WO2017159877A1 - Use of eribulin and 3-quinuclidinone derivatives in the treatment of cancer - Google Patents

Abstract

The invention features methods for treating and preventing cancer (e.g., a p53-mutated cancer) in a patient in need thereof by administering eribulin (e.g., eribulin mesylate) in combination with a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET).

Description

USE OF ERIBULIN AND 3-QUINUCLIDINONE DERIVATIVES IN THE TREATMENT OF CANCER BACKGROUND OF THE INVENTION

Cancer is a term used to describe a wide variety of diseases that are each characterized by the uncontrolled growth of a particular type of cell. It begins in a tissue containing such a cell and, if the cancer has not spread to any additional tissues at the time of diagnosis, may be treated by, for example, surgery, radiation, or another type of localized therapy. However, when there is evidence that cancer has metastasized from its tissue of origin, different approaches to treatment are typically used. Indeed, because it is not possible to determine with certainty the extent of metastasis, systemic approaches to therapy are usually undertaken when any evidence of spread is detected. These approaches can involve the administration of chemotherapeutic drugs that interfere with the growth of rapidly dividing cells, such as cancer cells. Other approaches involve the use of immunotherapy, in which an immune response against cancerous cells in a subject is elicited or enhanced.

Halichondrin B is a structurally complex, macrocyclic compound that was originally isolated from the marine sponge Halichondria okadai, and subsequently was found in Axinella sp., Phakellia carteri, and Lissodendoryx sp. A total synthesis of halichondrin B was published in 1992 (Aicher et al., J. Am. Chem. Soc. 114:3162-3164, 1992). Halichondrin B has been shown to inhibit tubulin polymerization, microtubule assembly, beta^S-tubulin crosslinking, GTP and vinblastine binding to tubulin, and tubulin-dependent GTP hydrolysis in vitro. This molecule has also been shown to have anti-cancer properties in vitro and in vivo. Halichondrin B analogs having anti-cancer activities are described in U.S. Patent No. 6,214,865 B1.

The chemical name for eribulin mesylate is 11,15:18,21:24,28-triepoxy-7,9-ethano-12,15-methano-9H,15H-furo[3,2-i]furo[2',3':5,6]pyrano[4,3-b][l,4]dioxacyclopentacosin-5(4H)-one, 2-[(2S)-3-amino-2-hydroxypropyl]hexacosahydro-3-methoxy-26-methyl-20,27-bis(methylene)-, (2R,3R,3aS,7R,8aS,9S,l0aR,11S,12R,13aR,13bS,15S,18S,21S,24S,26R,28R,29aS)-methanesulfonate (salt), and it can be depicted as follows:

PRIMA-1 and PRIMA-1^MET are 3-quinuclidinone derivatives that are able to restore the apoptosis-inducing function of mutant p53. PRIMA-1^MET (also known as APR-246) has been evaluated in a Phase I clinical trial and reported to be safe and to induce wild-type p53-mediated biological effects. The chemical name for PRIMA-1 is 2,2-bis(hydroxymethyl)quinuclidin-3-one, while the chemical name for PRIMA-1^MET is 2-(hydroxymethyl)-2-(methoxymethyl)quinuclidin-3-one. PRIMA-1 and PRIMA-1^MET are depicted as follows:

SUMMARY OF THE INVENTION

The present invention provides methods of treating and preventing cancer (e.g., p53-mutated cancer) by administration of eribulin, or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate), and a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET). The invention also provides methods of predicting the responsiveness of a cancer subject to therapy including eribulin, or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate), and a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET). When the term "eribulin" is used herein, it should be considered as indicating eribulin or a pharmaceutically acceptable salt thereof (such as eribulin mesylate), unless the context indicates otherwise.

Accordingly, in a first aspect, the invention provides methods for treating a subject (e.g., a human subject) having or at risk of developing a p53-mutated cancer by administering to the subject (a) eribulin, or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate), and (b) a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET). In some embodiments, (a) and (b) are administered substantially simultaneously. In some embodiments, (a) is administered first, followed by administration of (b). In some embodiments, (b) is administered first, followed by administration of (a). In some embodiments, (a) and (b) are administered substantially simultaneously, followed by administration of (a). In some embodiments, (a) and (b) are administered substantially simultaneously, followed by administration of (b). In some embodiments, the subject is diagnosed with the p53-mutated cancer, in treatment for the p53-mutated cancer, or in post-therapy recovery from the p53-mutated cancer.

The p53-mutated cancer may be a primary tumor, locally advanced, or metastatic. In various examples, the p53-mutated cancer is selected from the group consisting of breast cancer, lung cancer, stomach cancer, colon cancer, liver cancer, renal cancer, colorectal cancer, prostate cancer, pancreatic cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, testicular cancer, pelvic cancer, thyroid cancer, uterine cancer, rectal cancer, brain cancer, head and neck cancer, esophageal cancer, bronchus cancer, gallbladder cancer, ovarian cancer, bladder cancer, oral cancer, oropharyngeal cancer, larynx cancer, biliary tract cancer, skin cancer, a cancer of the central nervous system, a cancer of the respiratory system, and a cancer of the urinary system.

In certain examples, the p53-mutated cancer is breast cancer, for example, breast cancer selected from the group consisting of triple-negative breast cancer, triple-positive breast cancer, HER2-negative breast cancer, HER2-positive breast cancer, estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, progesterone receptor-positive breast cancer, progesterone receptor-negative breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, Paget disease of the nipple, and phyllodes tumor.

In other examples, the p53-mutated cancer is selected from the group consisting of B-cell leukemia, T-cell leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic (lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), erythroleukemia, angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, malignant fibrous cytoma, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, synovial sarcoma, vascular sarcoma, Kaposi's sarcoma, dermatofibrosarcoma, epithelioid sarcoma, leyomyosarcoma, neurofibrosarcoma, basal cell carcinoma, large cell carcinoma, small cell carcinoma, non-small cell lung carcinoma, renal carcinoma, hepatocarcinoma, gastric carcinoma, choriocarcinoma, adenocarcinoma, hepatocellular carcinoma, giant (or oat) cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastmic carcinoma, adrenocortical carcinoma, cholangiocarcinoma, Merkel cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, glioblastoma multiforme, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, multiple myeloma, plasmacytoma, localized myeloma, extramedullary myeloma, superficial spreading melanoma, nodular melanoma, lentigno maligna melanoma, acral lentiginous melanoma, amelanotic melanoma, ganglioneuroma, Pacinian neuroma, acoustic neuroma, astrocytoma, oligoastrocytoma, ependymoma, brainstem glioma, optic nerve glioma, oligoastrocytoma, pheochromocytoma, meningioma, malignant mesothelioma, and a virally induced cancer.

The eribulin (or pharmaceutically acceptable salt thereof, e.g., eribulin mesylate) may be administered by intravenous infusion. In some embodiments, the intravenous infusion is for about 1 to about 20 minutes (e.g., about 2 to about 5 minutes). The amount of eribulin (or pharmaceutically acceptable salt thereof, e.g., eribulin mesylate) administered can be in the range of about 0.1 mg/m² to about 20 mg/m² (e.g., about 1.1 mg/m² or 1.4 mg/m²).

The 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) may be administered by intravenous infusion. In some embodiments, the intravenous infusion is for about 0.5 to about 3 hours (e.g., about 2 hours). The amount of the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) administered can be in the range of about 2 mg/kg to about 90 mg/kg (e.g., about 2 mg/kg to about 60 mg/kg).

Treatment according to the methods of the invention may: (i) reduce the number of cancer cells; (ii) reduce tumor volume; (iii) increase tumor regression rate; (iv) reduce or slow cancer cell infiltration into peripheral organs; (v) reduce or slow tumor metastasis; (vi) reduce or inhibit tumor growth; (vii) prevent or delay occurrence and/or recurrence of the cancer and/or extends disease- or tumor-free survival time; (viii) increase overall survival time; (ix) reduce the frequency of treatment; and/or (x) relieve one or more of symptoms associated with the cancer.

The invention also provides methods for decreasing the size of a tumor (e.g., a tumor of a cancer type as described herein) in a subject having a p53-mutated cancer by administering to the subject (a) eribulin, or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate), and (b) a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET).

The invention further provides kits for use in treating p53-mutated cancer or decreasing tumor size in a subject having the p53-mutated cancer. The kits can includes (a) eribulin, or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate), and (b) a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET), optionally in dosage form.

Further, the invention includes methods of predicting the responsiveness of a cancer subject to a combination therapy including eribulin, or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate), and a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET). The methods can include: (a) collecting a sample (e.g., a tissue, blood, or tumor sample) from the cancer subject; and (b) determining the presence of a mutant p53 protein or gene in the sample from the cancer subject by measuring the level of expression of the mutant p53 protein or gene (e.g., using ELISA or PCR), wherein the presence of a mutant p53 protein or gene in the sample from the cancer subject indicates that the cancer subject may be responsive to the combination therapy.

The invention further includes the use of the combinations described herein for the prevention and treatment of the diseases and conditions described herein, as well as the use of these combinations for the preparation of medicaments for preventing and treating these diseases and conditions.

The methods of the invention provide improved efficacy against cancer. For example, the combination treatment methods described herein can be used to obtain synergistic effects in which, for example, the effects are greater than the sum of the effects of the drugs administered individually, as can be determined by those of skill in the art.

Other features and advantages of the invention will be apparent from the following detailed description, drawings, and claims.

Fig.1

Table listing 23 breast cell lines. Also included is information concerning their molecular subtype, ER status, HER2 status, p53 mutational status, p53 mutational subtype, p53 protein expression status, p63 protein expression status, p73 protein expression status, BRCA1 mutational status, and mean IC₅₀ values ± SEM for both PRIMA-1 and PRIMA-1^MET.

Fig.2

Effect of PRIMA-1 and PRIMA-1^MET on cell line growth. A, IC₅₀ values for cell lines treated with PRIMA-1. B, cell lines treated with PRIMA-1^MET. IC₅₀ values are means ± SEM of 3 separate experiments. Values were calculated using Calcusyn software following MTT assay. C, Bivariate Scatter plot representation of the relationship between IC₅₀ values of PRIMA-1 and PRIMA-1^MET. Data was analysed using Spearman rank test. Data points represent the mean of 3 independent experiments. D, Bivariate scatter plot representation of the relationship between IC₅₀ values from MTT assay and CFA (colony formation assay). Data was analysed using Spearman rank test. Data points represent the mean of 3 independent experiments. E-H, xCELLigence analyse of cell growth. Data was normalized to the point before addition of the compound, and monitored for up to 96 hr, in a real-time manner. Data was analysed using Paired t test. All experiments were carried out in triplicate.

Fig.3

Relationship between PRIMA-1^MET and p53 mutational status, protein levels, and cell line molecular subtype. A, Scatterplot representing the relationship between response to PRIMA-1^MET and p53 mutational status of cell lines. Data was analysed using Student's t test. Data points represent the mean of 3 independent experiments. Mut= mutant WT= wild-type. *2 of the 5 WT cells used were immortalized breast epithelial cells and shown as triangles, in contrast to cancer cell lines which are represented by squares. B, Scatterplot representing the relationship between response to PRIMA-1^MET and subtype of p53 mutation. The relationship between contact and structural mutations was analysed using the Student's t test. One-way ANOVA was used to analysed the relationship between all 3 subtypes. Data points represent the mean of 3 independent experiments. C, Scatter plot representing p53 protein status as determined by a visible band detected by Western blotting (p53+) or no band seen at the expected molecular mass of p53 (p53-), and IC₅₀ values. Data was analysed using Student t test. All experiments were carried out in triplicate. D, Bivariate scatterplot representing the correlation between p53 protein levels quantified by ELISA and PRIMA-1^MET IC₅₀ values, measured by MTT assay. Data was analysed by the Spearman rank test. E, Bivariate scatterplot representing the correlation between p63 protein levels determined by Western blot and semi-quantified by densitometry, and PRIMA-1^MET IC₅₀ values, measured by MTT assay. Data was analysed by the Spearman rank test. F, Bivariate scatterplot representing the correlation between p73 protein levels determined by Western blot and semi-quatified by densitometry and PRIMA-1^MET IC₅₀ values, measured by MTT assay. Data was analysed by the Spearman rank test. G, Relationship between response to PRIMA-1^MET and triple-negative (TN) status of cells. Data was analysed using Student's t test. Data points represent the mean of 3 independent experiments. H, Relationship between response to PRIMA-1^MET and molecular subtype. TN=triple negative. Data was analysed using One-way ANOVA. Data points represent the mean of 3 independent experiments.

Fig.4

Western blot analysis. A, a representative image of a p53 protein Western blot. Cells are in order left to right based on their IC₅₀ values (low to high). B and C, bar graphs showing p53 protein levels determined by Western blotting semiquantified by densitometry and by ELISA, respectively.

Fig.5

Effect of PRIMA-1^MET on apoptosis. A-C, MDA-MB-453 cells; D-F, MDA-MB-468 cells; G-I, BT549 cells; J-L, MCF7 cells. Cells were stained with annexin-V (AV) and propidium iodide (PI) and analysed by flow cytometry. Viable cell= AV-/PI-, e arly apoptotic= AV+/PI-, late apoptotic= AV+/PI+, dead cells=AV-/PI+. All experiments were carried out in triplicate. Data was analysed using Paired t test.

Fig.6

Effect of PRIMA-1^MET on cell migration. A-D, Bar chart representation of percentage cell migration determined by Transwell migration assay, and a representative image of Transwell insert (×20). Percent migration was determined by quantifying the number of cells on the underside of the insert, and normalising to vehicle control. All experiments were carried out in triplicate. Data was analysed using Paired t test. E-H, Bar chart representation of cell lines wound repair upon treatment with PRIMA-1^MET and picture representative of scratched wells. The % open area was analysed using Tscratch software. All experiments were carried out in triplicate. Data was analysed using the Paired t test.

Fig.7

Effect of combined treatment with PRIMA-1^MET and different cytotoxic agents on cell viability. Cells were treated with PRIMA-1^MET alone, cytotoxic drug alone or the combination at a fixed ratio for 5 days. Cell viability was assessed using the MTT assay. CI (combination index) values were calculated using Calcusyn software. All experiments were carried out in triplicate. P-1^MET= PRIMA-1^MET. Data points represent the mean of three independent experiments.

Fig.8

Table showing the effect of combined treatment with PRIMA-1^MET and Eribulin or Docetaxel on CI values for specific cell lines. CI (combination index) values were calculated using Compusyn software, based on the Chou-Talalay method. CI <0.9 indicates synergism, 0.9-1.1 indicates an additive effect and CI >1.1 indicates an absence of an enhanced effect from the combination.

Fig.9

Effect of combined treatment with Eribulin and PRIMA-1^MET. Cells were treated with eribulin alone, PRIMA-1^MET alone, or the combination at a fixed ratio for 5 days. Cell viability was assessed using the MTT assay. CI (combination index) values were calculated using Calcusyn software. P-1^MET= PRIMA-1^MET.

Fig.10

Effect of Eribulin alone or in combination with PRIMA-1^MET on breast cell line growth. A, Growth curve representation of the effects of eribulin monotherapy on cell line growth. Cells were treated with a range of concentrations of eribulin for 5 days. Cell viability was assessed by the MTT assay. B, Growth curve representation of the effects of eribulin in combination with PRIMA-1^MET on cell line growth. Cells were treated with a range of concentrations of eribulin plus PRIMA-1^MET for 5 days. Cell viability was assessed by the MTT assay. P-1^MET= PRIMA-1^MET.

Fig.11

Relationship between eribulin alone or in combination with PRIMA-1^MET and molecular subtype or p53 mutational status. A, Relationship between response to eribulin and molecular subtype of cell lines. Data was analyzed using Student's t test. B, Relationship between response to eribulin and p53 mutational status of cell lines. Data was analyzed using Student's t test. C, Relationship between the Combination Index and molecular subtype of cell lines. Data was analyzed using Student's t test. D, Relationship between Combination Index and p53 mutational status. Data was analyzed using Student's t test. P<0.05 was considered significant. ns= no significance.

Fig.12

Effect of combination treatments on apoptosis. Bar chart representing percentage apoptosis induced by PRIMA-1^MET, eribulin, docetaxel, or the combinations in a panel of cell lines. Cells were stained with annexin-V (AV) and propidium iodide (PI) and analyzed by flow cytometry. Viable cell=AV-/PI-, early apoptotic=AV+/PI-, late apoptotic=AV+/PI+, dead cells=AV-/PI+. All experiments were carried out in triplicate. Data was analyzed using Paired t test.

DETAILED DESCRIPTION

The invention provides methods for the treatment or prevention of cancer (e.g., a p53-mutated cancer) involving administration of eribulin or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate) in combination with a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET). The invention also provides methods of predicting the responsiveness of a cancer subject to therapy including eribulin, or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate), and a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) by (a) collecting a sample from the cancer subject, and (b) determining the presence of a mutant p53 protein or gene in the sample from the cancer subject by measuring the level of expression of the mutant p53 protein or gene, wherein the presence of a mutant p53 protein or gene in the sample from the cancer subject indicates that the cancer subject may be responsive to the therapy.

Treatment of a p53-mutated cancer according to the methods of the invention, can (i) reduce the number of cancer cells; (ii) reduce tumor volume; (iii) increase tumor regression rate; (iv) reduce or slow cancer cell infiltration into peripheral organs; (v) reduce or slow tumor metastasis; (vi) reduce or inhibit tumor growth; (vii) prevent or delay occurrence and/or recurrence of the cancer and/or extend disease- or tumor-free survival time; (viii) increase overall survival time; (ix) reduce the frequency of treatment; and/or (x) relieve one or more of symptoms associated with the cancer.

Pharmaceutical Compositions, Dosage, and Methods
Pharmaceutical compositions including eribulin and/or the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) described herein can be prepared using standard methods known in the art, or can be obtained from commercial sources. Typically, eribulin and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) used in the invention are included within separate pharmaceutical compositions but they can, optionally, be included within a single composition. Eribulin and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) are typically provided in liquid form, for intravenous administration.

Pharmaceutical compositions used in the invention can be prepared by, for example, mixing or dissolving the active ingredient(s), having the desired degree of purity, in a physiologically acceptable diluent, carrier, excipient, or stabilizer (see, e.g., Remington's Pharmaceutical Sciences (22^nd edition), ed. A. Gennaro, 2012, Lippincott, Williams & Wilkins, Philadelphia, PA). Acceptable diluents include water and saline, optionally including buffers such as phosphate, citrate, or other organic acids; antioxidants including butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagines, arginine or lysine; monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN^TM, PLURONICS^TM, or PEG.

In preparing compositions for oral dosage form, any of the usual pharmaceutical media can be employed, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents. In addition, carriers such as starches, sugars, microcristalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used in the case of oral solid preparations such as, for example, powders, capsules, and tablets.

Optionally, the formulations of the invention contain a pharmaceutically acceptable preservative. In some embodiments the preservative concentration ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts, such as benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben. Further, the eribulin and/or the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) formulations can optionally include a pharmaceutically acceptable salt, such as sodium chloride at, for example, about physiological concentrations. Thus, in one example, eribulin (e.g., eribulin mesylate) is formulated in 0.9% Sodium Chloride Injection (USP).

The formulations noted above (and others) can be used for parenteral administration of the drugs. Thus, the drugs can be administered by routes including intravenous, intra-tumoral, peri-tumoral, intra-arterial, intra-dermal, intra-vesical, ophthalmic, intramuscular, intradermal, intraperitoneal, pulmonary, subcutaneous, and transcutaneous routes. Other routes can also be used including, for example, transmucosal, transdermal, inhalation, intravaginal, rectal, and oral administration routes.

The dosage of eribulin and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) described herein administered can differ markedly depending on the type of target disease, the choice of delivery method, as well as the age, sex, and weight of the patient, the severity of the symptoms, along with other factors.

Eribulin and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) used in the methods of the invention are described below, in addition to regimens of administration.

As noted above, eribulin can optionally be used in the present invention in salt forms. There are no particular limitations as to the salt used, whether inorganic acid salt or organic acid salt. For example, the salt can be selected from mesylic acid salt (e.g., eribulin mesylate), hydrochloric acid salt, sulfuric acid salt, citrate, hydrobromic acid salt, hydroiodine acid salt, nitric acid salt, bisulfate, phosphoric acid salt, super phosphoric acid salt, isonicotinic acid salt, acetic acid salt, lactic acid salt, salicic acid salt, tartaric acid salt, pantotenic acid salt, ascorbic acid salt, succinic acid salt, maleic acid salt, fumaric acid salt, gluconic acid salt, saccharinic acid salt, formic acid salt, benzoic acid salt, glutaminic acid salt, methanesulfonic acid salt, ethanesulfonic acid salt, benzenesulfonic acid salt, p-toluenesulfonic acid salt, pamoic acid salt (pamoate), and so on. Moreover, it is acceptable to use salt of aluminum, calcium, lithium, magnesium, sodium, zinc, and diethanolamine.

The daily dosage of eribulin (e.g., eribulin mesylate) can be in the range of, e.g., 0.001 mg/m² to about 100 mg/m² (e.g., in the range of about 0.1 mg/m² to about 50 mg/m² or in the range of about 0.7 mg/m² to about 1.5 mg/m², or in any single amount within these ranges (e.g., 1.4 mg/m² or 1.1 mg/m²)). Eribulin can be administered as a single dose once per day, week, month, or year, or more than one dose of eribulin can be administered per day, week, month, or year. For example, in one administration protocol, eribulin can be administered once on days 1 and 8 of a 21-day cycle. More specifically, a recommended dose of eribulin mesylate is 1.4 mg/m² administered intravenously over 2 to 5 minutes on days 1 and 8 of a 21-day cycle. A recommended dose of eribulin mesylate in patients with mild hepatic impairment (Child-Pugh A) is 1.1 mg/m² administered intravenously over 2 to 5 minutes on days 1 and 8 of a 21-day cycle, while a recommended dose of eribulin mesylate in patients with moderate hepatic impairment (Child-Pugh B) is 0.7 mg/m² administered intravenously over 2 to 5 minutes on days 1 and 8 of a 21-day cycle. Further, a recommended dose of eribulin mesylate in patients with moderate renal impairment (creatinine clearance of 30-50 mL/min) is 1.1 mg/m² administered intravenously over 2 to 5 minutes on days 1 and 8 of a 21-day cycle. These or other lower doses of eribulin mesylate can optionally be used in the context of combination treatment, according to the methods of the present invention.

3-Quinuclidinone Derivative (e.g., PRIMA-1 or PRIMA-1^MET)
Methods for the synthesis of 3-quinuclidinone derivatives are described, for example, in U.S. Patent No. 7,759,361; U.S. Patent No. 6,921,765; U.S. Patent No. 7,348,330; U.S. Patent No. 7,659,278; and U.S. Patent No. 9,061,016, each of which is incorporated herein by reference.

A 3-quinuclidinone derivative includes compounds according to formula (I):

wherein R¹ is selected from H, -CH₂-O-R³, -CH₂-S-R³, and -CH₂-NR³ R⁴; R² is selected from -CH₂-O-R³, -CH₂-S-R³, and -CH₂-NR³ R⁴; R³ and R⁴ are the same or different and are independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or C1-C10 alkyl; substituted or non-substituted benzyl; substituted or non-substituted mono- or bicyclic aryl; substituted or non-substituted mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl containing one or several heteroatoms independently selected from N, O and S; or R³ and R⁴ in -CH₂-NR³R⁴ are bonded together and form, together with the nitrogen atom to which they are bonded, a substituted or non-substituted non-aromatic C2-C10 mono- or bicyclic heterocyclyl optionally containing one or several further heteroatoms independently selected from N, O and S and optionally comprising one or several cyclic keto groups; wherein the substituents of the substituted groups are independently selected from unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl or C1-C10 alkyl; halogen; halogen substituted C1-C10 alkyl, mono- or bicyclic aryl; mono-, bi- or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl containing one or several heteroatoms independently selected from N, O and S; C1-C10 alkoxy; amino; and C1-C10 alkylamino; or a pharmaceutically acceptable salts thereof.

The pharmaceutically acceptable salt of the compound of formula (I), e.g. may be an acid addition salt of an inorganic mineral acid or of an organic acid.

In a compound of formula (I), R¹ is selected from H, -CH₂-O-R³, -CH₂-S-R³, and -CH₂-NR³R⁴.

In some examples, R¹ is selected from H, -CH₂-O-R³, and -CH₂-S-R³. In some embodiments, R¹ is selected from H and -CH₂-O-R³. In other embodiments, R¹ is selected from -CH₂-O-R³ and -CH₂-S-R³. In some embodiments, R¹ is H.

R² in formula (I) is selected from -CH₂-O-R³, -CH₂-S-R³, and -CH₂-NR³R⁴. In some embodiments, R² is selected from -CH₂-O-R³ and -CH₂-S-R³. In still other embodiments, R² is -CH₂-O-R³.

In one embodiment, R¹ is selected from H, -CH₂-O-R³ and -CH₂-S-R³; and R² is selected from -CH₂-O-R³ and -CH₂-S-R³.

In one example, R¹ is selected from H and -CH₂-O-R³; and R² is -CH₂-O-R³.

In one example, both R¹ and R² are -CH₂-O-R³.

In one example, each R³ is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl. For example, each R³ may be independently selected from H and C1-C10 alkyl, e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.

In one example, R¹ is selected from H and -CH₂-O-R³, and R² is -CH₂-O-R³, and each R³ is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl, and benzyl, in particular from H and C1-C10 alkyl, e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.

In one example, R¹ and R² are both -CH_2--O-R³, and each R³ is independently selected from H; substituted or non-substituted, unbranched or branched, saturated or unsaturated C3-C12 cycloalkyl and C1-C10 alkyl; in particular from H and C1-C10 alkyl; e.g. from H and C1-C6 alkyl, from H and C1-C4 alkyl, or from H and C1-C3 alkyl, in particular from H and methyl.

In a compound of formula (I), as defined herein above, any C1-C10 alkyl e.g. may be a C1-C6 alkyl, or a C1-C4 alkyl, e.g. methyl, ethyl, propyl or butyl. Any C3-C12 cycloalkyl may be e.g. a C3-C8 cylcloalkyl, or a C3-C6 cycloalkyl. Any mono- or bicyclic aryl may be e.g. a monocyclic aryl, such as phenyl. Any mono-, bi- or tricyclic C2-C10 heteroaryl may be e.g. a monocyclic or bicyclic C2-C5 heteroaryl, e.g. a 5- or 6-membered moncyclic or a 9-membered bicyclic C2-C5 heteroaryl. Any mono-, bi- or tricyclic non-aromatic C2-C10 heterocyclyl may be e.g. a monocyclic or bicyclic C2-C5 heterocyclyl, e.g. a 5- or 6-membered monocyclic or 9- or 10-membered bicyclic C2-C5 heterocyclyl. Any halogen may be selected from F, Cl, Br and I, preferably from F and Cl. Any heterocycle, aromatic or not, containing one of several heteroatoms independently selected from N, O and S, e.g. may contain 1-5 heteroatoms, e.g. independently selected from N and O.

In one example, in a compound of formula (I) as defined herein above, any substituted or non-substituted C3-C12 cycloalkyl or C1-C10 alkyl is non-substituted.

In one example, any substituted or non-substituted benzyl is non-substituted.

In one example, any substituted or non-substituted mono- or bicyclic aryl is non-substituted.

In one example, any substituted or non-substituted mon-, bi or tricyclic C2-C10 heteroaryl or non-aromatic C2-C10 heterocyclyl is non-substituted.

In one example, when any of the above groups is substituted, each substituent is selected from C1-C10 alkyl, e.g. C1-C6 alkyl, C1-C4 alkyl, or C1-C3 alkyl, such as methyl; halogen, e.g. Cl; halogen-substituted C1-C10 alkyl, e.g. trifluoromethyl; monocyclic C2-C5 heteroaryl, e.g. pyridyl; C1-C10 alkoxy, e.g. C1-C6 alkoxy, C1-C4 alkoxy, or C1-C3 alkoxy, such as methoxy; and amino.

In one example, when any of the above groups is substituted, the number of substituents on each substituted group is 1, 2, or 3.

Some exemplary 3-quinuclidinone derivatives are listed below:

In some examples, the daily dosage of a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) can be in the range of, e.g., 0.001 mg/m² to about 100 mg/m² (e.g., in the range of about 0.1 mg/m² to about 50 mg/m²; e.g., 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg/m²). In some embodiments, a pharmaceutical composition may include a dosage of a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) ranging from 0.01 to 500 mg/kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg), or ranging from 1 mg/kg to 100 mg/kg (e.g., 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg/kg), or ranging from 2 mg/kg to 90 mg/kg (e.g., 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 mg/kg), or ranging from 2 mg/kg to 60 mg/kg (e.g., 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 mg/kg). The 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) can be administered as a single dose once per day, week, month, or year, or more than one dose of the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) can be administered per day, week, month, or year. For example, in one administration protocol, the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) can be administered as a 2-hour intravenous infusion once per day for 4 consecutive days at 2 mg/kg, 4 mg/kg, 6 mg/kg, 8 mg/kg, or 10 mg/kg. The 4-day administration cycle may be repeated as necessary either at the same dose as the previous cycle, or at a lower or higher dose. For example, the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) can be administered as a 2-hour intravenous infusion once per day from day 1 to day 4 at 2 mg/kg, from day 5 to day 8 at 4 mg/kg, and from day 9 to day 12 at 6 mg/kg. These or other doses of the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) can optionally be used in the context of combination treatment, according to the methods of the present invention.

Combination Administration Regimens
As noted above, according to the methods of the invention, eribulin (e.g., eribulin mesylate) and a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) are administered in combination. In some embodiments, eribulin (e.g., eribulin mesylate) and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) are administered substantially simultaneously. In some embodiments, eribulin (e.g., eribulin mesylate) and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) are administered separately, e.g., eribulin (e.g., eribulin mesylate) is administered first, followed by administration of the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET); or the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) is administered first, followed by administration of the eribulin (e.g., eribulin mesylate). In some embodiments, eribulin (e.g., eribulin mesylate) and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) are administered substantially simultaneously, followed by administration of eribulin (e.g., eribulin mesylate) or the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET). In some embodiments, eribulin (e.g., eribulin mesylate) or the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) is administered first, followed by administration of eribulin (e.g., eribulin mesylate) and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) substantially simultaneously. The administrations can begin on the same day or treatment using one agent can start, e.g., 1, 2, 3, 4, 5, or 6 weeks before treatment the other, as can be determined to be appropriate by those of skill in the art.

In addition to eribulin and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) noted above, the methods of the present invention can also include the administration of one or more additional therapeutic agents. Among these agents, immunomodulatory agents (e.g., antibodies or vaccines), chemotherapeutic/antitumor agents, antibacterial agents, anti-emetics, and anti-inflammatory agents are suitable. In other instances, eribulin (e.g., eribulin mesylate) and the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) can be used in a treatment regimen as the sole therapeutic (e.g., sole anti-cancer) agents. Thus, the methods of the invention can consist of administration of (a) eribulin or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate), and (b) a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET).

Cancers
The methods of the invention can be used to treat (including, e.g., delay progression) or prevent cancer (e.g., a p53-mutated cancer) in a subject (e.g., a human patient) and/or to decrease tumor size. A p53-mutated cancer refers to a cancer having a mutation the p53 gene or protein. The subject can be diagnosed with cancer (e.g., a p53-mutated cancer), at risk for developing cancer, in treatment for cancer, or in post-therapy recovery from cancer. Further, the methods can be used to treat or prevent metastases and/or recurrence. The treatment can be chemotherapeutic alone, although treatment in combination with a surgical procedure to remove or reduce the size of a tumor, radiation therapy, immunotherapy, and/or ablation therapy is also envisioned.

Examples of cancers that can be treated or prevented according to the methods of the invention include, but are not limited to, breast cancer, lung cancer, stomach cancer, colon cancer, liver cancer, renal cancer, colorectal cancer, prostate cancer, pancreatic cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, testicular cancer, pelvic cancer, thyroid cancer, uterine cancer, rectal cancer, brain cancer, head and neck cancer, esophageal cancer, bronchus cancer, gallbladder cancer, ovarian cancer, bladder cancer, oral cancer, oropharyngeal cancer, larynx cancer, biliary tract cancer, skin cancer, a cancer of the central nervous system, a cancer of the respiratory system, and a cancer of the urinary system. Examples of breast cancers include, but are not limited to, triple-negative breast cancer, triple-positive breast cancer, HER2-negative breast cancer, HER2-positive breast cancer, estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, progesterone receptor-positive breast cancer, progesterone receptor-negative breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, Paget disease of the nipple, and phyllodes tumor.

Other examples of cancers that can be treated or prevented according to the methods of the invention include, but are not limited to, leukemia (e.g., B-cell leukemia, T-cell leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic (lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), and erythroleukemia), sarcoma (e.g., angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, malignant fibrous cytoma, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, synovial sarcoma, vascular sarcoma, Kaposi's sarcoma, dermatofibrosarcoma, epithelioid sarcoma, leyomyosarcoma, and neurofibrosarcoma), carcinoma (e.g., basal cell carcinoma, large cell carcinoma, small cell carcinoma, non-small cell lung carcinoma, renal carcinoma, hepatocarcinoma, gastric carcinoma, choriocarcinoma, adenocarcinoma, hepatocellular carcinoma, giant (or oat) cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastmic carcinoma, adrenocortical carcinoma, cholangiocarcinoma, Merkel cell carcinoma, ductal carcinoma in situ (DCIS), and invasive ductal carcinoma), blastoma (e.g., hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, and glioblastoma multiforme), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, and Burkitt lymphoma), myeloma (e.g., multiple myeloma, plasmacytoma, localized myeloma, and extramedullary myeloma), melanoma (e.g., superficial spreading melanoma, nodular melanoma, lentigno maligna melanoma, acral lentiginous melanoma, and amelanotic melanoma), neuroma (e.g., ganglioneuroma, Pacinian neuroma, and acoustic neuroma), glioma (e.g., astrocytoma, oligoastrocytoma, ependymoma, brainstem glioma, optic nerve glioma, and oligoastrocytoma), pheochromocytoma, meningioma, malignant mesothelioma, and virally induced cancer.

The methods of the invention can be used to treat or prevent breast cancer (e.g., triple-negative breast cancer, triple-positive breast cancer, HER2-negative breast cancer, HER2-positive breast cancer, estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, progesterone receptor-positive breast cancer, progesterone receptor-negative breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, Paget disease of the nipple, and phyllodes tumor). In some embodiments, the methods of the invention can be used to treat or prevent triple-negative breast cancer. The methods of the invention can further be used to treat locally advanced or metastatic breast cancer. In some embodiments, the methods of the invention can be used to treat or prevent ovarian cancer. In various examples, the methods of the invention are carried out in the neoadjuvant setting, and thus are carried out before another, primary treatment, such as surgery.

Kits
The invention also provides kits that include a container with eribulin (e.g., eribulin mesylate) and/or a container with a 3-quinuclidinone derivative described herein (e.g., PRIMA-1 or PRIMA-1^MET). The eribulin and/or the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) in such kits can be provided in amounts sufficient to treat cancer (e.g., a p53-mutated cancer) in a patient in need thereof (e.g., amounts sufficient for a single administration or for multiple administrations). The kits can thus include multiple containers that each include effective amounts of single-dose eribulin and/or the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) pharmaceutical composition(s). Optionally, instruments and/or devices necessary for administering the pharmaceutical composition(s) can also be included in the kits. Furthermore, the kits can include additional components, such as instructions or administration schedules, for treating a patient with cancer (e.g., a p53-mutaed cancer) with the eribulin and/or the 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) described herein.

Diagnostic Methods
The invention also provides methods of predicting the responsiveness of a cancer subject to therapy including eribulin, or a pharmaceutically acceptable salt thereof (e.g., eribulin mesylate), and a 3-quinuclidinone derivative (e.g., PRIMA-1 or PRIMA-1^MET) by (a) collecting a sample (e.g., a tissue sample (e.g., one or more lymph nodes), a blood or serum sample (for detection of , e.g., circulating tumor cells or circulating DNA or RNA), or a tumor sample (e.g., a primary tumor biopsy or a metastatic tumor biopsy)) from the cancer subject, and (b) determining the presence of a mutant p53 protein or gene in the sample from the cancer subject by measuring the level of expression of the mutant p53 protein or gene, wherein the presence of a mutant p53 protein or gene in the sample from the cancer subject indicates that the cancer subject may be responsive to the therapy.

Expression of various proteins or genes in a sample can be detected and analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including but not limited to, immunohistochemical and/or western blot analysis, immunoprecipitation, molecular binding assays, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA), fluorescence activated cell sorting (FACS), quantitative blood based assays (e.g., serum ELISA), biochemical enzymatic activity assays, in situ hybridization, northern blot analysis, southern blot analysis, and/or PCR analysis of DNAs, RNAs, mRNAs, and/or cDNAs, as well as any one of the wide variety of assays that can be performed by gene and/or tissue array analysis (e.g., microarray analysis). Typical protocols for evaluating the status of genes and proteins are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting), and 18 (PCR Analysis).

The present invention is illustrated by the following examples, which are in no way intended to be limiting of the invention.

EXAMPLES

Example 1
Materials and Methods
Cell Culture
A panel of 21 breast cancer cell lines and 2 immortalized cell lines were used. The cancer cell lines consisted of all the main molecular subtypes of breast cancer. Fig. 1 lists 23 breast cell lines used in the experiments, their molecular subtype, ER status, HER2 status, p53 mutational status, p53 mutational subtype, p53 protein expression status, p63 protein expression status, p73 protein expression status, BRCA1 mutational status, and mean IC₅₀ values ± SEM for both PRIMA-1 and PRIMA-1^MET. The Hs578t(i8) cell line was derived from the parental Hs578t cell line by sequential selection through in vitro invasive chambers (Hughes et al., Clin Exp Metastasis 25: 549-57, 2008). CAL-85-1 cells were obtained from German Collection of Microorganisms and Cell (DSMZ), Germany. All other cell lines were purchased from the American Type Culture Collection (ATCC).

Cell lines were maintained through continued passaging at 37°C with a humidified atmosphere of 5% CO₂. All media was supplemented with 10% foetal bovine serum (FBS) (Invitrogen Life Technologies), 1% penicillin/streptomycin (Invitrogen Life Technologies), and 1% Fungizone (Invitrogen Life Technologies). BT549 and HCC70 cells were maintained in RPMI 1640 supplemented with 0.023 IU Insulin (Sigma-Aldrich), 10 mM Hepes (Sigma-Aldrich), 1.5 g/L sodium bicarbonate (Sigma-Aldrich), and 1 mM sodium pyruvate (Sigma-Aldrich). Cal-85-1 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) and 1 mM Na Pyruvate (Sigma-Aldrich). ZR-75-1 cells were maintained in DMEM supplemented with 1 nM estradiol (Sigma-Aldrich). UACC812 cells were maintained in Lebovitz L-15 media. MCF10A and MCF12A cells were maintained as previously described (Debnath et al., Methods 30:256-68, 2003). All other cell lines were maintained in RPMI 1640. Cell line identity was confirmed by analysis of Short Term Repeat Loci and cells were routinely tested for mycoplasma infection.

Cell viability assays
The effect of PRIMA-1 and PRIMA-1^MET (Tocris Bioscience) on cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as previously described (McGowan et al., Ann Oncol 24:362-369, 2013). The clonogenic assays were also carried out as previously described (McGowan et al., above). For the xCELLigence system, cells were seeded at a density of 1 × 10⁴/well of the E-plate. Following overnight incubation, cells were treated with varying concentrations of PRIMA-1^MET. The impedance value of each well was automatically monitored by the xCELLigence system and expressed as a CI (cell index) value (Dowling et al., Biosci Rep 34: e00126, 2014). CI values were recorded every minute for the first 10 hr and then every 15 min for up to 5 days.

Apoptosis assays
Cells were seeded in 6-well plates (Sigma-Aldrich) at a density of 2 × 10⁵ cells/well. Following overnight incubation, cells were treated for 1-5 days with concentrations ranging 0 to 50 μM, in low serum (2% FBS) supplemented RPMI 1640. Staining for annexin-V and propidium iodide was carried out using the Annexin V-FITC Apoptosis Detection Kit (eBioscience), according to the manufacturer's instructions. FACS analysis was immediately performed using a BD FACSCalibur^TM. Data was analysed using FlowJo_V10 software.

To investigate cell wound repair, cells were seeded in 24-well plates and grown to confluency. A sterile p1000 pipette tip was used to create a linear wound across the well. Cells were treated in low (2% FBS) serum media for 5 days. This was followed by fixation using 1% glutaraldehyde and staining with 0.1% crystal violet. Data was analyzed using Tscratch software (Geback et al., BioTechniques 46:265-274, 2009).

Statistical analysis
Data was analysed using Prism version 5.0b software (GraphPad Software). P values < 0.05 were considered significant. The IC₅₀ (concentration required to inhibit growth by 50%) for each compound and combination index (CI value) was determined using CalcuSyn software (Biosoft). For the experiments involving drug combinations, CI <0.9 indicated synergism, 0.9-1.1 indicated an additive effect and CI >1.1 indicated an absence of an enhanced effect from the combination (Chou et al., Cancer Res 70:440-446, 2010).

Results
Effect of PRIMA-1 and PRIMA-1^MET on Cell Viability in Breast Cell Lines
Using the MTT cell viability assay, the sensitivity of 23 cell lines for both PRIMA-1 and PRIMA-1^MET was investigated. Figs. 2A and 2B show that the sensitivity for both PRIMA-1 and PRIMA-1^MET was widely variable across the panel of the 23 cell lines investigated, with IC₅₀ values ranging from 1.4 μM to 15.1 μM for PRIMA-1 and from 0.9 μM to 31.1 μM for PRIMA-1^MET. Fig. 2C shows that the response to PRIMA-1 correlated significantly with that for PRIMA-1^MET (p<0.0001, r=0.84). Results from the MTT assay for PRIMA-1^MET were confirmed using the more biologically relevant colony formation assay (CFA). For this comparison, 12 randomly selected cell lines evaluated with the MTT assays were also assessed with the CFA. As shown in Fig. 2D, a significant correlation was found between the IC₅₀ values using the MTT and CFA assays ( r=0.78, p=0045).

In contrast to both the MTT and CFA assays, the xCELLigence system, which detects cellular impedance, monitors response to drugs in real-time (Dowling et al., Biosci Rep 34: e00126, 2014). Using this system, response to PRIMA-1^MET in MDA-MB-453 and MDA-MB-468 cells was found to be both time and concentration-dependent, as shown in Figs. 2E and 2F. Consistent with the relatively high IC₅₀ values obtained with Hs578T(i8) and MCF-7 cells using the MTT assay (11.9 μM and 31.1 μM, respectively), no response to PRIMA-1^MET was seen using the xCELLigence system, as shown in Figs. 2G and 2H.

Effect of p53 Mutational Status and Protein Levels on Response to PRIMA-1^MET
Since PRIMA-1^MET is believed to target mutant p53 (Brown et al., Trends Pharmacol Sci 32:53-62, 2011; Bykov et al., Nat Med 8:282-8, 2002), it was important to determine if response was related to the p53 mutational status of the cell lines. As shown in Fig. 3A, significantly lower IC₅₀ values were found in p53-mutant compared to p53-wild-type cells (p=0.014, Student's t test), i.e., mutant cells were more sensitive to PRIMA-1^MET than wild-type cells. As 2 of the p53 wild-type cells lines, i.e., MCF10A and MCF12A, were immortalized rather than tumorigenic, analysis was also carried out following exclusion of these cell lines. Following exclusion, the IC₅₀ values remained significantly higher for the p53 mutant cell lines vs the p53 wild-type cell lines (p=0.0008, Student's t test). Response to PRIMA-1^MET however, was independent of the type of p53 mutation, i.e., whether the mutation was of the contact type or structural. (p=0.35, Student's t test) (Fig. 3B).

Since mutant p53 tends to encode a stabilized protein as compared to the wild type gene (Suh et al., Cancer Res 71:7168-75, 2011), response to PRIMA-1^MET was related to p53 protein levels using Western blotting and ELISA (Figs. 4A-4C). As shown in Fig. 3C, IC₅₀ values for PRIMA-1^MET were significantly lower in the p53-positive cell lines compared to the p53-negative cell lines (p=0.006). Following quantitation by ELISA, a significant inverse correlation was found between IC₅₀ values and p53 absolute protein levels (p=0.0001, r=-0.76, n=22) (Fig. 3D). We also related response to PRIMA-1^MET with levels of the p53 family members, p63 and p73. A significant inverse correlation was found between p63 protein levels and IC₅₀ values (p=0.01, r=-0.55, n=22) (Fig. 3E). In contrast, no correlation was seen between p73 protein level and IC₅₀ values (p=0.55, r=-0.13, n=22) (Fig. 3F).

Comparative Effect of PRIMA-1^MET in TNBC and Non-TNBC Cell Lines
We also compared response in TN vs non-TN cells. However, no difference in response was found between the 2 groups of cell lines (p = 0.75) (Fig. 3G). Similarly, when the cell lines were separated into TN, luminal and HER2+ subgroups, no difference in response was seen between the subgroups (p=0.42) (Fig. 3H).

Effect of PRIMA-1 and PRIMA-1^MET on Induction of Apoptosis
As induction of apoptosis is one of the best-established roles for wild-type p53, we measured the effects of PRIMA-1 and PRIMA-1^MET on this process. For this phase of the work, we selected 3 p53 mutant cell lines that were sensitive to PRIMA-1^MET using the MTT assay (i.e., all had IC₅₀ values < 5 μM) and one p53 wild-type cell line that was resistant to PRIMA-1^MET, i.e., MCF7 cells which had an IC₅₀ value of 31.1 μM. Using the annexin-V and propidium iodide (PI) staining method, both PRIMA-1 and PRIMA-1^MET induced early and late apoptosis in the three p53 mutated cell lines used, i.e., MDA-MB-453 (Figs. 5A-5C), MDA-MB-468 (Figs. 5D-5F), and BT549 (Figs. 5G-5I). Induction of apoptosis was found to be both time and concentration dependent. In contrast, neither compound had an effect on apoptosis in the p53 wild-type cell line, MCF7, apart from PRIMA-1^MET at the highest concentration used (i.e., 20 μM) and following 5 days incubation (p=0.02; Paired t test) (Figs. 5J-5L).

Effect of PRIMA-1^MET on Cell Migration
Since mutant p53 promotes cell migration and invasion (Muller et al., J Cell Biol 192:209-18, 2011), we investigated if PRIMA-1^MET inhibited migration. Using the Transwell migration assay, treatment with PRIMA-1^MET for 5 day significantly inhibited migrations of the three p53 mutated breast cancer cell lines (Figs. 6A-6C) investigated (for MDA-MB-453, p=0.0009; for MDA-MB-468, p=0.014 and for BT549, p=0.002; Paired t test), but had no effect on the p53 WT cell line, MCF7 (Fig. 6D).

In order to confirm these results, we also investigated the effects of PRIMA-1^MET on migration using the scratch wound assay. Consistent with our findings with the modified Boyden Chambers, following 5 days treatment, we observed inhibition of migration of the three p53 mutant cell lines but not the p53 WT cells, by PRIMA-1^MET (Figs. 6E-H).

Effect of Combined Treatment with PRIMA-1^MET and Cytotoxic Compounds on Cell Viability
In an attempt to enhance response, PRIMA-1^MET was combined with a number of different cytotoxic agents. Cells were treated with PRIMA-1^MET alone, cytotoxic drug alone, or the combination at a fixed ratio for 5 days. Cell viability was assessed using the MTT assay. CI (combination index) values were calculated using Calcusyn software. All experiments were carried out in triplicate. P-1^MET= PRIMA-1^MET. Data points represent the mean of three independent experiments. As shown in Figs. 7A and 7B, synergistic growth inhibition was found when PRIMA-1^MET was combined with eribulin in the 2 different cell lines investigated (for MDA-MB-453 C1=0.43; for MDA-MB-468 CI=0.6). Both docetaxel and carboplatin showed an additive effect when used in combination with PRIMA-1^MET. However, this additive effect was cell line-dependent, i.e., docetaxel plus PRIMA-1^MET gave an additive effect in MDA-MB-453 cells (CI=0.92) (Fig. 7C), but not in MDA-MB-468 cells (CI=1.7) (Fig. 7D); while carboplatin plus PRIMA-1^MET was additive in MDA-MB-468 cells (CI=0.94) (Fig. 7F), but not in MDA-MB-453 cells (CI=1.2) (Fig. 7E). No enhanced benefit was seen when PRIMA-1^MET was combined with cisplatin (Figs. 7G and 7H).

Example 2
Effect of Combined Treatment with PRIMA-1^MET and Cytotoxic Compounds on Cell Viability and Apoptosis
To investigate further whether combined treatment with PRIMA-1^MET and different cytotoxic agents results in enhanced growth inhibition and apoptosis, additional cell lines and cytotoxic agents were tested. Cell viability was determined using the MTT assay. Combination index (CI) values were calculated using Calcusyn software, based on the Chou-Talalay method. Apoptosis was detected using Annexin V-FITC Apoptosis Detection Kit followed by FACs analysis.

Highly synergistic cell growth inhibition was found when PRIMA-1^MET was combined with eribulin in 6 different p53-mutated cell lines (mean CI values range from 0.38 to 0.77) (Figs. 8 and 9). In contrast, enhanced growth inhibition was not found using this combination in the 3 p53-WT cell lines investigated (mean CI values ranged from 1.13 to 2.9) (Figs. 8 and 9). Overall, p53 mutated cell lines had significantly lower CI values than p53 wild-type cells (p=0.008) (Figs. 10 and 11). No difference however, was seen between the CI values for triple-negative cells lines compared to non-triple-negative cells lines (Fig. 11). Furthermore, the eribulin IC₅₀ values were similar in triple-negative and non-triple negative cells, as well as in p53 mutated vs p53 wild-type cells (Fig. 11).

In all of the 4 p53-mutated cell lines investigated, a significant increase in apoptosis was also seen when PRIMA-1^MET was combined with eribulin (Fig. 12). This enhanced apoptosis appeared to result from increased expression of the pro-apoptotic factors PUMA and NOXA by the drug combination as compared to either compound alone. In contrast to our findings with eribulin, combined treatment with PRIMA-1^MET plus docetaxel, doxorubicin, cisplatin, or carboplatin was cell line-dependent. Thus, docetaxel plus PRIMA-1^MET was synergistic in 1/6 cell lines, while doxorubicin, cisplatin or carboplatin plus PRIMA-1^MET was synergistic in 3/6 cell lines. In particular, only the MDA-MD-231 cell line showed an enhanced apoptotic effect when PRIMA-1^MET was combined with docetaxel (Fig. 12).

The invention is further described in the following numbered paragraphs.

1. A method for treating a subject having or at risk of developing a p53-mutated cancer, the method comprises administering to the subject (a) eribulin, or a pharmaceutically acceptable salt thereof, and (b) a 3-quinuclidinone derivative.

2. The method of paragraph 1, wherein the pharmaceutically acceptable salt of eribulin is eribulin mesylate.

3. The method of paragraph 1 or 2, wherein the 3-quinuclidinone derivative is PRIMA-1 or PRIMA-1^MET.

4. The method of any one of paragraphs 1-3, wherein the method consists of administering to the subject (a) eribulin mesylate and (b) PRIMA-1.

5. The method of any one of paragraphs 1-3, wherein the method consists of administering to the subject (a) eribulin mesylate and (b) PRIMA-1^MET.

6. The method of any one of paragraphs 1-5, wherein (a) and (b) are administered substantially simultaneously.

7. The method of any one of paragraphs 1-5, wherein (a) is administered first, followed by administration of (b).

8. The method of any one of paragraphs 1-5, wherein (b) is administered first, followed by administration of (a).

9. The method of any one of paragraphs 1-5, wherein (a) and (b) are administered substantially simultaneously, followed by administration of (a).

10. The method of any one of paragraphs 1-5, wherein (a) and (b) are administered substantially simultaneously, followed by administration of (b).

11. The method of any one of paragraphs 1-10, wherein the subject is a human.

12. The method of any one of paragraphs 1-11, wherein the subject is diagnosed with the p53-mutated cancer, in treatment for the p53-mutated cancer, or in post-therapy recovery from the p53-mutated cancer.

13. The method of any one of paragraphs 1-12, wherein the p53-mutated cancer is a primary tumor.

14. The method of any one of paragraphs 1-12, wherein the p53-mutated cancer is locally advanced.

15. The method of any one of paragraphs 1-14, wherein the p53-mutated cancer is metastatic.

16. The method of any one of paragraphs 1-15, wherein the p53-mutated cancer is selected from the group consisting of lung cancer, stomach cancer, breast cancer, colon cancer, liver cancer, renal cancer, colorectal cancer, prostate cancer, pancreatic cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, testicular cancer, pelvic cancer, thyroid cancer, uterine cancer, rectal cancer, brain cancer, head and neck cancer, esophageal cancer, bronchus cancer, gallbladder cancer, ovarian cancer, bladder cancer, oral cancer, oropharyngeal cancer, larynx cancer, biliary tract cancer, skin cancer, a cancer of the central nervous system, a cancer of the respiratory system, and a cancer of the urinary system.

17. The method of paragraph 16, wherein the p53-mutated cancer is breast cancer.

18. The method of paragraph 17, wherein breast cancer is selected from the group consisting of triple-negative breast cancer, triple-positive breast cancer, HER2-negative breast cancer, HER2-positive breast cancer, estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, progesterone receptor-positive breast cancer, progesterone receptor-negative breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, Paget disease of the nipple, and phyllodes tumor.

19. The method of paragraph 18, wherein breast cancer is triple-negative breast cancer.

20. The method of paragraph 16, wherein the p53-mutated cancer is ovarian cancer.

21. The method of any one of paragraphs 1-15, wherein the p53-mutated cancer is selected from the group consisting of B-cell leukemia, T-cell leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic (lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), erythroleukemia, angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, malignant fibrous cytoma, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, synovial sarcoma, vascular sarcoma, Kaposi's sarcoma, dermatofibrosarcoma, epithelioid sarcoma, leyomyosarcoma, neurofibrosarcoma, basal cell carcinoma, large cell carcinoma, small cell carcinoma, non-small cell lung carcinoma, renal carcinoma, hepatocarcinoma, gastric carcinoma, choriocarcinoma, adenocarcinoma, hepatocellular carcinoma, giant (or oat) cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastmic carcinoma, adrenocortical carcinoma, cholangiocarcinoma, Merkel cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, glioblastoma multiforme, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, multiple myeloma, plasmacytoma, localized myeloma, extramedullary myeloma, superficial spreading melanoma, nodular melanoma, lentigno maligna melanoma, acral lentiginous melanoma, amelanotic melanoma, ganglioneuroma, Pacinian neuroma, acoustic neuroma, astrocytoma, oligoastrocytoma, ependymoma, brainstem glioma, optic nerve glioma, oligoastrocytoma, pheochromocytoma, meningioma, malignant mesothelioma, and a virally induced cancer.

22. The method of any one of paragraphs 1-21, wherein said eribulin or said pharmaceutically acceptable salt thereof is administered by intravenous infusion.

23. The method of paragraph 22, wherein said intravenous infusion is for about 1 to about 20 minutes.

24. The method of paragraph 23, wherein said intravenous infusion is for about 2 to about 5 minutes.

25. The method of any one of paragraphs 1-24, wherein said eribulin or said pharmaceutically acceptable salt thereof is administered in an amount in the range of about 0.1 mg/m² to about 20 mg/m².

26. The method of paragraph 25, wherein said eribulin or said pharmaceutically acceptable salt thereof is administered in an amount of about 1.1 mg/m² or 1.4 mg/m².

27. The method of any one of paragraphs 1-26, wherein said 3-quinuclidinone derivative is administered by intravenous infusion.

28. The method of paragraph 27, wherein said intravenous infusion is for about 0.5 to about 3 hours.

29. The method of paragraph 28, wherein said intravenous infusion is for about 2 hours.

30. The method of any one of paragraphs 1-29, wherein said 3-quinuclidinone derivative is administered in an amount in the range of about 2 mg/kg to about 90 mg/kg.

31. The method of paragraph 30, wherein said 3-quinuclidinone derivative is administered in an amount in the range of about 2 mg/kg to about 60 mg/kg.

32. The method of any one of paragraphs 1-31, wherein said treating: (i) reduces the number of cancer cells; (ii) reduces tumor volume; (iii) increases tumor regression rate; (iv) reduces or slows cancer cell infiltration into peripheral organs; (v) reduces or slows tumor metastasis; (vi) reduces or inhibits tumor growth; (vii) prevents or delays occurrence and/or recurrence of the cancer and/or extends disease- or tumor-free survival time; (viii) increases overall survival time; (ix) reduces the frequency of treatment; and/or (x) relieves one or more of symptoms associated with the cancer.

33. A method for decreasing the size of a tumor in a subject having a p53-mutated cancer, the method comprising administering to the subject (a) eribulin, or a pharmaceutically acceptable salt thereof, and (b) a 3-quinuclidinone derivative.

34. The method of paragraph 33, wherein the pharmaceutically acceptable salt of eribulin is eribulin mesylate.

35. The method of paragraph 33 or 34, wherein the 3-quinuclidinone derivative is PRIMA-1 or PRIMA-1^MET.

36. A kit for use in treating a p53-mutated cancer or decreasing tumor size in a subject having the p53-mutated cancer, the kit comprising (a) eribulin, or a pharmaceutically acceptable salt thereof, and (b) a 3-quinuclidinone derivative, optionally in dosage form.

37. The method of paragraph 36, wherein the pharmaceutically acceptable salt of eribulin is eribulin mesylate.

38. The method of paragraph 36 or 37, wherein the 3-quinuclidinone derivative is PRIMA-1 or PRIMA-1^MET.

39. A method of predicting the responsiveness of a cancer subject to a combination therapy comprising eribulin, or a pharmaceutically acceptable salt thereof, and a 3-quinuclidinone derivative, the method comprising:

(a) collecting a sample from the cancer subject; and

(b) determining the presence of a mutant p53 protein or gene in the sample from the cancer subject by measuring the level of expression of the mutant p53 protein or gene,

wherein the presence of a mutant p53 protein or gene in the sample from the cancer subject indicates that the cancer subject may be responsive to the combination therapy.

40. The method of paragraph 39, wherein the pharmaceutically acceptable salt of eribulin is eribulin mesylate.

41. The method of paragraph 39 or 40, wherein the 3-quinuclidinone derivative is PRIMA-1 or PRIMA-1^MET.

42. The method of any one of paragraphs 39-41, wherein said sample is a tissue sample, a blood sample, or a tumor sample.

43. Use of (a) eribulin, or a pharmaceutically acceptable salt thereof, and (b) a 3-quinuclidinone derivative for treating a subject having or at risk of developing a p53-mutated cancer.

Paragraphs 1-42, as set forth above, also apply in the context of the use of paragraph 43, as well as the use of the agents set forth in paragraph 43 in the preparation of a medicament for the use set forth in paragraph 43.

Other Embodiments
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features set forth herein.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated as being incorporated by reference in their entirety.

Use of singular forms herein, such as "a" and "the," does not exclude indication of the corresponding plural form, unless the context indicates to the contrary. Similarly, use of plural terms does not exclude indication of a corresponding singular form.

Other embodiments are within the scope of the following claims.

What is claimed is:

Claims (43)

1. A method for treating a subject having or at risk of developing a p53-mutated cancer, the method comprises administering to the subject (a) eribulin, or a pharmaceutically acceptable salt thereof, and (b) a 3-quinuclidinone derivative.
   
2. The method of claim 1, wherein the pharmaceutically acceptable salt of eribulin is eribulin mesylate.
   
3. The method of claim 1 or 2, wherein the 3-quinuclidinone derivative is PRIMA-1 or PRIMA-1^MET.
   
4. The method of any one of claims 1-3, wherein the method consists of administering to the subject (a) eribulin mesylate and (b) PRIMA-1.
   
5. The method of any one of claims 1-3, wherein the method consists of administering to the subject (a) eribulin mesylate and (b) PRIMA-1^MET.
   
6. The method of any one of claims 1-5, wherein (a) and (b) are administered substantially simultaneously.
   
7. The method of any one of claims 1-5, wherein (a) is administered first, followed by administration of (b).
   
8. The method of any one of claims 1-5, wherein (b) is administered first, followed by administration of (a).
   
9. The method of any one of claims 1-5, wherein (a) and (b) are administered substantially simultaneously, followed by administration of (a).
   
10. The method of any one of claims 1-5, wherein (a) and (b) are administered substantially simultaneously, followed by administration of (b).
    
11. The method of any one of claims 1-10, wherein the subject is a human.
    
12. The method of any one of claims 1-11, wherein the subject is diagnosed with the p53-mutated cancer, in treatment for the p53-mutated cancer, or in post-therapy recovery from the p53-mutated cancer.
    
13. The method of any one of claims 1-12, wherein the p53-mutated cancer is a primary tumor.
14. The method of any one of claims 1-12, wherein the p53-mutated cancer is locally advanced.
    
    
15. The method of any one of claims 1-14, wherein the p53-mutated cancer is metastatic.
    
16. The method of any one of claims 1-15, wherein the p53-mutated cancer is selected from the group consisting of lung cancer, stomach cancer, breast cancer, colon cancer, liver cancer, renal cancer, colorectal cancer, prostate cancer, pancreatic cancer, cervical cancer, anal cancer, vulvar cancer, penile cancer, vaginal cancer, testicular cancer, pelvic cancer, thyroid cancer, uterine cancer, rectal cancer, brain cancer, head and neck cancer, esophageal cancer, bronchus cancer, gallbladder cancer, ovarian cancer, bladder cancer, oral cancer, oropharyngeal cancer, larynx cancer, biliary tract cancer, skin cancer, a cancer of the central nervous system, a cancer of the respiratory system, and a cancer of the urinary system.
    
17. The method of claim 16, wherein the p53-mutated cancer is breast cancer.
    
18. The method of claim 17, wherein breast cancer is selected from the group consisting of triple-negative breast cancer, triple-positive breast cancer, HER2-negative breast cancer, HER2-positive breast cancer, estrogen receptor-positive breast cancer, estrogen receptor-negative breast cancer, progesterone receptor-positive breast cancer, progesterone receptor-negative breast cancer, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, invasive lobular carcinoma, inflammatory breast cancer, Paget disease of the nipple, and phyllodes tumor.
    
19. The method of claim 18, wherein breast cancer is triple-negative breast cancer.
    
20. The method of claim 16, wherein the p53-mutated cancer is ovarian cancer.
    
21. The method of any one of claims 1-15, wherein the p53-mutated cancer is selected from the group consisting of B-cell leukemia, T-cell leukemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic (lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), erythroleukemia, angiosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, malignant fibrous cytoma, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, synovial sarcoma, vascular sarcoma, Kaposi's sarcoma, dermatofibrosarcoma, epithelioid sarcoma, leyomyosarcoma, neurofibrosarcoma, basal cell carcinoma, large cell carcinoma, small cell carcinoma, non-small cell lung carcinoma, renal carcinoma, hepatocarcinoma, gastric carcinoma, choriocarcinoma, adenocarcinoma, hepatocellular carcinoma, giant (or oat) cell carcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastmic carcinoma, adrenocortical carcinoma, cholangiocarcinoma, Merkel cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, retinoblastoma, glioblastoma multiforme, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Burkitt lymphoma, multiple myeloma, plasmacytoma, localized myeloma, extramedullary myeloma, superficial spreading melanoma, nodular melanoma, lentigno maligna melanoma, acral lentiginous melanoma, amelanotic melanoma, ganglioneuroma, Pacinian neuroma, acoustic neuroma, astrocytoma, oligoastrocytoma, ependymoma, brainstem glioma, optic nerve glioma, oligoastrocytoma, pheochromocytoma, meningioma, malignant mesothelioma, and a virally induced cancer.
    
22. The method of any one of claims 1-21, wherein said eribulin or said pharmaceutically acceptable salt thereof is administered by intravenous infusion.
    
23. The method of claim 22, wherein said intravenous infusion is for about 1 to about 20 minutes.
    
24. The method of claim 23, wherein said intravenous infusion is for about 2 to about 5 minutes.
    
25. The method of any one of claims 1-24, wherein said eribulin or said pharmaceutically acceptable salt thereof is administered in an amount in the range of about 0.1 mg/m² to about 20 mg/m².
    
26. The method of claim 25, wherein said eribulin or said pharmaceutically acceptable salt thereof is administered in an amount of about 1.1 mg/m² or 1.4 mg/m².
    
27. The method of any one of claims 1-26, wherein said 3-quinuclidinone derivative is administered by intravenous infusion.
    
28. The method of claim 27, wherein said intravenous infusion is for about 0.5 to about 3 hours.
    
29. The method of claim 28, wherein said intravenous infusion is for about 2 hours.
    
30. The method of any one of claims 1-29, wherein said 3-quinuclidinone derivative is administered in an amount in the range of about 2 mg/kg to about 90 mg/kg.
    
31. The method of claim 30, wherein said 3-quinuclidinone derivative is administered in an amount in the range of about 2 mg/kg to about 60 mg/kg.
    
32. The method of any one of claims 1-31, wherein said treating: (i) reduces the number of cancer cells; (ii) reduces tumor volume; (iii) increases tumor regression rate; (iv) reduces or slows cancer cell infiltration into peripheral organs; (v) reduces or slows tumor metastasis; (vi) reduces or inhibits tumor growth; (vii) prevents or delays occurrence and/or recurrence of the cancer and/or extends disease- or tumor-free survival time; (viii) increases overall survival time; (ix) reduces the frequency of treatment; and/or (x) relieves one or more of symptoms associated with the cancer.
    
33. A method for decreasing the size of a tumor in a subject having a p53-mutated cancer, the method comprising administering to the subject (a) eribulin, or a pharmaceutically acceptable salt thereof, and (b) a 3-quinuclidinone derivative.
    
34. The method of claim 33, wherein the pharmaceutically acceptable salt of eribulin is eribulin mesylate.
    
35. The method of claim 33 or 34, wherein the 3-quinuclidinone derivative is PRIMA-1 or PRIMA-1^MET.
    
36. A kit for use in treating a p53-mutated cancer or decreasing tumor size in a subject having the p53-mutated cancer, the kit comprising (a) eribulin, or a pharmaceutically acceptable salt thereof, and (b) a 3-quinuclidinone derivative, optionally in dosage form.
    
37. The method of claim 36, wherein the pharmaceutically acceptable salt of eribulin is eribulin mesylate.
    
38. The method of claim 36 or 37, wherein the 3-quinuclidinone derivative is PRIMA-1 or PRIMA-1^MET.
    
39. A method of predicting the responsiveness of a cancer subject to a combination therapy comprising eribulin, or a pharmaceutically acceptable salt thereof, and a 3-quinuclidinone derivative, the method comprising:
    
    (a) collecting a sample from the cancer subject; and
    
    (b) determining the presence of a mutant p53 protein or gene in the sample from the cancer subject by measuring the level of expression of the mutant p53 protein or gene,
    
    wherein the presence of a mutant p53 protein or gene in the sample from the cancer subject indicates that the cancer subject may be responsive to the combination therapy.
    
    
40. The method of claim 39, wherein the pharmaceutically acceptable salt of eribulin is eribulin mesylate.
    
41. The method of claim 39 or 40, wherein the 3-quinuclidinone derivative is PRIMA-1 or PRIMA-1^MET.
    
42. The method of any one of claims 39-41, wherein said sample is a tissue sample, a blood sample, or a tumor sample.
    
43. Use of (a) eribulin, or a pharmaceutically acceptable salt thereof, and (b) a 3-quinuclidinone derivative for treating a subject having or at risk of developing a p53-mutated cancer.
    
    

Images