Patent Application
20220047596
The sequence listing that is contained in the file named “UTFCP1379WO_ST25.txt”, which is 6 KB (as measured in Microsoft Windows®) and was created on Sep. 12, 2019, is filed herewith by electronic submission and is incorporated by reference herein.
The present invention relates generally to the fields of medicine and immunology. More particularly, it concerns the combination therapy of PARP and BRD4 inhibition for cancer therapy.
DNA double-strand breaks (DSBs) can lead to mutation, chromosomal aberration, or cell death. DSBs are repaired by two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR) (Hoeijmakers, 2001; Jackson and Bartek, 2009). Mutation-prone NHEJ ligates broken DNA ends without requiring sequence complementarity. In contrast, HR mediates high fidelity DNA repair using sister chromatids as the repair template. The different DSB repair pathways are tightly controlled (Huertas, 2010). HR is instigated by DSB end resection, which generates a long single-stranded DNA (ssDNA) that is protected by replication protein A (RPA) (Broderick et al., 2016; Kaidi et al., 2010). C-terminal binding protein (CtBP) interacting protein (CtIP) physically interacts with the MRE11-RAD50-NBS1 (MRN) complex at DSBs, promoting DNA end resection, ssDNA generation, and nuclease activity of the MRN complex (Davies et al., 2015; Yun and Hiom, 2009).
CtIP downregulation abolishes ssDNA formation, and impairs HR function (Sartori et al., 2007; Yun and Hiom, 2009). Bromodomain containing 4 (BRD4), a member of the bromo-domain and extraterminal (BET) protein family, maintains and facilitates oncogenic transcription directly by recruiting transcriptional machinery or indirectly by binding to enhancers, contributing to cancer cell proliferation and survival (Yang et al., 2005). BRD4 can be selectively targeted with small-molecule inhibitors, such as JQ1 (Filippakopoulos et al., 2010), GSK1210151A (I-BET151 [Dawson et al., 2011]), GSK525762A (I-BET-762 [Nicodeme et al., 2010]), GSK1324726A (I-BET-726 [Gosmini et al., 2014]), and AZD5153 (Rhyasen et al., 2016). BRD4i are active in preclinical models of hematological malignancies and solid tumors (Asangani et al., 2014; Delmore et al., 2011; Filippakopoulos et al., 2010; Yokoyama et al., 2016). Multiple BRD4i have entered clinical trials (NCT01587703, NCT03059147, NCT02419417, NCT01949883, NCT03068351, and NCT02259114).
BRD4 is frequently amplified and correlates with poor prognosis in patients with high-grade serous ovarian carcinoma (HGSOC) (Zhang et al., 2016). In addition, at least half of HGSOCs exhibit aberrations in the HR pathway (Cancer Genome Atlas Research Network, 2011). Tumor cells that lack functional BRCA1, BRCA2, or other key components of the HR pathway, are highly sensitivity to poly(ADP-ribose) polymerase inhibitor (PARPi) (Bryant et al., 2005; Ledermann et al., 2016), leading to regulatory approval of three different PARPi for ovarian cancer treatment (Kaufman et al., 2015; Mirza et al., 2016; Swisher et al., 2017). Although high response rates are achieved, most tumors rapidly become resistant, including BRCA1/2 mutant cancers. Therefore, the development of strategies to prevent or reverse PARPi resistance to increase the duration of response and expand the utility of PARPi to HR-competent tumors is needed.
In one embodiment, the present disclosure provides a method for treating cancer in a subject comprising administering an effective amount of a poly-ADP-ribose polymerase (PARP) inhibitor in combination with a bromodomain-containing protein 4 (BRD4) inhibitor to the subject. In some aspects, the administration of the PARP inhibitor and BRD4 inhibitor results in greater reduction in tumor growth or greater reduction in tumor mass relative to administration of PARP inhibitor or BRD4 inhibitor alone. In certain aspects, the subject is human.
In some aspects, the subject is PARP inhibitor resistant. In other aspects, the subject is PARP inhibitor sensitive. In particular aspects, the administration of the PARP inhibitor in combination with the BRD4 inhibitor prevents emergence of PARP inhibitor resistance.
In certain aspects, the cancer is a RAS/BRAF, BRCA1/2, and/or p53 mutant cancer. In some aspects, the RAS/BRAF mutation is KRAS or NRAS. In particular aspects, the cancer is homologous recombination (HR) competent. In specific aspects, the HR competent cancer is a RAS/BRAF, BRCA1/2, and/or p53 wild-type cancer. In some aspects, the cancer is breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, or melanoma. In some aspects, the subject has increased expression of C-terminal binding protein interacting protein (CtIP).
In certain aspects, the PARP inhibitor is Olaparib, BMN673, Niraparib, Rucaparib, or ABT888 (Veliparab). In particular aspects, the PARP inhibitor is BMN673. In some aspects, the BRD4 inhibitor is JQ1, GSK1210151A (I-BET151), GSK1324726A (I-BET-726), or AZD5153. In particular aspects, the BRD4 inhibitor is JQ1.
In some aspects, the PARP inhibitor and/or BRD4 inhibitor are administered orally, intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. In particular aspects, the PARP inhibitor and/or BRD4 inhibitor are administered orally. In some aspects, the PARP inhibitor is administered at a dose of 200-400 mg/day. In certain aspects, the BRD4 inhibitor is administered at a dose of 10-40 mg/day. In some aspects, the PARP inhibitor and BRD4 inhibitor are administered more than once. In particular aspects, the PARP inhibitor and BRD4 inhibitor are administered daily. In some aspects, the PARP inhibitor and BRD4 inhibitor are administered concurrently. In certain aspects, the PARP inhibitor is administered before the BRD4 inhibitor. In specific aspects, the BRD4 inhibitor is administered before the PARP inhibitor.
In some aspects, the administration results in induction of homologous repair deficiency. In particular aspects, the induction of homologous repair deficiency results in an increase in DNA damage and checkpoint defects. In some aspects, the administration results decreased expression of WEE1 and/or TOPBP1. In particular aspects, the administration results in decreased expression of C-terminal binding protein interacting protein (CtIP).
In additional aspects, the method further comprises the step of administering at least one additional therapeutic agent to the subject. In some aspects, the subject receives at least one additional type of therapy. In particular aspects, the at least one additional type of therapy is selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, and immunotherapy.
In another embodiment, there is provided a method for treating a PARP-resistant cancer or preventing PARP resistance in a subject comprising administering an effective amount of a BRD4 inhibitor to the subject. In some aspects, BRD4 inhibition resensitizes PARP resistant cells to PARP inhibition.
In additional aspects, the method further comprises administering an effective amount of a PARP inhibitor to the subject.
In some aspects, the cancer is breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, or melanoma. In some aspects, the subject has increased expression of C-terminal binding protein interacting protein (CtIP).
In particular aspects, the PARP inhibitor is Olaparib, BMN673, Niraparib, Rucaparib, or ABT888 (Veliparab). In some aspects, the BRD4 inhibitor is JQ1, GSK1210151A (I-BET151), GSK1324726A (I-BET-726), or AZD5153. In certain aspects, the PARP inhibitor and/or BRD4 inhibitor are administered orally, intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. In certain aspects, the PARP inhibitor and/or BRD4 inhibitor are administered intravenously. In some aspects, the PARP inhibitor and BRD4 inhibitor are administered more than once. In certain aspects, the PARP inhibitor and BRD4 inhibitor are administered daily. In some aspects, the PARP inhibitor and BRD4 inhibitor are administered concurrently. In certain aspects, the PARP inhibitor is administered before the BRD4 inhibitor. In some aspects, the BRD4 inhibitor is administered before the PARP inhibitor.
In certain aspects, the administration results decreased expression of WEE1 and/or TOPBP1. In some aspects, the administration results in decreased expression of C-terminal binding protein interacting protein (CtIP).
In additional aspects, the method further comprised the step of administering at least one additional therapeutic agent to the subject. In some aspects, the subject receives at least one additional type of therapy. In certain aspects, the at least one additional type of therapy is selected from the group consisting of chemotherapy, radiotherapy, and immunotherapy.
Another embodiment provides a method of predicting response to a PARP inhibitor comprising measuring the expression of CtIP in said subject, wherein low CtIP expression identifies a PARP sensitive cancer and high CtIP expression identifies a PARP resistant cancer. In some aspects, a subject with the PARP sensitive cancer is administered an effective amount of a PARP inhibitor. In certain aspects, a subject with the PARP resistant cancer is administered an effective amount of a BRD4 inhibitor to induce PARP sensitivity. In additional aspects, the subject is further administered an effective amount of a PARP inhibitor.
In some aspects, the cancer is breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, or melanoma. In particular aspects, the PARP inhibitor is Olaparib, BMN673, Niraparib, Rucaparib, or ABT888 (Veliparab). In specific aspects, the BRD4 inhibitor is JQ1, GSK1210151A (I-BET151), GSK1324726A (I-BET-726), or AZD5153.
In specific aspects, the PARP inhibitor and/or BRD4 inhibitor are administered orally, intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, regionally, or by direct injection or perfusion. In some aspects, the PARP inhibitor and/or BRD4 inhibitor are administered intravenously. In certain aspects, the PARP inhibitor and BRD4 inhibitor are administered more than once. In particular aspects, the PARP inhibitor and BRD4 inhibitor are administered daily. In some aspects, the PARP inhibitor and BRD4 inhibitor are administered concurrently. In particular aspects, the PARP inhibitor is administered before the BRD4 inhibitor. In some aspects, the BRD4 inhibitor is administered before the PARP inhibitor.
In additional aspects, the method further comprises the step of administering at least one additional therapeutic agent to the subject. In some aspects, the subject receives at least one additional type of therapy. In specific aspects, the at least one additional type of therapy is selected from the group consisting of chemotherapy, radiotherapy, targeted therapy, and immunotherapy.
A further embodiment provides a method of treating cancer in a subject comprising administering a BRD4 inhibitor to the subject, wherein the patient has been determined to be resistant to PARP inhibitors. In some aspects, the cancer is breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, or melanoma. In particular aspects, the PARP inhibitor is Olaparib, BMN673, Niraparib, Rucaparib, or ABT888 (Veliparab). In specific aspects, he BRD4 inhibitor is JQ1, GSK1210151A (I-BET151), GSK1324726A (I-BET-726), or AZD5153.
In yet another embodiment, there is provided a method of inhibiting CtIP expression in a subject comprising administering an effective amount of BRD4 inhibitor to said subject. In some aspects, the subject has cancer. In particular aspects, the cancer is breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, non-small cell lung cancer, or melanoma. In some aspects, the BRD4 inhibitor is JQ1, GSK1210151A (I-BET151), GSK1324726A (I-BET-726), or AZD5153. In additional aspects, the method further comprises administering an effective amount of a PARP inhibitor to the subject. In some aspects, the PARP inhibitor is Olaparib, BMN673, Niraparib, Rucaparib, or ABT888 (Veliparab).
Further provided herein is a pharmaceutical composition comprising a PARP inhibitor and a BRD4 inhibitor. Also provided herein is the pharmaceutical composition comprising a PARP inhibitor and a BRD4 inhibitor for use in the treatment of cancer. Further embodiments provide the use of a therapeutically effective amount of a PARP inhibitor and a BRD4 inhibitor for the treatment of cancer. In some aspects, the cancer is breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, or melanoma. In certain aspects, the BRD4 inhibitor is JQ1, GSK1210151A (I-BET151), GSK1324726A (I-BET-726), or AZD5153. In specific aspects, the PARP inhibitor is Olaparib, BMN673, Niraparib, Rucaparib, or ABT888 (Veliparab).
A further embodiment provides a composition comprising a therapeutically effective amount of a PARP inhibitor and a BRD4 inhibitor for the treatment of cancer in a subject. Also provided herein is the use of a PARP inhibitor and a BRD4 inhibitor in the manufacture of a medicament for the treatment of cancer. In some aspects, the PARP inhibitor is Olaparib, BMN673, Niraparib, Rucaparib, or ABT888 (Veliparab). In specific aspects, the BRD4 inhibitor is JQ1, GSK1210151A (I-BET151), GSK1324726A (I-BET-726), or AZD5153. In some aspects, the cancer is breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, or melanoma.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Poly (ADP-ribose) polymerase inhibitors (PARPi) are selectively active in cells with homologous recombination (HR) deficiency (HRD) caused by mutations in BRCA1, BRCA2, and other pathway members. The present studies sought small molecules that induce HRD in HR-competent cells to induce synthetic lethality with PARPi and extend the utility of PARPi. It was demonstrated that inhibition of bromodomain containing 4 (BRD4) induced HRD and sensitized cells across multiple tumor lineages to PARPi regardless of BRCA1/2, TP53, RAS, or BRAF mutation status through depletion of the DNA double-stand break resection protein CtIP (C-terminal binding protein interacting protein). Importantly, BRD4 inhibitor (BRD4i) treatment reversed multiple mechanisms of resistance to PARPi. Furthermore, PARPi and BRD4i were synergistic in multiple in vivo models. Therefore, the combination of BRD4 and PARP inhibitors has the potential to reverse or prevent the emergence of PARPi resistance and to increase the spectrum of patients who may benefit from the antitumor activity of PARP inhibitors.
Accordingly, in certain embodiments, the present disclosure provides compositions and methods for the treatment of cancer by a combination treatment of a PARP inhibitor and a BRD4 inhibitor. The present disclosure further provides methods for the prevention or reversal of PARP inhibitor resistance. In addition, the expression of CtIP in a subject can identify whether a patient is sensitive to PARP inhibition. For example, a subject with low CtIP expression may have a PARP sensitive cancer and a patient with high CtIP expression may have a PARP resistant cancer.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
The term “about” refers to the stated value plus or minus 5%.
As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
As used herein, a composition that is “substantially free” of a specified substance or material contains 30%, 20%, 15%, more preferably 10%, even more preferably 5%, or most preferably 1% of the substance or material.
As used herein, the term “patient” or “subject” refers to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or transgenic species thereof. In certain embodiments, the patient or subject is a primate. Non-limiting examples of human patients are adults, juveniles, infants and fetuses.
“Treating” or treatment of a disease or condition refers to executing a protocol, which may include administering one or more drugs to a patient, in an effort to alleviate signs or symptoms of the disease. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. Alleviation can occur prior to signs or symptoms of the disease or condition appearing, as well as after their appearance. Thus, “treating” or “treatment” may include “preventing” or “prevention” of disease or undesirable condition. In addition, “treating” or “treatment” does not require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only a marginal effect on the patient.
The term “effective,” as that term is used in the specification and/or claims, means adequate to accomplish a desired, expected, or intended result. “Effective amount,” “Therapeutically effective amount” or “pharmaceutically effective amount” when used in the context of treating a patient or subject with a compound means that amount of the compound which, when administered to a subject or patient for treating or preventing a disease, is an amount sufficient to effect such treatment or prevention of the disease.
“Prevention” or “preventing” includes: (1) inhibiting the onset of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease, and/or (2) slowing the onset of the pathology or symptomatology of a disease in a subject or patient which may be at risk and/or predisposed to the disease but does not yet experience or display any or all of the pathology or symptomatology of the disease.
As generally used herein “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
“Pharmaceutically acceptable salts” means salts of compounds disclosed herein which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, 2-naphthalenesulfonic acid, 3-phenylpropionic acid, 4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid, aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids, aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoic acid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, tartaric acid, tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. It should be recognized that the particular anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as a whole, is pharmacologically acceptable. Additional examples of pharmaceutically acceptable salts and their methods of preparation and use are presented in Handbook of Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag Helvetica Chimica Acta, 2002).
A “pharmaceutically acceptable carrier,” “drug carrier,” or simply “carrier” is a pharmaceutically acceptable substance formulated along with the active ingredient medication that is involved in carrying, delivering and/or transporting a chemical agent. Drug carriers may be used to improve the delivery and the effectiveness of drugs, including for example, controlled-release technology to modulate drug bioavailability, decrease drug metabolism, and/or reduce drug toxicity. Some drug carriers may increase the effectiveness of drug delivery to the specific target sites. Examples of carriers include: liposomes, microspheres (e.g., made of poly(lactic-co-glycolic) acid), albumin microspheres, synthetic polymers, nanofibers, protein-DNA complexes, protein conjugates, erythrocytes, virosomes, and dendrimers.
In some embodiments, provided herein are methods for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a PARP inhibitor and a BRD4 inhibitor.
Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, liver, gallbladder, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, uterus, melanoma, prostate, and breast. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.
The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; B-cell lymphoma; low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia.
BRD4 bromodomains can be selectively targeted with small-molecule inhibitors, such as JQ1, GSK1210151A (I-BET151), GSK1324726A (I-BET-726) and AZD5153, which compete with acetyl-lysine recognition to displace BRD4 from chromatin. The BRD4 inhibitor may be administered at a dose of from about 1 mg/day to about 100 mg/day. In some embodiments, the BRD4 inhibitor is administered once or twice daily at a dose of from about 10 mg to about 40 mg. In some embodiments, the BRD4 inhibitor is administered at doses of about 1 mg/kg per day, about 2 mg/kg per day, about 5 mg/kg per day, about 10 mg/kg per day, about 15 mg/kg per day, about 20 mg/kg per day, about 25 mg/kg per day, about 30 mg/kg per day, about 35 mg/kg per day, about 40 mg/kg per day, about 45 mg/kg per day, or about 50 mg/kg per day. The BRD4 inhibitor may be administered orally at a dose of 10 mg, 20 mg, or 40 mg tablets or capsules.
In some embodiments, the PARP inhibitor is administered at a dose of from about 20 mg/day to about 800 mg/day. In some embodiments, the PARP inhibitor is administered once or twice daily at a dose of from about 20 mg to about 400 mg. In some embodiments, the PARP inhibitor is administered at doses of about 1 mg/kg per day, about 2 mg/kg per day, about 5 mg/kg per day, about 10 mg/kg per day, about 15 mg/kg per day, about 20 mg/kg per day, about 25 mg/kg per day, about 30 mg/kg per day, about 35 mg/kg per day, about 40 mg/kg per day, about 45 mg/kg per day, about 50 mg/kg per day, about 60 mg/kg per day, about 70 mg/kg per day, about 80 mg/kg per day, about 90 mg/kg per day, about 100 mg/kg per day, about 125 mg/kg per day, about 150 mg/kg per day, about 175 mg/kg per day, about 200 mg/kg per day, about 250 mg/kg per day, or about 300 mg/kg per day. The PARP inhibitor may be administered in doses of 50, 100, or 150 oral tablets or capsules, such as at a daily dose of 300 or 400 mg/day. In some embodiments the PARP inhibitor is selected from the group consisting of talazoparib, niraparib, olaparib, veliparib, rucaparib, CEP 9722, talazoparib and BGB-290.
In certain embodiments, the BRD4 inhibitor and the PARP inhibitor are administered orally, intravenously, intraperitoneally, directly by injection to a tumor, topically, or a combination thereof. In some embodiments, the BRD4 inhibitor and the PARP inhibitor are administered as a combination formulation. In certain embodiments, the BRD4 inhibitor and the PARP inhibitor are administered as individual formulations. In some embodiments, the inhibitors are administered sequentially. In other embodiments, the inhibitors are administered simultaneously.
A. Combination Therapies
In certain embodiments, the methods provided herein further comprise a step of administering at least one additional therapeutic agent to the subject. All additional therapeutic agents disclosed herein will be administered to a subject according to good clinical practice for each specific composition or therapy, taking into account any potential toxicity, likely side effects, and any other relevant factors.
In certain embodiments, the additional therapy may be immunotherapy, radiation therapy, surgery (e.g., surgical resection of a tumor), chemotherapy, bone marrow transplantation, or a combination of the foregoing. The additional therapy may be targeted therapy. In certain embodiments, the additional therapy is administered before the primary treatment (i.e., as adjuvant therapy). In certain embodiments, the additional therapy is administered after the primary treatment (i.e., as neoadjuvant therapy).
A PARP inhibitor and BRD4 inhibitor may be administered before, during, after, or in various combinations relative to an additional cancer therapy, such as immune checkpoint therapy. The administrations may be in intervals ranging from concurrently to minutes to days to weeks. In embodiments where the immune cell therapy is provided to a patient separately from an additional therapeutic agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient. In such instances, it is contemplated that one may provide a patient with the antibody therapy and the anti-cancer therapy within about 12 to 24 or 72 h of each other and, more particularly, within about 6-12 h of each other. In some situations it may be desirable to extend the time period for treatment significantly where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) lapse between respective administrations.
Various combinations may be employed. For the example below PARP inhibitor and/or BRD4 inhibitor therapy is “A” and an anti-cancer therapy is “B”:
Administration of any compound or therapy of the present embodiments to a patient will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the agents. Therefore, in some embodiments there is a step of monitoring toxicity that is attributable to combination therapy.
1. Chemotherapy
A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PS Kpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine, plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.
2. Radiotherapy
Other factors that cause DNA damage and have been used extensively include what are commonly known as γ-rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation, and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
3. Immunotherapy
The skilled artisan will understand that immunotherapies may be used in combination or in conjunction with methods of the embodiments. In the context of cancer treatment, immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is such an example. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing. The antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells
Antibody-drug conjugates (ADCs) comprise monoclonal antibodies (MAbs) that are covalently linked to cell-killing drugs and may be used in combination therapies. This approach combines the high specificity of MAbs against their antigen targets with highly potent cytotoxic drugs, resulting in “armed” MAbs that deliver the payload (drug) to tumor cells with enriched levels of the antigen. Targeted delivery of the drug also minimizes its exposure in normal tissues, resulting in decreased toxicity and improved therapeutic index. Exemplary ADC drugs include ADCETRIS® (brentuximab vedotin) and KADCYLA® (trastuzumab emtansine or T-DM1).
In one aspect of immunotherapy, the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present embodiments. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, erb b2 and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects. Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.
Examples of immunotherapies include immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds); cytokine therapy, e.g., interferons α, β, and γ, IL-1, GM-CSF, and TNF; gene therapy, e.g., TNF, IL-1, IL-2, and p53; and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185. It is contemplated that one or more anti-cancer therapies may be employed with the antibody therapies described herein.
In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either turn up a signal (e.g., co-stimulatory molecules) or turn down a signal. Inhibitory immune checkpoints that may be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer-cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAGS), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA). In particular, the immune checkpoint inhibitors target the PD-1 axis and/or CTLA-4.
The immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligand or receptors, or, in particular, are antibodies, such as human antibodies. Known inhibitors of the immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used. As the skilled person will know, alternative and/or equivalent names may be in use for certain antibodies mentioned in the present disclosure. Such alternative and/or equivalent names are interchangeable in the context of the present disclosure. For example, it is known that lambrolizumab is also known under the alternative and equivalent names MK-3475 and pembrolizumab.
In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are PDL1 and/or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partners. In a specific aspect, PDL1 binding partners are PD-1 and/or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partners. In a specific aspect, a PDL2 binding partner is PD-1. The antagonist may be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody that may be used. Pembrolizumab, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an exemplary anti-PD-1 antibody. CT-011, also known as hBAT or hBAT-1, is also an anti-PD-1 antibody. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor.
Another immune checkpoint that can be targeted in the methods provided herein is the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has the Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an “off” switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of Helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to the T-cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86, also called B7-1 and B7-2 respectively, on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells and may be important to their function. T cell activation through the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for B7 molecules.
In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.
Anti-human-CTLA-4 antibodies (or VH and/or VL domains derived therefrom) suitable for use in the present methods can be generated using methods well known in the art. Alternatively, art recognized anti-CTLA-4 antibodies can be used. An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen binding fragments and variants thereof. In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Accordingly, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2 and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding with and/or binds to the same epitope on CTLA-4 as the above-mentioned antibodies. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above-mentioned antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).
4. Surgery
Approximately 60% of persons with cancer will undergo surgery of some type, which includes preventative, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies. Tumor resection refers to physical removal of at least part of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).
Upon excision of part or all of cancerous cells, tissue, or tumor, a cavity may be formed in the body. Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may be of varying dosages as well.
5. Other Agents
It is contemplated that other agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin.
B. Pharmaceutical Compositions
In another aspect, provided herein are pharmaceutical compositions and formulations comprising a PARP inhibitor, BRD4 inhibitor and a pharmaceutically acceptable carrier.
Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (such as an antibody or a polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012), in the form of aqueous solutions, such as normal saline (e.g., 0.9%) and human serum albumin (e.g., 10%). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); 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, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e g Zinc-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
An article of manufacture or a kit is provided comprising a PARP inhibitor and BRD4 inhibitor is also provided herein. The article of manufacture or kit can further comprise a package insert comprising instructions for using the inhibitors to treat or delay progression of cancer in an individual. Any of the PARP and/or BRD4 inhibitors described herein may be included in the article of manufacture or kits. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture further includes one or more of another agent (e.g., a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
BRD4 Inhibition Induces an HRD Signature: The HR defect (HRD) gene signature (Peng et al., 2014) was applied to publicly available transcriptional profiling data with or without BRD4 inhibition to determine whether BRD4 inhibition impaired HR. BRD4i (JQ1) and BRD4 small hairpin RNA (shRNA) significantly elevated HRD scores in human THP-1 cells and in murine MLL-AF9/NrasG12D acute myeloid leukemia cells (Zuber et al., 2011) (
Ingenuity Pathway Analysis revealed that BRD4 inhibition altered expression of genes involved in DNA replication, BRCA1 in DNA damage response, hereditary breast cancer signaling, DNA damage checkpoint, cell cycle, and DNA repair pathway (
BRD4 Inhibition Decreases CtIP Expression: To identify mechanisms underlying the effect of BRD4 inhibition on HR, reverse phase protein arrays (RPPA) was used to assess signaling pathway perturbations in response to a clinical candidate (GSK525762A) and three experimental (GSK1210151A, GSK1324726A, and JQ1) BRD4i in five cancer cell lines. Replicates for each treatment condition (2D, spheroid 3D, and two time points [24 and 48 hr]) were averaged for each line (
CtIP is required for MRE11 to mediate DNA end resection, with loss of CtIP markedly decreasing DNA DSB repair through HR (Sartori et al., 2007; Yun and Hiom, 2009). Notably, JQ1 decreased CtIP and phosphorylated RPA32 (pRPA32 (S4/8)) protein in a dose- and time-dependent manner (
BRD4 Binds CtIP Promoter and Enhancers, Regulating CtIP Transcription: Transcription profiling demonstrated that RBBP8 is decreased by BRD4 inhibition (
Downregulation of CtIP Is Sufficient to Impair DNA End Resection, Generation of ssDNA, and HR Function: CtIP is essential for efficient DNA end processing during DSB repair, with cells depleted for CtIP showing a defect in generation of ssDNA and subsequent formation of RPA foci (Polato et al., 2014; Yun and Hiom, 2009). It was thus hypothesized that BRD4 inhibition would block DNA end resection and HR through down-regulation of CtIP. Indeed, BRD4 protein and RBBP8 are negatively correlated with HRD score in both NCI60 and CCLE (
RAD51 loading onto DNA requires ssDNA created by the CtIP/MRN complex. Compared with vehicle, JQ1 and AZD5153 retained RAD51 in the cytosol and decreased RAD51 nuclear foci after PARPi (
A comet assay was used to directly examine whether BRD4i would increase PARPi-induced DNA damage. Whereas JQ1 or BMN673 monotherapy modestly induced DNA damage, the combination increased accumulation of damaged DNA (
DNA resection is the key commitment step for DSB repair by HR (Ira et al., 2004). These results suggested that BRD4 inhibition leading to loss of CtIP would decrease HR competency. Indeed, similar to BRD4i (
PARPi were developed to capitalize on synthetic lethality with HRD (Bryant et al., 2005; Farmer et al., 2005). Since BRD4 inhibition induced HRD, at least in part, through loss of CtIP, it was reasoned that knock down of BRD4 or CtIP would sensitize cells to PARPi. Indeed, knock down of BRD4 or CtIP markedly sensitized cells to PARPi (
CtIP, but Not RAD51 or BRCA1, Partially Rescues BRD4 Inhibition Induced Defects in DNA End Resection and HR: To evaluate whether suppression of CtIP is necessary for BRD4 inhibition-induced defects in DNA end resection and HR function, Dox-inducible stable cell lines were generated expressing wild-type (WT) CtIP or inactive CtIP (T847A). CDK-mediated phosphorylation of CtIP on T847 was required for optimal CtIP function, thus conversion of threonine 847 to alanine (T847A) creates an inactive CtIP that is compromised for CtIP catalytic, ssDNA-, and RPA-binding activities (Huertas and Jackson, 2009; Polato et al., 2014). Ectopic expression of WT, but not inactive, CtIP increased ssDNA formation 4 hr after 10 Gy IR in the presence of JQ1 (
Ectopic expression of BRCA1 or RAD51 did not rescue cells from the effects of combination treatment (
PARPi and BRD4i Demonstrate Synergy in Multiple Cancer Lineages: Based on the ability of BRD4 inhibition to compromise HR, the effects of combination treatment with PARPi and BRD4i was assessed. Of 55 cancer cell lines tested, 40 lines demonstrated synergy as assessed by the CalcuSyn model (combination index <0.5,
As demonstrated previously (Sun et al., 2017), KRAS mutation is a potent inducer of PARPi resistance (
In addition to JQ1, three other BRD4i demonstrated similar patterns of synergy with PARPi (
In 55 cell lines tested for response, CtIP expression was much lower in PARPi-sensitive cells, indicating that CtIP may serve as a marker of PARPi sensitivity (
Six of the lines tested were resistant to PARPi alone as well as to the combination. Strikingly, BRD4i failed to alter CtIP levels in the three resistant cell lines tested. In contrast, RAD51 was decreased in IGROV1, and BRCA1 decreased in EFE184 (
It was further sought to identify mechanisms by which KRAS mutant cell lines would be selectively sensitive to PARPi and BRD4i combinations. Induction of activated KRASG12D in HPDE cells induced both BRD4 and CtIP (
BRD4i Resensitizes Acquired PARPi Resistance: Although many patients benefit from PARPi, acquired PARPi resistance is an almost universal occurrence. To explore whether BRD4i could resensitize PARPi-resistant cells to PARPi, several PARPi-resistant models representing different mechanisms of PARPi resistance were used. First, PARPi-resistant cells were developed by culturing sensitive cells (A2780CP, OAW42, and OC316) in the continued presence of BMN673. It was demonstrated previously that A2780CP_R has acquired mutations in KRAS, as well as in MAP2K1 (Sun et al., 2017). JQ1 resensitized A2780CP_R, OAW42_R and OC316_R to PARPi (
Taken together, BRD4i resensitizes multiple mechanisms of acquired PARPi resistance that have been observed in patients and model systems to PARPi. Thus, BRD4i and PARPi combinations may prevent emergence of PARPi resistance, or may be effective in the emerging population of patients where PARPi are initially active and then fail.
Inhibition of PARP Enzyme Activity Appears Sufficient for Synergy with BRD4i: The different PARPi currently available in the clinic effectively inhibit the enzyme activity of PARP, but vary in their ability to trap PARP on DNA. BMN673 is most active, Olaparib, Niraparib, and Rucaparib intermediate in activity, whereas ABT888/Veliparib has the weakest PARPi trapping activity (Murai et al., 2012). High levels of PARP1 are required for trapping activity of PARPi to be manifest and, thus, a role for trapping activity can be elucidated by testing activity of different PARPi, as well as by determining the effects of partial knock down of PARP1 (Murai et al., 2012). Indeed, as noted above, the synergistic effects of PARPi and BRD4i are not altered by partial knock down of PARP1 (note residual PARP1 remains) (
BRD4i and PARPi Are Synergistic In Vivo: On the basis of synergy of BRD4i and PARPi in vitro, BRD4i and PARPi combinations were explored in five different in vivo models. OVCAR8 is a KRASP121H mutant (the mutant is a variant of unknown significance, but the line has an activated RAS/MAPK pathway [Sun et al., 2017]) ovarian cancer line, OVCAR3 is a TP53 mutant, RAS WT ovarian cancer line, WU-BC3 is a breast cancer PDX (HER2-E subtype with WT TP53) (Ma et al., 2012), PATX53 is a KRASG12D and TP53 mutant pancreatic PDX, and LPA1-T127 is an MMTV-LPA receptor transgene-induced transplantable tumor that acquired a spontaneous KRASQ61H mutation (Federico et al., 2017). Similar to human PDX, the LPA1-T127 tumor has never been cultured on plastic and may thus be more representative of the heterogeneity of human breast cancers. Furthermore, LPA receptor transgene-induced tumors are late onset, heterogeneous, and are associated with an inflammatory response similar to human cancers (Liu et al., 2009). Strikingly, in OVCAR8, WU-BC3, and LPA1-127, the JQ1 and PARPi combination induced prolonged tumor control (
IHC of OVCAR8 and WU-BC3 PDX tumors at study termination recapitulated the in vitro studies. JQ1 increased gH2AX, which was further increased by combination with BMN673. As expected, CtIP and its direct downstream effector pRPA32 (S4/8) were decreased in JQ1-treated tumors, which was not reversed by addition of BMN673 (
It was demonstrated that decreased CtIP transcription appears to be a major contributor to the effects of BRD4 inhibition on HR function and to be necessary and sufficient for much of the synergy between PARPi and BRD4i. CtIP inhibition has previously been associated with PARPi sensitivity (Lin et al., 2014; Wang et al., 2016). Importantly, enforced expression of CtIP was sufficient to, at least in part, reverse the effects of BRD4i on DNA end resection, HR function, and PARPi sensitivity. DNA replication fork reversal and fork stability are emerging mechanisms of PARPi resistance independent of HR repair (Ray Chaudhuri et al., 2016). CtIP has also been demonstrated to induce replication fork recovery in a FANCD2-dependent manner (Yeo et al., 2014). The effects of CtIP on DNA repair as well as replication stress induced by tumorigenesis may contribute to DNA damage observed in cells treated with BRD4i herein. Thus, BRD4 inhibition induced CtIP loss may contribute to PARPi sensitivity through multiple CtIP-dependent mechanisms. However, as BRD4 regulates the expression of many molecules, there may be additional effects of BRD4i that contribute to sensitization to PARPi either independent of CtIP loss or in cooperation with CtIP loss.
Clinical Specimens: Use of ovarian cancer samples was approved by the Ethics or Institutional Review Board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, China, in accordance with the Declaration of Helsinki. Informed consent was obtained from all subjects. 102 serous ovarian cancer stage IIIC or IV (International Federation of Gynecology and Obstetrics staging) samples were collected between January 2009 and October 2013. Samples were routinely fixed immediately after surgery in 10% formalin for approximately 24 hr at room temperature. After fixation, samples were dehydrated, incubated in xylene, infiltrated with paraffin, and finally embedded in paraffin.
WU-BC3 PDX, which was established in Washington University (Li et al., 2013), was obtained from Dr. Helen Piwnica-Worms in Department of Experimental Radiation Oncology in MDACC (MD Anderson Cancer Center) (Ma et al., 2012). PATX53 was obtained from Dr. Michael P. Kim in Department of Surgical Oncology in MDACC. WU-BC3 and PATX53 PDX were under IRB approved protocol by the ethics committee of the Washington University, or the MDACC respectively, with written informed consent for formation and use of PDX.
Animal Studies: 6 week old female NCRNU-F sp/sp mice were purchased from Taconic and were used for OVCAR8 xenografts, WU-BC3 PDX and PATX53 PDX experiments. 6 week old female FVB mice were purchased from Taconic and were used for LPA-T127 syngeneic breast cancer model experiments. Tumors were injected or transplanted into female mice of approximately 8-10 weeks of age. All mice were housed under pathogen-free conditions at MDACC AAALAC (Association for the Assessment and Accreditation of Laboratory Animal Care) accredited facility. All animal experiments with these models were conducted in compliance with the National Institute of Health guidelines for animal research and approved by the Institutional Animal Care and Use Committee of the MDACC.
6 weeks old female C.B-17 scid mice were purchased from Charles River Laboratories and used for OVCAR3 xenografts. Tumor cells were injected into female mice of approximately 8-10 weeks of age. All mice were housed under pathogen-free conditions at AstraZeneca AAALAC accredited facility. All animal experiments were conducted in compliance with the National Institute of Health guidelines for animal research and approved by the Institutional Animal Care and Use Committee of AstraZeneca.
Cell Lines: All human cell lines were authenticated by fingerprinting using short tandem repeat testing and were verified to be free of mycoplasma contamination. All cell lines were maintained in a 5% CO2 incubator at 37° C. Detail information about cells are provided in Table 1. pCW-GFP-CtIP was a gift from Daniel Durocher (Addgene plasmid #71109) (Orthwein et al., 2015). pCW-GFP-CtIP (T847A) was generated by site-directed mutagenesis. Cells infected with viruses expressing these cDNAs were maintained in 2 mg/mL puromycin to generate stable cell lines. GFP-CtIP and GFP-CtIP (T847A) expression were induced with 100 nM doxycycline (Dox). HOC1, SKOV3, HOC1-GFP-CtIP stably expressing RAD51 and BRCA1 were established through standard procedural.
Generation of PARPi Resistant Cells: To generate PARPi resistant cells, A2780CP and OAW42 were subjected to gradual increases in BMN673 concentrations until cells grew in the presence of 10 mM of BMN673 (3-4 months from initial exposure).
For PARPi resistant OC316 clones, cells were subjected to gradual increases in BMN673 concentrations until cells grew in the presence of 5 mM of the BMN673 (3-4 months from initial exposure). Monoclonal cell populations of the OC316 resistant cells are isolated by limiting dilution. Individual clones demonstrated different degrees of resistance to PARPi.
Cells were cultured in the absence of BMN673 for a minimum of 1 month before they were used for experiments.
RPPA: Five breast and ovarian cancer cell lines, [BT474 (PIK3CA_Mut, HER2_Amp), HCC1954 (PIK3CA_Mut and HER2_Amp), MDA-MB-468 (EGFR_Overexpression and PTEN_Mut), SKBR3 (HER2_Amp), SKOV3 (PIK3CA_Mut and HER2_Amp)], were cultured in Matrigel (3D) or monolayer (2D) and treated for 24 hr or 48 hr, respectively, with DMSO or BRD4i (GSK1210151A, GSK1324726A, GSK525762A, and JQ1). Median inhibitory concentration (IC50) was determined experimentally for JQ1 for each line for 2D and 3D conditions with other inhibitors being used at 2 concentrations (100 nM and 1000 nM). Protein lysates were analyzed by RPPA in MDACC CCSG (The Cancer Center Support Grant) supported RPPA Core. Antibodies and approaches are described at the RPPA website (https://www.mdanderson.org/research/research-resources/core-facilities/functional-proteomics-rppa-core.html). For visualization, 2D and 3D, concentrations and time were averaged for each cell line. Heat map represents “rank-ordered” changes induced by BRD4i treatment, calculated by summing median-centered protein amount normalized to DMSO. Western Blot: To prepare whole cell lysates, cells were lysed with RIPA buffer (Thermo Fisher Scientific) supplemented with Halt™ Protease and Phosphatase Inhibitor (EDTA-free) Cocktail (Thermo Fisher Scientific). After thorough mixing and incubation at 4° C. for 10 min, lysates were centrifuged at 15,000 g at 4° C. for 15 min, and supernatants were collected. To prepare subcellular fraction of nuclear soluble and chromatin-bound fraction, cells were treated with indicated drugs, and then cells were collected. For fractionation, a Subcellular Protein Fractionation kit (Thermo Fisher Scientific) was used following the manufacturer's instructions. The protein content of the cell was determined, and the cellular lysates were separated by 10% SDS-PAGE, and electro-transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% non-fat milk in TBST, the membranes were incubated with primary antibodies at 4° C. overnight, followed by 1:2000 horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam) for 1 hr. Bands were visualized using a Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific). Primary antibodies used are listed in Key Resources Table.
qRT-PCR: Total RNA was isolated using RNeasy Plus Mini Kit (Qiagen) according to the manufacturer's protocol. RNA was treated with RNase-free DNase set (Qiagen) to remove contaminating genomic DNA. cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed using SYBR Green Master Mix (Applied Biosystems). Data were analyzed by the DDCT method using GAPDH as a housekeeping gene. The sequences of primers used are listed in Table 2.
Site-directed Mutagenesis: pCW-GFP-CtIP was a gift from Daniel Durocher (Addgene plasmid #71109) (Orthwein et al., 2015). Mutant pCW-GFP-CtIP (T847A) was generated by targeting WT pCW-GFP-CtIP using QuikChange II XL Site-Directed Mutagenesis kit (Agilent Technologies) with primers list in Table 2. Mutagenesis reactions were prepared in PCR tubes on ice: 5 mL of 10× reaction buffer, 2 mL pCW-GFP-CtIP plasmid DNA (10 ng), 1.25 mL of mutagenic primer (CtIP_T847A_F at 100 ng/mL), 1.25 mL of mutagenic primer (CtIP_T847A_R at 100 ng/mL), 1 mL of dNTP mix, 3 mL of QuickSolution reagent, 36.5 mL PCR-quality water to a final volume of 50 mL were mixed then 1.0 mL PfuUltra HF DNA polymerase (2.5 U/mL) was fused. Tubes were placed in the cycler to begin the PCR reaction for 18 cycles. 1 mL of the Dpn I restriction enzyme (10 U/ml) was added directly to amplification reaction and mixed thoroughly and incubated at 37° C. for 1 hour. Then 2 ml of the Dpn I-treated DNA was transformed to XL10-Gold Ultracompetent Cells. Mutation was confirmed by sequencing.
RNA Interference: All siRNAs employed in this study were ON-TARGET plus siRNA SMARTpools purchased from GE Dharmacon (Table 2). RNA interference (RNAi) transfections were performed using Lipofectamine™ 3000 Transfection Reagent (Invitrogen) in a forward transfection mode using manufacturer's guidelines. Except when stated otherwise, siRNAs were transfected with the amounts of siRNA oligos at 40 nM final concentration.
CCLE and NCI60 Dataset Gene expression profiles (Gene transcript level z score) for correlations analysis in NCI60 human tumor cell lines were obtained using the web-based tool provided by CellMiner. Gene expression data for Cancer Cell Line Encyclopedia (CCLE) (CCLE_expression_CN_muts_GENEE_2010-04-16) were downloaded. The correlations between gene expressions were determined by Pearson's correlation test with R.
Microarray Analysis and IPA Analysis: Gene expression datasets of GSE29799 (Zuber et al., 2011), GSE66048 (Ambrosini et al., 2015), GSE44929 (Love′ n et al., 2013), GSE85840 (Rhyasen et al., 2016), GSE31365 (Delmore et al., 2011), and GSE43392 (Puissant et al., 2013) were downloaded from Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/geo). Raw data were subjected to intensity normalization using affy package in R (Bioconductor), followed by log transformation and quantile normalization. Normalized data were checked for quality and determined to be free of outliers by analysis using box plots, density plots and MA plots. Differential expression genes after BRD4 inhibition were calculated using a linear model provided by the limma package in R based on the cutoffs: 2 for absolute fold change, 0.05 for p value. Then, results were imported into Ingenuity Pathway Analysis (IPA) and a core analysis feature was used to reveal dysregulated canonical pathway after BRD4 inhibition.
HRD Score Acquisition from HRD Signature HRD signature consisting of 230 differentially expressed genes was obtained as previously described (Peng et al., 2014). Normalized gene expression data (GSE29799, GSE66048, GSE44929, GSE85840, GSE31365, and GSE43392) after BRD4 inhibition were subjected to unsupervised clustering with these 230 genes. HRD scores were determined by calculating the Pearson's correlations between median centered gene expression levels for HRD signature and gene expression levels for a given sample (Peng et al., 2014).
ChIP-Seq Analysis: ChIP-seq data for T47D and HCC1935 cells from GSE63581 (Shu et al., 2016) were aligned versus hg19 human genome for mapping using bowtie. For peaking calling, MACS2 was used to get the bam files, which were converted to bigwig files later in deeptools and loaded in Intergrative Genomics Viewer (IGV) for final visualization and cross comparison. Specifically, ChIP-seq for T47D and HCC1935 cells treated with JQ1 and vehicle control were compared with input.
CtIP Co-expression Signature and GSEA Analysis: CtIP co-expression signature was constructed base on genes whose expressions are correlated with RBBP8 levels in the CCLE dataset at cBioPortal. 326 genes were selected using Pearson's correlation coefficient R 0.3 as cutoff. Then these 326 genes were imported into Ingenuity Pathway Analysis (IPA) for network and pathway analysis.
For GSEA analysis against CtIP co-expression signature, these 326 genes were incorporated into the GSEA Desktop v3.0 as the CtIP co-expression signature. Then normalized gene expression data (GSE29799, GSE66048, GSE44929, GSE85840, GSE31365, and GSE43392) were used to calculate enrichment of CtIP co-expression signature after BRD4 inhibition by Benjamin-Hochberg (B-H) method.
Viability Measurements: Five thousand cells were seeded into sterile 96-well plates and treated with indicated drug combinations for 96 hr. DMSO was used as a vehicle. PrestoBlue® Cell Viability Reagent (Thermo Fisher Scientific) was used to assess cell viability. Background values from empty wells were subtracted and data normalized to vehicle-treated control. Synergistic effects between both compounds were calculated using the Chou-Talalay equation in CalcuSyn software, which takes into account both potency (IC50) and shape of the dose-effect curve. CI<0.5 indicates synergism, CI between 0.5 to 1 indicates additive effects, and CI>1 indicates antagonism.
Immunohistochemical Staining (IHC) Tissues were fixed in 10% formalin overnight and embedded in paraffin. 4 mm paraffin embedded sections were first deparaffinized in xylene. IHC were carried out with EnVision Detection Systems HRP. Rabbit/Mouse (DAB+) kit (Agilent) following manufacturer's instructions. Endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide for 15 min. Antigen retrieval was performed by boiling the slides in citrate buffer (10 mM, pH 6.0) in a water bath for 20 mM. Slides were rinsed in PBS Tween 0.05% and blocked for 30 mM with 5% bovine serum albumin (BSA). Slide were incubated overnight at 4° C. with primary antibodies (anti-BRD4,
#13440S, 1:200; anti-CtIP, #9201S, 1:200 from Cell Signaling Technology; anti-RAD51, PC130, 1:100; anti-g-H2AX (Ser139), clone JBW301, 1:500 from Millipore Corp; and anti-pRPA32(S4/8), A300-245A, 1:1000 from Bethyl Laboratories), followed by 1 hr with Labelled Polymer-HRP at room temperature. Negative controls were treated identically, but without primary antibody. Subsequently, slides were incubated with DAB+ Chromogen. Slides were counterstained with hematoxylin. After mounting, slides were observed under microscope and photographed.
The IHC score for BRD4 and CtIP staining are the average of the score of tumor-cell staining multiplied by the score of staining intensity. Tumor cell staining was assigned a score using a semi-quantitative five-category grading system: 0, no tumor-cell staining; 1, 1-10% tumor-cell staining; 2, 11-25% tumor-cell staining; 3, 26-50% tumor-cell staining; 4, 51-75% tumor-cell staining; and 5, >75% tumor-cell staining Staining intensity was assigned a score using a semi-quantitative four-category grading system: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining Every core was assessed individually and the mean of three readings was calculated for every case. Tumor cell staining score was determined separately by two independent experts simultaneously under the same conditions. In rare cases, discordant scores were reevaluated and scored on the basis of consensus opinion.
Alkaline Single-Cell Agarose Gel Electrophoresis (Comet) Assays: Alkaline comet assays were performed with Comet Assay Kit (Trevigen) using manufacturer's instructions. Briefly, cell suspensions were embedded in LM (low melting) Agarose and deposited on comet slides. Slides were incubated for 1 hr at 4° C. in lysis solution, followed by immersing slides in freshly prepared alkaline unwinding solution (pH >13) for 20 min at room temperature in the dark. Electrophoresis was carried out for 30 min at 21 V in electrophoresis solution (pH >13). Slides were then stained with SYBR™ Gold (Thermo Fisher Scientific). Tail DNA content was analyzed with Comet score 1.5 software. DNA strand breakage was expressed as “comet tail moment”. The tail moment was measured for a minimum of 50 cells per sample, and average damage from 3 independent experiments was calculated.
Clonogenic Assay: Five thousand cells were seeded in triplicate into six-well plates and allowed to adhere overnight. Cells were then cultured in absence or presence of drug for 7-10 days as indicated. Remaining cells were fixed with formaldehyde (4%), stained with Crystal violet solution (sigma), and photographed using a digital scanner.
Chromatin Immunoprecipitation (ChIP-qPCR): ChIP assays were performed with EZ-Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore Corp) as described in manufacturer's instructions. Briefly, cells were crosslinked with 1% formaldehyde. After cell lysis, isolated nuclei were subjected to sonication for chromatin fragmentation. Sheared chromatin was diluted in diluted buffer, and divided into aliquots for immunoprecipitation. Anti-BRD4 antibody (1:50, #13440S, Cell Signaling), anti-H3K27ac antibody (1:100, ab4729, Abcam), anti-H3K4M1 antibody (1:200, ab8895, Abcam), anti-Pol II antibody (1:100, sc-47701, Santa Cruz) or normal Rabbit IgG control (1:200, #2729, Cell Signaling) were added to chromatin samples, followed by overnight incubation at 4° C., with rotation. Antibody-chromatin complexes were captured using magnetic protein A/G beads. Purified DNAs were subjected to quantitative PCR (qPCR). All primers are list in Table 2.
Detection of ssDNA by Immunofluorescence: Cells were grown in 50 mg/ml BrdU for two doubling times before irradiation. Where indicated, 200 nM JQ1 was added 4 hr before irradiation. Cells were placed on ice 10 min before irradiation and kept on ice during the irradiation with 10 Gy. Warm media with or without JQ1 was added for 4 hr at 37° C. BrdU was stained (anti-BrdU, ab8152, 1:100 from Abcam) in non-denaturing conditions which enables detection of BrdU incorporated in ssDNA. TE-2000 imaging acquisition system (Nikon) equipped with a 60× objective lens was used to capture images. Stained was quantified by ImageJ.
Immunofluorescence Staining and Microscopy: Briefly, cells were washed with PBS and fixed with 4% paraformaldehyde for 5 min, followed by permeabilization with 0.5% NP-40 and 1% Triton X-100 for 10 min Cells were then blocked with 5% FBS for 30 min and incubated with primary antibody (anti-RAD51, PC130, 1:100; anti-g-H2AX (Ser139), clone JBW301, 1:1000 from Millipore Corp; anti-RPA32, ab2175, 1:500 from Abcam) for 2 hr, followed by secondary antibody incubation for 1 hr at room temperature. Slides were sealed in mounting medium containing DAPI (Vector Laboratories, H1200) for further image acquisition. TE-2000 imaging acquisition system (Nikon) equipped with a 60× objective lens was used to capture images. Stained was quantified by ImageJ.
HR Repair Analysis: U2OS DR-GFP cells contain a single copy of the HR repair reporter substrate DR-GFP, which contains two nonfunctional GFP open reading frames, including one GFP-coding sequence that is interrupted by a recognition site for the I-SceI endonuclease. Expression of I-SceI leads to formation of a DSB in the I-SceI GFP allele, which can be repaired by HR using the nearby GFP sequence lacking the N- and C-termini, thereby producing functional GFP that can be detected by flow cytometry. To examine the role of JQ1 or individual genes in DSB repair, cells were treated with JQ1 (100 nM), AZD5153 (100 nM) or transfected with CtIP or BRD4 siRNA for 24 hr. Then, cells were transfected with a plasmid expressing I-SceI (pCBASce) for 48 hr. Cells transfected with an empty vector were used as a negative control. GFP-expressing plasmid (pEGFP-C1) was used for transfection efficiency control. Flow cytometry analysis was performed to detect GFP+ cells using FACScalibur with CellQuest software (Becton Dickinson). The repair efficiency was scored as the percentage of GFP+ cells.
In Vivo Drug Studies:
OVCAR8 Xenografts: 3×106 OVCAR8 cells were injected s.c. into mouse flanks in a 1:1 mix of PBS and Matrigel. When tumors reached 50 to 200 mm3, drugs were administered daily by [vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80, oral gavage), BMN673 (0.333 mg/kg, oral gavage), and JQ1 (40 mg/kg, I.P.), or combinations of BMN673 and JQ1, n=6 per group]. Mice were treated for 28 day and sacrificed for tissue analysis. Tumor volumes were calculated using volume=length*width/2.
WU-BC3 PDX: 3×106 WU-BC3 cells (Ma et al., 2012) were injected subcutaneously into flanks mice in a 1:1 mix of PBS and Matrigel. After palpable tumors formed, drugs were administered daily by [vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80, oral gavage), BMN673 (0.333 mg/kg, oral gavage), and JQ1 (40 mg/kg, I.P), or combinations of BMN673 and JQ1, n=6 per group]. Mice were treated until Day 28 and sacrificed for tissue harvest.
PATX53 PDX: Minced fresh tumor tissue (0.1-0.2 cm3 per mouse) was transplanted subcutaneously into flanks of mice. After palpable tumors formed, drugs were administered daily by [vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80, oral gavage), Olaparib (100 mg/kg, oral gavage), AZD5153 (2.5 mg/kg, oral gavage), or combinations of Olaparib and AZD5153, n=6 per group]. Mice were treated until Day 28 and sacrificed for tissue harvest.
LPAJ-T127 Syngeneic Breast Cancer Models: LPA-T127 is a primary invasive and metastatic mammary cancer from transgenic mice, with expression of LPA1 receptor in mammary epithelium and a spontaneous KRASQ61H mutation (Liu et al., 2009; Federico et al., 2017). Minced fresh tumor tissue (0.1-0.2 cm3 per mouse) was transplanted into mammary fat pads of FVB mice. After palpable tumors formed, drugs were administered daily by [vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80, oral gavage), BMN673 (0.333 mg/kg, oral gavage), and JQ1 (40 mg/kg, I.P.), or combinations of BMN673 and JQ1, n=6 per group]. Mice were sacrificed when tumor diameter reach maximum limit of 2.5 cm at Day 22.
LPA-T127 was also repeated with daily [vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80, oral gavage), Olaparib (100 mg/kg, oral gavage), AZD5153 (2.5 mg/kg, oral gavage), or combinations of Olaparib and AZD5153, n=6 per group]. Mice were sacrificed when tumor diameter reached maximum limits of 2.5 cmat Day 22 for tissue harvest and blood collection 3 hr after the final treatment.
OVCAR3 Xenograft: 2×3 107 OVCAR3 cells were injected subcutaneously in the right flank of mice. Mice were randomized based on tumor volumes using stratified sampling and enrolled into control and treatment groups. Dosing began when mean tumor size reached approximately 200 mm3 and continued for 35 days. Drugs were administered daily by [vehicle (0.5% hydroxypropylmethylcellulose and 0.2% Tween 80, oral gavage), oral gavage), Olaparib (100 mg/kg, oral gavage), AZD5153 (2.5 mg/kg, oral gavage), or combinations of Olaparib and AZD5153], n=10 per group].
Quantification and statistical analysis: Two-sided Student's t test was used to compare differences between two groups of cells in vitro. Data are presented as means±SEM, and p<0.05 is considered significant. The correlation between groups was determined by Pearson's correlation test. Analysis of variance was used to compare differences among multiple groups. All statistical analyses were done using SPSS 17.0 (SPSS Inc.). Data were analyzed and plotted using GraphPad Prism 6 software and Microsoft Excel.
Data and software availability: Following GEO datasets of BRD4 inhibition were used for gene expression analysis: GSE29799, GSE66048, GSE44929, GSE85840, GSE31365, and GSE43392. CHIP-seq data after treatment with JQ1 with BRD4 antibody were obtained from GSE63581.
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
This application claims the benefit of U.S. Provisional Patent Application No. 62/730,171, filed Sep. 12, 2018, the entirety of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/050887 | 9/12/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62730171 | Sep 2018 | US | |
| 62730171 | Sep 2018 | US |
