Methods for treating solid cancer patients with undefined potential clonal hematopoiesis

Abstract

The present invention relates to methods for treating solid cancers in patients suffering from undefined potential clonal hematopoietic (CHIP). The method introduces therapeutic intervention of dapam nitrile to improve the treatment of solid cancers, such as breast cancer, in CHIP patients. The method comprises first determining whether a patient has a CHIP disorder by detecting the presence or absence of a Tet2 or DNMT3A mutation in a biological sample from the patient, and then subsequently performing a therapeutic intervention in a patient having a CHIP disorder using dapam nitrile. The application of the invention shows the effect of CHIP in driving breast cancer. This data provides a fundamental principle of screening for Tet2 or DNMT3A mutations in breast cancer patients, and provides dapam nitrile as a therapeutic treatment.

Description

Technical Field

[0001] The present invention relates to a method for treating solid cancer in a patient with clonal hematopoiesis of indeterminate potential (CHIP). The method comprises first determining whether the patient has CHIP by detecting the presence or absence of Tet2 or DNMT3A mutations, and then subsequently performing therapeutic intervention on the patient with CHIP using dapamoate. Background Art

[0002] As people age, their tissues accumulate more and more somatic mutations. When this process occurs in the hematopoietic system, a significant proportion of circulating blood cells can be derived from a single mutated stem cell. This growth is called "clonal hematopoiesis" and is highly common in the elderly population. Clonal hematopoiesis (CH) refers to the gradual, selective expansion of hematopoietic stem cells (HSCs) that usually carry somatic mutations in one allele of a gene. CH is characterized by the overexpression of blood cells derived from a single clone. Blood cancers such as chronic myeloid leukemia or myelodysplastic syndrome are archetypal examples of clonal hematopoiesis. However, the same mutations present in these cancers are also found in a large proportion of healthy elderly populations.

[0003] Clonal hematopoiesis of undetermined potential (CHIP) refers to the presence of clonal molecular genetic or cytogenetic changes in blood or bone marrow cells in the absence of a hematologic neoplasm and in the absence of cytopenias. CHIP is a nonmalignant condition characterized by mutation and clonal expansion of blood cells. The incidence of CHIP increases with age. CHIP is diagnosed when testing of a person's blood or bone marrow sample shows that blood cells carry one of the genetic mutations associated with the condition.

[0004] CHIP is associated with a higher risk of developing hematological malignancies and cardiovascular disease, as well as a reduced lifespan.

[0005] Tumorigenesis is initiated by genomic alterations including point mutations, gene deletions, chromosomal rearrangements leading to cell transformation, self-sufficient proliferation, insensitivity to antiproliferative signals, escape from apoptosis, and unlimited replicative potential, ultimately leading to tissue invasion and metastasis of cancer cells. However, the expansion of tumor cells is associated with a complex network of events involving both cancer cells and non-cancerous cells. Chronic inflammation is a typical example of such a promoting condition (1, 2).

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

[0007] Types of breast cancer include 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, among other types. Triple-negative breast cancer tumors are characterized by the absence of estrogen receptors (ER), progesterone receptors (PR), and elevated levels of human epidermal growth factor receptor 2 (HER2) protein (7).

[0008] NLRP3 (NOD-like receptor family pyrin domain-containing protein 3, also known as NLRP3 or cryopyrin) 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 dangers during intracellular infection (bacterial and viral proteins) or tissue damage (ischemia). NLRP3 activation leads to the recruitment of ASC (apoptosis-associated speck-like protein containing a carboxyl-terminal caspase recruitment domain) and caspase-1, resulting in inflammasome formation and ultimately cell death.

[0009] Dapanchonil is a synthetic small molecule β-sulfonyl nitrile that has been shown to selectively inhibit the NLRP3 inflammasome and is safe when orally administered to healthy subjects (8). BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1A Shown are germline Tet2+ / + (wild type) or Tet2 from orthotopically implanted E0771 cells. + / - Tumor growth curves in mice. Tet2 + / - Dapanchonil for mice Orally treated with a control diet without dapoxetine (N=8, two independent experiments).

[0011] Tet2+ / - driven breast cancer progression is dependent on NLRP3 activation. Figure 1B Shown are the mean ± SEM of IL-1β production from bone marrow adherent cells isolated from tumor-bearing mice.

[0012] Figure 2 Bone marrow (BM) chimeras were generated by transplanting 75% WT:25% Tet2+ / + or Tet2+ / - mixed BM. BM chimera mice were orthotopically implanted with E0771 breast tumors and fed a standard diet or a dapoxetine diet. Figure 2 Shown are tumor growth curves of Tet2+ / + (WT) or Tet2+ / - chimeric mice implanted with E0771 cells that received a standard diet or a dalazine diet (N=10, two independent experiments).

[0013] FIG. 3A to FIG. 3B Shown from Figure 2 Total percentage of donor-derived cells in peripheral blood (3A) and tumors (3B) of BM chimera mice.

[0014] FIG. 4A to FIG. 4B Shown from Figure 2 The frequency of tumor-infiltrating granulocytes (4A) and gMFI of MHC-II+ expression (4B) in BM chimeric mice.

[0015] FIG. 5A to FIG. 5B Shown from Figure 2 Plasma cytokine production of chemokine C-C motif ligand 2 (CCL2, 5A) and keratinocyte chemoattractant (KC, 5B) in BM chimeric mice.

[0016] In all figures: ***p<0.001, **p<0.01, *p<0.05. DETAILED DESCRIPTION

[0017] Clonal hematopoiesis (CH) is associated with an increase in hematological malignancies. CH can be defined as somatic mutations in hematopoietic stem cells that lead to clonal expansion of myeloid cells with an increased inflammatory phenotype. Mutations in genes involved in DNA methylation, ten-eleven translocation 2 (Tet2) and / or DNA methyltransferase 3a (DNMT3A), drive the clonal expansion. Tet2 is one of three enzymes responsible for catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine and additional intermediates that can ultimately lead to demethylation. The DNMT3A gene provides instructions for making DNA methyltransferase 3alpha, one of two enzymes responsible for de novo methylation of the fifth position in the cytosine base of DNA, a marker that affects gene expression.

[0018] The inventors have found the role of CHIP in driving solid cancers such as breast cancer and prostate cancer. In the myeloid cells infiltrated by solid tumors, the immunosuppressive tumor microenvironment (TME) is promoted by dysregulated cytokine signaling. The connection between CHIP and solid tumors is that infiltrated myeloid cells appear in the TME and have a high inflammatory response, which promotes tumor-promoting signal transduction. For cancer patients with CHIP, the disease may be more serious or the patient is at greater risk. In breast cancer, CHIP has a deleterious effect in all advanced breast cancers, because when breast cancer progresses, IL-1β increases with the severity of the disease.

[0019] The proinflammatory cytokine IL-1β promotes breast cancer progression and is associated with disease severity. Activation of the NLRP3 inflammasome in myeloid cells is a major source of IL-1β processing.

[0020] Activation of the NLRP3 inflammasome amplifies the inflammatory response to tissue injury and mediates additional injury. Dapamocil is a selective NLRP3 inflammasome inhibitor; Dapamocil reduces inflammation by preventing activation of the NLRP3 inflammasome.

[0021] According to the mechanism of action of dapoxetine, which prevents the production and / or release of IL-1β and IL-18 and inhibits the formation of NLRP3 inflammasomes in animals and human subjects, the inventors have found a method for introducing therapeutic intervention of dapoxetine to improve the treatment of solid cancers (such as breast cancer or prostate cancer) in CHIP patients. The method includes first determining whether the patient suffers from CHIP disease by detecting the presence or absence of Tet2 or DNMT3A mutations, and then subsequently using dapoxetine to perform therapeutic intervention on patients with CHIP disease.

[0022] Treatment

[0023] The present invention relates to a method for treating a patient with a solid cancer such as breast cancer or prostate cancer. The method comprises the following steps: (a) determining a clonal mutation of TET2 or DNMT3A in a sample of a patient having a solid cancer and being treated with fidaxolotl, and (b1) if the patient has a variant allele frequency (VAF) of TET2 or DNMT3A greater than 0.02, treating the patient with an effective amount of fidaxolotl in addition to fidaxolotl treatment, or (b2) if the patient has a VAF of TET2 or DNMT3A ≤ 0.02, continuing to treat the patient with fidaxolotl without fidaxolotl treatment.

[0024] In the present method, a patient with solid cancer who is treated with fidapanoxanil 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. A clonal mutation of TET2 or DNMT3A is then determined in the patient sample.

[0025] TET2 and DNMT3A are the most commonly mutated CHIP driver genes. Monocytes and T cells with TET2 or DNMT3A mutations show very similar increases in pro-inflammatory gene expression.

[0026] Identifying clonal mutations of TET2 or DNMT3A in cancer patient samples provides prognosis for the patient. The inventors have found that patients with high Tet2 or DNMT3A VAFs are at greater risk of developing advanced disease and benefit from dapoxetine treatment. Dapoxetine intervention reverses the CHIP phenotype and may provide some synergy with other non-dapoxetine treatments by inhibiting NLRP3 activation that attenuates the efficacy of other treatments.

[0027] To identify clonal mutations, the threshold for CHIP was set to a variant allele fraction (VAF) of 2% for Tet2 or DNMT3A (meaning that 2% of sequenced alleles contain the mutation, or approximately 4% of cells contain the mutation, assuming the mutation is heterozygous). VAF for Tet2 or DNMT3A can be determined from patient blood samples by next-generation sequencing or error-corrected sequencing (9, 10). For example, VAF can be determined by sample preparation, DNA isolation, target enrichment, high-throughput sequencing, and variant calling as described in Dorsheimer (10).

[0028] After determining the clonal mutation of TET2 or DNMT3A, if the patient has a variant allele frequency (VAF) of TET2 or DNMT3A greater than 0.02, the patient is treated with an effective amount of dalazine in addition to treatment with fidaxolotl. If the patient has a VAF of TET2 or DNMT3A ≤ 0.02, the patient continues treatment with fidaxolotl without adding dalazine.

[0029] As used herein, an "effective amount of dapoxetine" is an amount of dapoxetine that is effective in treating a disease by ameliorating the pathological condition and / or alleviating, ameliorating and / or eliminating disease symptoms. For example, an effective amount is an amount that reduces cancer growth and / or reduces tumor size.

[0030] Solid cancers suitable for treatment by the present method 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, among other types.

[0031] Non-dapoxetine treatments for breast cancer suitable for dapoxetine intervention include, but are not limited to, checkpoint inhibitor therapy, which is a form of cancer immunotherapy. Checkpoint inhibitor therapy targets immune checkpoints, which are key regulators of the immune system that, when stimulated, can attenuate immune responses to immune stimulation. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint inhibitors currently used to treat breast cancer and suitable for dapoxetine intervention include, but are not limited to, inhibitors of programmed cell death protein 1 (PD-1), programmed 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 a receptor for PD-L1. PD-1 plays a role in downregulating immune responses by inhibiting inflammatory T cell activity. This mechanism helps the body prevent autoimmune diseases, however, it can also prevent cancer cells from being killed (11).

[0032] Non-dapoxetine treatments for breast cancer suitable for dapoxetine intervention may also include chemotherapy such as 5-fluorouridine and gemcitabine.

[0033] Non-dapamoxil treatments for prostate cancer that are suitable for dapamoxil intervention include but are not limited to radiotherapy, chemotherapy and / or long-term androgen deprivation therapy. The chemotherapeutic agents currently approved for prostate cancer are docetaxel or cabazitaxel, which together with androgen deprivation have been shown to be effective in increasing survival. The hormone strategy of targeted androgen production or signal transduction includes abiraterone or enzalutamide.

[0034] Dapamoate intervention reverses the CHIP phenotype and may provide some synergy with chemotherapy by inhibiting NLRP3 activation that attenuates the efficacy of other treatments. For example, Dapamoate intervention reverses the CHIP phenotype and may provide some synergy with checkpoint inhibitor treatments such as anti-PD-1 and anti-PD-L1.

[0035] If the patient is determined to have a variant allele frequency (VAF) of TET2 or DNMT3A greater than 0.02 and is suitable for combination therapy with dalazine, dalazine therapy and non-dalazine therapy may be administered simultaneously or sequentially.

[0036] Compound

[0037] The present invention uses purified dapamoic acid nitrile (3-methanesulfonyl-propionitrile) or a pharmaceutically acceptable solvate thereof as a therapeutic intervention.

[0038]

[0039] As used herein, a "pharmaceutically acceptable solvate" is a solvate that retains the desired biological activity of the parent compound and does not impart undesirable toxicological effects. As used herein, a "solvate" is an addition complex in which a compound is combined with an acceptable co-solvent in a certain fixed ratio. Co-solvents include, but are not limited to, water, acetic acid, ethanol, and other suitable organic solvents.

[0040] Pharmaceutical composition

[0041] The amount of the active compound dapoxetine or a pharmaceutically acceptable solvate thereof in the pharmaceutical composition is generally about 0.1% to 5% for injectable preparations, about 1% to 90% for tablet preparations, 1% to 100% for capsule preparations, about 0.01% to 20%, 0.05% to 20%, 0.1% to 20%, 0.2% to 15%, 0.5% to 10%, or 1% to 5% (w / w) for topical preparations, and about 0.1% to 5% for patch preparations.

[0042] As used in this application, "about" means ± 10% of the recited value.

[0043] Pharmaceutically acceptable carriers are inactive ingredients that 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 may also contain a variety of ingredients, including, but not limited to, saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers such as salts of hydroxides, phosphates, citrates, acetates, borates; and triethanolamine; antioxidants such as salts, acids, and / or bases of bisulfites, sulfites, metabisulfites, thiosulfites, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and palmitic acid ascorbyl ester; surfactants such as lecithin, phospholipids, including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylcholine. Inositol; poloxamers and poloxamines, polysorbates (such as polysorbate 80, polysorbate 60 and polysorbate 20), polyethers (such as polyethylene glycol and polypropylene glycol); polyethylenes such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose and hydroxypropylmethylcellulose and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, soybean oil; mono-, di- and triglycerides; acrylic acid polymers such as carboxypolymethylene gel, and hydrophobically modified cross-linked acrylate copolymers; polysaccharides (such as dextran) and glycosaminoglycans (such as sodium hyaluronate). Such pharmaceutically acceptable carriers may be preserved to prevent bacterial contamination with well-known preservatives including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or may be formulated as non-preserved preparations for single or multiple use.

[0044] For example, the tablet preparation or capsule preparation of dapanoxanil may contain other excipients that do not have biological activity and do not react with the active compound. The excipient of tablet may include filler, adhesive, lubricant and glidant, disintegrant, wetting agent and release rate regulator. Adhesive promotes the adhesion of preparation particles, and is important for tablet preparation. The example of adhesive includes but is not limited to carboxymethyl cellulose, cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose, karaya gum, starch, starch and tragacanth gum, poly (acrylic acid) and polyvinyl pyrrolidone.

[0045] For example, the patch preparation of dapanoxanil can include some inactive ingredients, such as 1,3-butylene glycol, dihydroxy aminoacetic acid aluminum, disodium edetate, D-sorbitol, gelatin, clay, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide and purified water. The patch preparation can also contain a skin permeability enhancer, such as lactic acid ester (e.g., lauryl lactate) or diethylene glycol monoethyl ether.

[0046] The topical preparation including dapanoxanil can be in the form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution and suspension. Inactive ingredients in topical preparations 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), octyl salicylate (emollient / UV filter), silicone fluid (emollient / diluent), squalene (emollient), sunflower oil (emollient) and silicon dioxide (thickening agent). In one embodiment, diethylene glycol monoethyl ether is included in the topical gel preparation.

[0047] The pharmaceutical composition of dapanoxanil can be applied by systemic administration or partial administration. Systemic administration includes but is not limited to oral, parenteral (such as intravenous, intramuscular, subcutaneous or rectal) and inhalation administration. In systemic administration, the active compound first reaches the plasma and is then distributed to the target tissue. Oral administration is the preferred route of administration of the present invention. Partial administration includes topical administration.

[0048] The dosage of the composition may vary based on the extent of breast cancer in the subject and the individual response of each patient. For systemic administration, the plasma concentration of the delivered active compound may vary; but is generally 1x10 -10 mol / L to 1x10 -4 mol / L, and preferably 1x10 -8 mol / L to 1x10 -5 mol / L.

[0049] In one embodiment, dapanoxanil is orally administered to a subject. The dosage for oral administration is generally at least 1mg / kg / day and less than 100mg / kg / day, preferably 5mg / kg / day to 100mg / kg / day, depending on the age and condition of the subject. For example, for human subjects, the dosage for oral administration is 1mg / kg / day to 10mg / kg / day or 1mg / kg / day to 50mg / kg / day or 1mg / kg / day to 100mg / kg / day or 5mg / kg / day to 50mg / kg / day or 5mg / kg / day to 100mg / kg / day or 10mg / kg / day to 50mg / kg / day or 10mg / kg / day to 100mg / kg / day. For example, for human subjects, the dosage for oral administration is 100 mg / day to 10,000 mg / day, and preferably 100 mg / day to 2500 mg / day, 500 mg / day to 2500 mg / day, 500 mg / day to 4000 mg / day, 1000 mg / day to 5000 mg / day, 2000 mg / day to 5000 mg / day, 2000 mg / day to 6000 mg / day or 2000 mg / day to 8000 mg / day. The drug can be taken orally once, twice, three times or four times a day. The patient is treated every day for 14 days, up to 1 month, 2 months or 3 months or for life.

[0050] In one embodiment, dapoxetine is administered to the subject intravenously. The dose for intravenous push or intravenous infusion is typically 0.03 mg / kg / day to 5 mg / kg / day or 0.03 mg / kg / day to 1 mg / kg / day.

[0051] In one embodiment, dapoxetine is administered subcutaneously to the subject. The dosage for subcutaneous administration is typically 0.3 mg / kg / day to 20 mg / kg / day, 0.3 mg / kg / day to 3 mg / kg / day, or 0.1 mg / kg / day to 1 mg / kg / day.

[0052] In one embodiment, dapoxetine is applied topically. Depending on the medical problem and disease pathology, the topical dapoxetine composition is topically applied at least once or twice a day, or 3 to 4 times a day. Typically, the topical composition comprises about 0.01% to 20%, or 0.05% to 20%, or 0.1% to 20%, or 0.2% to 15%, 0.5% to 10%, or 1% to 5% (w / w) of the active compound. Typically, 0.2mL to 10mL of the topical composition is applied to an individual per dose.

[0053] Those skilled in the art will recognize that a variety of delivery mechanisms for dapoxetine may be suitable for use in the present invention.

[0054] When a breast cancer patient or a prostate cancer patient is determined to be additionally treated with dapamoxil, the patient's treatment with fedapamoxil will follow the same regimen already established for that patient. Alternatively, the dose or treatment interval of the patient's treatment with fedapamoxil may be reduced.

[0055] The present invention can be used to treat mammalian subjects, such as humans, horses, dogs and cats. The present invention is particularly useful for treating humans.

[0056] This patent application has demonstrated that dapoxetine treatment reduced Tet2 expression compared to those mice not treated with dapoxetine. + / - Tumor growth of E0771 TNBC in mice.

[0057] Furthermore, the present inventors have demonstrated that compared with wild-type mice, + / - Cultured bone marrow cells from mice showed increased IL-1β production. + / - Compared with mice treated with dapoxetine, mice bearing Tet2 + / - The mice's bone marrow cells secreted significantly less IL-1β.

[0058] In triple-negative breast cancer, IL-1β promotes an immunosuppressive TME. The inventors have demonstrated that tumors bearing Tet2 + / - Mice produced more IL-1β in bone marrow adherent cells, indicating that the increase in Tet2+ / - mutations renders the host more susceptible to the immunosuppressive TME. CHIP patients with increased Tet2 VAF are at greater risk of developing advanced disease, and intervention with dapamoate inhibited IL-1β production and improved patient symptoms. Dapamoate effectively reversed the CHIP phenotype by inhibiting IL-1β production and inhibiting NLRP3 inflammasome activity.

[0059] This patent application has demonstrated the tumor promoting consequences of loss-of-function TET2 mutations in metastatic breast cancer. Tet2-CH itself does not represent a specific disease, but rather acts as a pre-existing trigger that exacerbates IL-1β mediated disease. The inventors have demonstrated a 2-fold increase in tumor growth in Tet2+ / - germline mice. To determine whether this observation was simply an artifact of Tet2 heterozygosity in the whole organism or a true Tet2 CH phenomenon, the inventors generated BM chimeric mice using mixed BM with 25% Tet2 CH and showed that Tet2+ / - bone marrow chimeras had a 2-fold increase in tumor growth compared to Tet2+ / + controls, demonstrating that this was in fact a CH consequence.

[0060] In addition, the present patent application shows that the NLRP3-dependent recruitment of myeloid cells to the tumor microenvironment (TME) by the induction of plasma CCL2 and KC in Tet2+ / - chimeras leads to an increase in tumor infiltrating granulocytes. Consistently, the inventors observed a significant increase in donor-derived Tet2+ / - mutants in the TME, thereby placing these highly inflammatory cells in the TME. These data indicate that the recruitment of highly inflammatory Tet2+ / - myeloid cells to the breast cancer TME promotes breast cancer progression.

[0061] Supplementation of the diet of Tet2+ / - tumor-bearing mice normalized granulocyte levels to those present in Tet2+ / + mice, reflecting a dependence on IL-1β. These data implicate granulocytes in tumor progression and highlight a cycle in which IL-1β-induced granulopoiesis drives tumor growth. Thus, dapamoic acid presents a therapeutic approach to neutralize inflammatory diseases mediated by Tet2+ / - CH or DNMT3A CH.

[0062] The following examples further illustrate the present invention. These examples are intended only to illustrate the present invention and should not be construed as limiting.

[0063] Example

[0064] The following materials and protocols were used in the examples described below.

[0065] Mouse

[0066] B6(Cg)-Tet2tm1.2Rao / J(JAX) female mice, 6 to 8 weeks old, heterozygous for Tet2 alleles or wild-type for Tet2 alleles were purchased from Jackson Laboratories.

[0067] Cell lines

[0068] 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 environment.

[0069] Tumor Models

[0070] 2x10 5E0771 cells were mixed with matrigel and implanted in situ into the mammary fat pad (day 0). Starting on the 3rd day after E0771 cell implantation, mice treated with dapoxetine were fed ad libitum with food pellets containing 7.5g dapoxetine / kg food, and continued until they were killed. For the germline mouse model, mice were killed on the 18th day, and for the chimeric mouse model, mice were killed on the 15th day. Mice usually consume about 4g of food per day, resulting in an approximate daily dose of 0mg / kg / day for the control group and 1,000mg / kg / day for the treatment group. This food particle concentration in the mouse feed (7.5g / kg of dapoxetine in the food) results in blood levels that are almost the same as the blood levels of humans treated orally with 1000mg / day doses of dapoxetine (40μg / mL blood levels) (14). Wild-type and Tet2+ / - mice were fed with control food pellets without dapoxetine. Mice were sacrificed 15 to 18 days after E0771 cell implantation. Tumor volume was assessed using 1 / 2 (LxWxH) measured by digital calipers.

[0071] Mice with chimeric bone marrow (BM)

[0072] To create a mouse model that is more similar to human CHIP, we generated mice with chimeric bone marrow (BM) of 75% wild type (WT) and 25% Tet2 homozygous (Tet2+ / +) or heterozygous (Tet2+ / -). To identify the contribution of WT or Tet2+ / - cells to hematopoiesis, we used the pan-hematopoietic marker CD45. We used two syngeneic mouse strains with variants in CD45 C57BL / 6 and Boy / J (CD45.2 and CD45.1, respectively), which can be identified using fluorescently labeled antibodies using flow cytometric analysis. To generate chimeric BM, we lethally irradiated Boy / J recipient mice (CD45.1), with 11gy fractionated doses administered every three hours. The irradiated recipient mice were then transplanted with two million unfractionated BM cells, which were a mixture of 75% CD45.1WT and 25% CD45.2.

[0073] Tet2+ / + or Tet2+ / -. Cells were administered by retro-orbital (RO) sinus injection. To monitor transplantation of donor-derived cells, mice were bled every four weeks through the submandibular vein. The blood was then treated by hemolysis to remove red blood cells, and then leukocytes were stained with fluorescent antibodies for flow cytometric analysis.

[0074] Six weeks after transplantation, mice were given mammary tumors by orthotopic transplantation of breast cancer cells. Tumor progression was assessed by measuring the tumor volume of mice using a caliper. After 15 days, mice were sacrificed and their bone marrow, peripheral blood, and tumors were harvested for flow cytometric analysis to determine the level of immune cell populations and the relative contribution from WT or Tet2+ / + or Tet2+ / - bone marrow cells.

[0075] Bone marrow cytokine production

[0076] Bone marrow cells from tumor-bearing mice were collected and filtered through a 40 μM cell strainer. 3×10 6 The cells were placed in a 24-well plate and incubated overnight. The next day, the non-adherent portion was removed and the adherent portion was treated with 10 μM dapamoate and cultured for 24 hours in RPMI supplemented with 10% FBS, 100 units / ml penicillin and 0.1 mg / ml streptomycin. Cytokines in cell culture supernatants were measured using DuoSet ELISA (R&D Systems).

[0077] Flow cytometry

[0078] Bone marrow isolation and cell staining were performed on ice in staining medium (SM; Hanks balanced salt solution [HBSS1x, Corning 1-022-CM], supplemented with heat-inactivated fetal bovine serum [FBS, VWR 97068-05], to a final concentration of 2%). Femurs and tibiae were rinsed with 3 mL of SM, and then RBC lysis was performed with ACK lysis buffer. Peripheral blood was separated by heart and placed in RBC lysis buffer. After RBC lysis, cells were incubated with antibody cocktail on ice for 30 minutes, then washed and resuspended in SM containing propidium iodide to stain dead cells. Flow cytometry was performed on a BD FACSCelesta.

[0079] Adherent bone marrow isolation, culture and stimulation

[0080] Bone marrow cells were collected from four long bones as described above and filtered through a 40 μM cell strainer before counting. 1x10 6 The cells were plated in flat-bottom tissue culture plates and incubated overnight. The next day, the non-adherent fraction was removed and the adherent fraction was used for downstream experiments.

[0081] Cytokine analysis

[0082] For Western blot and cytokine analysis, adherent bone marrow cells were stimulated with ±LPS (100 ng / mL) and cultured for an additional 24 hours. Cells were cultured in RPMI supplemented with 10% FBS, 100 units / ml penicillin, and 0.1 mg / ml streptomycin. For conditioned medium stimulation, cells were cultured by incubating 1×10 6 The E0771 conditioned medium was collected from the cells for 72 hours, 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 for 24 hours. Cytokines in the cell culture supernatant were measured using DuoSet ELISA (R&D Systems).

[0083] Example 1A: Dapanoxanil reduces mammary tumor volume in Tet2+ / - mice (strain)

[0084] Myeloid 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. As myeloid cells from Tet2-deficient mice have increased IL-1β gene expression following inflammatory stimulation, we assessed whether mice heterozygous for Tet2 exhibit a more aggressive breast cancer phenotype. We orthotopically implanted the mouse metastatic luminal B breast cancer cell line E0771 into Tet2+ / + (WT) or Tet2+ / - germline mice. Three days after implantation, mice were placed on a control chow diet or an enriched The mice were fed the same nutritional diet as E0771 cells (dapamil) for 15 days. The mice were sacrificed 21 days after E0771 cell implantation.

[0085] The results of tumor volume are shown in Figure 1. Compared with Tet2+ / + (WT) mice, Tet2 + / - Mice showed a 2-fold increase in tumor growth (p < 0.05). + / - Compared with mice on a standard diet, tumor growth was significantly inhibited in Tet2+ / - mice that received a dapoxetine diet (68% reduction, p<0.01). Tumor growth was also significantly reduced in Tet2+ / + (WT) mice on dapoxetine compared with a standard diet (p<0.05). No changes in tumor growth were observed between Tet2+ / + (WT) or Tet2+ / - mice on a dapoxetine diet (Figure 1).

[0086] Example 1B: Dapanoxanil reduces IL-1β production in Tet2+ / - mice

[0087] Next, we evaluated the inflammatory potential of myeloid cells from tumor-bearing mice. Bone marrow adherent cells were cultured from tumor-bearing mice of Example 1 and stimulated with LPS overnight. Bone marrow cells cultured from tumor-bearing Tet2+ / - mice showed increased IL-1β production compared to WT (p<0.01, Figure 1BIn addition, bone marrow cells from tumor-bearing Tet2+ / - mice treated with dapasconit secreted significantly less IL-1β than untreated Tet2+ / - mice (p<0.05, Figure 2 ).

[0088] Example 2: Dapansutamide reduces breast tumor volume in Tet2+ / - bone marrow chimeric model

[0089] CH is characterized by clonal expansion of somatic mutations in the hematopoietic compartment. In this example, we determined whether the tumor-promoting effects of Tet2 deficiency are maintained in a bone marrow chimera model, an experimental model of clonal hematopoiesis.

[0090] We mixed unfractionated bone marrow (BM) cells from Tet2+ / + and Tet2+ / - donor mice with CD45.1+Boy / J control group BM in a 1:3 donor:control ratio and transplanted the cells into lethally irradiated Boy / J recipient mice. Six weeks after transplantation, E0771 cells were orthotopically implanted as described above. On day 3 after implantation, mice were given a control diet or a diet enriched with dapoxetine. We observed essentially the same pattern of accelerated tumor growth in Tet2+ / - chimeric mice (p<0.05), which was again inhibited in Tet2+ / - chimeric mice receiving a dapoxetine diet (p<0.001). Similarly, dapoxetine also inhibited tumor growth in Tet2+ / + chimeric animals to levels equivalent to baseline levels in Tet2+ / - chimeric mice treated with dapoxetine ( Figure 2 ). The results of Example 1 and Example 2 indicate that Tet2+ / - promotes breast cancer progression, and dapoxetine can effectively reduce tumor growth.

[0091] Example 3. Accelerated TET2+ / - driven breast tumor growth is reduced by dapoxetine

[0092] Granulocytes and monocytes, collectively referred to as myeloid cells, are differentiated descendants of common progenitor cells derived from hematopoietic stem cells in the bone marrow.

[0093] In this example, we first evaluated the infiltration of Tet2-deficient myeloid cells into the peripheral blood ( Figure 3A ) and tumors ( Figure 3B ) in the degree. Figure 3A It was shown that the frequency of donor-derived cells was significantly increased in the peripheral blood of Tet2+ / - chimeric mice compared to Tet2+ / + mice. Figure 3B It was shown that the frequency of Tet2+ / - donor-derived cells was significantly increased in tumors of Tet2+ / - chimeric mice compared to Tet2+ / + mice.

[0094] Figure 4AShown is a significant increase in total tumor infiltrating granulocytes in Tet2+ / - chimeras receiving a standard diet compared to Tet2+ / + chimeras receiving a standard diet (p<0.05). Figure 4A It was also shown that tumor infiltrating granulocytes were significantly reduced in Tet2+ / - chimeras receiving the dalpanacitide diet compared to Tet2+ / - chimeras on a standard diet (p<0.05).

[0095] Figure 4B It was shown that tumor infiltrating granulocytes expressed significantly less MHC-II protein (gMFI) in Tet2+ / - chimeras receiving a standard diet compared to Tet2+ / + chimeras receiving a standard diet (p<0.05). The reduced MHC-II expression present in Tet2+ / - chimeras was alleviated after the use of the dapoxetine diet (p<0.001).

[0096] Figure 5 shows the Figure 2 Plasma chemokines and cytokines in BM chimeric mice. As shown in Figure 5, Tet2+ / - chimeric mice receiving a standard diet showed increased circulating CCL2 and KC compared to Tet2+ / + chimeras on a standard diet (p<0.05). In addition, circulating CCL2 and KC were significantly reduced in Tet2+ / - chimeras receiving a dapoxetine diet compared to Tet2+ / - chimeras on a standard diet (p<0.05).

[0097] Taken together, these data demonstrate how breast cancer-promoting expansion of Tet2+ / - clones maintains the inflammatory environment of the TME, which in turn promotes breast cancer progression, and that dapagliflozin is able to reduce the expansion of Tet2+ / - clones.

[0098] References

[0099] 1. Y. Guo, et al. Cancer Res 77, 6429-6441 (2017).

[0100] 2.S.Shalapour,et al.J Clin Invest 125,3347-3355(2015).

[0101] 3.CADinarello.Blood 117,3720-3732(2011).

[0102] 4. RNApte, et al. Cancer Metastasis Rev 25, 387-408 (2006).

[0103] 5.CADinarello.Cancer Metastasis Rev 29,317-329(2010).

[0104] 6. B. Guo, et al. Sci Rep 6, 36107 (2016).

[0105] 7. KLLee, et al. Cancers 11(9), 1334(2019).

[0106] 8. C. Marchetti, et al. Proc Natl Acad Sci USA 115, E1530-E1539 (2018).

[0107] 9.A.Young,et al.Nature Communications.DOI:10.1038(2016)

[0108] 10. L. Dorsheimer, et al. JAMA Cardiol. 4: 25-33 (2019)

[0109] 11.NLSyn, et al. Lancet Oncology 18(12), PE731-E741(2017).

[0110] From the foregoing it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without departing from the scope of the invention. Accordingly, the invention is not to be limited by the appended claims.

Claims

1. A method for treating a solid cancer in a patient, the method comprising the steps of: (a) determining a clonal mutation in TET2 or DNMT3A in a sample of a patient having solid breast or prostate cancer and being treated with fidaxonitrile therapy, and (b1) if the patient is determined to have a variant allele frequency (VAF) of TET2 or DNMT3A greater than 0.02, treating the patient with an effective amount of fidaxonitrile in addition to the fidaxonitrile therapy, or (b2) if the patient is determined to have a VAF of TET2 or DNMT3A ≤ 0.02, continuing to treat the patient with the fidaxonitrile therapy.

2. The method of claim 1, wherein the solid cancer is breast cancer and the fidapanoxic acid treatment is a checkpoint inhibitor treatment.

3. The method of claim 2, wherein the checkpoint inhibitor is an inhibitor of programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

4. The method of claim 3, wherein the checkpoint inhibitor is an inhibitor of PD-1.

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

6. The method of claim 2, wherein the fidapamosulfuron treatment is chemotherapy.

7. The method of claim 2, wherein 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.

8. The method of claim 1, further comprising the step of selecting a patient having breast cancer and being treated with fidapamosulfuron therapy prior to step (a).

9. The method of claim 1, wherein dapoxetine is administered by oral administration.

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