representative photographs / bioluminescence images with pseudocolor scale (E) taken 5 min after retro-orbital injection of 1 mg D-luciferin. Data in (D) are presented as raw data (circles), medians and quartiles (lines), kernel density distributions (violin plots), and probability values ​​(P), two-way ANOVA. ** and ***, P<0.01 and P<0.001, respectively, compared with mice receiving BMT from NGL donors, Bonferroni post-hoc test. [Figure 17](Figure S11). Pharmacological and genetic macrophage ablation abolishes pleural metastases. (A) C57BL / 6 mice (n = 5 mice / treatment / time point) received intrapleural liposomal clodronate or empty liposomes (500 μg / mouse) and were subjected to pleural lavage at different time points after clodronate for analysis of the number of macrophages expressing F4 / 80 (encoded by Adgre1, the mouse orthologue of human ADGRE1 from Figure S1). P, probability, two-way ANOVA; *** and ****, P < 0.001 and P < 0.0001, respectively, compared with empty liposomes, Bonferroni post-hoc test. (B) Pleural fluid volume of C57BL / 6 mice 15 days after intrapleural delivery of 500 μg liposomal clodronate or empty liposomes, followed by 2×105 intrapleural MC38 or LLC cells 1 day later, and rechallenge of 500 μg liposomal clodronate or empty liposomes into the pleural cavity 3 days later. n=5 mice / group, P, probability, two-way ANOVA; * and **, P<0.05 and P<0.01, respectively, compared with empty liposomes, Bonferroni post-hoc test. (C, D) Pleural fluid nucleated cell counts (C) and malignant pleural fluid volumes (D) of C57BL / 6 mice carrying one or both Lyz2.Cre and Dta alleles 14 days after intrapleural delivery of 2×105 MC38 or LLC cells. From left to right, n=22, 14, 16, and 7 mice / group; P, probability, two-way ANOVA; * and **, P<0.05 and P<0.01, respectively, for Lyz2.Cre;Dta macrophage-deficient mice compared to single transgenic controls, Bonferroni post-hoc test. All data are presented as raw data (circles), medians and quartiles (lines), and kernel density distributions (violin plots). [Figure 18](Figure S12). Uncut immunoblots. Shown are uncut immunoblots with the areas (dashed rectangles) indicated in the main figure. (A-C) Blots shown in Figure 3C with IKKα (A), IKKβ (B), and β-actin (C). (D, E) Inverted blots shown in Figure 4E with versican (VCAN; D) and α-tubulin (TUBA; E). (F-H) Inverted blots shown in Figure 4H with IKKα (H), IKKβ (F), and β-actin (G). (I, J) Inverted blots shown in Figure 4I with VCAN (I) and TUBA (J). [Figure 19] (Figure S13). Toll-like receptor (TLR) expression by mouse bone marrow-derived macrophages (BMDM) by microarray. Mouse BMDMs from C57BL / 6 mice were isolated from whole bone marrow cells using 1 week exposure to 20 ng / mL macrophage colony-stimulating factor (MCSF). Total cellular RNA was extracted from BMDMs under various conditions and from different cancer cell lines (n=9 / group) and hybridized to Affymetrix Mo Gene ST2.0 microarrays (data from GEO datasets GSE94847, GSE94880, GSE130624, and GSE130716). Shown are unsupervised clustering of all differentially expressed genes (A) and TLR genes (B), as well as a summary of the results for TLR gene expression (C). Data in (C) are shown as mean with SD. P, probability, two-way ANOVA; * and ****, P<0.05 and P<0.0001, respectively, compared to cancer cells, Bonferroni post-hoc test. Microarrays compared were GSM3744950, GSM3744952, GSM3744954, GSM3744955, GSM3744958, GSM3744961, GSM3744962, GSM3744957, and GSM3744960 (cancer cells) versus GSM2486425, GSM2486426, GSM2487750, GSM2487751, GSM3752396, GSM3752397, GSM3752398, GSM3752399, and GSM3752400 (BMDM). [Figure 20](Figure S14). Versican as a potential diagnostic and prognostic biomarker for KRAS mutant human cancers. (A, B) Data summary of VCAN expression normalized by ACTB transcript in various human cancer types with different KRAS mutation frequencies (KRASMUT%; data from COSMIC; https: / / cancer.sanger.ac.uk / cosmic). (A) VCAN / ACTB expression in GEO dataset GSE43458, which encompasses lung adenocarcinoma (LUAD) from smokers (KRASMUT%=36.5%) and non-smokers (KRASMUT%=11.8%), and normal lung tissue from non-smokers (KRASMUT%<1.0%). (B) VCAN / ACTB expression in GEO dataset GSE103512, which encompasses prostate (KRASMUT%=2.8%), colorectal (KRASMUT%=32.4%), and lung (KRASMUT%=14.9%) cancers. (A,B) n, sample size; P, probability, one-way ANOVA; *** and ****, P<0.001 and P<0.0001, respectively, compared to lung tissue from non-smokers (A) or prostate cancer (B); #, P<0.05, compared to LUAD from non-smokers, Bonferroni post-hoc test. (C) Kaplan-Meier survival analysis from all patients in the KMplot database stratified by VCAN transcript expression with optimal cutoffs (data from KMplot; http: / / kmplot.com / analysis / index.php?p=service&cancer=pancancer_rnaseq. Shown is a summary of hazard ratios (HRs) obtained for VCAN and ACTB control expression in 7,462 patients with 18 different tumor types. P, probability, one-sample Wilcoxon test for comparisons with HR=1.0. All data are presented as raw data (circles), medians and quartiles (lines), and kernel density distributions (violin plots). [Figure 21](Figure S15). Versican overexpression by KRAS mutant human cancers predicts poor survival. Kaplan-Meier survival plots with median overall survival (OS), hazard ratios (HR) with 95% confidence intervals, and univariate log-rank probability values ​​(P) from 504 patients with lung adenocarcinoma (A), 373 patients with ovarian cancer (B), 371 patients with gastric cancer (C), 177 patients with pancreatic cancer (D), 370 patients with liver cancer (E), and 304 patients with cervical cancer (F), stratified into low (black) and high VCAN transcript expression by the optimal cutoffs indicated. RMA, robust microarray averages (data from KMplot; http: / / kmplot.com / analysis / index.php?p=service&cancer=pancancer_rnaseq) [Figure 22](Figure S16). KRAS, VCAN, and IKBKB Alterations in Human Cancers. KRAS, VCAN, and IKBKB mutation frequencies in The Cancer Genome Atlas (TCGA) pan-cancer dataset (n=10,967 samples from 10,953 patients). Data are from https: / / www.cbioportal.org / (link: https: / / bit.ly / 3yGys8i). Shown are mutation plots with alteration frequency (A), lollipop plot (B), co-occurrence Venn diagram (C), and most frequently mutated tumor types (D). In (A), columns represent patients and rows represent genes. In (C), shown is the sample number (n). P, probability, hypergeometric test. In (D), shown are raw data points (circles), rotated kernel density distributions (violins), medians (dashed lines), quartiles (dotted lines), top quartile altered cancer types with P, two-way ANOVA, and Spearman's correlation coefficient (ρ) and P across 32 cancer types. The TCGA acronym is from https: / / gdc.cancer.gov / resources-tcga-users / tcga-code-tables / tcga-study-abbreviations. Note the alteration enrichment (poisoning) of VCAN to KRAS and IKBKB to VCAN along the pathways proposed here. Also note that lung adenocarcinoma (LUAD), colon adenocarcinoma (COAD), and uterine corpus endometrial carcinoma (UCEC) are among the top 25% mutated cancers for all three genes and are among the most frequent cancer types metastasizing to the pleural cavity. [Figure 23](Figure S17). VCAN and IKBKB alterations in lung adenocarcinoma (LUAD), colon adenocarcinoma (COAD), rectal adenocarcinoma (READ), and uterine corpus endometrial carcinoma (UCEC). KRAS, VCAN, and IKBKB mutation frequencies in The Cancer Genome Atlas (TCGA) LUAD, COAD, READ, and UCEC datasets (n=1,689 specimens / patient). Data are from https: / / www.cbioportal.org / and can be retrieved at https: / / bit.ly / 3wzrbFF. Shown are alteration frequencies (A), co-occurrence Venn diagrams (B), and mutation plots with altered vs. non-altered patient features (C). In (A), columns represent patients and rows represent genes. In (B), shown is the specimen number (n). P, probability, hypergeometric test. In (C), shown are rotated kernel density distributions (violins), medians (dashed lines), quartiles (dotted lines), and numbers (n) and percentages (%) of UCEC patients. TCGA acronyms are from https: / / gdc.cancer.gov / resourcestcga-users / tcga-code-tables / tcga-study-abbreviations. P, probability, Kruskal-Wallis test (graphs), or overall and paired χ2 test for numbers and percentages of patients before and within brackets (tables). *, ***, and ****: P<0.05, P<0.001, and P<0.0001, compared with unchanged patients, Dunn's post hoc test. In (B), note the alteration enrichment from IKBKB to VCAN (oncogene poisoning), and oncogene repulsion from IKBKB to KRAS. Also note the significant enrichment of VCAN and IKBKB alterations in selected KRAS-mutant tumor types. Note in (C) that VCAN-, IKBKB-, and VCAN / IKBKB-altered patients exhibit varying degrees of cachexia (not documented for LUAD), hypermutation, microsatellite instability (MSI), hypoxia, and aggressive tumor grade. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0188] Example 1 - Non-oncogene addiction of KRAS mutant cancers to IL-1β via Veriscan and mononuclear IKKβ KRAS mutant cancers are frequent, metastatic, fatal, and largely undruggable. Interleukin (IL)-1β and nuclear factor (NF)-κB inhibition show promise against cancer, but non-targeted treatments are ineffective. Here, we show that human KRAS mutant cancers are addicted to IL-1β through inflammatory versican signaling to macrophage inhibitor of NF-κB kinase (IKK)β.

[0189] Human pan-cancer and experimental NF-κB reporter, transcriptome, and proteome screens reveal that KRAS mutant tumors trigger macrophage IKKβ activation and IL-1β release via secreted versican. Tumor-specific versican silencing and macrophage-restricted IKKβ deletion prevent myeloid NF-κB activation and metastasis. Versican and IKKβ are mutually addicted and / or overexpressed in human cancers and have diagnostic and prognostic potential. Non-oncogene KRAS / IL-1β addiction is abolished by IL-1β and TLR1 / 2 inhibition, indicating a critical and actionable role for versican and IKKβ in metastasis.

[0190] Tumor-associated inflammation is closely associated with tumor progression and therapeutic response. (1) Interleukin (IL)-1β is a key mediator of tumor-associated inflammation, and its inhibition via the monoclonal antibody canakinumab was recently shown to have a strong protective effect against incident lung cancer in an exploratory analysis of the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) (2). Unexpectedly, the phase III CANOPY-2 trial (ClinicalTrials.gov NCT03626545), investigating second / third line canakinumab with docetaxel for non-small cell lung cancer (NSCLC), regardless of histological subtype and driver mutations, was negative for unknown reasons (https: / / www.novartis.com / news / media-releases / novartis-provides-update-phase-iii-studyevaluating-canakinumab-acz885-second-or-third-line-treatment-combination-chemotherapy-nonsmall-cell-lung-cancer). To this end, the protective effect of canakinumab in the CANTOS trial NSCLC exploratory study was significantly stronger for current and past smokers, as well as for early stage lung adenocarcinoma (LUAD) histological subtype, both of which have a high mutation rate of the KRAS proto-oncogene GTPase (encoded by the KRAS / Kras gene in humans / mice).

[0191] Multiple lines of evidence determine that tumor genomic alterations primarily define tumor-associated inflammation and the efficacy of immune-directed therapies (1). To this end, NSCLC with high mutational burden and smoking-associated trinucleotide signatures were found to exhibit more favorable and durable responses to the immune checkpoint inhibitor pembrolizumab, which targets programmed cell death-1 (PD-1) (3). Furthermore, STK11 / LKB1 alterations were reported to be the major drivers of primary resistance to PD-1 inhibitors in KRAS mutant LUAD, the most frequent and deadly histological subtype of NSCLC (4). Experimental evidence supports that the immune landscape and vulnerability of various tumor types may depend on single mutational driver oncogenes, such as KRAS and MYC, that orchestrate distinct transcriptional programs and determine the specific proinflammatory mediator secretion profile of tumors, primarily defining the cellular composition of the tumor microenvironment (5, 6). In this regard, oncogenic KRAS is known to cooperate with proinflammatory nuclear factor (NF)-κB signaling in cancer cells to drive stemness, elaboration of proinflammatory mediators, and responsiveness to IL-1β signaling ( 7 – 10 ), ideally positioning it as a biomarker of therapeutic response to anti-IL-1β therapy.

[0192] Here, we show that KRAS mutant cancers display a specific non-oncogene addiction to host-provided IL-1β in humans and mice. We further elucidate how these tumors activate NF-κB in tumor-associated macrophages to induce IL-1β, which is necessary for sustained growth. Mutant KRAS-IL-1β addiction is mediated through secretion of the glycoprotein versican (VCAN) by tumor cells, which induces an inhibitor of NF-κB kinase (IKK)β in macrophages and releases IL-1β into the tumor microenvironment. Importantly, the VCAN-IKKβ axis is shown to be necessary for the sustained growth of KRAS mutant tumors and constitutes a diagnostic and prognostic biomarker for these tumors. Finally, we show how the non-oncogene addiction of KRAS mutant cancers to myeloid IL-1β can be abolished by pharmacological inhibition of IL-1β or VCAN targeting Toll-like receptor (TLR)2. Our findings can be directly translated and tested in clinical trials of IL-1β inhibition for genomically stratified LUAD.

[0193] Non-oncogene addiction of KRAS mutant human and mouse cancers to IL-1β Puzzled by the negative results of the CANOPY-2 trial, we focused on published mutation data from incident LUAD from the CANTOS trial (11) and cross-referenced it with the Cancer Genome Atlas (TCGA) LUAD dataset (12) to hypothesize that IL-1β neutralization with canakinumab would specifically prevent the development of early KRAS mutant (MUT) LUAD. Indeed, mutations in KRAS, but not TP53, EGFR, and BRAF, were statistically significantly less enriched in CANTOS compared to TCGA patients (Figure 1A,B). We then analyzed TCGA pancreatic transcriptome data to find elevated and amplified IL1B mRNA levels in KRASMUT and performed IL-1β immunohistochemistry in our own patients with resected LUAD (13) to find increased IL-1β protein expression in KRASMUT LUAD compared to KRAS wild-type (WT) LUAD and adjacent lung tissue (Figure 1C,D). We next used syngeneic cancer cell lines carrying KrasWT and KrasMUT alleles (9, 10) to inject competent (WT) and diploid incompetent for the Il1b allele (Il1b- / -) (14) C57BL / 6 mice (9, 10). Because we previously identified that malignant pleural effusion (MPE) in mice is exclusively caused by KrasMUT tumor cells (9, 10), we used both subcutaneous (sc) and pleural routes of tumor cell injection. All cell lines were validated multiple times for Kras, Mycoplasma spp., and identity status during these investigations (Fig. S1A, B). These experiments specifically showed that KrasMUT tumors were dependent on host IL-1β (Fig. 1E). Collectively, these results demonstrate that IL-1β neutralization prevents the development of early KRASMUT LUAD in humans, that KRASMUT human cancers contain elevated levels of IL-1β, and that murine KrasMUT cancers are specifically dependent on host IL-1β signaling, supporting the hypothesis of selective non-oncogene addiction of KRASMUT cancers to IL-1β.

[0194] Tumor-associated macrophages as a source of tumorigenic IL-1β We next investigated the source of increased IL-1β in KRASMUT cancer, as both host immune cells and tumor cells enable IL-1β production (15-17). We also built on previous work demonstrating that the IL-1β promoter is under the transcriptional control of NF-κB (18), a fact we verified in a ChIP-X Enrichment Analysis (CHEA) dataset (https: / / maayanlab.cloud / Harmonizome / dataset / CHEA+Transcription+Factor+Targets) and a ChIPseq dataset from the ENCyclopedia Of DNA Elements (ENCODE) portal (https: / / www.encodeproject.org / ) (Figure S1C). To this end, we first searched the TCGA pan-cancer transcriptome (n=10,071) for associations between IL1B mRNA levels and established cancer and immune cell lineage markers. IL1B mRNA levels did not correlate with the mRNA levels of the neutrophil marker ELANE, the mast cell marker KIT, the fibroblast marker ACTA2, and the endothelial marker F8, but were significantly associated with the mRNA levels of KRAS itself, the pan-lymphocyte marker CD3D, and the cancer cell marker KRT18, but showed the closest correlation (coefficient = 0.4; P < 10-300) with the mRNA levels of the macrophage marker ADGRE1 (figs. S2, S3). To test this further, we sought to identify host cells that respond to KRAS tumor cells with NF-κB activation, as this transcription factor controls IL-1β transcription (18) and is central to the innate immune response (19). To this end, we initiated an in vivo screening of mouse tumor cell lines with known Kras mutation status [(9), Figures 2A, S1] by transplanting them into two lines of bioluminescent NF-κB reporter mice expressing either the ubiquitous HIV-LTR.luciferase (HLL mice) (20) or NF-κB.GFP.luciferase (NGL mice) (21) transgenes. Pleural injection was chosen for tumor cell inoculation because it generates MPE with obvious cancer-induced inflammation (9, 10, 15).Sequential imaging showed time-dependent NF-κB activation in host cells of recipient mice, conditional on the presence of Kras mutation in tumor cells (Figs. 2B–E, S4A,B). NF-κB reporter signals emanated from pleural tumors and fluids containing both cancer and immune cells (Figs. 2F, S4C–F) (9, 10, 15). Histological and flow cytometric analysis and quantification localized NF-κB reporter signals to tumor-infiltrating macrophages in mice bearing KrasMUT pleural tumors and effusions (Figs. 2G–I, S5–S7). Mast cells, which promote MPE onset (15), were not involved in the observed NF-κB response (Fig. S8A,B). Time-dependent NF-κB activation in host cells was stronger in the pleura compared to the sc tumor model and required expression of mutant Kras by tumor cells (Figs. S8C,D and S9A–C). Adoptive bone marrow transfer confirmed bone marrow cells as the origin of tumor-induced NF-κB activation, and pharmacological killing of pleural macrophages prevented host NF-κB activation and pleural carcinomatosis (figs. S10A–E and S11A,B). The protumor function of pleural macrophages was also consistent with the phenotype of macrophage-depleted Lyz2.Cre;Dta mice (22) (fig. S11C,D). Tumor-secreted solute factors are involved in NF-κB activation in macrophages, as murine RAW264.7 macrophages stably expressing the NGL reporter responded with robust in vitro NF-κB activation to cell-free medium conditioned by KrasMUT, but not KrasWT or Krassilenced, tumor cells (Fig. 3A,B and S9A,D,E). This NF-κB response requires canonical NF-κB signaling, as it involves IKKβ and was attenuated by the proteasome inhibitor bortezomib (Figures 3C,D, S9A,D,E, and S12A-C). Proteasome-dependent canonical NF-κB activity was also demonstrated in bone marrow-derived macrophages (BMDMs) derived from NGL mice (Figures 3E,F).Differential gene expression (ΔGE) analysis (GEO datasets GSE94847, GSE94880, GSE130624, and GSE130716; total n = 32) identified 13 BMDM-specific transcripts that were further induced by incubation with tumor-conditioned medium (ΔGE > 5; ANOVA P < 0.05), including Il1b, but not Il6 and Tnf, reported elsewhere (23) (Figure 3G and Table 1). In addition, NGL mice diploid insufficient in the Il1b allele (14) were resistant to tumor-induced NF-κB activation (Figure 3H). Incubation of BMDMs with KrasMUT tumor-conditioned medium promoted their differentiation, as assessed by flow cytometry for the markers MHCII and CD206, as well as Il1b mRNA and IL-1β protein expression (Figure 2I-L). These data directly demonstrate that KRASMUT tumor cells can activate NF-κB in macrophages via solute mediator(s) that induce IKKβ-mediated NF-κB activation, differentiation, and IL-1β elaboration.

[0195] Tumor-secreted versican as a major macrophage effector We next compared KrasMUT to KrasWT cancer cells for secreted molecules that induce macrophage NF-κB activation. Microarrays identified transcripts over-represented in Kras25MUT tumor cells, and proteomic screening of tumor cell-conditioned media detected 226 proteins secreted 10-fold more by KrasMUT versus KrasWT cells, with the glycoprotein versican (VCAN; encoded by the human / mouse VCAN / Vcan genes) emerging from both screens and withstanding validation (Figures 4A–E, S12D–E, Table 2). We also screened multiple NF-κB ligands using pNGL-expressing RAW264.7 macrophages, revealing that the Toll-like receptor (TLR) 2 ligand VCAN potently activated macrophage NF-κB-driven transcription to the same extent as the TLR4 ligand lipopolysaccharide (LPS) (Figures 4F,G). VCAN also induced IKKβ in primary mouse BMDMs, which was verified by microarrays to be overexpressed >10-fold on cancer cells TLR1, TLR2, TLR6-9, and TLR13 (Fig. 4H, S12F-G, and S13A-C). Importantly, shRNA-mediated Vcan silencing in LLC cells reduced their ability to induce NF-κB activation and precipitate MPE in NGL mice (Fig. 4I-M and S12I-J). VCAN overexpression is not limited to mouse KrasMUT cancers, as VCAN transcripts are also overrepresented in human cancers with high KRASMUT frequency (derived from Catalog of Somatic Mutations in Cancer, COSMIC), such as LUAD from smokers (GEO dataset GSE43458), as well as NSCLC and colorectal adenocarcinoma (COAD / READ; GEO dataset GSE103512) (Fig. S14A-B) (24-26). High VCAN mRNA expression also predicted poor survival in several human cancers from the KMplot pan-cancer RNAseq dataset ( http: / / kmplot.com / ; figs. S14C and S15A–F) ( 27 ).Analysis of specimens from two of our own clinical studies (13, 28) showed that VCAN protein expression was significantly increased in LUAD compared to adjacent lung tissue, and VCAN mRNA expression was significantly increased in human MPE compared to benign pleural effusion (BPE) (Figure 4N and O). To test whether the proposed inflammatory loop could serve as a diagnostic tool to distinguish MPE from BPE, an unmet clinical need (28), we exposed pNGL-expressing RAW264.7 macrophages to cell-free supernatants from human pleural effusion. After 4 h, a robust NF-κB reporter signal was selectively induced by MPE supernatants (Figure 4P). Taken together, these data indicate that VCAN secreted by cancer cells induces IKKβ-mediated NF-κB activation in tumor-associated macrophages and promotes metastasis. Furthermore, VCAN is overexpressed in human KRASMUT cancer and may serve as a diagnostic and prognostic biomarker.

[0196] Bone marrow IKKB as VCAN accessory To identify the IKKs involved in NF-κB signaling in macrophages, we silenced the four major IKKs (encoded by mouse Chuk, Ikbkb, Ikbke, and Tbk1 genes) in RAW264.7 macrophages and identified IKKβ as the major mediator of NF-κB activation in these cells (Figure 5A-B). To further define myeloid IKKβ function, we obtained BMDMs from intercrossing Lyz2.Cre mice with mice carrying conditional deletion alleles of IKKα (Chukf / f) and IKKβ (Ikbkbf / f), as well as Cre-reporter mice that switch from red to green fluorescence upon Cre-mediated recombination (mT / mG), all of which have been previously reported (22, 29). Treatment of bone marrow cells from mT / mG;Lyz2.Cre mice with macrophage colony-stimulating factor (M-CSF; 100 ng / mL) drove them towards macrophage differentiation and lysozyme 2 (LYZ2) expression, resulting in efficient Cre-mediated recombination (Figure 5C). Flow cytometric assessment of BMDM derived from these mice showed that intact IKKβ signaling in primary macrophages was essential for differentiation and expression of key proinflammatory genes, including Lyz2, Il1b, and C3 (Figure 5D-F and Table 3). Finally, two different syngeneic KrasMUT tumor cell lines, characterized by VCAN overexpression, were inoculated into the pleural cavity of the myeloid IKK-deficient mice described above to reveal that intact IKKβ signaling in macrophages is required for MPE (Figure 5G). Accordingly, VCAN-driven IKKβ activation mediates NF-κB signaling, IL-1β expression, differentiation, and pro-tumor functions in macrophages (Figure 5H). To further query the proposed KRAS-VCAN-IKKβ connection, we investigated mutations, copy number alterations, and fusions of coding genes in the TCGA pan-cancer dataset. Interestingly, VCAN and IKBKB alterations (mostly missense mutations) each occurred in 5% of all cancer patients and were significantly mutually enriched (VCAN in KRAS and IKBKB in VCAN mutations), suggesting mutual toxicity (Figure S16A-C).In addition, the alteration frequencies of KRAS, IKBKB, and VCAN across 32 human cancer types were closely correlated, with the highest in LUAD, COAD / READ, and uterine endometrial carcinoma (UCEC), cancers that commonly cause MPE (Figure S16D). In the latter tumor types characterized by alteration frequencies of KRAS, IKBKB, and VCAN, the addiction of IKBKB and VCAN mutations persisted, and patients with cancers with alterations in VCAN and / or IKBKB exhibited weight loss (cachexia), higher mutation burden, microsatellite instability, and hypoxia indices (Figures S17A-C). Collectively, these data support that tumor cell VCAN cooperates with myeloid IKKβ in mouse and human cancers.

[0197] Non-oncogene addiction of KRAS mutant tumors to IL-1B is actionable To block the proposed inflammatory loop, we used the novel IL-1 receptor antagonist, isunakinra (30). Systemic delivery of isunakinra to mice bearing established tumors specifically inhibited sc growth of KrasMUT tumors (Figure 6A). In addition, isunakinra limited nuclear factor (NF)-κB activation in KrasMUT cancer cells in vivo, a phenomenon we have previously shown to be driven by myeloid IL-1β and their ability to induce lethal MPE (Figure 6B-D). As VCAN is a known TLR2 ligand (23), the proinflammatory loop proposed here was also targeted with the TLR1 / 2 inhibitor Cu-CPT22 (31). This drug effectively inhibited VCAN-induced NF-κB activation and cell survival in RAW264.7 macrophages in the low micromolar range and blocked tumor growth in vivo at clinically relevant concentrations (Figure 6E-H). Thus, VCAN-IKKβ-mediated addiction of KRASMUT cancers to host IL-1β can be used to indirectly target these tumors.

[0198] Consideration Here, we show how KRAS mutant tumors are dependent on IL-β provided by tumor-associated macrophages. Importantly, we show that tumor-secreted versican triggers IKKβ activation in myeloid cells to drive this proinflammatory circuit. And, despite cancers with other mutations that can drive tumors with IL-1β, as well as other myeloid cells such as neutrophils and mast cells, we define here a non-oncogene addiction of KRAS and IL-1β in parallel with their partners in crime VCAN and IKKβ. This finding highlights the need for molecular stratification of current clinical trials of IL-1β inhibition for lung cancer. A unique experimental model for the study of tumor genome-host immune interactions is provided, and novel diagnostic platforms and prognostic biomarkers are described for further validation.

[0199] Although sotorasib was recently approved in the United States for KRASG12C-mutated NSCLC (32), KRAS mutant cancers from multiple sites of origin remain notoriously aggressive and undruggable (33), and direct KRAS inhibition is associated with some toxicity that likely renders such treatment unsuitable for chemoprevention (34). In contrast, based on their excellent safety profile, anti-IL-1β-directed therapies have potential for chemoprevention, as shown by the CANTOS study (2). We identify cancer cell VCAN and myeloid IKKβ as associates of KRAS that induce the secretion of IL-1β in the setting of KRAS mutant cancers. The results position these cancers as favorable candidates for anti-IL-1β therapy and versican as a diagnostic and prognostic biomarker and therapeutic target, alone or in combination with anti-IL-1β agents, in this tumor category that comprises 9% of all human cancers.

[0200] NF-κB signaling in cancer and myeloid cells influences the mode of tumor progression and metastasis in various tumor types and is closely dependent on oncogenic KRAS signaling (35, 36). However, lessons learned from clinical trials of proteasome (and therefore also canonical NF-κB pathway) inhibitors for multiple myeloma indicate that therapeutic intervention in the NF-κB pathway is also associated with significant toxicity because the pathway acts simultaneously in epithelial and immune cells in opposing modes (37, 38). In addition to previous studies elucidating the oncogenic functions of IKKβ in tumor cells (5, 10, 16, 29, 35, 36, 39), here we show how myeloid IKKβ functions to promote tumor cell NF-κB signaling by IL-1β, further highlighting the complex and multifaceted protumor functions of NF-κB and the need for its therapeutic targeting in cancer.

[0201] In conclusion, KRAS mutant cancers depend on host IL-1β, which they induce from host macrophages via secreted versican that activates myeloid IKKβ. This inflammatory loop offers multiple opportunities to improve the diagnosis, prognosis, and identification of therapeutic vulnerabilities of KRAS mutant cancers.

[0202] Materials and Methods Approval for mouse and human studies All mice used in these studies were bred at the Faculty of Medicine, University of Patras, Greece. The experiments were prospectively approved by the Veterinary Administration of the Prefecture of Western Greece (approval number 276134 / 14873 / 2) and performed in accordance with the European Union Directive 2010 / 63 / EU (https: / / eur-lex.europa.eu / legalcontent / EN / TXT / ?uri=celex%3A32010L0063). Male and female experimental mice were matched for sex, weight (20–25 g), and age (6–12 weeks). Exact sample sizes (n) are included in the figures and their legends. Animals were assigned to experimental groups by randomization (if n ≥ 20) or alternation (if n < 20), control and experimental mice were always litters, and transgenic animals were enrolled by case-control. Data were collected by at least two blinded investigators from specimens coded by an unblinded investigator.The Munich lung adenocarcinoma and Patras pleural effusion (13, 28) clinical studies were conducted in accordance with the Declaration of Helsinki (https: / / www.wma.net / policies-post / wma-declaration-of-helsinkiethical-principles-for-medical-research-involving-human-subjects / ), approved by the Ludwig-Maximilians-University Munich Ethics Committee (approval number 623-15) and the University of Patras Ethics Committee (approval number 22699 / 21.11.2013), and published in the German Clinical Trials Register (Deutsches Register Klinischer The trials were registered with ClinicalTrials.gov (pleural effusion used to diagnose cancer; NCT03319472; https: / / clinicaltrials.gov / ct2 / show / NCT03319472?term=NCT03319472&rank=1), respectively, and prior written informed consent was obtained from all patients.

[0203] reagent D-luciferin potassium salt [(S)-4,5-dihydro-2-(6-hydroxy-2-benzothiazolyl)-4-thiazole carboxylic acid potassium salt, Chemical Abstracts Service number, CAS number 115144-35-9] was from Biosynth (Lake Constance, Switzerland). Clodronate (dichloromethylene diphosphonic acid disodium salt, CAS number 22560-50-5) and Hoechst 33258 nuclear dye (CAS number 23491-45-4) were from Sigma-Aldrich (St. Louis, MO). Egg-phosphatidylcholine (CAS number 97281-44-2) was from Avanti Polar Lipids (Alabaster, AL). Lentiviral shRNA, puromycin (CAS number 58-58-2) and lipopolysaccharide (LPS; catalog number sc-3535) were from Santa Cruz (Dallas, TX). Geneticin (G418; catalog number 10131035) was from Thermo Fisher Scientific (Waltham, MA). Recombinant human active versican (VCAN; catalog number RPB817Mu01) and osteopontin (secreted phosphoprotein 1, SPP1; catalog number APA899Hu61) were from Cloud-Clone Corp (Houston, TX), and all other recombinant proteins were from Immunotools (Friesoythe, Germany). Bortezomib (CAS number 179324-69-7) was from Selleckchem (Houston, TX). IL-1β ELISA (cat. no. 900-K47) was from Peprotech (London, UK). Primers were from VBC Biotech (Vienna, Austria).Isunakinra (EBI-005), a recombinant protein that binds to interleukin-1 receptor 1 (IL1R1) and potently blocks IL-1α and IL-1βbeta (30), was from Buzzard Pharmaceutical (Stockholm, Sweden), and the TLR1 / TLR2 antagonist Cu-CPT22 or 3,4,6-trihydroxy-2-methoxy-5-oxo-5H-benzocycloheptene-8-carboxylic acid hexyl ester (CAS number 1416324-85-0) (31) was from Merck (Darmstadt, Germany). Primer and lentiviral shRNA pool sequences are listed in Tables 4 and 5, and antibodies are listed in the respective Methods sections.

[0204] cell Lewis lung carcinoma (LLC, RRID:CVCL_4358), B16F10 cutaneous melanoma (male, RRID:CVCL_0159), and PAN02 pancreatic adenocarcinoma cells (male, RRID:CVCL_D627) were from the National Cancer Institute Tumor Repository (Frederick, MD). RAW264.7 murine myelomonocytic leukemia (male, RRID:CVCL_0493) and murine lung epithelial 12 (MLE12, female, RRID:CVCL_3751) cells were from ATCC (Manassas, VA). MC38 colon adenocarcinoma (female, RRID:CVCL_B288) and AE17 mesothelioma (female, RRID:CVCL_4408) cells were kindly provided by Dr. Barbara Fingleton (Vanderbilt University, Nashville, TN) and Dr. YC Gary Lee (University of Western Australia, Perth, Australia), respectively. FVB urethane-induced lung adenocarcinoma (FULA1) cells were produced in our laboratory (female, RRID:CVCL_A9KV). Cells were cultured at 37°C in 5% CO2-95% air using Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM pyruvate, 100 U / mL penicillin, and 100 mg / mL streptomycin. For in vivo injections, cells were trypsinized, incubated with trypan blue, and counted in a grid hemocytometer according to the Neubauer method, and only 95% of viable cells were used for the experiments. All in vitro experiments were repeated at least three times independently, and the stated n always reflects the biological sample size, not the technical specimen size.All cell lines were repeatedly resequenced for Kras mutations, their status was confirmed to be the same as previously reported, and tested annually for identification by short tandem repeats by PCR using primers GGGAGCAAACAGGATTAGATACCCT (SEQ ID NO: 14) and TGCACCATCTGTCACTCTGTTAACCTC (SEQ ID NO: 15) (amplicon size 270 bp) and for Mycoplasma Spp. ( 9 , 10 , 15 , 22 , 29 , 40 ).

[0205] Experimental mice NGL and HLL NF-κB reporter mice have been described elsewhere ( 20 , 21 , 39 ). Mice were obtained from Jackson Laboratories (Bar Harbor, MN): wild-type (WT) C57BL / 6J mice (C57BL / 6; no. 000664), B6.129(Cg)-Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo / J double-membrane fluorescent Cre-recombinase reporter mice (mT / mG; no. 007676) ( 41 ), and mice expressing Cre-recombinase under the control of the Lyz2 promoter. These mice were B6.129P2-Lyz2tm1(cre)Ifo / J mice (Lyz2.Cre; no. 004781) expressing diphtheria toxin by Cre-mediated recombination resulting in cell suicide (22, 43), and B6;129S-Tnftm1Gkl / J Tnfdeficient mice (Tnf- / -; no. 005540) (10, 44). B6.B4B6-Chuk harboring conditional Chuk and Ikbkb alleles deleted by Cre recombinase expression. <tm1mpa> / Cgn (Chukf / f) and B6.B4B6-Ikbkb<tm2.1Mpa> / Cgn(Ikbkbf / f) mice (45, 46), as well as Il1btm1Yiw Il1b-deficient mice (Il1b- / -; MGI no. 215739631) (14), and Cpa3.Cre+ / - mast cell-deficient mice (Cpa3.Cre), in which mast cells undergo Trp53-mediated apoptosis (47), have been described elsewhere and were kindly donated by their founders. All mice used in these studies were derived or backcrossed onto a C57BL / 6 background from generations beyond F12. For these studies, n = 929 mice were used.

[0206] Mouse tumor models For generation of solid tumors, mice were inoculated with 5 × 10 cells in 100 μl of phosphate-buffered saline (PBS) as described elsewhere. 5 Tumor cells were injected subcutaneously (sc) into the shaved hind flank dermis (9, 10, 15, 29). Mice were examined weekly for tumor volume (V) by measuring three perpendicular tumor diameters (d1, d2, d3) using the formula V = π*d1*d2*d3 and were sacrificed when the tumor volume reached 1 cm3 (PANO2 cells) or 2 cm3 (all other cell lines). For induction of malignant pleural effusion (MPE), mice were injected with 2 × 106 cells suspended in 100 μL of PBS. 5 Mice received an intrapleural injection of cancer cells and were sacrificed when they showed signs of disease or at the indicated time points (14–28 days after tumor cell delivery, depending on the cell line used) (9, 10, 15). In all models, both mice and inoculated cancer cells were always syngeneic to avoid inflammatory allograft rejection and artificial NF-κB activation.

[0207] Bioluminescence and Biofluorescence Imaging Mice were imaged for NF-κB reporter bioluminescence signal daily, starting from day 10 after tumor cell injection, until sacrifice. For this, mice were anesthetized by isoflurane inhalation and imaged for bioluminescence on a Xenogen Lumina II (Perkin-Elmer, Waltham, MA) 5–20 min after delivery of 1 mg of D-luciferin potassium salt diluted in 100 μL of sterile water into the retro-orbital vein. Pleural tumors isolated from NGL mice were also imaged ex vivo for green biofluorescence on a Xenogen Lumina II using background control excitation of 410–440 nm, experimental excitation of 445–490 nm, and emission passbands of 515–575 nm. Cells were imaged for bioluminescence on a Xenogen Lumina II at 0, 4, 8, 16, and 24 h after a single addition of 300 μg / mL (equivalent to 1 mM) D-luciferin to the culture medium. Data were analyzed using Living Image v.4.2 (Perkin-Elmer, Waltham, MA) as previously described ( 9 , 10 , 15 , 15 , 22 , 29 , 39 ).

[0208] Sequencing Genomic DNA was extracted from cell lines using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). Kras exons 1-3 were amplified by PCR using Phusion Polymerase (New England Biolabs, Ipswich, MA) and an annealing temperature of 60°C. Primers are listed in Table 4. PCR products were analyzed on a 1% agarose gel, purified by QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), and sequenced by Eurofins Genomics (Ebersberg, Germany).

[0209] Constructs and transfection Control shRNA (shC, sc-108080-V; target sequence is proprietary to the manufacturer) and anti-mouse Vcan shRNA (shVcan, sc-41904-V) pools were from Santa Cruz. pNGL constructs and lentiviral shRNA pools for silencing Kras, Chuk, Ikbkb, Ikbke, and Tbk1 have been described previously (10). Lentiviral shRNA catalog numbers and target sequences are listed in Table 5. For stable plasmid transfection, 105 RAW264.7 cells were transfected with 5 μg of DNA using Xfect (Takara, Mountain View, CA) followed by selection with G418 (400-800 μg / mL). For stable shRNA transfection, 105 tumor cells were transfected with lentiviral particles and clones were selected with puromycin (2-10 μg / mL) (9, 10).

[0210] Intrapleural Catheter For in vivo MPE drainage, a 1.2 cm long catheter with serial fenestrations spaced 1 mm apart was used, following a detailed model description previously reported (48). Mice were anesthetized using isofluorane, the catheter insertion site was shaved and disinfected using 70% ethanol and 10% povidone iodide, and the catheter was then attached to the pleural cavity and sutured under the skin. Mice were imaged before and after MPE drainage and then sacrificed.

[0211] cytology MPE fluids were treated with red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) and MPE cells were centrifuged and stained with May-Grunwald-Giemsa. Slides were then mounted with Entellan (Merck Millipore, Darmstadt, Germany) and analyzed microscopically for differential counts of pleural cells. Pleural lavage was performed by injecting 1 ml of saline into the pleura and withdrawing it after 30 seconds. Pleural cells were enumerated in a hemocytometer, centrifuged, stained with May-Grunwald-Giemsa or anti-rabbit F4 / 80 antibody (ab111101; Abcam, London, UK; RRID:AB_10859466) and hematoxylin, and analyzed microscopically for differential counts of pleural cells.

[0212] Flow cytometry Pleural fluid cells were treated with red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA), counted, and 0.5–1.0 × 106 cells were processed for antibody staining. Pleural tumors were dissociated using a 70 μm strainer (BD Bioscience, San Jose, CA), counted, and 0.5–1.0 × 106 cells were processed for antibody staining. BMDMs were counted and 0.5–1.0 × 106 cells were processed for antibody staining. All samples were suspended in 50 μL of PBS containing 2% FBS and 0.1% NaN3 and stained with the following antibodies at a concentration of 0.1 μg / 106 cells for 20 min in the dark: anti-CD45 (11-0451-85; eBioscience, Santa Clara, CA; RRID:AB_465051), anti-CD11b (12-0112-82; eBioscience; RRID:AB_2734869), anti-Ly6C (45-5932-82; eBioscience; RRID:AB_2723343), anti-F4 / 80 (123128; Biolegend, San Diego, CA; RRID:AB_893484), anti-Ly6G (127624; Biolegend; AB_10640819), and anti-GFP. eFluor® 660 (50-6498-82; eBioscience; RRID:AB_11043268), anti-MHC class II (17-5321; eBioscience; RRID:AB_469454), Alexa Fluor® 647 anti-CD206 (141712; Biolegend; RRID:AB_10900420), biotinylated anti-firefly luciferase (ab634; Abcam, London, UK; RRID:AB_305434), and streptavidin (17-4317-82; eBioscience).Samples were analyzed on a CyFlowML flow cytometer using FloMax Software (Partec, Darmstadt, Germany; RRID:SCR_014437), Flowing Software v.2.5.1 (http: / / flowingsoftware.btk.fi / ; RRID:SCR_015781) and FlowJo Software v10.6.2 (BD Bioscience, San Jose, CA; RRID:SCR_008520).

[0213] immunohistochemistry For dark-field immunofluorescence, pleural tumors were fixed overnight in 4% paraformaldehyde at 4°C, cryoprotected in 30% sucrose, embedded in Tissue-Tek (Sakura, Tokyo, Japan), and stored at -80°C. 10 μm frozen sections were then fixed in 4% paraformaldehyde for 10 min, treated with 0.3% Triton X-100 for 5 min, blocked in 1× phosphate-buffered saline (PBS) containing 10% fetal bovine serum (FBS), 3% bovine serum albumin (BSA), and 0.1% Tween 20 for 1 h, and then incubated overnight at 4°C with the indicated primary antibodies. Sections were then treated with fluorescent secondary antibodies, counterstained with Hoechst33258 (CAS number 23491-45-4), and mounted with Mowiol 4-88 (Calbiochem, Darmstadt, Germany; CAS number 9002-89-5). The following primary antibodies were used: mouse anti-GFP (1:200 dilution; sc-9996; Santa Cruz, Dallas, TX; RRID:AB_627695), rat anti-CD68: Alexa Fluor® 488 (MCA1957A488T; AbD Serotec, Kidlington, UK; RRID:AB_1102282), mouse anti-CD45 FITC (11-0451-85; eBioscience; RRID:AB_465051), and rabbit anti-PCNA (1:3000 dilution; ab18197; Abcam, London, UK; RRID:AB_444313). Alexa Fluor donkey anti-mouse 488 (A21202; RRID:AB_141607), Alexa Fluor goat anti-rat 568 (A11077; RRID:AB_141874), and Alexa Fluor donkey anti-rabbit 568 (A10042; RRID:AB_2534017) secondary antibodies used at 1:500 dilution were from Thermo Fisher Scientific (Waltham, MA). For isotype controls, primary antibodies were omitted. Fluorescence microscopy was performed on either an AxioObserver D1 inverted fluorescence microscope (Zeiss, Jena, Germany) or a TCS SP5 confocal microscope (Leica, Wetzlar, Germany) using 20x, 40x, and 63x lenses.Digital images were processed with Fiji academic freeware (RRID:SCR_002285) (49). All quantifications of cell populations were obtained by counting at least five random non-overlapping tumor-containing fields per section. Bright-field immunohistochemistry was performed as previously described (9,22,29) using the following antibodies: rabbit anti-versican (1:100; E-AB-36300; Elabscience, Wuhan, China), mouse secondary anti-rabbit (1:5000; ab191866; Abcam, London, UK; RRID:AB_2650595). All quantifications of cell populations were obtained by counting at least five random non-overlapping tumor-containing fields per section.

[0214] Bone Marrow Transplantation (BMT) and Liposomal Clodronate In adoptive BMT experiments described in detail elsewhere (9, 10, 15), wild-type (WT) and NF-κB.eGFP.LUC (NGL) recipient mice on a C57BL / 6 background received total body irradiation (1100 Rad) followed 12 h later by intravenous (via retro-orbital injection) 107 total bone marrow cells obtained from WT and NGL donors. One irradiated mouse per group did not receive BMT to control for effective ablation of endogenous bone marrow and died 5–15 days after radiation. One month later, allowing complete bone marrow reconstitution by chimeric bone marrow cells, liposomal clodronate was prepared as previously described (21, 50) and 500 μg was administered intrapleurally. Another month was required for replacement of pleural bone marrow cells by transplanted bone marrow cells (50), after which mice were injected with tumor cells.

[0215] Bone marrow derived macrophages (BMDM) For BMDM generation, 107 bone marrow cells were plated and cultured for 7 days in the presence of 100ng / mL macrophage colony-stimulating factor (M-CSF). Optionally, recombinant human versican (1nM) was added to the culture medium on day 6 of culture, or alternatively, the culture medium was removed and BMDMs were exposed to cancer cell-conditioned medium for 4 hours. Culture supernatants were then isolated for ELISA, and cells were processed for Western blot, flow cytometry, or qPCR.

[0216] Immunoblotting Nuclear and cytoplasmic protein extracts were prepared using the NEPER Extraction Kit (Thermo Fisher Scientific, Waltham, MA), separated by SDS-PAGE, and electroblotted onto PVDF membranes (Merck Millipore, Darmstadt, Germany). The membrane was incubated with the following primary antibodies: anti-IKKα (1:1000 dilution; 2682; Cell Signaling, Danvers, MA; RRID:AB_331626), anti-IKKβ (1:1000 dilution; 2684; Cell Signaling;RRID:AB_2122298), anti-VCAN (1:200 dilution;ab19345;Abcam, London, UK;RRID:AB_444865), anti-β-actin (1:500 dilution;sc-47778;Santa Cruz, Dallas, TX; RRID:AB_2714189) and anti-α-tubulin (TUBA; 1:4000 dilution; T5168; Sigma Aldrich, St. Louis, MO; RRID:AB_477579) followed by incubation with secondary goat anti-mouse (1:8000 dilution; 1030-05; Southern Biotech, Birmingham, AL; RRID:AB_2619742) or goat anti-rabbit (1:8000 dilution; 4030-05; Southern Biotech; RRID:AB_2687483) HRP-conjugated antibodies. Membranes were visualized by chemiluminescence film exposure after incubation with enhanced chemiluminescence substrate (Merck Millipore, Darmstadt, Germany).

[0217] qPCR and microarrays 10 6 Triplicate cultures of cells were subjected to RNA extraction using Trizol (Thermo Fisher Scientific, Waltham, MA) followed by column purification and DNA removal (RNeasy Mini Kit, Qiagen, Hilden, Germany). Pooled RNA (5 μg) was quality tested on an ABI 2000 bioanalyzer (Agilent Technologies, Sta. Clara, CA), labeled, and hybridized to GeneChip Mouse Gene 2.0 ST arrays (Affymetrix, Sta. Clara, CA). All data were analyzed on the Affymetrix Expression and Transcriptome Analysis Console (RRID:SCR_018718). RNA was reverse transcribed using Superscript III (Thermo Fisher Scientific) and qPCR was performed using first strand synthesis and SYBR FAST qPCR Kit (Kapa Biosystems, Wilmington, MA) in a StepOne cycler (Applied Biosystems, Carlsbad, CA). Primers for qPCR are listed in Table 4. Ct values ​​from triplicate reactions were analyzed using the relative quantification method 2-ΔCT compared to mouse Gusb or human ACTB transcripts (51).

[0218] Shotgun Proteomics Mouse KrasMUT (LLC, MC38, AE17) and KrasWT (B16F10 and PANO2) cell cultures (pooled triplicate cultures for each cell line; 5 million cells / 175 cm 2 Supernatants from culture flasks / 24 h in complete DMEM followed by 24 h in FBS-free DMEM were analyzed. For this, 600 μL of cell culture supernatant was enzymatically digested using a modified filtration-assisted sample preparation (FASP) protocol (52, 53). Peptides were stored at -20 °C until mass spectrometry (MS) measurements. MS data were acquired in data-dependent acquisition (DDA) mode on a Q Exactive (QE) high-field (HF) mass spectrometer (Thermo Fisher Scientific). Approximately 0.5 μg per sample was automatically loaded onto an online-coupled RSLC (Ultimate 3000, Thermo Fisher Scientific) HPLC system. A Nanotrap column was used (300 μm inner diameter (ID) × 5 mm, Acclaim PepMap100 C18, 5 μm, 100 Å (LC Packings (Sunnyvale, CA). Peptides were eluted with a 95 min gradient from 3% to 40%. MS spectra were acquired in the mass range of 300–1500 m / z at a resolution of 60000 with an AGC set to 3 × 106 and an IT of up to 50 ms. From the MS prescan, the 10 most abundant peptide ions were selected for fragmentation (MSMS) with dynamic exclusion of 30 s if at least doubly charged. MSMS spectra were recorded at a resolution of 15000 with an AGC set to 1 × 105 and an IT of up to 100 ms. CE was set to 28 and all spectra were recorded with a profile type. Peptides were analyzed using Sequest HT (as node for PD) and searching against the UniProtKB / Swiss-Prot mouse database (publication 2017_2, 16872 sequences) using Proteome discoverer (version 2.3; Thermo Fisher Scientific). Label-free quantification of DDA-MS data was performed using the Quantitative Analysis of Fluorescence Spectroscopy (Quantitative Analysis of Fluorescence Spectroscopy) Scientific. Searches were performed with a precursor mass tolerance of 10 ppm and a fragment mass tolerance of 0.02 Da.Carbamidomethylation (C) was set as a static modification, while deamidation (N, Q), oxidation (M), and N-terminal Met loss + acetyl were selected as dynamic modifications, with two missing cleavages allowed. Percolator (54) was used to validate peptide-spectrum matches and peptides, accepting only the top-scoring hits for each spectrum and fulfilling cutoff values ​​for FDR < 1% and posterior error probability < 0.01. The final list of proteins adhered to a strict parsimony principle. Protein quantification after precursor recalibration was based on abundance values ​​(area under the curve) for unique peptides. Abundance values ​​were normalized in a retention-time-dependent manner. Protein abundance was calculated by summing abundance values ​​for accepted peptides. Comparison between KrasMUT (LLC, MC38, AE17) and KrasWT (B16F10 and PANO2) cell lines was performed using only proteins detected in all five cell lines.

[0219] Cell treatment Cells were exposed to tumor-conditioned medium diluted 1:1 in DMEM. Bortezomib pretreatment was applied 1 h prior to exposure to conditioned medium at 1 μg / mL (equivalent to 3 μM). Cells were exposed to potential NF-κB ligands at the following concentrations: lipopolysaccharide, LPS, 1 μg / mL (equivalent to 10-20 nM); secreted phosphoprotein 1, SPP1, 100 ng / mL (equivalent to 1.25-2.5 nM); tumor necrosis factor, TNF, 20 ng / mL (equivalent to 1 nM); versican, VCAN, 360 ng / mL (equivalent to 1 nM); interleukin (IL)-1β, 30 ng / mL (equivalent to 1 nM); and CC motif chemokine ligand 2, CCL2, 20 ng / mL (equivalent to 1.5 nM) and after 4 h were imaged for bioluminescence or processed for other assays. pNGL RAW264.7 macrophages were exposed to 1 nM VCAN and then treated with increasing concentrations of the TLR1 / TLR2 antagonist Cu-CPT22.

[0220] Mouse treatment The IL-1 receptor antagonist Isunakinra (30) was given by daily intraperitoneal injection of 20-50 mg / Kg of drug diluted in 100 μL of PBS. Treatment was initiated 10-17 days after sc tumor cells or 5 days after intrapleural tumor cells to allow efficient tumor uptake and therapeutic study design. Treatment with the TLR1 / TLR2 antagonist Cu-CPT22 (31) was initiated 3 days after intrapleural cancer cell injection and consisted of daily intraperitoneal injections of 20 mg / kg Cu-CPT22 diluted in 100 μl of corn oil 10% DMSO or 100 μl of corn oil 10% DMSO.

[0221] Data availability Survival data were obtained from the Kaplan-Meier Plotter pan-cancer RNA-seq dataset (https: / / kmplot.com / analysis / ) using the search term VCAN. TCGA pan-cancer data were downloaded from https: / / www.cbioportal.org / .

[0222] Transcription factor binding site analysis We downloaded RELA and RELB binding sequence motifs from the ENCODE portal (https: / / www.encodeproject.org / ) using the identifiers: ENCFF507YCV (CHIP-seq on HuH-7.5 cells) and ENCFF615HZF (CHIP-seq on 8988T cells), respectively, and queried the ChIPseq dataset from the ChIP-X Enrichment Analysis (CHEA) transcription factor target dataset (https: / / maayanlab.cloud / Harmonizome / dataset / CHEA+Transcription+Factor+Targets) (55, 56).

[0223] statistics Sample sizes were calculated using power analysis with G*power (57), assuming α=0.05, β=0.05, and an effect size of d=1.5. No data were excluded from the analysis. Pooled data from replicate in vivo experiments are shown. All in vitro experiments were repeated at least three times independently, and the n stated always reflects the biological sample size, not the technical sample size. Animals were assigned to treatments by randomization (if n ≥ 20) or alternation (if n < 20), and transgenic animals were enrolled by case-control. Data were collected by at least two blinded investigators from samples coded by an unblinded investigator. All data were tested for normality of distribution by the Kolmogorov-Smirnov test and are given as violin plots or mean ± SD, and sample sizes (n) always refer to biological replicates, not technical replicates. Differences in frequencies were examined by Fisher's exact test and χ2 test in medians by Mann-Whitney or Kruskal-Wallis tests with Dunn's post-hoc test, and in means by t-test or one-way ANOVA with Bonferroni post-hoc test. Changes over time and interactions between two variables were examined by two-way ANOVA with Bonferroni post-hoc test. Hypergeometric tests were performed at the Graeber Lab website (https: / / systems.crump.ucla.edu / hypergeometric / index.php). All probability (P) values ​​were two-sided and considered significant when P<0.05. All analyses and plots were performed in Prism v8.0 (GraphPad, La Jolla, CA; RRID:SCR_002798).

[0224] Table 1. Differential gene expression of BMDM-specific transcripts following incubation with tumor-conditioned medium. As assessed by microarray (Mouse Gene ST2.0, Affymetrix, Sta. Clara, CA), the transcript was statistically significantly (overall ANOVA P<0.05) over-represented by >5-fold in bone marrow-derived macrophages (BMDMs) and induced by >5-fold in BMDMs compared with MC38 and LLC cancer cells after incubation with CM. Note the significant induction of Il1b (shaded grey). The microarrays compared were GSM3744950, GSM3744952, GSM3744954, GSM3744955, and GSM3744958 (cancer cells) versus GSM2486425, GSM2487750, GSM3744964, GSM3752400, and GSM2486426 (naive BMDMs) versus GSM3752396, GSM3752397, GSM3752398, GSM3752399, and GSM2487751 (tumor-adapted BMDMs). [Table 3]

[0225] Table 2. Differential gene expression in Kras mutant cancer cells. As assessed by microarrays (Mouse Gene ST2.0, Affymetrix, Sta. Clara, CA), transcripts were over-represented by more than 10-fold in MC38, LLC, and AE17 cells compared to PANO2 and B16F10 cells. Only Nid1 and Can (shaded grey) were also identified by proteomic screening of tumor cell supernatants. Microarrays compared were GSM3744958 and GSM3752395 (Kras wild-type cancer cells) versus GSM3744954, GSM3744950, and GSM3744952 (Kras mutant cancer cells). [Table 4]

[0226] Table 3. Differential gene expression in BMDMs lacking NF-κB signaling. As assessed by microarrays (Mouse Gene ST2.0, Affymetrix, Sta. Clara, CA), transcripts were under-represented by >5-fold in bone marrow-derived macrophages (BMDMs) from Lyz2.Cre;Chukf / f and Lyz2.Cre;Ikbkbf / f ​​mice compared to Lyz2.Cre controls. Note the significant downregulation of Lyz2 and Il1b. Microarrays compared were GSM2486425 (pooled BMDMs from Chukf / f, Ikbkbf / f, and Lyz2.Cre mice) vs. GSM3752396 (BMDMs from Chukf / f;Lyz2.Cre mice) vs. GSM3752397 (BMDMs from Ikbkbf / f;Lyz2.Cre mice). [Table 5] [Table 6] [Table 7]

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Claims

1. 1. An inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling for use in treating cancer in a subject, wherein the cancer comprises a KRAS mutation and elevated levels and / or activity of veriscan (VCAN).

2. The inhibitor for use according to claim 1, wherein the cancer is determined to contain a KRAS mutation.

3. The inhibitor for use according to claim 1 or 2, wherein the cancer comprises elevated levels of VCAN mRNA and / or VCAN protein.

4. The inhibitor for use according to any one of claims 1 to 3, wherein the elevated level and / or activity of VCAN is a level and / or activity of VCAN that is elevated relative to the level and / or activity of VCAN in a reference sample.

5. The inhibitor for use according to any one of claims 1 to 4, wherein the cancer comprises elevated levels and / or activity of IL-1β.

6. The inhibitor for use according to claim 5, wherein the cancer comprises elevated levels of IL-1β mRNA and / or IL-1β protein.

7. The inhibitor for use according to claim 5 or 6, wherein the elevated level and / or activity of IL-1β is elevated relative to the level and / or activity of IL-1β in a reference sample.

8. The inhibitor for use according to claim 4 or 7, wherein the reference sample is a non-cancerous sample.

9. The inhibitor for use according to claim 8, wherein the non-cancerous sample is a sample comprising non-cancerous cells or tissues derived from a subject.

10. The inhibitor for use according to claim 8 or 9, wherein the non-cancerous samples are derived from the same subject or different subjects.

11. The inhibitor for use according to any one of claims 1 to 10, wherein the cancer is selected from the group comprising lung cancer (such as non-small cell lung cancer (NSCLC)), kidney cancer, skin cancer, thymus cancer, breast cancer, pancreatic cancer, thyroid cancer, bladder cancer, liver cancer, cervical cancer, endometrial cancer, colorectal cancer, and gastric cancer.

12. The inhibitor for use according to any one of claims 1 to 11, wherein the cancer is selected from the group comprising lung adenocarcinoma (LUAD), colon adenocarcinoma (COAD), rectal adenocarcinoma (READ), and uterine endometrial carcinoma (UCEC).

13. 13. The inhibitor for use according to any one of claims 1 to 12, wherein said inhibitor is selected from the group comprising peptide IL-1 receptor antagonists, nucleotide IL-1 receptor antagonists, peptide fragments of IL-1R1, anti-IL-1 antibodies, decoy IL-1 receptor (optionally soluble IL-1 receptor or IL-1 TRAP).

14. 14. The inhibitor for use according to any one of claims 1 to 13, wherein the inhibitor is at least 90% identical to a sequence selected from any one of SEQ ID NO: 1 (P01), SEQ ID NO: 2 (P02), SEQ ID NO: 3 (P03), SEQ ID NO: 4 (P04), and SEQ ID NO: 5 (P05).

15. The inhibitor for use according to any one of claims 1 to 14, wherein the inhibitor is Isunakinra.

16. The inhibitor for use according to any one of claims 1 to 15, wherein the subject is also administered an inhibitor of VCAN.

17. The inhibitor for use according to claim 16, wherein the inhibitor of VCAN is a toll-like receptor 1 / 2 (TLR1 / 2) inhibitor.

18. The inhibitor for use according to any one of claims 1 to 17, wherein the subject is also administered one or more chemotherapeutic agents.

19. 1. Use of an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling in the manufacture of a medicament for treating cancer in a subject, wherein the cancer comprises a KRAS mutation and elevated levels and / or activity of veriscan (VCAN).

20. 1. A method of treating cancer in a subject, wherein the cancer comprises a KRAS mutation and elevated levels and / or activity of veriscan (VCAN), the method comprising administering to the subject an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

21. 1. A method for selecting a subject having cancer, the cancer being predicted to be therapeutically responsive to a treatment comprising an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling, the method comprising: d) determining whether the cancer contains a KRAS mutation; e) determining whether the cancer has elevated levels and / or activity of veriscan (VCAN); wherein, if the cancer from the subject comprises a KRAS mutation and has elevated levels and / or activity of VCAN, the subject is selected as having a cancer that is predicted to respond therapeutically to a treatment comprising an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

22. 22. The method of claim 21, wherein determining whether the cancer has an elevated level and / or activity of VCAN comprises determining whether the level and / or activity of VCAN in the cancer is elevated relative to the level and / or activity of VCAN in a reference sample.

23. 23. The method of claim 21 or 22, further comprising the step of: f) determining whether the cancer has elevated levels and / or activity of IL-1β, wherein if the cancer from the subject comprises a KRAS mutation, elevated levels and / or activity of VCAN, and has elevated levels and / or activity of IL-1β, the subject is selected as having a cancer that is predicted to respond therapeutically to a treatment comprising an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

24. 24. The method of claim 23, wherein determining whether the cancer has elevated levels and / or activity of IL-1β comprises determining whether the level and / or activity of IL-1β in the cancer is elevated relative to the level and / or activity of IL-1β in a reference sample.

25. 25. The method of any one of claims 21 to 24, wherein any of steps (a), (b), and (c) are performed in vitro and / or on a sample provided by the subject.

26. 26. The method of any one of claims 21 to 25, further comprising treating the subject selected as having a cancer predicted to be therapeutically responsive to a treatment comprising an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling with an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

27. Use of veriscan (VCAN) as a biomarker to determine whether a subject with a cancer containing a KRAS mutation is suitable for treatment with an inhibitor of interleukin-1 receptor type 1 (IL-1R1) signaling.

28. A kit comprising: (i) a reagent for detecting the presence of a KRAS mutation; (ii) a reagent for determining the level and / or activity of VCAN.

29. 29. The kit of claim 28, further comprising (iii) a reagent for determining the level and / or activity of IL-1β.

30. 10. An inhibitor for use, use, method or kit substantially as herein described with reference to the accompanying description and / or drawings.

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