Methods for treating patients with solid tumors of indeterminate clonal hematopoietic potential - Patent Application 20070223333

Abstract

The present invention relates to a method for treating solid cancers in patients with clonal hematopoiesis of indeterminate potential (CHIP), which introduces a therapeutic intervention with dapanstril to improve the treatment of solid cancers, such as breast cancer, in CHIP patients. The method includes first detecting the presence or absence of Tet2 or DNMT3A mutations in a patient's biological sample to determine whether the patient has a CHIP condition, and then administering a therapeutic intervention with dapanstril to patients with the CHIP condition. The present application shows the role of CHIP in the progression of breast cancer. The present data provides a rationale for screening for Tet2 or DNMT3A mutations in breast cancer patients and proposes dapanstril as a therapeutic treatment.

Description

[Technical field]

[0001] The present invention relates to a method for treating solid tumors in patients with clonal hematopoiesis of indeterminate potential (CHIP), comprising first determining whether the patient has the CHIP condition by detecting the presence or absence of a Tet2 or DNMT3A mutation, and then administering therapeutic intervention with dapansutrile to the patient with the CHIP condition. [Background technology]

[0002] As people age, an increasing number of somatic mutations accumulate in tissues. If this process occurs in the hematopoietic system, a significant proportion of circulating blood cells may originate from a single mutated stem cell. This expansion, called "clonal hematopoiesis", is highly prevalent in the elderly population. Clonal hematopoiesis (CH) refers to the gradual selective expansion of hematopoietic stem cells (HSCs) that often carry somatic mutations in one allele of a gene. CH is characterized by the appearance of an excess of blood cells derived from a single clone. Blood cancers such as chronic myeloid leukemia and myelodysplastic syndromes are classic examples of clonal hematopoiesis. However, the same mutations found in these cancers are also found in the majority of healthy elderly individuals.

[0003] Clonal hematopoiesis of potential indeterminate (CHIP) refers to the presence of clonal molecular genetic or cytogenetic changes in blood or bone marrow cells in the absence of evidence of hematologic neoplasms or cytopenias. CHIP is a non-malignant condition characterized by mutations and clonal proliferation of blood cells. The incidence of CHIP increases with age. CHIP is diagnosed when testing of a blood or bone marrow sample shows that blood cells have one of the genetic mutations associated with the condition.

[0004] CHIP is also associated with a higher risk of developing hematologic malignancies and cardiovascular disease, as well as a shorter life expectancy.

[0005] Tumor development is initiated by genomic alterations, such as point mutations, gene deletions, and chromosomal rearrangements, that result in cellular transformation, self-sufficient growth, insensitivity to anti-growth signals, evasion of apoptosis, and unlimited replicative capacity, ultimately leading to tissue invasion and metastasis. However, tumor cell expansion is associated with a complex network of events involving both cancer and non-cancerous cells. Chronic inflammation is a classic example of such a promoting state (1, 2).

[0006] The proinflammatory cytokine IL-1β is a potent mediator of many chronic inflammatory diseases. (3) Consistent with the link between cancer and chronic inflammation, IL-1β has been shown to be overexpressed in several tumors and to function as an inducer of tumor-promoting mechanisms, such as angiogenesis, immunosuppression, tumor-associated macrophage (TAM) recruitment, and metastasis. (4-6)

[0007] Types of breast cancer include, among others, ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), triple-negative breast cancer (TNBC), inflammatory breast cancer (IBC), metastatic breast cancer, and breast cancer during pregnancy. Triple-negative breast cancer tumors are characterized by the absence of estrogen receptors (ER), progesterone receptors (PR), and elevated levels of the human epidermal growth factor receptor 2 (HER2) protein (7).

[0008] NLRP3 (NOD-like receptor family, pyrin domain containing 3), also called NLRP3 or cryopirin, is one of the sensors of the inflammasome, a macromolecular structure involved in the processing of interleukin 1β (IL-1β) and IL-18. NLRP3 senses intracellular danger during intracellular infection (bacterial and viral proteins) or tissue injury (ischemia). Activation of NLRP3 recruits ASC (apoptosis-associated speck-like protein containing a carboxy-terminal caspase recruitment domain) and caspase-1, leading to the formation of the inflammasome and ultimately cell death.

[0009] Dapansutrile, a small synthetic β-sulfonylnitrile molecule, selectively inhibits the NLRP3 inflammasome and has been proven safe when administered orally to healthy subjects (8). [Brief description of the drawings]

[0010] [Figure 1A] Figure 1A shows tumor growth curves of germline Tet2+ / + (wild type) or Tet2+ / - mice orthotopically implanted with E0771 cells. Tet2+ / - mice were orally administered dapanstril (OLT1177®) or a control diet without dapanstril (N=8, two independent experiments). Tet2+ / - breast cancer progression is dependent on NLRP3 activation. [Figure 1B] FIG. 1B shows the mean±SEM of IL-1β production from bone marrow adherent cells isolated from tumor-bearing mice. [Diagram 2] Figure 2: Bone marrow (BM) chimeras were generated by transplanting 75% WT:25% Tet2+ / + or Tet2+ / - mixed BM. BM chimeric mice were orthotopically transplanted with E0771 breast cancer and fed a standard or dapanstril diet. Figure 2 shows tumor growth curves of Tet2+ / + (WT) or Tet2+ / - chimeric mice transplanted with E0771 cells and fed a standard or dapanstril diet (N=10, two independent experiments). [Diagram 3] 3A-3B show the total percentage of donor-derived cells in the peripheral blood (3A) and tumors (3B) of the BM chimeric mice of FIG. [Figure 4] 4A-4B show the frequency of MHC-II+ expression (4A) and gMFI (4B) in tumor-infiltrating granulocytes of the BM chimeric mice of FIG. [Diagram 5] Figures 5A-5B show plasma cytokine production of chemokine CC motif ligand 2 (CCL2, 5A) and keratinocyte chemoattractant (KC, 5B) in the BM chimeric mice of Figure 2. In all figures: ***p<0.001, **p<0.01, *p<0.05.

[0011] Detailed Description Clonal hematopoiesis (CH) is associated with an increase in hematologic malignancies. CH can be defined as a somatic mutation of hematopoietic stem cells, resulting in clonal expansion of bone marrow cells with an increased inflammatory phenotype. Inducing mutations in 10-11 translocation 2 (Tet2) and / or DNA methyltransferase 3a (DNMT3A), genes involved in DNA methylation, leads to clonal expansion. Tet2 is one of three enzymes that catalyze the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and further intermediates, which may ultimately lead to demethylation. The DNMT3A gene provides the instructions to make DNA methyltransferase 3α, which is one of two enzymes responsible for de novo methylation of the fifth position of cytosine bases in DNA, a mark that affects gene expression.

[0012] The inventors have discovered the role of CHIP in the induction of solid tumors, such as breast and prostate cancer. In solid tumor-infiltrating myeloid cells, dysregulated cytokine signaling promotes an immunosuppressive tumor microenvironment (TME). The association of CHIP with solid tumors is that infiltrating myeloid cells appear in the TME, exhibiting an excessive inflammatory response and promoting tumor-promoting signaling. For cancer patients with CHIP, the disease becomes more severe or the patient is at higher risk. In the case of breast cancer, CHIP has a negative effect on all advanced stage breast cancers. This is because as breast cancer progresses, IL-1β increases with the severity of the disease.

[0013] The proinflammatory cytokine IL-1β promotes breast cancer progression and correlates with disease severity. Activation of the NLRP3 inflammasome in myeloid cells is a major driver of IL-1β processing.

[0014] NLRP3 inflammasome activation amplifies the inflammatory response to tissue injury and mediates further damage. Dapanstril is a selective NLRP3 inflammasome inhibitor that reduces inflammation by preventing NLRP3 inflammasome activation.

[0015] Considering the mechanism of action of dapanstril, which prevents the production and / or release of IL-1β and IL-18 and inhibits the formation of the NLRP3 inflammasome in animal and human subjects, the present inventors have discovered a method for introducing dapanstril therapeutic intervention to improve the treatment of solid cancers, such as breast and prostate cancer, in patients with CHIP, which comprises first determining whether a patient is in the CHIP state by detecting the presence or absence of a Tet2 or DNMT3A mutation, and then administering dapanstril therapeutic intervention to patients in the CHIP state.

[0016] Treatment method The present invention relates to a method of treating a solid tumor, such as breast or prostate cancer, in a patient, comprising the steps of: (a) determining a clonal mutation in TET2 or DNMT3A in a patient sample having a solid tumor and being treated with a non-dapanstril therapy, and (b1) treating the patient with an effective amount of dapanstril in addition to the non-dapanstril therapy if the patient's TET2 or DNMT3A variant allele frequency (VAF) is greater than 0.02, or (b2) continuing to treat the patient with the non-dapanstril therapy without the dapanstril therapy if the patient's TET2 or DNMT3A VAF is less than or equal to 0.02.

[0017] In this method, a patient having a solid tumor and being treated with a non-dapanstril therapy is identified and a biological sample is obtained from the patient. The biological sample can be, for example, a blood sample or bone marrow cells from the patient. A blood sample is preferred. Clonal mutations in TET2 or DNMT3A are then determined in the patient sample.

[0018] TET2 and DNMT3A are the most commonly mutated CHIP driver genes. Monocytes and T cells with TET2 or DNMT3A mutations show highly similar increases in proinflammatory gene expression.

[0019] Determining clonal mutations in TET2 or DNMT3A in cancer patient samples can inform patient prognosis. We found that patients with high VAF of Tet2 or DNMT3A are at high risk of progressing to end-stage disease and would benefit from dapanstril treatment. Dapanstril intervention therapy may reverse the CHIP phenotype and provide synergy with other non-dapanstril treatments by inhibiting NLRP3 activation, which weakens the efficacy of other treatments.

[0020] To determine clonal mutations, the threshold for CHIP is set at a variant allele fraction (VAF) of 2% for Tet2 or DNMT3A (i.e., 2% of sequenced alleles contain a mutation or, assuming the mutation is heterozygous, approximately 4% of cells contain a mutation). Tet2 or DNMT3A VAF can be detected from patient blood samples by next-generation sequencing or error-correction sequencing (9, 10). For example, VAF can be detected by sample preparation, DNA isolation, target enrichment, high-throughput sequencing, and variant calling as described in Dorsheimer (10).

[0021] After TET2 or DNMT3A clonal mutations are determined, if the patient's TET2 or DNMT3A variant allele frequency (VAF) is greater than 0.02, the patient is treated with an effective amount of dapanstril in addition to a non-dapanstril treatment. If the patient's TET2 or DNMT3A VAF is ≦0.02, the patient continues the non-dapanstril treatment without adding dapanstril treatment.

[0022] As used herein, an "effective amount of dapanstril" is an amount of dapanstril effective to treat a disease by ameliorating the condition and / or reducing, ameliorating, and / or eliminating the symptoms of the disease. For example, an effective amount is an amount that inhibits cancer growth and / or reduces tumor size.

[0023] Solid cancers suitable for treatment by the present methods include, for example, breast cancer and prostate cancer. Breast cancer includes triple-negative breast cancer (TNBC), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), inflammatory breast cancer (IBC), metastatic breast cancer, and breast cancer during pregnancy.

[0024] Non-dapanstril treatments for breast cancer that are suitable for dapanstril intervention include, but are not limited to, checkpoint inhibitor therapy, a form of cancer immunotherapy. Checkpoint inhibitor therapy targets immune checkpoints, which are key regulators of the immune system that, when stimulated, can dampen the immune response to immune stimulation. Some cancers can defend themselves against attack by stimulating immune checkpoint targets. Checkpoint inhibitors are currently used to treat breast cancer, and checkpoint inhibitors suitable for dapanstril intervention include, but are not limited to, inhibitors against programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). PD-1 is present on the surface of T cells and is the receptor for PD-L1. PD-1 plays a role in downregulating the immune response by suppressing the activity of inflammatory T cells. This mechanism helps the body prevent autoimmune diseases, but this mechanism can also prevent cancer cells from dying (11).

[0025] Non-dapanstril treatments for breast cancer suitable for dapanstril intervention may also include chemotherapy such as 5-fluorouridine or gemcitibine.

[0026] Non-dapanstril treatments for prostate cancer suitable for dapanstril intervention include, but are not limited to, radiation therapy, chemotherapy, and / or long-term androgen deprivation therapy.Currently approved chemotherapy agents for prostate cancer are docetaxel or caboxitaxel, which have been shown to be effective in increasing survival rates in conjunction with androgen deprivation therapy.Hormonal strategies that target androgen production or signaling include abiraterone or enzalutamide.

[0027] Dapanstril intervention therapy may reverse the CHIP phenotype and provide some synergy with chemotherapy by inhibiting NLRP3 activation, which reduces the efficacy of other therapies. For example, dapanstril intervention therapy may reverse the CHIP phenotype and provide some synergy with checkpoint inhibitor therapy, such as anti-PD-1 and anti-PD-L1.

[0028] If a patient's TET2 or DNMT3A variant allele frequency (VAF) is greater than 0.02 and the patient is deemed suitable for combination dapanstril therapy, dapanstril therapy and non-dapanstril therapy can be administered simultaneously or sequentially.

[0029] compound The present invention uses purified dapanstril (3-methanesulfonylpropionitrile), or a pharma- ceutical acceptable solvate thereof, as a therapeutic intervention. [ka]

[0030] As used herein, a "pharmaceutical acceptable solvate" is a solvate that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. As used herein, the term "solvate" refers to an addition complex in which the compound is combined with an acceptable cosolvent in a fixed ratio. Cosolvents include, but are not limited to, water, acetic acid, ethanol, and other suitable organic solvents.

[0031] Pharmaceutical Compositions The active compound dapanstril or a pharma- ceutically acceptable solvate thereof in the pharmaceutical composition is generally present in an amount of about 0.1-5% for injections, about 1-90% for tablets, 1-100% for capsules, about 0.01-20%, 0.05-20%, 0.1-20%, 0.2-15%, 0.5-10%, or 1-5% (w / w) for topicals, and about 0.1-5% for patches.

[0032] As used in this application, "about" refers to ±10% of the stated value.

[0033] Pharmaceutically acceptable carriers for inactive ingredients can be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments. Pharmaceutically acceptable carriers also include, but are not limited to, saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents, such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusting agents and buffers, such as hydroxides, phosphates, citrates, acetates, borates, and trolamine; antioxidants, such as bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, and ascorbyl palmitate salts, acids, and / or bases; surfactants, such as lecithin, phospholipids, including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol; poloxamers and poloxamines. , polysorbates (e.g., polysorbate 80, polysorbate 60, polysorbate 20), polyethers (e.g., polyethylene glycol and polypropylene glycol); polyvinyls, e.g., polyvinyl alcohol, povidone; cellulose derivatives, e.g., methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose, and their salts; petroleum derivatives, e.g., mineral oil, white petrolatum; fats, e.g., lanolin, peanut oil, palm oil, soybean oil; monoglycerides, diglycerides, triglycerides; polymers of acrylic acid, e.g., carboxypolymethylene gel, and hydrophobically modified crosslinked acrylate copolymers; polysaccharides such as dextran, and glycosaminoglycans such as sodium hyaluronate.Such pharma- ceutically acceptable carriers may be preserved against microbial contamination through the use of well-known preservatives, including but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, phenylethyl alcohol, or may be formulated as preservative-free preparations for single or multiple use.

[0034] For example, tablet formulations or capsules of dapanstril may contain other excipients that are not biologically active and do not react with the active compound. Tablet excipients may include fillers, binders, lubricants and glidants, disintegrants, wetting agents, and release rate modifiers. Binders are important for tablets because they promote the adhesion of the particles of the formulation. Examples of binders include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, tragacanth gum, poly(acrylic acid), polyvinylpyrrolidone.

[0035] For example, a patch formulation of dapanstril may contain several inactive ingredients, such as 1,3-butylene glycol, dihydroxyaluminum aminoacetate, disodium edetate, D-sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. The patch formulation may also contain a skin permeation enhancer, such as a lactate ester (e.g., lauryl lactate) or diethylene glycol monoethyl ether.

[0036] Topical formulations containing dapanstril can be in the form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution, suspension.Inactive ingredients in topical formulations include, but are not limited to, lauryl lactate (emollient / penetration enhancer), diethylene glycol monoethyl ether (emollient / penetration enhancer), DMSO (solubility enhancer), silicone elastomer (rheology / texture modifier), caprylic / capric triglyceride (emollient), octisalate (emollient / UV filter), silicone fluid (emollient / diluent), squalene (emollient), sunflower oil (emollient), silicon dioxide (thickener).In one embodiment, diethylene glycol monoethyl ether is included in topical gel formulations.

[0037] The pharmaceutical composition of dapanstril can be applied by systemic or local administration. Systemic administration includes, but is not limited to, oral, parenteral (e.g., intravenous, intramuscular, subcutaneous, or rectal), and inhalation administration. In systemic administration, the active compound first reaches the plasma and then distributes to the target tissue. Oral administration is the preferred administration route for the present invention. Local administration includes topical administration.

[0038] The dosage of the composition may vary based on the extent of breast cancer in the subject and the individual response of each patient. When administered systemically, the plasma concentration of the active compound delivered may vary, but is generally in the range of 1×10 -10 ~1×10 -4 moles / liter, preferably 1 x 10 -8 ~1×10 -5 It is in moles per liter.

[0039] In one embodiment, dapanstril is orally administered to a subject. The dosage for oral administration is generally at least 1 mg / kg / day and less than 100 mg / kg / day, preferably 5-100 mg / kg / day, depending on the age and condition of the subject. For example, the dosage for oral administration is 1-10, or 1-50, or 1-100, or 5-50, or 5-100, or 10-50, or 10-100 mg / kg / day for a human subject. For example, the dosage for oral administration is 100-10,000 mg / day for a human subject, preferably 100-2500, 500-2500, 500-4000, 1000-5000, 2000-5000, 2000-6000, or 2000-8000 mg / day. The drug can be administered orally once, twice, three times, or four times daily. Patients are treated daily for 14 days, up to 1 month, 2 months, or 3 months, or for the rest of their lives.

[0040] In one embodiment, dapanstril is administered intravenously to a subject. The dosage for intravenous bolus injection or intravenous infusion is generally 0.03 to 5 or 0.03 to 1 mg / kg / day.

[0041] In one embodiment, dapanstril is administered subcutaneously to a subject. The dosage for subcutaneous administration is generally 0.3 to 20, 0.3 to 3, or 0.1 to 1 mg / kg / day.

[0042] In one embodiment, dapanstril is applied topically. The topical dapanstril composition is applied topically at least once or twice a day, or 3-4 times a day, depending on the medical problem and disease pathology. Typically, the topical composition contains about 0.01-20%, or 0.05-20%, or 0.1-20%, or 0.2-15%, 0.5-10, or 1-5% (w / w) of the active compound. Typically, 0.2-10 mL of the topical composition is applied to an individual per dose.

[0043] Those skilled in the art will recognize that a wide variety of delivery mechanisms for dapanstril may be suitable for the present invention.

[0044] If it is decided that a breast cancer or prostate cancer patient should be further treated with dapanstril, the non-dapanstril treatment for the patient will follow the same protocol as that already established for the patient, or the dosage or treatment interval of the patient's non-dapanstril treatment may be reduced.

[0045] The present invention is useful for treating mammalian subjects, including humans, horses, dogs, cats, etc. The present invention is particularly useful for treating humans.

[0046] The present application discloses that dapanstril treatment reduces Tet2 expression in mice compared to mice not treated with dapanstril. + / +- It was demonstrated that it reduced the tumor growth of E0771TNBC in mice.

[0047] Furthermore, the present inventors have demonstrated that tumor-bearing Tet2 + / - We demonstrated that bone marrow cells cultured from mice with Tet2+ tumors treated with dapanstril increased IL-1β production compared to wild-type mice. + / - Bone marrow cells from mice were cultured with untreated Tet2 + / - Compared to mice, they secreted significantly less IL-1β.

[0048] In triple-negative breast cancer, IL-1β promotes an immunosuppressive TME. We show that tumor-bearing Tet2 + / - We have demonstrated that mice produce more IL-1β in bone marrow adherent cells, which is consistent with Tet2 + / - These results indicate that increased mutations increase the host's susceptibility to the immunosuppressive TME. CHIP patients with increased Tet2VAF are at high risk of progressing to end-stage disease, and dapanstril intervention therapy inhibits IL-1β production and improves the patient's condition. Dapanstril is effective in reversing the CHIP phenotype by inhibiting IL-1β production and inhibiting NLRP3 inflammasome activity.

[0049] This application demonstrated the tumor-promoting consequences of loss-of-function TET2 mutations in metastatic breast cancer. Tet2-CH does not represent a specific disease in itself, but acts as a preloaded trigger that exacerbates IL-1β-mediated disease. We demonstrated that Tet2 + / - We demonstrated a two-fold increase in tumor growth in germline mice. To determine whether this observation was simply an artifact of whole-organism Tet2 heterozygosity or truly a Tet2 CH phenomenon, we generated BM chimeric mice using mixed BM containing 25% Tet2 CH and then compared the Tet2 + / - Tet2 in bone marrow chimeras + / + They showed a two-fold increase in tumor growth compared to the control group, demonstrating that this was indeed the result of CH.

[0050] Furthermore, the present application relates to Tet2 + / - We show that NLRP3-dependent recruitment of bone marrow cells to the tumor microenvironment (TME) through induction of plasma CCL2 and KC in chimeras leads to an increase in tumor-infiltrating granulocytes. Consistently, we show that donor-derived Tet2 expression in the TME increases the expression of NLRP3-dependent myeloid cells. + / - We observed a significant increase in the Tet2 mutant, which places these hyperinflammatory cells in the TME. These data support the inclusion of hyperinflammatory Tet2 in the breast cancer TME. + / - There is evidence that mobilization of bone marrow cells promotes breast cancer progression.

[0051] Tet2 + / - Adding dapanstril to the diet of tumor-bearing mice increased granulocyte levels of Tet2. + / + These data implicate granulocytes in tumor progression and highlight the IL-1β-induced granulopoietic cycle that drives tumor growth. Thus, dapanstril inhibits Tet2 + / - A therapeutic approach to counteract inflammatory diseases mediated by CH or DNMT3A CH.

[0052] The following examples further illustrate the invention. These examples are intended to be merely illustrative of the invention and should not be construed as limiting the invention. EXAMPLES

[0053] In the examples below, the following materials and protocols were used:

[0054] mouse Six- to eight-week-old B6(Cg)-Tet2tm1.2Rao / J(JAX) female mice, either heterozygous for the Tet2 allele or wild-type for the Tet2 allele, were purchased from Jackson Laboratories.

[0055] cell line The mouse metastatic breast cancer cell line E0771 was purchased from ATCC. E0771 cells were cultured in RPMI (Corning) supplemented with 10% FBS, 1% Hepes, 100 units / ml penicillin, and 0.1 mg / ml streptomycin. Cells were maintained at 37°C in a humidified 5% CO2 atmosphere.

[0056] Tumor models 2×10 5 E0771 cells were mixed with Matrigel and orthotopically implanted into the mammary fat pad (day 0). Dapanstril-treated mice were fed food pellets containing 7.5 g dapanstril / kg of feed ad libitum from day 3 after E0771 cell implantation until sacrifice. Mice were sacrificed on day 18 in the germline mouse model and on day 15 in the chimeric mouse model. Mice typically consume approximately 4 g of food per day, resulting in a dose of approximately 0 mg / kg / day in the control group and approximately 1,000 mg / kg / day in the treated group. This food pellet concentration in the mouse diet (7.5 g dapanstril / kg in food) was similar to the blood concentration (40 μg / ml blood level) of humans orally administered dapanstril at a dose of 1,000 mg per day (14). Wild-type and Tet2 mice + / -Mice were fed control food pellets that did not contain dapanstril. Mice were sacrificed 15–18 days after E0771 cell implantation. Tumor volumes were assessed using ½ (length x width x height) measurements with digital calipers.

[0057] Chimeric bone marrow (BM)-bearing mice To generate a mouse model that more closely resembles human CHIP, we used 75% wild-type (WT) and 25% Tet2 homozygous (Tet2 + / + ) or heterozygous (Tet2 + / - We generated mice with chimeric bone marrow (BM) of either WT or Tet2. + / - To identify the contribution of cells to hematopoiesis, we used the pan-hematopoietic marker CD45. Two congenic mouse strains were used, harboring variants of CD45 C57BL / 6 and Boy / J (CD45.2 and CD45.1, respectively), which can be identified with fluorescently labeled antibodies using flow cytometry analysis. To generate chimeric BM, Boy / J recipient mice (CD45.1) were lethally irradiated with a split dose of 11 Gy, 3 hours apart. Irradiated recipient mice were enriched for 75% CD45.1WT and 25% CD45.2 Tet2. + / + or Tet2 + / - Two million unfractionated BM cells, a mixture of 100% erythrocytes, ...

[0058] Six weeks after implantation, mice were implanted with breast tumors by orthotopic implantation of breast cancer cells. Tumor progression was assessed by measuring the tumor volume in mice using calipers. After 15 days, mice were sacrificed and bone marrow, peripheral blood, and tumors were collected for flow cytometry analysis to determine the levels of immune cell populations and the expression of WT or Tet2. + / + or Tet2 + / - The relative contribution of bone marrow cells was determined.

[0059] Bone marrow cytokine production Bone marrow cells were harvested from tumor-bearing mice and filtered through a 40 μM cell strainer. 6 Cells were seeded in 24-well plates and cultured overnight. The next day, the non-adherent fraction was removed and the adherent fraction was treated with 10 μM dapanstril and cultured for 24 h in RPMI supplemented with 10% FBS, 100 units / ml penicillin, and 0.1 mg / ml streptomycin. Cytokines were measured in cell culture supernatants using DuoSet ELISA (R&D Systems).

[0060] Flow cytometry Bone marrow isolation and cell staining were performed on ice in staining medium (SM, Hank's balanced salt solution [HBSS1x, Corning 1-022-CM] supplemented with heat-inactivated fetal bovine serum [FBS, VWR97068-05] at a final concentration of 2%). Femurs and tibias were flushed with 3 mL of SM, followed by lysis of red blood cells with ACK lysis buffer. Peripheral blood was isolated from the heart and placed in red blood cell lysis buffer. After red blood cell lysis, cells were incubated with antibody cocktail for 30 min on ice, then washed and resuspended in SM containing propidium iodide to stain for dead cells. Flow cytometry was performed on a BD FACSCelesta.

[0061] Isolation, culture, and stimulation of adherent bone marrow Bone marrow cells were harvested from 4 long bones as described above and counted after filtering through a 40 μM cell strainer. 6 Cells were seeded onto flat-bottom tissue culture plates and cultured overnight. The next day, the non-adherent fraction was removed and the adherent fraction was used for further experiments.

[0062] Cytokine analysis For Western blot and cytokine analysis, adherent bone marrow cells were stimulated with ± LPS (100 ng / mL) and further cultured for 24 h. Cells were cultured in RPMI supplemented with 10% FBS, 100 units / ml penicillin, and 0.1 mg / ml streptomycin. For conditioned medium stimulation, 1 × 10 6The cells were cultured for 72 h to harvest E0771-conditioned medium, and the supernatant was collected and centrifuged to remove cell debris. The conditioned medium was then added to normal medium at a ratio of 1:2 and cultured for 24 h. Cytokines were measured in the cell culture supernatants using DuoSet ELISA (R&D Systems).

[0063] Example 1A: Dapanstril is a Tet2 + / - Reduced breast tumor volume in mice (germline) Bone marrow cells are the major source of tumor-promoting IL-1β in the breast cancer TME, and TET2 mutations have been reported in tumor-infiltrating leukocytes. Because bone marrow cells from Tet2-deficient mice show increased IL-1β gene expression after inflammatory stimulation, we assessed whether Tet2 heterozygous mice exhibit a more aggressive breast cancer phenotype. We used the murine metastatic luminal B breast cancer cell line E0771 as a model for the development of Tet2-deficient mice. + / + (WT) or Tet2 + / - Germline mice were orthotopically transplanted. Three days after transplantation, mice were fed a control diet or a nutritionally identical diet enriched with OLT1177® (dapanstril) for 15 days. Mice were sacrificed 21 days after E0771 cell transplantation.

[0064] The results of tumor volume are shown in Figure 1. Tet2 + / - Mice are Tet2 + / + Tumor growth was increased 2-fold compared to WT mice (p<0.05). Furthermore, Tet2 mice fed the dapanstril diet showed a significant increase in tumor growth compared to WT mice. + / - In mice, Tet2 + / - Tumor growth was significantly suppressed in mice given dapanstril compared to Tet2 mice (68% reduction, p<0.01). + / + Tumor growth in WT mice was also significantly reduced compared to standard chow (p<0.05). + / + (WT) mice and Tet2 + / - No changes in tumor growth were observed among the mice (Figure 1).

[0065] Example 1B: Dapanstril is a Tet2+ / - Decreased IL-1β production in mice Next, the inflammatory potential of bone marrow cells from tumor-bearing mice was evaluated. Bone marrow adherent cells from tumor-bearing mice of Example 1 were cultured and stimulated with LPS overnight. + / - Bone marrow cells cultured from mice showed increased IL-1β production compared to WT (p<0.01, Figure 1B). Furthermore, Tet2 cells bearing tumors treated with dapanstril showed increased IL-1β production compared to WT (p<0.01, Figure 1B). + / - Adherent mouse bone marrow cells express untreated Tet2 + / - The secretion of IL-1β was significantly less in mice compared to those in controls (p<0.05, Fig. 2).

[0066] Example 2: Dapanstril is a Tet2 + / - Reduced breast tumor volume in a bone marrow chimera model CH is characterized by the clonal expansion of somatic mutations in the hematopoietic compartment. In this example, we determined whether the tumor-promoting role of Tet2 deficiency in a bone marrow chimera model, an experimental model of clonal hematopoiesis, is maintained.

[0067] Tet2 + / + and Tet2 + / - Unfractionated bone marrow (BM) cells from donor mice were mixed with CD45.1+Boy / J competitor BM at a 1:3 donor:competitor ratio and the cells were transplanted into lethally irradiated Boy / J recipient mice. Six weeks after transplantation, E0771 cells were orthotopically transplanted as described above. Three days after transplantation, mice were fed a control diet or a dapanstril-enriched diet. Tet2 + / - An essentially identical pattern of accelerated tumor growth was observed in chimeric mice (p<0.05), which was consistent with the Tet2 + / - The Tet2 expression was again suppressed in the chimeric mice (p<0.001). + / + Tumor growth in chimeric animals was also significantly improved by Tet2 + / - The results of Examples 1 and 2 show that Tet2 + / -promotes breast cancer progression and dapanstril is effective in inhibiting tumor growth.

[0068] Example 3. Accelerated TET2 + / - Driven breast cancer growth is inhibited by dapanstril Granulocytes and monocytes, collectively called myeloid cells, are differentiated progeny of a common progenitor cell derived from hematopoietic stem cells in the bone marrow.

[0069] In this example, we first assessed the extent to which Tet2-deficient bone marrow cells infiltrated into peripheral blood (Figure 3A) and tumors (Figure 3B). + / + Compared with mice, Tet2 + / - The frequency of donor-derived cells in the peripheral blood of chimeric mice was significantly increased. + / - Tet2 in tumors of chimeric mice + / - The frequency of donor-derived cells is + / + The results show that the increase was significant compared to mice.

[0070] FIG. 4A shows Tet2 + / - In the chimeras, Tet2 + / + Figure 4A also shows that the total number of tumor-infiltrating granulocytes was significantly increased in the Tet2 mice fed a standard diet compared to the chimeras (p<0.05). + / - Tet2 fed the dapanstril diet compared to chimeras + / - The chimeras also showed a significant reduction in tumor-infiltrating granulocytes (p<0.05).

[0071] FIG. 4B shows Tet2 + / - In the chimeras, Tet2 + / + The tumor-infiltrating granulocytes showed significantly less expression of MHC-II protein (gMFI) compared to the chimeras (p<0.05). + / - The decrease in MHC-II expression observed in the chimeras was restored by the dapanstril diet (p<0.001).

[0072] FIG. 5 shows plasma chemokines and cytokines in the BM chimeric mice of FIG. 2. As shown in FIG. 5, Tet2 + / - In chimeric mice, Tet2 + / + Circulating CCL2 and KC were increased (p<0.05) in the dapanstril-fed Tet2 mice compared to the chimeras. + / - In the chimeras, Tet2 + / - Circulating CCL2 and KC were significantly reduced compared to chimeras (p<0.05).

[0073] Taken together, these data support the breast cancer-promoted Tet2 + / - We further explore how clonal expansion perpetuates an inflammatory environment in the TME, thereby promoting breast cancer progression, and how dapanstril inhibits Tet2 + / - It is shown whether the proliferation of the clones could be reduced.

[0074] References 1. Y. Guo, et al. Cancer Res 77, 6429-6441 (2017). 2. S. Shalapour, et al. J Clin Invest 125, 3347-3355 (2015). 3. CA Dinarello. Blood 117, 3720-3732 (2011). 4. RN Apte, et al. Cancer Metastasis Rev 25, 387-408 (2006). 5. CA Dinarello. Cancer Metastasis Rev 29, 317-329 (2010). 6. B. Guo, et al. Sci Rep 6, 36107 (2016). 7. KL Lee, et al. Cancers 11(9), 1334 (2019). 8. C. Marchetti, et al. Proc Natl Acad Sci USA 115, E1530-E1539 (2018). 9. A. Young, et al. Nature Communications. DOI: 10.1038 (2016) 10. L. Dorsheimer, et al. JAMA Cardiol. 4:25-33 (2019) 11. NL Syn, et al. Lancet Oncology 18(12), PE731-E741 (2017).

[0075] From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited only by the scope of the appended claims.

Claims

1. 1. A pharmaceutical composition comprising dapanstril for use in a method for treating solid tumors, the method comprising the steps of: (a) determining clonal mutations in TET2 or DNMT3A in patient samples from patients with solid tumors of the breast or prostate who are being treated with a non-dapanstril therapy; and (b1) treating the patient with the pharmaceutical composition in addition to the non-dapanstril treatment if the patient's TET2 or DNMT3A variant allele frequency (VAF) is determined to be greater than 0.02, or (b2) continuing to treat the patient with the non-dapanstril treatment if the patient's TET2 or DNMT3A VAF is determined to be less than or equal to 0.

02.

2. 2. The pharmaceutical composition of claim 1, wherein the solid tumor is breast cancer and the non-dapanstril treatment is a checkpoint inhibitor treatment.

3. The checkpoint inhibitors are inhibitors of programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). The pharmaceutical composition of claim 2.

4. The pharmaceutical composition according to claim 3, wherein the checkpoint inhibitor is an inhibitor against PD-1.

5. The pharmaceutical composition of claim 4, wherein the checkpoint inhibitor is an anti-PD-1 antibody.

6. The pharmaceutical composition of claim 2, wherein the non-dapanstril treatment is chemotherapy.

7. The breast cancer is selected from the group consisting of ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), triple-negative breast cancer (TNBC), inflammatory breast cancer (IBC), metastatic breast cancer, and breast cancer during pregnancy. The pharmaceutical composition of claim 2.

8. The pharmaceutical composition described in claim 1, wherein the method further comprises a step of selecting a patient having breast cancer and being treated with a non-dapanstril therapy before step (a).

9. A pharmaceutical composition described in claim 1 for oral administration.

10. A method for providing an indication of whether or not to administer dapanstril to a patient with a solid tumor, comprising the steps of: (a) determining clonal mutations in TET2 or DNMT3A in patient samples from patients with solid tumors of the breast or prostate who are being treated with a non-dapanstril therapy; and (b1) if the patient's TET2 or DNMT3A variant allele frequency (VAF) is determined to be greater than 0.02, providing the result of the determination as an indication to treat the patient with dapanstril in addition to the non-dapanstril treatment, or (b2) if the patient's TET2 or DNMT3A VAF is determined to be less than 0.02, providing the result of the determination as an indication to continue treating the patient with the non-dapanstril treatment.

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