Patent Application
20210220471
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jan. 11, 2021, is named 115872-2056_SL.txt and is 92,480 bytes in size.
The present technology relates to methods for treating or preventing pancreatic cancer using inhibitors of Type 2 cytokine signaling. Kits for use in practicing the methods are also provided.
The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.
Pancreatic cancer was the 12th most common type of cancer in the U.S. in 2014, representing about 2.8% of all new cancer cases. However, pancreatic cancer was the 4th most common cause of cancer-related deaths (Schneider G et al., Gastroenterology 128(6):1606-1625 (2005)). In 2014, about 46,420 new cases and 39,590 deaths were attributable to pancreatic cancer in the United States, of which pancreatic ductal adenocarcinoma (PDAC) represents the vast majority. The fact that the annual number of pancreatic cancer-related deaths nearly equals the annual number of new pancreatic cancer cases highlights the lethality of this disease. PDAC, the most common malignancy of the pancreas, is both aggressive and difficult to treat. Complete surgical removal of the tumor remains the only chance for cure, however 80-90% of patients have disease that is surgically incurable at the time of clinical presentation deaths (Schneider G et al., Gastroenterology 128(6):1606-1625 (2005)). Accordingly, there is an urgent need for effective therapies for pancreatic cancer.
In one aspect, the present disclosure provides a method for treating or preventing pancreatic cancer in a subject in need thereof comprising administering to the subject an effective amount of an inhibitor of Type 2 cytokine signaling, wherein the subject harbors a KRAS mutation. In some embodiments, the KRAS mutation is selected from the group consisting of G12C, G12D, G12V, G12A, G12S, G12R, G13D, G13C, G13S, G13R, G13A, G13V, Q61H, Q61L, Q61R, Q61K, Q61P, and Q61E. Additionally or alternatively, the inhibitor of Type 2 cytokine signaling inhibits a Type 2 cytokine or Type 2 cytokine receptor signaling protein selected from the group consisting of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1. The inhibitor of Type 2 cytokine signaling may be a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), a tyrosine kinase inhibitor, a decoy cytokine receptor, or an antibody (e.g., a neutralizing antibody). Additionally or alternatively, in some embodiments, the small molecule, the inhibitory nucleic acid, the tyrosine kinase inhibitor, the decoy cytokine receptor, or the antibody specifically binds to and inhibits/neutralizes the expression or activity of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, or IL1R1. Examples of inhibitors of Type 2 cytokine signaling include, but are not limited to dupilumab, Etokimab (ANB020), Mepolizumab, reslizumab, benralizumab, lebrikizumab, risankizumab, tocilizumab, tralokinumab, anrukinzumab, AMG317, STATE inhibitors, STAT3 inhibitors, Secukinumab, ustekinumab, guselkumab, gevokizumab, or any other agent that inhibits the expression or activity of any of the Type 2 cytokines or Type 2 cytokine receptor signaling proteins disclosed herein. The pancreatic cancer may comprise exocrine tumors. In certain embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma.
Additionally or alternatively, in some embodiments, the methods further comprise administering to the subject an effective amount of a Brd4 inhibitor. The Brd4 inhibitor may be a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), or an antibody (e.g., a neutralizing antibody). Examples of Brd4 inhibitors include, but are not limited to, (+)-JQ1, I-BET762, OTX015, I-BET151, CPI203, PFI-1, MS436, CPI-0610, RVX2135, FT-1101, BAY1238097, INCB054329, TEN-010, GSK2820151, ZEN003694, BAY-299, BMS-986158, ABBV-075, GS-5829, and PLX51107. Additionally or alternatively, in some embodiments, the method further comprises sequentially, simultaneously, or separately administering one or more additional therapeutic agents to the subject.
In any and all embodiments of the methods disclosed herein, the subject harbors a mutation in TP53. The subject may have a family history of pancreatic ductal adenocarcinoma or exhibits chronic pancreatitis, Type 2 diabetes or other risk factors for developing pancreatic cancer. Additionally or alternatively, in some embodiments, the subject exhibits elevated expression levels of at least one of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1 compared to that observed in a healthy control subject or a predetermined threshold.
In another aspect, the present disclosure provides a method for selecting pancreatic cancer patients for treatment with an inhibitor of Type 2 cytokine signaling comprising (a) detecting expression levels or chromatin accessibility levels of a Type 2 cytokine or Type 2 cytokine receptor signaling protein in biological samples obtained from pancreatic cancer patients, wherein the Type 2 cytokine or Type 2 cytokine receptor signaling protein is selected from the group consisting of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1; (b) identifying pancreatic cancer patients that exhibit (i) mRNA/protein expression levels of Type 2 cytokine or Type 2 cytokine receptor signaling protein that are elevated compared to that observed in a healthy control subject or a predetermined threshold, and/or (ii) chromatin accessibility levels of Type 2 cytokine or Type 2 cytokine receptor signaling protein that are elevated compared to that observed in a healthy control subject or a predetermined threshold; and (c) administering an inhibitor of Type 2 cytokine signaling to the pancreatic cancer patients of step (b). The inhibitor of Type 2 cytokine signaling may be a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), a tyrosine kinase inhibitor, a decoy cytokine receptor, or an antibody (e.g., a neutralizing antibody). Additionally or alternatively, in some embodiments, the small molecule, the inhibitory nucleic acid, the tyrosine kinase inhibitor, the decoy cytokine receptor, or the antibody specifically binds to and inhibits/neutralizes the expression or activity of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, or IL1R1. Examples of inhibitors of Type 2 cytokine signaling include, but are not limited to dupilumab, Etokimab (ANB020), Mepolizumab, reslizumab, benralizumab, lebrikizumab, risankizumab, tocilizumab, tralokinumab, anrukinzumab, AMG317, STATE inhibitors, STAT3 inhibitors, Secukinumab, ustekinumab, guselkumab, gevokizumab, or any other agent that inhibits the expression or activity of any of the Type 2 cytokines or Type 2 cytokine receptor signaling proteins disclosed herein.
Additionally or alternatively, in some embodiments, the methods further comprise administering a Brd4 inhibitor to the pancreatic cancer patients of step (b). The Brd4 inhibitor may be a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), or an antibody (e.g., a neutralizing antibody). Examples of Brd4 inhibitors include, but are not limited to, (+)-JQ1, I-BET762, OTX015, I-BET151, CPI203, PFI-1, MS436, CPI-0610, RVX2135, FT-1101, BAY1238097, INCB054329, TEN-010, GSK2820151, ZEN003694, BAY-299, BMS-986158, ABBV-075, GS-5829, and PLX51107.
In any and all embodiments of the methods disclosed herein, the pancreatic cancer patients harbor a KRAS mutation selected from the group consisting of G12C, G12D, G12V, G12A, G12S, G12R, G13D, G13C, G13S, G13R, G13A, G13V, Q61H, Q61L, Q61R, Q61K, Q61P, and Q61E. Additionally or alternatively, in some embodiments, the pancreatic cancer patients harbor a mutation in TP53. The pancreatic cancer patients may exhibit exocrine tumors. In certain embodiments, the pancreatic cancer patients suffer from or are at risk for pancreatic ductal adenocarcinoma.
In any of the preceding embodiments of the methods disclosed herein, the expression levels or chromatin accessibility levels of the Type 2 cytokine or Type 2 cytokine receptor signaling protein are detected via ChIP, MNase, FAIRE, DNAse, ATAC-seq, RT-PCR, Northern Blotting, RNA-Seq, microarray analysis, High-performance liquid chromatography (HPLC), mass spectrometry, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), Western Blotting, immunoprecipitation, flow cytometry, Immuno-electron microscopy, immunoelectrophoresis, enzyme-linked immunosorbent assays (ELISA), or multiplex ELISA antibody arrays. In some embodiments, the biological samples are pancreatic cancer specimens, blood, serum, or plasma.
Also disclosed herein are kits comprising at least one inhibitor of Type 2 cytokine signaling and instructions for using the at least one inhibitor of Type 2 cytokine signaling to treat or prevent pancreatic cancer. The inhibitor of Type 2 cytokine signaling may be a small molecule, an inhibitory nucleic acid, a tyrosine kinase inhibitor, a decoy cytokine receptor, or an antibody. In certain embodiments, the inhibitor of Type 2 cytokine signaling inhibits a Type 2 cytokine or Type 2 cytokine receptor signaling protein selected from the group consisting of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1. Additionally or alternatively, in some embodiments of the kits of the present technology, the inhibitor of Type 2 cytokine signaling is selected from the group consisting of dupilumab, Etokimab (ANB020), Mepolizumab, reslizumab, benralizumab, lebrikizumab, risankizumab, tocilizumab, tralokinumab, anrukinzumab, AMG317, STATE inhibitors, STAT3 inhibitors, Secukinumab, ustekinumab, guselkumab, gevokizumab, and any other agent that inhibits the expression or activity of any of the Type 2 cytokines or Type 2 cytokine receptor signaling proteins disclosed herein.
In any of the preceding embodiments, the kits further comprise at least one Brd4 inhibitor, wherein the at least one Brd4 inhibitor is a small molecule, an inhibitory nucleic acid, or an antibody. Examples of Brd4 inhibitors include, but are not limited to, (+)-JQ1, I-BET762, OTX015, I-BET151, CPI203, PFI-1, MS436, CPI-0610, RVX2135, FT-1101, BAY1238097, INCB054329, TEN-010, GSK2820151, ZEN003694, BAY-299, BMS-986158, ABBV-075, GS-5829, and PLX51107. Additionally or alternatively, in some embodiments, the kits further comprise reagents for detecting mRNA or protein expression levels or chromatin accessibility levels of a Type 2 cytokine or Type 2 cytokine receptor signaling in a biological sample.
It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present technology are described below in various levels of detail in order to provide a substantial understanding of the present technology.
In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).
The present disclosure identifies the shared and specific transcriptional programs that underlie normal tissue regeneration and early neoplasia. Thus, while there are notable similarities between the cell fate transitions accompanying neoplastic transformation and regeneration, the underlying transcriptional programs and chromatin states are distinct. Specifically, IL-33, an ‘alarmin’ cytokine that plays a central role in triggering inflammation and tissue remodeling in response to injury, was identified to be a specific mediator of pancreatic tumorigenesis downstream of the described epigenetic alterations associated with KRAS mutation, showing features of enhancer-dependent activation in early neoplasia but not in normal tissue regeneration. Along with the activation of IL33, tumor-specific epigenetic alterations also induce aberrant expression of Th2 cytokine receptors (e.g., IL4RA, IL13RA1, IL13RA2, IL17RE, IL18R1, IL18RAP, IL31RA) in cells undergoing mutant KRAS-driven neoplastic transformation and in advanced pancreatic cancer cells. The present disclosure demonstrates that the inhibitors of Type 2 cytokine signaling disclosed herein are useful in methods for detecting, treating, and/or preventing pancreatic cancer in a subject in need thereof.
Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.
As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), intratumorally, or topically. Administration includes self-administration and the administration by another.
As used herein, a “control” is an alternative sample used in an experiment for comparison purpose. A control can be “positive” or “negative.” For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease or condition, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.
As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of pancreatic cancer. As used herein, a “therapeutically effective amount” of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.
As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
As used herein, the terms “individual”, “patient”, or “subject” are used interchangeably and refer to an individual organism, a vertebrate, a mammal, or a human. In certain embodiments, the individual, patient or subject is a human.
As used herein, “prevention”, “prevent”, or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample. As used herein, preventing PDAC, includes preventing or delaying the initiation of symptoms of PDAC. As used herein, prevention of PDAC also includes preventing a recurrence of one or more signs or symptoms of PDAC.
As used herein, a “sample” or “biological sample” refers to a body fluid or a tissue sample isolated from a subject. In some cases, a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and/or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like. The term “sample” may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids. Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art. A blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.
As used herein, the term “separate” therapeutic use refers to an administration of at least two active ingredients at the same time or at substantially the same time by different routes.
As used herein, the term “sequential” therapeutic use refers to administration of at least two active ingredients at different times. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several minutes, hours, or days before administering the other active ingredient or ingredients. There is no simultaneous treatment in this case.
As used herein, the term “simultaneous” therapeutic use refers to the administration of at least two active ingredients by the same route and at the same time or at substantially the same time.
“Treating”, “treat”, or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
As used herein “type 2 cytokines” refer to cytokines that promote a strong T helper type 2 (Th2) immune response, characterized by the production of interleukin-4 (IL-4), IL-5 and IL-13, and classically drive recruitment and activation of mast cells, basophils and eosinophils, and goblet cell hyperplasia in airway and intestinal epithelia. Type 2 cytokines are generally produced by Th2 T-helper cells, CD8+ T cells, and non-T cell leukocytes such as monocytes, ILC2, B cells, eosinophils, mast cells, and basophils. See Lucey et al., Clinical Microbiology Reviews 9(4): 532-562 (1996). Examples of type 2 cytokines include but are not limited to IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL17E, IL-31, IL-33 etc.
The present disclosure provides inhibitors of Type 2 cytokine signaling. In some embodiments, the inhibitor of Type 2 cytokine signaling inhibits the activity or expression of a Type 2 cytokine or Type 2 cytokine receptor signaling protein selected from the group consisting of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1.
Exemplary mRNA sequences of Type 2 cytokines or Type 2 cytokine receptor signaling proteins are provided below, represented by SEQ ID NOs: 23-38 and 46-51:
Also disclosed herein are Brd4 inhibitors. An exemplary mRNA sequence of Brd4 is provided below, represented by SEQ ID NO: 39:
In some embodiments, the inhibitor of Type 2 cytokine signaling is a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), a tyrosine kinase inhibitor, a decoy cytokine receptor, or an antibody (e.g., a neutralizing antibody). Additionally or alternatively, in some embodiments, the Brd4 inhibitor is a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), or an antibody (e.g., a neutralizing antibody).
In one aspect, the present disclosure provides Type 2 cytokine-specific, Type 2 cytokine receptor signaling protein-specific, or Brd4-specific inhibitory nucleic acids comprising a nucleic acid molecule which is complementary to a portion of a Type 2 cytokine nucleic acid sequence or a Brd4 nucleic acid sequence selected from the group consisting of SEQ ID NOs: 23-39 and 46-51.
The present disclosure also provides an antisense nucleic acid comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 23-39 or SEQ ID NOs: 46-51 (mRNA of Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4), thereby reducing or inhibiting expression of Type 2 cytokine, Type 2 cytokine receptor signaling protein, or Brd4. The antisense nucleic acid may be antisense RNA, or antisense DNA. Antisense nucleic acids based on the known Type 2 cytokine, Type 2 cytokine receptor signaling protein, or Brd4 gene sequence can be readily designed and engineered using methods known in the art. In some embodiments, the antisense nucleic acid comprises the nucleic acid sequence of any one of SEQ ID NOs: 3, 4, 5, 6, or a complement thereof.
Antisense nucleic acids are molecules which are complementary to a sense nucleic acid strand, e.g., complementary to the coding strand of a double-stranded DNA molecule (or cDNA) or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid. The antisense nucleic acid can be complementary to an entire Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4 coding strand, or to a portion thereof, e.g., all or part of the protein coding region (or open reading frame). In some embodiments, the antisense nucleic acid is an oligonucleotide which is complementary to only a portion of the mRNA coding region of Type 2 cytokine, Type 2 cytokine receptor signaling protein, or Brd4. In certain embodiments, an antisense nucleic acid molecule can be complementary to a noncoding region of the Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4 coding strand. In some embodiments, the noncoding region refers to the 5′ and 3′ untranslated regions that flank the coding region and are not translated into amino acids. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-hodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thouridine, 5-carboxymethylaminometh-yluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-metnylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v), wybutosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thlouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-cxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
The antisense nucleic acid molecules may be administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding the protein of interest to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can occur via Watson-Crick base pairing to form a stable duplex, or in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
In some embodiments, the antisense nucleic acid molecules are modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. In some embodiments, the antisense nucleic acid molecule is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res. 15:6625-6641(1987)). The antisense nucleic acid molecule can also comprise a 2′-O -methylribonucleotide (Inoue et al., Nucleic Acids Res. 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).
The present disclosure also provides a short hairpin RNA (shRNA) or small interfering RNA (siRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 23-39 or SEQ ID NOs: 46-51 (mRNA of Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd), thereby reducing or inhibiting expression of a Type 2 cytokine or Brd4. In some embodiments, the shRNA or siRNA is about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 base pairs in length. Double-stranded RNA (dsRNA) can induce sequence-specific post-transcriptional gene silencing (e.g., RNA interference (RNAi)) in many organisms such as C. elegans, Drosophila, plants, mammals, oocytes and early embryos. RNAi is a process that interferes with or significantly reduces the number of protein copies made by an mRNA. For example, a double-stranded siRNA or shRNA molecule is engineered to complement and hydridize to a mRNA of a target gene. Following intracellular delivery, the siRNA or shRNA molecule associates with an RNA-induced silencing complex (RISC), which then binds and degrades a complementary target mRNA (such as mRNA of a Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4). In some embodiments, the shRNA or siRNA comprises the nucleic acid sequence of any one of SEQ ID NOs: 3-6.
The present disclosure also provides a ribozyme comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 23-39 or SEQ ID NOs: 46-51 (mRNA of Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4), thereby reducing or inhibiting expression of Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a complementary single-stranded nucleic acid, such as an mRNA. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, Nature 334:585-591 (1988))) can be used to catalytically cleave Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4 transcripts, thereby inhibiting translation of a Type 2 cytokine, Type 2 cytokine receptor signaling protein, or Brd4.
A ribozyme having specificity for a Type 2 cytokine-, Type 2 cytokine receptor signaling protein- or Brd4-encoding nucleic acid can be designed based upon nucleic acid sequence of Brd4, a Type 2 cytokine, or Type 2 cytokine receptor signaling protein disclosed herein. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a Type 2 cytokine-, Type 2 cytokine receptor signaling protein- or Brd4-encoding mRNA. See, e.g., U.S. Pat. Nos. 4,987,071 and 5,116,742. Alternatively, mRNA of a Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4 can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418, incorporated herein by reference.
The present disclosure also provides a synthetic guide RNA (sgRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 23-39 or SEQ ID NOs: 46-51 (mRNA of Type 2 cytokine, Type 2 cytokine receptor signaling protein or Brd4). Guide RNAs for use in CRISPR-Cas systems are typically generated as a single guide RNA comprising a crRNA segment and a tracrRNA segment. The crRNA segment and a tracrRNA segment can also be generated as separate RNA molecules. The crRNA segment comprises the targeting sequence that binds to a portion of any one of SEQ ID NOs: 23-39 or SEQ ID NOs: 46-51, and a stem portion that hybridizes to a tracrRNA. The tracrRNA segment comprises a nucleotide sequence that is partially or completely complementary to the stem sequence of the crRNA and a nucleotide sequence that binds to the CRISPR enzyme. In some embodiments, the crRNA segment and the tracrRNA segment are provided as a single guide RNA. In some embodiments, the crRNA segment and the tracrRNA segment are provided as separate RNAs. The combination of the CRISPR enzyme with the crRNA and tracrRNA make up a functional CRISPR-Cas system. Exemplary CRISPR-Cas systems for targeting nucleic acids, are described, for example, in WO2015/089465.
In some embodiments, a synthetic guide RNA is a single RNA represented as comprising the following elements:
where X1 and X2 represent the crRNA segment, where X1 is the targeting sequence that binds to a portion of any one of SEQ ID NOs: 23-39 or SEQ ID NOs: 46-51, X2 is a stem sequence the hybridizes to a tracrRNA, Z represents a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to X2, and Y represents a linker sequence. In some embodiments, the linker sequence comprises two or more nucleotides and links the crRNA and tracrRNA segments. In some embodiments, the linker sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. In some embodiments, the linker is the loop of the hairpin structure formed when the stem sequence hybridized with the tracrRNA.
In some embodiments, a synthetic guide RNA is provided as two separate RNAs where one RNA represents a crRNA segment: 5′-X1-X2-3′ where X1 is the targeting sequence that binds to a portion of any one of SEQ ID NOs: 23-39 or SEQ ID NOs: 46-51, X2 is a stem sequence the hybridizes to a tracrRNA, and one RNA represents a tracrRNA segment, Z, that is a separate RNA from the crRNA segment and comprises a nucleotide sequence that is partially or completely complementary to X2 of the crRNA.
Exemplary crRNA stem sequences and tracrRNA sequences are provided, for example, in WO/2015/089465, which is incorporated by reference herein. In general, a stem sequence includes any sequence that has sufficient complementarity with a complementary sequence in the tracrRNA to promote formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the stem sequence hybridized to the tracrRNA. In general, degree of complementarity is with reference to the optimal alignment of the stem and complementary sequence in the tracrRNA, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the stem sequence or the complementary sequence in the tracrRNA. In some embodiments, the degree of complementarity between the stem sequence and the complementary sequence in the tracrRNA along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the stem sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the stem sequence and complementary sequence in the tracrRNA are contained within a single RNA, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In some embodiments, the tracrRNA has additional complementary sequences that form hairpins. In some embodiments, the tracrRNA has at least two or more hairpins. In some embodiments, the tracrRNA has two, three, four or five hairpins. In some embodiments, the tracrRNA has at most five hairpins.
In a hairpin structure, the portion of the sequence 5′ of the final “N” and upstream of the loop corresponds to the crRNA stem sequence, and the portion of the sequence 3′ of the loop corresponds to the tracrRNA sequence. Further non-limiting examples of single polynucleotides comprising a guide sequence, a stem sequence, and a tracr sequence are as follows (listed 5′ to 3′), where “N” represents a base of a guide sequence (e.g. a modified oligonucleotide provided herein), the first block of lower case letters represent stem sequence, and the second block of lower case letters represent the tracrRNA sequence, and the final poly-T sequence represents the transcription terminator:
Selection of suitable oligonucleotides for use in as a targeting sequence in a CRISPR Cas system depends on several factors including the particular CRISPR enzyme to be used and the presence of corresponding proto-spacer adjacent motifs (PAMs) downstream of the target sequence in the target nucleic acid. The PAM sequences direct the cleavage of the target nucleic acid by the CRISPR enzyme. In some embodiments, a suitable PAM is 5′-NRG or 5′-NNGRR (where N is any Nucleotide) for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively. Generally the PAM sequences should be present between about 1 to about 10 nucleotides of the target sequence to generate efficient cleavage of the target nucleic acid. Thus, when the guide RNA forms a complex with the CRISPR enzyme, the complex locates the target and PAM sequence, unwinds the DNA duplex, and the guide RNA anneals to the complementary sequence on the opposite strand. This enables the Cas9 nuclease to create a double-strand break.
A variety of CRISPR enzymes are available for use in conjunction with the disclosed guide RNAs of the present disclosure. In some embodiments, the CRISPR enzyme is a Type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme is any Cas9 protein, for instance any naturally-occurring bacterial Cas9 as well as any chimeras, mutants, homologs or orthologs. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cash, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified variants thereof. In some embodiments, the CRISPR enzyme cleaves both strands of the target nucleic acid at the Protospacer Adjacent Motif (PAM) site. In some embodiments, the CRISPR enzyme is a nickase, which cleaves only one strand of the target nucleic acid.
Other exemplary inhibitors of Type 2 cytokine signaling include, but are not limited to, dupilumab, Etokimab (ANB020), Mepolizumab, reslizumab, benralizumab, lebrikizumab, risankizumab, tocilizumab, tralokinumab, anrukinzumab, AMG317, STAT6 inhibitors, STAT3 inhibitors, Secukinumab, ustekinumab, guselkumab, and gevokizumab. Examples of STAT6 inhibitors are described in Nat Rev Drug Discov. 12(8): 611-629 (2013), and include, but are not limited to AS1517499, Niflumic acid, AS1810722, YM-341619, TMC-264, Leflunomide, berbamine, (R)-76, (R)-84, STAT6BP, and STAT6-IP. Examples of STAT3 inhibitors are described in Nat Rev Drug Discov. 12(8): 611-629 (2013), and include, but are not limited to ISS-610, PM-73G, CJ-1383, ISS-840, peptide inhibitors (e.g., PY*LKTK (SEQ ID NO: 52), Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH2 (SEQ ID NO: 53)), 531-M2001, STA-21, Stattic, LLL12, FLLL32, S3I-201, BP-1-102, S3I-201.1066, SC-1, SC-49, Indirubin, Berbamine, Honokiol, Cryptotanshinone, Evodiamine, paclitaxel, Vinorelbine, Oleanolic acid/CDDO-Me, Cucurbitacin E, Emodin, Resveratrol, Capsaicin, Avicin D, Piceatannol, Sanguarine, Celastrol, Withaferin A, Cucurbitacin I, Cucurbitacin B, 3,3′-diindolyl-methane, Caffeic acid, and the like. Other Type 2 cytokine inhibitors include, but are not limited to, anti-mouse IL-4 (clone 11B11) (Bio X Cell, West Lebanon N.H.), Mouse IL-13 Neutralizing antibody (clone 8H8) (Invivogen, San Diego Calif.), Mouse IL-33 MAb (Clone 396118) (R&D Systems, Minneapolis, Minn.), anti-mouse/human IL-5 (Clone TRFK5) (Bio X Cell, West Lebanon N.H.) or Mouse ST2/IL-33R antibody (Clone 245707) (R&D Systems, Minneapolis, Minn.).
Exemplary Brd4 inhibitors include but are not limited to, (+)-JQ1, I-BET762, OTX015, I-BET151, CPI203, PFI-1, MS436, CPI-0610, RVX2135, FT-1101, BAY1238097, INCB054329, TEN-010, GSK2820151, ZEN003694, BAY-299, BMS-986158, ABBV-075, GS-5829, and PLX51107.
In one aspect, the present disclosure provides a method for treating or preventing pancreatic cancer in a subject in need thereof comprising administering to the subject an effective amount of an inhibitor of Type 2 cytokine signaling, wherein the subject harbors a KRAS mutation. The three major isoforms of RAS (KRAS, NRAS, and HRAS) together are mutated in about 20% of human cancers, primarily in the active site at residues G12, G13, and Q61 near the g-phosphate of the guanosine triphosphate (GTP) substrate (See Marcus & Mattos, Clin Cancer Res 21(8): 1810-1818 (2015)). In certain embodiments, the KRAS mutation selected from the group consisting of G12C, G12D, G12V, G12A, G12S, G12R, G13D, G13C, G13S, G13R, G13A, G13V, Q61H, Q61L, Q61R, Q61K, Q61P, and Q61E.
Additionally or alternatively, the inhibitor of Type 2 cytokine signaling inhibits a Type 2 cytokine or Type 2 cytokine receptor signaling protein selected from the group consisting of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1. The inhibitor of Type 2 cytokine signaling may be a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), a tyrosine kinase inhibitor, a decoy cytokine receptor, or an antibody (e.g., a neutralizing antibody). Additionally or alternatively, in some embodiments, the small molecule, the inhibitory nucleic acid, the tyrosine kinase inhibitor, the decoy cytokine receptor, or the antibody specifically binds to and inhibits/neutralizes the expression or activity of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, or IL1R1. Examples of inhibitors of Type 2 cytokine signaling include, but are not limited to dupilumab, Etokimab (ANB020), Mepolizumab, reslizumab, benralizumab, lebrikizumab, risankizumab, tocilizumab, tralokinumab, anrukinzumab, AMG317, STATE inhibitors, STAT3 inhibitors, Secukinumab, ustekinumab, guselkumab, gevokizumab, or any other agent that inhibits the expression or activity of any of the Type 2 cytokines or Type 2 cytokine receptor signaling proteins disclosed herein. The pancreatic cancer may comprise exocrine tumors. In certain embodiments, the pancreatic cancer is pancreatic ductal adenocarcinoma.
Additionally or alternatively, in some embodiments, the methods further comprise administering to the subject an effective amount of a Brd4 inhibitor. The Brd4 inhibitor may be a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), or an antibody (e.g., a neutralizing antibody). Examples of Brd4 inhibitors include, but are not limited to, (+)-JQ1, I-BET762, OTX015, I-BET151, CPI203, PFI-1, MS436, CPI-0610, RVX2135, FT-1101, BAY1238097, INCB054329, TEN-010, GSK2820151, ZEN003694, BAY-299, BMS-986158, ABBV-075, GS-5829, and PLX51107. Additionally or alternatively, in some embodiments, the method further comprises sequentially, simultaneously, or separately administering one or more additional therapeutic agents to the subject.
In any and all embodiments of the methods disclosed herein, the subject harbors a mutation in TP53. The subject may have a family history of pancreatic ductal adenocarcinoma or exhibits chronic pancreatitis, Type 2 diabetes or other risk factors for developing pancreatic cancer. Additionally or alternatively, in some embodiments, the subject exhibits elevated expression levels of at least one of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1 compared to that observed in a healthy control subject or a predetermined threshold.
In another aspect, the present disclosure provides a method for selecting pancreatic cancer patients for treatment with an inhibitor of Type 2 cytokine signaling comprising (a) detecting expression levels or chromatin accessibility levels of a Type 2 cytokine or Type 2 cytokine receptor signaling protein in biological samples obtained from pancreatic cancer patients, wherein the Type 2 cytokine or the Type 2 cytokine receptor signaling protein is selected from the group consisting of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1; (b) identifying pancreatic cancer patients that exhibit (i) mRNA/protein expression levels of Type 2 cytokine or Type 2 cytokine receptor signaling protein that are elevated compared to that observed in a healthy control subject or a predetermined threshold, and/or (ii) chromatin accessibility levels of Type 2 cytokine or Type 2 cytokine receptor signaling protein that are elevated compared to that observed in a healthy control subject or a predetermined threshold; and (c) administering an inhibitor of Type 2 cytokine signaling to the pancreatic cancer patients of step (b). The inhibitor of Type 2 cytokine signaling may be a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), a tyrosine kinase inhibitor, a decoy cytokine receptor, or an antibody (e.g., a neutralizing antibody). Additionally or alternatively, in some embodiments, the small molecule, the inhibitory nucleic acid, the tyrosine kinase inhibitor, the decoy cytokine receptor, or the antibody specifically binds to and inhibits/neutralizes the expression or activity of IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, or IL1R1. Examples of inhibitors of Type 2 cytokine signaling include, but are not limited to dupilumab, Etokimab (ANB020), Mepolizumab, reslizumab, benralizumab, lebrikizumab, risankizumab, tocilizumab, tralokinumab, anrukinzumab, AMG317, STATE inhibitors, STAT3 inhibitors, Secukinumab, ustekinumab, guselkumab, gevokizumab, or any other agent that inhibits the expression or activity of any of the Type 2 cytokines or Type 2 cytokine receptor signaling proteins disclosed herein.
Additionally or alternatively, in some embodiments, the methods further comprise administering a Brd4 inhibitor to the pancreatic cancer patients of step (b). The Brd4 inhibitor may be a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), or an antibody (e.g., a neutralizing antibody). Examples of Brd4 inhibitors include, but are not limited to, (+)-JQ1, I-BET762, OTX015, I-BET151, CPI203, PFI-1, MS436, CPI-0610, RVX2135, FT-1101, BAY1238097, INCB054329, TEN-010, GSK2820151, ZEN003694, BAY-299, BMS-986158, ABBV-075, GS-5829, and PLX51107.
In any and all embodiments of the methods disclosed herein, the pancreatic cancer patients harbor a KRAS mutation selected from the group consisting of G12C, G12D, G12V, G12A, G12S, G12R, G13D, G13C, G13S, G13R, G13A, G13V, Q61H, Q61L, Q61R, Q61K, Q61P, and Q61E. Additionally or alternatively, in some embodiments, the pancreatic cancer patients harbor a mutation in TP53. The pancreatic cancer patients may exhibit exocrine tumors. In certain embodiments, the pancreatic cancer patients suffer from or are at risk for pancreatic ductal adenocarcinoma.
In any of the preceding embodiments of the methods disclosed herein, the expression levels or chromatin accessibility levels of the Type 2 cytokine or the Type 2 cytokine receptor signaling proteins are detected via ChIP, MNase, FAIRE, DNAse, ATAC-seq, RT-PCR, Northern Blotting, RNA-Seq, microarray analysis, High-performance liquid chromatography (HPLC), mass spectrometry, immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), Western Blotting, immunoprecipitation, flow cytometry, Immuno-electron microscopy, immunoelectrophoresis, enzyme-linked immunosorbent assays (ELISA), or multiplex ELISA antibody arrays. In some embodiments, the biological samples are pancreatic cancer specimens, blood, serum, or plasma.
Any method known to those in the art for contacting a cell, organ or tissue with an inhibitor of Type 2 cytokine signaling and/or a Brd4 inhibitor may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of an inhibitor of Type 2 cytokine signaling and/or a Brd4 inhibitor, such as those described above, to a mammal, suitably a human. When used in vivo for therapy, the inhibitors of Type 2 cytokine signaling and/or the Brd4 inhibitors are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the infection in the subject, the characteristics of the particular inhibitor of Type 2 cytokine signaling and/or Brd4 inhibitor used, e.g., its therapeutic index, the subject, and the subject's history.
The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of an inhibitor of Type 2 cytokine signaling and/or a Brd4 inhibitor useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The inhibitor of Type 2 cytokine signaling and/or the Brd4 inhibitor may be administered systemically or locally.
By way of an example, inhibitors of Type 2 cytokine signaling and/or Brd4 inhibitors of the present technology may be formulated in a simple delivery vehicle. In certain embodiments, inhibitors of Type 2 cytokine signaling and/or Brd4 inhibitors of the present technology may be lyophilized or incorporated in a gel, cream, biomaterial, sustained release delivery vehicle.
Inhibitors of Type 2 cytokine signaling and/or Brd4 inhibitors of the present technology are generally combined with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity. Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids and amino acid copolymers. Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g. mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
The inhibitor of Type 2 cytokine signaling and/or the Brd4 inhibitor may be formulated as a pharmaceutically acceptable salt. The term “pharmaceutically acceptable salt” means a salt prepared from a base or an acid which is acceptable for administration to a patient, such as a mammal (e.g., salts having acceptable mammalian safety for a given dosage regime). However, it is understood that the salts are not required to be pharmaceutically acceptable salts, such as salts of intermediate compounds that are not intended for administration to a patient. Pharmaceutically acceptable salts can be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids. Salts derived from pharmaceutically acceptable inorganic bases include ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, and zinc salts, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally-occurring amines and the like, such as arginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperadine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. Salts derived from pharmaceutically acceptable inorganic acids include salts of boric, carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric or hydroiodic), nitric, phosphoric, sulfamic and sulfuric acids. Salts derived from pharmaceutically acceptable organic acids include salts of aliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic, lactobionic, malic, and tartaric acids), aliphatic monocarboxylic acids (e.g., acetic, butyric, formic, propionic and trifluoroacetic acids), amino acids (e.g., aspartic and glutamic acids), aromatic carboxylic acids (e.g., benzoic, p-chlorobenzoic, diphenylacetic, gentisic, hippuric, and triphenylacetic acids), aromatic hydroxyl acids (e.g., o-hydroxybenzoic, p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylic and 3-hydroxynaphthalene-2-carboxylic acids), ascorbic, dicarboxylic acids (e.g., fumaric, maleic, oxalic and succinic acids), glucuronic, mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids (e.g., benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic, isethionic, methanesulfonic, naphthalenesulfonic, naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic and p-toluenesulfonic acids), xinafoic acid, and the like.
The inhibitors of Type 2 cytokine signaling and/or the Brd4 inhibitors of the present technology described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment or prevention of a disorder described herein. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.
Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).
Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
The inhibitor of Type 2 cytokine signaling compositions and/or Brd4 inhibitor compositions can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, isotonic agents are included, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the inhibitors of Type 2 cytokine signaling and/or the Brd4 inhibitors of the present technology can be delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Pat. No. 6,468,798.
Systemic administration of an inhibitor of Type 2 cytokine signaling and/or a Brd4 inhibitor of the present technology as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.
An inhibitor of Type 2 cytokine signaling and/or a Brd4 inhibitor of the present technology can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the inhibitor of Type 2 cytokine signaling and/or the Brd4 inhibitor is encapsulated in a liposome while maintaining integrity. As one skilled in the art would appreciate, there are a variety of methods to prepare liposomes. (See Lichtenberg et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem et al., Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother 34(7-8):915-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.
The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the inhibitor of Type 2 cytokine signaling and/or the Brd4 inhibitor of the present technology can be embedded in the polymer matrix, while maintaining protein integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly α-hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).
Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy et al.), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale et al.), PCT publication WO 96/40073 (Zale et al.), and PCT publication WO 00/38651 (Shah et al.). U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.
In some embodiments, the inhibitors of Type 2 cytokine signaling and/or the Brd4 inhibitors of the present technology are prepared with carriers that will protect the inhibitors of Type 2 cytokine signaling and/or the Brd4 inhibitors of the present technology against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
The inhibitors of Type 2 cytokine signaling and/or the Brd4 inhibitors of the present technology can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995). Mizguchi et al., Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.
Dosage, toxicity and therapeutic efficacy of the inhibitor of Type 2 cytokine signaling and/or the Brd4 inhibitor of the present technology can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. In some embodiments, the inhibitor of Type 2 cytokine signaling and/or the Brd4 inhibitor of the present technology exhibit high therapeutic indices. While an inhibitor of Type 2 cytokine signaling and/or a Brd4 inhibitor of the present technology that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any an inhibitor of Type 2 cytokine signaling and/or a Brd4 inhibitor of the present technology used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
Typically, an effective amount of the inhibitor of Type 2 cytokine signaling and/or the Brd4 inhibitors of the present technology, sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of an inhibitor of Type 2 cytokine signaling and/or a Brd4 inhibitor ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, inhibitor of Type 2 cytokine signaling and/or Brd4 inhibitor concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
In some embodiments, a therapeutically effective amount of an inhibitor of Type 2 cytokine signaling and/or a Brd4 inhibitor of the present technology may be defined as a concentration of the inhibitor of Type 2 cytokine signaling and/or the Brd4 inhibitor at the target tissue of 1012 to 10′ molar, e.g., approximately 10′ molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue. In some embodiments, the doses are administered by single daily or weekly administration, but may also include continuous administration (e.g., parenteral infusion or transdermal application). In some embodiments, the dosage of the inhibitor of Type 2 cytokine signaling and/or the Brd4 inhibitor of the present technology is provided at a “low,” “mid,” or “high” dose level. In one embodiment, the low dose is provided from about 0.0001 to about 0.5 mg/kg/h, suitably from about 0.001 to about 0.1 mg/kg/h. In one embodiment, the mid-dose is provided from about 0.01 to about 1.0 mg/kg/h, suitably from about 0.01 to about 0.5 mg/kg/h. In one embodiment, the high dose is provided from about 0.5 to about 10 mg/kg/h, suitably from about 0.5 to about 2 mg/kg/h.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.
The mammal treated in accordance present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human.
In any and all embodiments of the methods disclosed herein, one or more inhibitors of Type 2 cytokine signaling of the present technology and/or one or more Brd4 inhibitors of the present technology may be separately, sequentially or simultaneously administered with at least one additional therapeutic agent. The at least one therapeutic agent may be selected from the group consisting of immunotherapeutic agents, alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducing agents, antimetabolites, mitotic inhibitors, nitrogen mustards, nitrosoureas, alkylsulfonates, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF/VEGFR inhibitors, EGF/EGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, endocrine/hormonal agents, bisphosphonate therapy agents, phenphormin and targeted biological therapy agents (e.g., therapeutic peptides described in U.S. Pat. No. 6,306,832, WO 2012007137, WO 2005000889, WO 2010096603 etc.). In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent.
Specific chemotherapeutic agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5-fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), cladribine, midostaurin, bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, chlorambucil, ifosfamide, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, altretamine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, hydroxyurea, pemetrexed, epirubicin, idarubicin, SN-38, ARC, NPC, campothecin, 9-nitrocamptothecin, 9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, amsacnne, etoposide phosphate, teniposide, azacitidine (Vidaza), decitabine, accatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetyl cephalomannine, streptozotocin, nimustine, ranimustine, bendamustine, uramustine, estramustine, mannosulfan, camptothecin, exatecan, lurtotecan, lamellarin D9-aminocamptothecin, amsacrine, ellipticines, aurintricarboxylic acid, HU-331, or combinations thereof.
Examples of antimetabolites include 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, and mixtures thereof.
Examples of taxanes include accatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetyl cephalomannine, and mixtures thereof.
Examples of DNA alkylating agents include cyclophosphamide, chlorambucil, melphalan, bendamustine, uramustine, estramustine, carmustine, lomustine, nimustine, ranimustine, streptozotocin; busulfan, mannosulfan, and mixtures thereof.
Examples of topoisomerase I inhibitor include SN-38, ARC, NPC, camptothecin, topotecan, 9-nitrocamptothecin, exatecan, lurtotecan, lamellarin D9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, and mixtures thereof. Examples of topoisomerase II inhibitors include amsacrine, etoposide, etoposide phosphate, teniposide, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, doxorubicin, and HU-331 and combinations thereof.
Examples of immunotherapeutic agents include immune checkpoint inhibitors (e.g., antibodies targeting CTLA-4, PD-1, PD-L1), ipilimumab, 90Y-Clivatuzumab tetraxetan, pembrolizumab, nivolumab, trastuzumab, cixutumumab, ganitumab, demcizumab, cetuximab, nimotuzumab, dalotuzumab, sipuleucel-T, CRS-207, and GVAX.
In any case, the multiple therapeutic agents may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.
The present technology provides kits containing components suitable for treating or preventing pancreatic cancer in a patient in need thereof. In one aspect, the kits comprise at least one inhibitor of Type 2 cytokine signaling disclosed herein and/or at least one a Brd4 inhibitor disclosed herein, in combination with instructions for using the same to treat or prevent pancreatic cancer. Optionally, the above described components of the kits of the present technology are packed in suitable containers and labeled for the prevention and/or treatment of pancreatic cancer.
The above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution. The kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition and a saline solution. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and/or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts. The kits may optionally include instructions customarily included in commercial packages of therapeutic or diagnostic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products.
The kit can also comprise, e.g., a buffering agent, a preservative or a stabilizing agent. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit. In certain embodiments, the use of the reagents can be according to the methods of the present technology.
In some embodiments, the kit contains additional reagents suitable for detecting mRNA, chromatin accessibility, or protein expression levels of a Type 2 cytokine or Type 2 cytokine receptor signaling (e.g., IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1) including an antibody that specifically binds to a Type 2 cytokine or Type 2 cytokine receptor signaling protein, primers and/or probes that specifically hybridize to a nucleic acid sequence that encodes a Type 2 cytokine or Type 2 cytokine receptor signaling protein, or any combination thereof, in biological samples obtained from a patient diagnosed with, or suspected of having pancreatic cancer. Additionally or alternatively, the kits of the present technology may also include instructions for performing assays that measure chromatin accessibility at gene loci (e.g., at intergenic or intronic regions) encoding a Type 2 cytokine or Type 2 cytokine receptor signaling protein (e.g., ChIP, MNase, FAIRE, DNAse, ATAC-based approaches). For example, the kits may contain a positive control sample that contains a reference level of a particular Type 2 cytokine or Type 2 cytokine receptor signaling protein (e.g., IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1) and/or a negative control sample that lacks a particular Type 2 cytokine or Type 2 cytokine receptor signaling protein (e.g., IL-33, IL-4, IL-13, IL-5, IL-23A, IL-17E, IL-9, IL9R, IL1RL1, IL4RA, IL13RA1, IL13RA2, IL5RA, IL1RL2, IL17RA, IL31RA, IL17RE, IL18R1, IL18RAP, and IL1R1). The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed.
The kit components, (e.g., reagents) can be packaged in a suitable container. The kit can also contain, e.g., a buffering agent, a preservative or a protein-stabilizing agent. The kit can further comprise, or alternatively consist essentially of, or yet further consist of components necessary for detecting the detectable-label, e.g., an enzyme or a substrate. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present disclosure may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit.
As amenable, these suggested kit components may be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit components may be provided in solution or as a liquid dispersion or the like.
The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way.
Generation and authentication of KC-shBrd4 ESC clones. KC-shBrd4 ESCs (Ptf1a-Cre;LSL-KrasG12D;RIK;CHC (Saborowski et al., Genes Dev 28: 85-97 (2014)) were targeted with 2 independent GFP-linked Brd4-shRNAs (shBrd4.552 and shBrd4.1448) (Tasdemir et al., Cancer Discov 6: 612-629 (2016); Zuber et al., Nature 478: 524-528 (2011)) cloned into mir30-based targeting constructs (Dow et al. Nat Protoc 7: 374-393 (2012)), as previously described (Dow et al. Nat Protoc 7: 374-393 (2012); Saborowski et al., Genes Dev 28: 85-97 (2014)). Targeted ESCs were selected and functionally tested for single intregation of the GFP-linked shRNA element into the CHC locus as previously described (Livshits et al., Elife 7: e35216 (2018)). The KC-shRen ESC control clone used in this study has been previously described (Livshits et al., Elife 7: e35216 (2018); Saborowski et al., Genes Dev 28: 85-97 (2014)). Before injection, ESCs were cultured briefly for expansion in KOSR+2i medium (Gertsenstein et al., PLoS One 5: e11260 (2010)). The identity and genotype of the ESC, resulting chimeric mice and their progeny was authenticated by genomic PCR using a common Col1a1 primer CACCCTGAAAACTTTGCCCC (SEQ ID NO: 1) paired with a transgene specific primer: shRen.713: GTATAGATAAGCATTATAATTCCTA (SEQ ID NO: 2); shBrd4.552: TATTGTTCCCATATCCAT (SEQ ID NO: 3); shBrd4.1448: CTAGTTTAGACTTGATTGTG (SEQ ID NO: 4), yielding an ˜250-bp product. ESC were confirmed to be negative for mycoplasma and other microorganisms before injection.
Animal models. All animal experiments in this study were performed in accordance with a protocol approved by the Memorial Sloan-Kettering Institutional Animal Care and Use Committee. Mice were maintained under specific pathogen-free conditions, and food and water were provided ad libitum. All mice strains have been previously described. p48-Cre, LSL-KrasG12D, CHC, CAGs-LSL-RIK, and TRE-GFP-shRen strains were interbred and maintained on mixed background. Chimeric cohorts of KC-shRNA mice derived from the ESCs above described were generated by the Center for Pancreatic Cancer Research (CPCR) at MSKCC or the Rodent Genetic Engineering Core at NYU as previously described (Saborowski et al., Genes Dev 28: 85-97 (2014)). ESC-derived KC-shMyc mice have been previously described (Saborowski et al., Genes Dev 28: 85-97 (2014)). Only KC-shRNA mice with a coat color chimerism of >95% were included for experiments. For induction of shRNA expression, mice were switched to a doxycycline diet (625 mg/kg, Harlan Teklad) that was changed twice weekly.
To compare the effects of tissue injury in the transcriptional and chromatin accessibility landscapes of mutant Kras-expressing or Kras wild-type pancreatic epithelial cells, KC;RIK (p48-Cre;RIK;LSLKrasG12D) or C;RIK (p48-Cre;RIK) male mice were treated with 8 hourly intraperitoneal injections of 80 μg/kg caerulein (Bachem) or PBS for 2 consecutive days, using littermates when possible. To characterize invasive disease, pancreatic ductal adenocarcinoma (PDAC) cells were isolated from cancer lesions arising in autochthonous transgenic models (KPflC;RIK (p48Cre;RIK;LSL-KrasG12D;p53fl/+) that were macro-dissected away from pre-malignant tissue. As an orthogonal approach, C57Bl/6 female mice (Harlan) were subjected to for orthotopic transplantations with syngeneic ductal organoids harboruing mutant Kras and inactivated Trp53 gene (see below). Prior to transplantation, organoid cultures were dissociated with TrypLE (Gibco) after mechanical dissociation by pipetting and 1-2×105 cells in serum-free advanced DMEM/F12 (Life Technologies) supplemented with 2 mM glutamine and penn-strep were mixed 1:1 with growth factor reduced matrigel (Corning) and injected into the exposed pancreas of 8-10 weeks old C57B16/N mice using a Hamilton syringe fitted with a 26 gauge needle. For treatment with recombinant Il-33, 5 weeks-old C or KC mice were injected intraperitoneally once daily doses with 1 ug of murine recombinant Il-33 (#580504, R&D Systems) or vehicle (PBS) for 5 consecutive days.
Pancreatic epithelial cell isolation. For RNA-seq and ATAC-seq analyes in lineage-traced epithelial cells isolated directly from KC, KPflC, or KC-shRNA mice, pancreata were finely chopped with scissors and incubated with digestion buffer containing 1 mg/ml Collagenase V (C9263, Sigma-Aldrich), 2 U/mL Dispase (17105041, Life Technologies) dissolved in HBSS with Mg2+ and Ca2+ (14025076, Thermo Fisher Scientific) supplemented with 0.1 mg/ml DNase I (Sigma, DN25-100MG) and 0.1 mg/ml Soybean Trypsin Inhibitor (STI) (T9003, Sigma), in gentleMACS C Tubes (Miltenyi Biotec) for 42 min at 37° C. using the gentleMACS Octo Dissociator. Normal (non-fibrotic) pancreas samples were dissociated as above, except that the digestion buffer contained 1 mg/mL Collagease D (11088858001, Sigma-Aldrich). After enzymatic dissociation, samples were washed with PBS and further digested with a 0.05% solution of Trypsin-EDTA (Ser. No. 15/400,054, Thermo Fisher Scientific) diluted in PBS for 5 min at 37° C. Trypsin digestion was neutralized with FACS buffer (10 mM EGTA and 2% FBS in PBS) containing STI. Samples were then washed in FACS buffer containing DNase I and STI, filtered through a 100 μm strainer. Cell suspensions were blocked for 5 min at room temperature with rat anti-mouse CD16/CD32 with Fcblock (Clone 2.4G2, BD Biosciences) in FACS buffer containing DNase I and STI, and APC-conjugated CD45 antibody was then added (Clone 30-F11,BD Biosciences) and incubated for 10 min at 4° C. Cells were then washed once with in FACS buffer containing DNase I and STI, filtered through a 40 μm strainer, and resuspended in FACS buffer containing DNase I and STI and 300 nM DAPI as live-cell marker. Sorts were performed on a BD FACSAria III cell sorter (Becton Dickinson) for mKate2 (co-expressing GFP for on dox-shRNA mice), excluding CD45+ cells. Cells were sorted directly into Trizol LS (Thermo Fisher Scientific) for RNA-seq or collected in 2% FBS in PBS for ATAC-seq.
Immunofluorescence, immunohistochemistry and histological analyses. Tissues were fixed overnight in 10% neutral buffered formalin (Richard-Allan Scientific), embedded in paraffin and cut into 5 μm sections. Slides were heated for 30 min at 55° C., deparaffinized, rehydrated with an alcohol series and subjected to antigen retrieval with citrate buffer (Vector Laboratories Unmasking Solution, H-3300) for 25 min in a pressure cooker set on high. Sections were treated with 3% H2O2 for 10 min followed by a wash in dionized water (for immunohistochemistry only), washed in PBS, then blocked in PBS/0.1% Triton X-100 containing 1% BSA. Primary antibodies were incubated overnight at 4° C. in blocking buffer. The following primary antibodies were used: mKate2 (Evrogen, AB233), GFP (ab13970, Abcam and 2956S, Cell Signaling Technology), Brd4 (HPA015055, Sigma-Aldrich), Myc (ab32072, Abcam), Cpa1 (R&D, AF2765), Clusterin (SCBT sc-6419), SOX9 (Millipore AB553), Amylase (sc-31869, Santa Cruz), Krt19 (Troma III, Developmental Studies Hybridoma Bank) and Il-33 (AF3626, R&D). For mKate2, GFP and cMyc immunohistochemistry, Vector ImmPress HRP kits and ImmPact DAB (Vector Laboratories) were used for secondary detection. Tissues were then counterstained with Haematoxylin or when indicated Alcian blue (pH 2.5) and 0.1% Nuclear Fast Red Solution, dehydrated and mounted with Permount (Fisher). The immunohistochemistry detection of Brd4 was performed at the Molecular Cytology Core Facility of Memorial Sloan Kettering Cancer Center using Discovery XT processor (Ventana Medical Systems-Roche). The tissue sections were blocked for 30 min in 10% normal goat serum, 2% BSA in PBS. A rabbit polyclonal anti-Brd4 antibody (HPA015055, Sigma-Aldrich) was used in 1 ug/ml (1:100) concentrations. The incubation with the primary antibody was done for 6 hours, followed by 60 minutes incubation with biotinylated goat anti-rabbit IgG (PK610, Vector labs) in 5.75 ug/mL concentration. Blocker D, Streptavidin-HRP and DAB detection kit (760-124, Ventana Medical Systems-Roche) were used according to the manufacturer instructions. Slides were counterstained with Hematoxylin (760-2021, Ventana), Bluing Reagent (760-2037, Ventana) and coverslipped with Permount (Fisher Scientific).
For immunofluorescence, the following secondary antibodies were used: donkey anti-chicken CF488 (SAB4600031, Sigma-Aldrich), goat anti-chicken AF488 (A11039, Invitrogen), donkey anti-rabbit AF594 (A21207, Invitrogen), goat anti-rabbit AF594 (A11037, Thermo Fisher Scientific), donkey anti-goat AF488 (A11055, Invitrogen) and donkey anti-goat AF594 (A32758, Thermo Fisher Scientific). Slides were counterstained with DAPI and mounted in ProLong Gold (Life Technologies). Hematoxylin and eosin (H&E) was performed using standard protocols.
Images were acquired on a Zeiss AxioImager microscope using a 10×(Zeiss NA 0.3) or 20×(Zeiss NA 0.17) objective, an ORCA/ER CCD camera (Hamamatsu Photonics, Hamamatsu, Japan), and Axiovision software. For histological analysis of lesions in KC-shRen or KC-shBrd4 mice, lesions were classified and graded by a veterinary pathologist blinded to genotype into ADM (ductal metaplasia) or mucinous (Alcian Blue+) PanIN lesions using established criteria. The GFP+ area was quantified using “SpotR software”. All lesions in at least 3 representative 20× fields per section were measured and counted. The results were averaged and normalized to total tissue area analyzed. Statistical analyses were performed using unpaired t-test in Prism 7. Graphs displayed averages±SEM of independent biological replicates (mice).
Quantitative Real-Time polymerase chain reaction (qRT-PCR) analysis Total RNA was isolated from mKate2+,CD45-DAPI-sorted primary pancreatic epithelial cells using the Trizol LS (Thermo Fisher Scientific), and cDNA was obtained from 500 ng of RNA using the Transcriptor First Strand cDNA Synthesis Kit (Roche) after treatment with DNAse I (Invitrogen) following manufacturer's instructions. The following primer sets for mouse sequences were used: Il-33_F GCTGCGTCTGTTGACACATT (SEQ ID NO: 5), Il-33_R GACTTGCAGGACAGGGAGAC (SEQ ID NO: 6), Agr2_F ACAACTGACAAGCACCTTTCTC (SEQ ID NO: 7), Agr2_R GTTTGAGTATCGTCCAGTGATGT (SEQ ID NO: 8), Muc6_F AGCCCACATTCCCTATCAGC (SEQ ID NO: 9), Muc6_R CACAGTGGAAGATTGCGAGAG (SEQ ID NO: 10), Cpa1_F CAGTCTTCGGCAATGAGAACT (SEQ ID NO: 11), Cpa1_R GGGAAGGGCACTCGAACATC (SEQ ID NO: 12), Sox9_F CGTGCAGCACAAGAAAGACCA (SEQ ID NO: 13), Sox9_R GCAGCGCCTTGAAGATAGCAT (SEQ ID NO: 14), Hprt_F TCAGTCAACGGGGGACATAAA (SEQ ID NO: 15), Hprt_R GGGGCTGTACTGCTTAACCAG (SEQ ID NO: 16), Rplp0_F GCTCCAAGCAGATGCAGCA (SEQ ID NO: 17) and Rplp0_R CCGGATGTGAGGCAGCAG (SEQ ID NO: 18) qRT-PCR was carried out in triplicate (5 cDNA ng/reaction) using SYBR Green PCR Master Mix (Applied Biosystems) on the ViiA 7 Real-Time PCR System (Life technologies). Hprt or Rplp0 (aka 36b4) served as endogenous normalization controls.
RNA-Seq Analysis.
RNA extraction, RNA-seq library preparation and sequencing: Total RNA was isolated from primary mKate2+,CD45-DAPI-pancreatic epithelial cells isolated from normal, regenerating (Reg-ADM), early neoplastic (Kras*, Kras*-ADR) and cancer (PDAC) tissues into TRIzolLS and assessed using a Agilent 2100 Bioanalyzer. Sequencing and library preparation was performed at the Integrated Genomics Operation (IGO) at MSKCC. RNA-seq libraries were prepared from total RNA. After RiboGreen quantification and quality control by Agilent BioAnalyzer, 100-500 ng of total RNA underwent polyA selection and TruSeq library preparation according to instructions provided by Illumina (TruSeq Stranded mRNA LT Kit, RS-122-2102), with 8 cycles of PCR. Samples were barcoded and run on a HiSeq 4000 or HiSeq 2500 in a 50 bp/50 bp paired end run, using the HiSeq 3000/4000 SBS Kit or TruSeq SBS Kit v4 (Illumina). An average of 41 million paired-end reads was generated per sample. At the most the ribosomal reads represented 0.01% of the total reads generated and the percent of mRNA bases averaged 53%.
RNA-seq read mapping and differential expression analysis: Resulting RNA-Seq data was analyzed by removing adaptor sequences using Trimmomatic. RNA-Seq reads were then aligned to GRCm38.91 (mm10) with STAR and transcript count was quantified using featureCounts to generate raw count matrix. Differential gene expression analysis was performed using DESeq2 package between experimental conditions, using 3-5 independent biological replicates (individual mouse) per condition, implemented in R. Principal component analysis (PCA) was performed using the DESeq2 package in R. Differentially expressed genes (DEGs) were determined by >2-fold change in gene expression with adjusted P-value<0.05.
RNA-Seq heatmap clustering and pathway enrichment analysis of gene clusters: The DEGs of regeneration and early neoplasia (determined by comparing Reg-ADM, Kras*, and Kras*-ADR conditions to Normal pancreas; FC>2, padj<0.05) were clustered using kmeans clustering of 5 and plotted using pheatmap package in R, with Euclidean measure to obtain distance matrix and ward.D2 agglomeration method for clustering. Pathway enrichment analysis was performed in the resulting gene clusters with the Reactome database using enrichR. Significance of the test was assessed using combined score, described as c=log(p)*z, where c is the combined score, p is Fisher exact test p-value, and z is z-score for deviation from expected rank.
Gene set enrichment analysis (GSEA): GSEA was performed using the GSEAPreranked tool for conducting gene set enrichment analysis of data derived from RNA-seq experiments (version 2.07) against signatures in the MSigDB database and published expression signatures in organoid models and human samples. The metric scores were calculated using the sign of the fold change multiplied by the inverse of the p-value.
Overlap with human gene expression datasets: 2 independent publicly datasets of microarray data from human PDAC and normal pancreas samples (GSE71729, Moffitt et al., Nat Genet 47, 1168-1178 (2015); and GSE62452, Yang et al., Cancer Res 76, 3838-3850 (2016)) were used. Differential expression analysis was then applied using the limma package to define differentially expressed genes (DEGs) between PDAC vs normal samples, using>2-fold change and adjusted P-value<0.05 cut-off. The overlap between human DEGS with DEGs identified in GEMMS is summarized in
ATAC-Seq Analysis
Cell preparation, transpositon reaction, ATAC-seq library construction and sequencing: 65,000 mKate2+ cells isolated by FACS, washed once with 50 uL of cold PBS, and resuspended in 50 ul cold lysis buffer (Buenrostro et al., 2015). Cells were then centrifuged immediately for 10 min at 500 g, 4° C. and nuclei pellet was subjected to transposition with Nextera Tn5 transposase (FC-121-1030, Illumina) for 30 min at 37° C., according to manufacturer's instructions. DNA was eluted using MinElute PCR Purification Kit in 11.5 ul elution buffer (Qiagen). ATAC-seq libraries were prepared using the NEBNext High-Fidelity 2× PCR Master Mix (NEB M0541) as previously described (Livshits et al., 2018). Purified libraries were assessed using a Bioanalyzer High-Sensitivity DNA Analysis kit (Agilent). Approximately 200 million paired-end 50 bp reads were sequenced per replicate on a HiSeq 2500 (High Output) at the New York Genome Center.
Mapping, peak calling and dynamic peak calling: Fastq files were trimmed with trimGalore and cutadapt, and the filtered, pair-ended reads were aligned to mm9 with bowtie2. Peaks were called over input using MACS2, and only peaks with a p-value of <=0.001 and outside the ENCODE blacklist region were kept. All peaks from all samples were merged by combining peaks within 500 bp of each. The featureCount was used to count the mapped reads for each sample. The resulting peak atlas was normalized using DESeq2 (PeakNorm). For comparison to DepthNorm, samples were normalized to 10 million mapped reads (
ATAC-seq heatmap clustering and motif enrichment analysis: The dynamic peaks of regeneration and early neoplasia determined by comparing Normal, Reg-ADM, Kras*, and Kras*ADR conditions (as defined in
Reg-ADM and Kras*-ADR Unique peak signatures: To find dynamic peaks unique and shared between regenerative or pro-neoplastic metaplasia compared to normal pancreas, bedtools was used remove, or keep, the unique/shared peaks between Reg-ADM vs normal and Kras*-ADR conditions vs normal pancreas. The unique peaks were sorted by log 2FC, and the top and bottom 500 up- and down-regulated peaks were investigated for motif enrichment using HOMER findMotifGenome.
Integrative analysis of RNA-seq-ATAC-seq data: To investigate chromatin accessibility changes associated with DEGs found in the RNA-Seq analysis, dynamic ATAC-Seq peaks were first averaged across samples within each tissue state. Each peak was separated based on ChIPSeeker annotations (TSS, Promoter, 5′UTR, etc). Peak signals were then first summarised on individual gene level, followed by averaging across individual RNA-Seq clusters (Z1-Z5). Data was z-score normalized for each of the indicated tissue states. RNA-Seq data were averaged over genes within each Z1-Z5 cluster. Data was z-score normalized across each Z1-Z5 cluster. The final average data was represented as heat map using pheatmap package in R.
Isolation, culture and genetic manipulation of pancreatic organoids. To isolate untransformed ductal organoids for the transplantable PDAC tumorigenesis model, normal pancreas from LSL-KrasG12D mice (pure Bl/6N background) minced and digested with 0.012% collagenase XI (C9407, Sigma-Aldrich) and 0.012% Dispase (17105041, Life Technologies) in in HBSS with Mg2+ and Ca2+ (14025076, Thermo Fisher Scientific) at 37° C. for a maximum of 30 mins. The material was further digested with TrypLE (GIBCO) for 5 min at 37° C., washed twice with DMEM/F12 (Life Technologies) supplemented with 2 mM glutamine and penn-strep, embedded in growth factor reduced matrigel (Corning), and cultured in complete medium, as described in Boj et al 2014. For activation of mutant Kras, organoids haroburing the LSL-KrasG12D allele were transduced with Ad-mCherry-Cre (Vector Biolabs), and Cherry+ cells were sorted from single cell organoid suspension by flow cytometry 36h thereafter. Resulting clones were assessed for LSL-KrasG12D recombination by genotyping PCR in genomic DNA using the following primers: 5′ gtc ttt ccc cag cac agt gc 3′ (SEQ ID NO: 19), 5′ ctc ttg cct acg cca cca get c 3′ (SEQ ID NO: 20), and 5′ agc tag cca cca tgg ctt gag taa gtc tgc a 3′ (SEQ ID NO: 21). Validated Cre-recombined clones were then subjected to CRISPR-based inactivation of Trp53 using the PX458 vector (Addgene #48138) and gRNA AGTGAAGCCCTCCGAGTGTC sequence (SEQ ID NO: 22). PX458-sgTrp53 was transduced into organoids by transient transfection using the spinoculation method previously described (O'Rourke et al., 2017), with the modification of using the Effectene transfection reagent (Qiagen). PX458-sgTrp53 introduced cells were sorted by GFP positivity with flow cytometry 36h post-transfection. p53 null status of targeted clones was validated by western blot, using anti-p53 antibody (CMS, Leica Microsystem) and anti-β-actin-peroxidase antibody (Sigma-Aldrich) as normalization control.
Statistical analysis. Statistical analyses were performed using Prism 7 by unpaired two-tailed t-test for all other experimental data. Grouped data are expressed as mean+SEM for the number of biological replicates indicated in the figures or associated legend. Statistically significant differences are indicated with asterisks in figures with the accompanying p-values in the legend. In RNA-seq data, significance for differential gene expression between groups was based on adjusted p-value<0.05. For pathway enrichment analysis of RNA-seq gene clusters, the significance of gene lists was assessed by adjusted p-value and Z-score (Chen et al., 2013) Significance of gene sets from GSEA was based on the normalized enrichment score (NES) and the false discovery rate q-value (FDR q-val). In ATAC-seq data, dynamic peaks were called if they had an absolute log 2FC>=0.58 and a FDR<=0.1. For experiments using chimeric KC-shRen mice, only animals with coat chimerism>95% were included.
All RNA-seq and ATAC have been deposited to GEO under series GSE132330.
To define the contribution of chromatin mechanisms controlling cell identity to mutant KRAS-driven neoplastic transformation in vivo, autochthonous mouse models that enable lineage-tracing and spatiotemporally-controlled perturbation of the chromatin reader Brd4 in vivo were developed (
To this end, a flexible embryonic stem cell (ESC)-based mouse modeling strategy that incorporates lineage tracing and inducible control of gene expression into multi-allelic mice predisposed to PDAC was exploited. To attain tight control of Brd4 activity specifically in the pancreatic epithelium, mice harboring: (i) a pancreas-specific Cre driver (Ptf1a-Cre), (ii) a Cre-activatable LSL-KrasG12D allele and (iii) two additional alleles [LSL-rtTA3-IRES-mKate (RIK) and the collagen homing cassette, (CHC)] that allow for inducible expression of a GFP-linked shRNA targeting Brd4 (shBrd4) in Cre-recombined cells labeled by the fluorescent reporter mKate2 were generated (
In addition, analogous models harboring the Brd4 shRNA without the LSL-KrasG12D allele (referred to as C-shBrd4 below) were generated to compare and contrast the epigenetic requirements of neoplastic transformation vs injury-driven regeneration (FIGS. 1A-1B). To control for on-target effects of RNAi, mice were produced that harbor two different well-validated shRNAs targeting Brd4 (named shBrd4.552 and shBrd4.1448). To control for potential phenotypes linked to dox treatment and off-target perturbations of the RNAi machinery, mice harboring a highly potent but phenotypically neutral shRNA targeting Renilla Luciferase (shRen.713) were produced. Animals derived from all of these models were studied in parallel.
First, histological and molecular analyses were performed to confirm that acute Brd4 knockdown disrupts the expression of genes driven by lineage-specific enhancers in vivo. As expected, histological analysis of pancreata from 4-week-old KC- and C-GEMM mice harboring control (shRen) or Brd4 shRNAs (
Taking advantage of the above systems, experiments were performed to determine the requirement for Brd4-regulated programs in mutant Kras-driven pancreatic neoplasia, both in settings where oncogenic Kras induces stochastic development of neoplastic cell fate and where transformation is accelerated by tissue injury. To track the effects on stochastic Kras driven neoplasia, KC-shRen or KC-shBrd4 mice were placed on a diet containing doxyxycline (the dox diet) at 10 days of age and analyzed the induction and evolution of metaplasia in 6 week and 1 year-old mice. By 6 weeks, pancreata of KC-shRen mice displayed features of acinar-to-ductal metaplasia (ADM), as assessed by the appearance of duct-like structures with decreased expression of acinar markers (e.g. Cpa1, Amylase) and emergent expression of ductal markers (e.g. Sox9 and Krt19) not normally expressed in acinar cells (
In contrast, Brd4 suppression prevented the subsequent neoplastic progression of metaplastic lesions into pancreatic intraepithelial neoplasias (PanINs). At both the 6 week and 1 year old time-points, KC-shBrd4 mice exhibited a marked reduction in PanIN lesions, evidenced by significantly reduced positive co-staining of the acidic mucin marker Alcian Blue and the shRNA-linked GFP (
To explore the requirement for Brd4 in the context of injury-accelerated tumorigenesis, a well-established model of pancreatic injury produced by treatment with the synthetic cholecystokinin analogue caerulein was used. Caerulein triggers acinar autolysis and inflammation, resulting in accelerated neoplasia associated with ADM within 48 h post-treatment and progression into PanIN lesions by 2-3 weeks. 4-week-old KC-shRen or KC-shBrd4 mice were placed on dox diet to acutely induce shRNA expression and, 6 days later, treated with caerulein, such that ADM and PanIN formation were triggered synchronously throughout the pancreas in the presence or absence of epithelial Brd4 (
Pancreatic metaplasia is not an exclusive feature of early pancreatic neoplasia but also occurs as part of a physiological regenerative response to tissue injury. To compare the epigenetic requirements of neoplastic vs regenerative epithelial plasticity, GEMMS permitting inducible Brd4 suppression on the background of wild-type Kras (C-shBrd4 and C-shRen) were generated by strain intercrossing, and 4-week-old mice were subjected to dox administration followed by caerulein treatment to induce synchronous ADM in normal pancreas in the presence or absence of epithelial Brd4 (
In contrast to controls, Brd4-suppressed mice exhibited a rapid reduction of mKate2/GFP+ labeled shRNA-expressing cells and pancreatic tissue size between day 2 to day 7 post-caerulein (
Given the established role of BRD4 in reading chromatin regulatory programs that control cell fate, the transcriptional and chromatin landscapes of lineage-traced pancreatic epithelial cells undergoing regenerative or pro-neoplastic cell fate transitions were next characterized in vivo. Specifically, the mKate2-positive populations isolated by fluorescence-activated cell sorting (FACS) from normal, regenerating, early neoplastic and malignant pancreatic tissues representing the following tissue states (n>=3/each): (i) normal healthy pancreas (Normal), (ii) normal pancreas undergoing regenerative injury-driven ADM (Reg ADM); (iii) Kras-mutated pancreata undergoing stochastic neoplastic transformation (Kras*), and (iv) Kras-mutated pancreata undergoing synchronous neoplastic reprogramming (Kras*-ADR) accelerated by induction of tissue injury were profiled (see
Principal component analysis (PCA) of RNA-seq data revealed that populations undergoing regeneration or mutant Kras-dependent neoplastic transformation exhibit transcriptional changes reminiscent of established PDAC, with injury and mutant Kras effects accounting for 74% of total variance vs 9% related to late stage transitions associated with p53 loss (
To compare gene expression programs linked to regeneration and early neoplasia (herein referred to as R/N-DEGs,
Integration of the in vivo transcriptional profiling data with ATAC-seq data at R/N-DEG loci revealed that 60% of the genes that undergo changes in expression during regeneration or early neoplasia are associated with consistent changes in chromatin accessibility (
The results disclosed herein identify the shared and specific transcriptional programs that underlie regeneration and early neoplasia. Thus, while there are notable similarities between the cell fate transitions accompanying neoplastic transformation and regeneration, the underlying transcriptional programs and chromatin states are distinct.
Next the global analysis of chromatin accessibility dynamics across the spectrum of epithelial states captured in the ATAC-Seq data was performed to define the chromatin accessibility landscapes characteristic of normal, regenerating, pre-malignant, and PDAC (
While the effects of mutant Kras and injury in inducing accessibility-LOSS were additive, their ability to lead to accessibility-GAIN at a large set of regions appeared synergistic (
Peaks uniquely gained or lost during early neoplasia versus regeneration (
While the above data indicate that oncogenic Kras and tissue injury cooperate to promote PDAC-relevant chromatin alterations prior to the establishment of PDAC precursor lesions, they do not establish functional causality of the downsteam effector programs. Given that Brd4 was required for both neoplastic and regenerative pancreatic plasticity, it was reasoned that the gene expression programs sensitive to Brd4 perturbation would be highly enriched for factors that mediate these cell fate transitions. Therefore, RNA-seq and ATAC-seq analyses were performed in lineage-traced pancreatic epithelial cells (mKate2/GFP+) undergoing regenerative (Reg ADM) or pro-neoplastic (Kras*ADR) metaplasia after acute dox-induced Brd4 suppression. In these experiments, metaplastic transitions were synchronously induced by caerulein treatment initiated 6 days after dox addition, and shRNA-expressing cells were analyzed 2 days later, a timepoint with maximal Brd4 knockdown but prior to the presentation of PanIN or regenerative defects (
Consistent with the divergent chromatin states that occur during normal regeneration and early neoplasia, the Brd4-sensitive transcriptional programs observed in each condition were associated with distinct biological processes (
To rule out that the Brd4-dependent transcriptional programs observed could be produced by depeletion of particular epithelial cell sub-types hypersensitive to Brd4 suppression, parallel ATAC-seq analyses were performed in shRen and shBrd4 cells in KC-GEMM mice at the same time point after dox and injury. This showed retained ATAC peaks associated with the abovementioned downregulation of Brd4-target gene expression (examples in
Accordingly, these results demonstrate that inhibition of factors regulated by Brd4 and tumor-specific chromatin alterations are useful in methods for treating or preventing pancreatic cancer in a subject in need thereof.
To identify specific mediators of pancreatic tumorigenesis downstream of the described epigenetic alterations, the datasets were then examined for genes showing features of enhancer-dependent activation in early neoplasia but not regeneration (
To determine whether the activation of Il-33 by the synergistic effects of mutant Kras and tissue injury functionally contributes to neoplasia, the extent to which exogenous Il-33 could recapitulate the effects of injury in Kras mutant mice was examined. Recombinant mouse Il-33 (rIl-33) was introduced into KC-GEMM (mutant Kras) and C-GEMM (wt Kras) mice by intraperitoneal injection, and mice were analyzed by histology and molecular analyses of cell fate markers in FACS-sorted mKate2+ cells 3 weeks later. Remarkably, rIl-33 was sufficient to trigger many of the tumor-promoting outputs of injury in the mutant Kras setting, facilitating the transition of acinar cells into a ductal, mucinous state and the rapid establishment of mucinous pancreatic intraepithelial neoplasia (PanIN) lesions (see “KC” panels in
These results demonstrate that elevated IL-33 signaling serves as a marker for pancreatic tumorigenesis, and that blockade of IL-33 signaling may be useful in treating pancreatic cancer. Accordingly, the inhibitors of Type 2 cytokine signaling disclosed herein are useful in methods for treating or preventing pancreatic cancer in a subject in need thereof.
Prompted by the functional experiment revealing mutant Kras-specific effects of IL-33 in the pancreatic epithelium, and in line with the ATAC- and RNA-seq profiling data, specific Th2 receptors (e.g., Il4ra, Il13ra1, Il13ra2, Il17re, Il18r1, Il18rap, Il4ra, Il31ra) become transcriptionally activated in Kras-mutant but not wild-type pancreatic epithelial cells (
These data demonstrate that injury drives a coordinated activation of an IL-33-Th2 cytokine signaling axis in the mutant Kras (but not wild-type) pancreatic epithelium that is pharmacologically actionable. Accordingly, the inhibitors of Type 2 cytokine signaling disclosed herein are useful in methods for treating or preventing pancreatic cancer in a subject in need thereof.
Normal pancreas expressing Brd4-shRNAs effectively undergo injury-driven ADM, as assessed by GFP and CK19 (Krt19) (ADM/dedifferentiation marker) co-IF (
Accordingly, these results demonstrate that inhibition of factors regulated by Brd4 are useful in methods for treating or preventing pancreatic cancer in a subject in need thereof.
In order to gain mechanistic insight into the epigenetic control of pancreatic tumorigenesis vs regeneration, the chromatin and transcriptional landscapes of sorted pancreatic epithelial cells undergoing mutant KRAS- and/or injury-driven cell identity changes via ATAC-Seq and RNA-Seq were mapped.
A synergistic interaction between mutant KRAS and inflammatory insults in shaping the chromatin landscape of pancreatic epithelial cells was identified (
As shown in
Activation of epithelial IL-33 signaling selectively promoted KRAS-driven tumorigenesis. As shown in
As shown in
Taken together, these results demonstrate that elevated IL-33 signaling serves as a marker for pancreatic tumorigenesis, and that blockade of IL-33 signaling may be useful in treating or preventing pancreatic cancer. Accordingly, the inhibitors of Type 2 cytokine signaling disclosed herein are useful in methods for treating or preventing pancreatic cancer in a subject in need thereof.
KC-GEMM and KPC-GEMM (advanced pancreatic cancer model that includes a p53 mutation) mice will be treated with one or more inhibitors of Type 2 cytokine signaling at varying doses. Exemplary inhibitors of Type 2 cytokine signaling that will be tested include one or more of dupilumab, Etokimab (ANB020), Mepolizumab, reslizumab, benralizumab, lebrikizumab, risankizumab, tocilizumab, tralokinumab, anrukinzumab, AMG317, STATE inhibitors, STAT3 inhibitors, Secukinumab, ustekinumab, guselkumab, and gevokizumab, or mouse-compatible analogs for the murine models of pancreatic cancer. Mouse-compatible Type 2 cytokine inhibitors that will be tested include anti-mouse IL-4 (clone 11B11) (Bio X Cell, West Lebanon N.H.), Mouse IL-13 Neutralizing antibody (clone 8H8) (Invivogen, San Diego Calif.), Mouse IL-33 MAb (Clone 396118) (R&D Systems, Minneapolis, Minn.), anti-mouse/human IL-5 (Clone TRFK5) (Bio X Cell, West Lebanon N.H.) or Mouse ST2/IL-33R antibody (Clone 245707) (R&D Systems, Minneapolis, Minn.). For in vitro treatment experiments, anti-tumor efficacy of the above mentioned inhibitors will be evaluated in human and mouse pancreatic cancer cell lines or organoids systems.
It is anticipated that KC-GEMM mice that are treated with one or more inhibitors of Type 2 cytokine signaling will exhibit reduced fibrosis and/or delayed progression of mutant KRAS-driven pancreatic intraepithelial neoplasias compared to untreated KC-GEMM control animals. In experiments treating advanced pancreatic cancer (KPC-GEMM and transplantable models), it is anticipated that mice treated with one or more inhibitors of Type 2 cytokine signaling will exhibit reduced fibrosis, enhanced anti-tumor immunity, tumor regressions and/or delayed progression. In in vitro drug studies, pancreatic cancer cell lines or organoids treated with one or more inhibitors of Type 2 cytokine signaling are anticipated to exhibit reduced stem-like properties, reduced proliferation and/or undergo cell death.
Taken together, these results will demonstrate that inhibitors of Type 2 cytokine signaling are useful for treating or preventing pancreatic cancer. Accordingly, the inhibitors of Type 2 cytokine signaling disclosed herein are useful in methods for treating or preventing pancreatic cancer in a subject in need thereof.
The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
This application is a US National Stage Application under 35 U.S.C. § 371 of International Patent Appl. No. PCT/US2019/041670, filed Jul. 12, 2019, which claims the benefit of and priority to U.S. Provisional Appl. No. 62/697,941, filed Jul. 13, 2018, the disclosure of each of which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/041670 | 7/12/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62697941 | Jul 2018 | US |
