Patent Application
20190185559
Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are two prevalent lymphoid malignancies that share the phenotype of small, mature, non-germinal center B-cells, but demonstrate distinctive clinical and biological features. Somatic mutations of the NOTCH1 gene are seen in 8-15% of CLL and MCL patients, while recurrent NOTCH2 mutations have also been reported in MCL. Notch gene mutations are associated with decreased overall survival and reduced time to treatment in both CLL and MCL, while in CLL, NOTCH1 mutations also appear to increase the risk of high-grade transformation, and reduce responsiveness to anti-CD20 monoclonal antibody therapy. In recent years, the clinical development of drugs targeting B-cell receptor (BCR) signaling and anti-apoptotic pathways have provided new options for patients with small B-cell lymphomas, but new approaches are still needed to improve response rate and prevent development of secondary drug resistance.
The invention provides therapeutic combinations comprising an agent that inhibits Notch signaling and an agent that inhibits B cell receptor signaling, and methods of using such agents to inhibit the survival or proliferation of a neoplastic cell.
In one aspect, the invention provides a pharmaceutical composition containing an effective amount of an agent that inhibits the expression or activity of a Notch polynucleotide or polypeptide and an effective amount of an agent that inhibits the expression or activity of a functional component of a B cell receptor polypeptide or polynucleotide.
In another aspect, the invention provides a method of inhibiting the survival or proliferation of a neoplastic cell, the method involving contacting the cell with an agent that inhibits expression or activity of a Notch polynucleotide or polypeptide and an effective amount of an agent that inhibits expression or activity of a functional component of a B cell receptor polypeptide or polynucleotide
In yet another aspect, the invention provides a method of inhibiting the survival or proliferation of a neoplastic cell, the method involving contacting the cell with a gamma secretase inhibitor and ibrutinib, thereby inhibiting the survival or proliferation of the neoplastic cell.
In still another aspect, the invention provides a method of treating a neoplasia in a subject, the method involving administering to the subject an agent that inhibits the expression or activity of a Notch polynucleotide or polypeptide and an effective amount of an agent that inhibits the expression or activity of a functional component of a B cell receptor polypeptide or polynucleotide, thereby treating cancer in the subject.
In still another aspect, the invention provides a method of treating a subject having a leukemia or lymphoma, the method involving administering to the subject a gamma secretase inhibitor and ibrutinib.
In still another aspect, the invention provides a method of treating a subject having a leukemia or lymphoma that has developed resistance to a B cell receptor signaling inhibitor, the method involving administering a gamma secretase inhibitor and an agent that inhibits expression or activity of a functional component of the B cell receptor.
In various embodiments of any of the above aspects or any other aspect of the invention delineated herein, the agent is a small compound, polypeptide, or polynucleotide. In various embodiments of any of the above aspects or any other aspect of the invention delineated herein, the agent that inhibits Notch expression or activity is a gamma secretase inhibitor (e.g., Compound E, MK-0752, PF03084014, RO-4929097, DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, tetralin imidazole PF-03084014, LY3039478, and BMS906-024), a Notch signaling pathway inhibitory antibody (e.g., anti-Delta-like-4 antibody), or an anti-Notch1 antibody (e.g., OMP-52M521). In various embodiments of any of the above aspects, the agent that inhibits Notch expression or activity is an inhibitory nucleic acid molecule. In various embodiments of any of the above aspects, the agent that inhibits B cell receptor signaling is a PI3 kinase inhibitor (e.g., idelalisib), BTK inhibitor (e.g., ibrutinib, ACP-196, ONO/GS-4059, BGB-3111, and CC-292), SRC family kinase inhibitor (e.g., Dasatinib), SYK inhibitor (e.g., Fostamatinib), or a protein kinase C inhibitor (e.g., Midostaurin, Enzastuarin, or Sotrasturin). In embodiments of any of the above aspects, the agents are formulated together or are formulated separately for simultaneous, separate or sequential co-administration. In embodiments of any of the above aspects or any other aspect of the invention delineated herein, a composition of the invention contains an agent that inhibits Notch expression or activity, an agent that inhibits B cell receptor expression or activity, and one or more additional therapeutic agents. In embodiments of any of the above aspects, the Notch activity is signaling. In embodiments of any of the above aspects, B cell receptor activity is signaling. The method further involves administration of one or more additional therapeutic agents. In embodiments of any of the above aspects, the neoplastic cell is derived from a leukemia or lymphoma. In embodiments of any of the above aspects, the leukemia is any one or more of a chronic lymphocytic leukemia, B cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, and early T cell acute lymphoblastic leukemia. In embodiments of any of the above aspects, the lymphoma is any one or more of small B-cell lymphomas, mantle cell lymphoma, small lymphocytic lymphoma, diffuse large B cell lymphoma, splenic marginal zone lymphoma, follicular lymphoma, splenic red pulp lymphoma, and MALT lymphoma. In embodiments of any of the above aspects, the neoplastic cell is a murine, rat, or human cell. In embodiments of any of the above aspects, the cell is in vitro or in vivo.
Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person of ordinary skill in the art to which this invention belongs. The following references provide a person of ordinary skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.
By “B cell receptor activity” is meant activation of proteins within the B-cell receptor (BCR) pathway that result in B cell activation. Such activation can take the form of tyrosine kinase phosphorylation (e.g., phosphorylation by a Src family kinase, Lyn, spleen tyrosine kinase (Syk), Bruton tyrosine kinase (Btk), Phospholipase C gamma 2 (PLCG2)), as well as activation or modulation of proteins in downstream pathways as a result of BCR signaling (e.g. phosphoinositol-3-kinase (PI3K)/AKT pathway protein phosphorylation, mitogen-activated protein kinase (MAPK) pathway protein phosphorylation, or protein kinase C/nuclear factor kappa B (NF-κB) phosphorylation, altered proteolysis, altered ubiquitination, or altered subcellular localization). In one embodiment, B cell receptor activity is B cell receptor signaling.
By “Notch activity” is meant activation of proteins within the Notch pathway that results in modifications in cell growth or proliferation. Such protein activation can take the form of proteolytic cleavage of Notch receptor proteins (or chimaeric proteins incorporating a portion of a Notch receptor protein), altered subcellular localization of Notch receptor proteins or a portion thereof from cellular membranes to the nucleus, cytoplasm, or other organelles, binding of Notch receptor proteins or a portion thereof to DNA (either directly or via binding of Notch proteins to other DNA-bound proteins), or binding of Notch proteins to transcriptional regulatory proteins independendent of association with DNA. In one embodiment, Notch activity is Notch signaling.
By “B cell receptor” is meant a transmembrane receptor protein complex present on B cells comprising a membrane bound immunoglobulin, CD79A and CD79B as functional components.
By “CD79A protein” is meant a polypeptide having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: P11912, or a fragment thereof, and having signal transduction activity.
sapiens GN = CD79A PE = 1 SV = 2
By “CD79A polynucleotide” is meant a nucleic acid molecule encoding the CD79A protein.
By “CD79B protein” is meant a polypeptide having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: P40259, or a fragment thereof, and having signal transduction activity.
sapiens GN = CD79B PE = 1 SV = 1
By “CD79B polynucleotide” is meant a nucleic acid molecule encoding the CD79B protein.
By “Bruton's tyrosine kinase (BTK) polypeptide” is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: Q06187.3, or a fragment thereof, and having tyrosine kinase activity. An exemplary BTK amino acid sequence is provided below:
By “BTK polynucleotide” is meant a nucleic acid molecule encoding a BTK polypeptide. An exemplary BTK polynucleotide sequence is provided at NCBI Reference Sequence: NM 000061.2, and reproduced herein below.
By “myc proto-oncogene protein (MYC of c-MYC) polypeptide” is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference
Sequence: NP_002458.2, or a fragment thereof, and having growth regulatory activity. Growth regulatory activity includes, but is not limited to, cell division or increase in cell size. An exemplary MYC amino acid sequence is provided below:
By “MYC polynucleotide” is meant a nucleic acid molecule encoding a MYC polypeptide. An exemplary MYC polynucleotide sequence is provided at NCBI Reference Sequence: V00568.1, and reproduced herein below.
By “Notch protein” or “Notch receptor” is meant any one of Notch 1, 2, 3, or 4.
By “Neurogenic locus notch homolog protein 1 (Notch1) polypeptide” is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: P46531.4, or a fragment thereof, and having Notch receptor activity. Examples of Notch receptor activity include interaction with Notch ligands at the cell surface, proteolytic cleavage of the Notch protein by ADAM family metalloproteases and/or gamma secretase (either following interaction with Notch ligands, or through ligand-independent mechanisms), altered sub-cellular localization of an intracellular portion of the Notch protein following a proteolytic cleavage event, binding of a Notch protein (or portion thereof) to other transcriptional regulatory proteins in the nucleus or cytoplasm, or binding of a Notch protein (or portion thereof) to DNA-bound chromatin complexes. An exemplary Notch1 amino acid sequence is provided below:
By “Notch1 polynucleotide” is meant a nucleic acid molecule encoding a Notch1 polypeptide. An exemplary Notch1 polynucleotide sequence is provided at NCBI Reference Sequence: NM 017617.4, and reproduced herein below.
By “Neurogenic locus notch homolog protein 2 (Notch2) polypeptide” is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: AAG37073.1, or a fragment thereof, and having Notch receptor activity. An exemplary Notch2 amino acid sequence is provided below:
By “Notch2 polynucleotide” is meant a nucleic acid molecule encoding a Notch2 polypeptide. An exemplary Notch2 polynucleotide sequence is provided at NCBI Reference Sequence: AF315356.1, and reproduced herein below.
By “Neurogenic locus notch homolog protein 3 (Notch3) polypeptide” is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: AAB91371.1, or a fragment thereof, and having Notch receptor activity. An exemplary Notch3 amino acid sequence is provided below:
By “Notch3 polynucleotide” is meant a nucleic acid molecule encoding a Notch3 polypeptide. An exemplary Notch3 polynucleotide sequence is provided at NCBI Reference Sequence: U97669.1, and reproduced herein below.
By “Neurogenic locus notch homolog protein 4 (Notch4) polypeptide” is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: AAC32288.1, or a fragment thereof, and having Notch receptor activity. An exemplary Notch4 amino acid sequence is provided below:
By “Notch4 polynucleotide” is meant a nucleic acid molecule encoding a Notch4 polypeptide. An exemplary Notch4 polynucleotide sequence is provided at NCBI Reference Sequence: U95299.1, and reproduced herein below.
By “Notch inhibitor” is meant an agent capable of inhibiting the expression or activity of a Notch protein. Notch proteins include, but are not limited to, Notch1, Notch2, Notch3 and/or Notch4. In one embodiment, a Notch inhibitor reduces Notch signaling, for example by disrupting the receptor: ligand interaction or any other signaling event downstream of the Notch1, Notch2, Notch3 and/or Notch4 receptor, such as proteolytic cleavage of the Notch protein. In one embodiment, the Notch inhibitor is a gamma-secretase inhibitor (GSI). Notch inhibitors can include, for example, MK-0752, PF03084014, RO-4929097, DAPT, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, tetralin imidazole PF-03084014, LY3039478 and BMS906-024. In some embodiments, inhibition is by at least about 10%, 25%, 50%, 75% or more. In another embodiment, a Notch inhibitor is any inhibitory nucleic acid that inhibits, for example, the expression of a Notch protein. In another embodiment, a Notch inhibitor is an antibody against Notch that inhibits Notch activity. Exemplary inhibitory Notch antibodies are known in the art, and include, for example, anti-Notch 1 (OMP-52M521) and anti-delta-like-4. In another embodiment, a
Notch inhibitor is a CRISPR-based therapeutic that depletes Notch (e.g., results in the conditional depletion of Notch).
By “B cell receptor inhibitor” is meant an agent capable of reducing B cell receptor signaling, including signaling by downstream pathways that are functionally regulated by B cell receptor signaling. In one embodiment, the B cell receptor inhibitor interrupts the receptor: ligand interaction or any other signaling event downstream of the B cell receptor. In one embodiment, the inhibitor is a Bruton tyrosine kinase (BTK) inhibitor. B cell receptor inhibitors can include, for example, ibrutinib (PCI-32765), acalabrutinib (ACP-196), ONO-4059 (e.g., GS-4059 or NCT02457598), spebrutinib (e.g., AVL-292, CC-292), and BGB-3111. In some embodiments, inhibition is by at least about 10%, 25%, 50%, 75% or more.
In another embodiment, a B cell receptor inhibitor is any inhibitory nucleic acid that inhibits, for example, the expression of a B cell receptor component, e.g., any protein that forms a functional part of the B cell receptor. In another embodiment, a B cell receptor inhibitor is an antibody that inhibits B cell receptor activity. In another embodiment, a B cell receptor inhibitor is a CRISPR-based therapeutic that depletes a B cell receptor component (e.g., results in the conditional depletion of a B cell receptor component).
By “Neural precursor cell expressed developmentally down-regulated protein 9 (Nedd9) polypeptide” is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: AAH40207.1, or a fragment thereof, and having cell cycle or growth regulatory activity. An exemplary Nedd9 amino acid sequence is provided below:
By “Nedd9 polynucleotide” is meant a nucleic acid molecule encoding a Nedd9 polypeptide. An exemplary Nedd9 polynucleotide sequence is provided at NCBI Reference Sequence BC040207.1, and reproduced herein below.
By “Phospholipase C Gamma 2, (PLCG2, 1-Phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) polypeptide” is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: AAQ76815.1, or a fragment thereof, and having phospholipase activity. An exemplary PLCG2 amino acid sequence is provided below:
By “PLCG2 polynucleotide” is meant a nucleic acid molecule encoding a PLCG2 polypeptide. An exemplary PLCG2 polynucleotide sequence is provided at NCBI Reference Sequence: NM 002661.4, and reproduced herein below.
By “recombining binding protein suppressor of hairless isoform 1 (RBPJ) polypeptide” is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: NP 005340.2, or a fragment thereof, and having transcriptional regulatory activity. An exemplary RBPJ amino acid sequence is provided below:
By “RBPJ polynucleotide” is meant a nucleic acid molecule encoding a RBPJ polypeptide. An exemplary RBPJ polynucleotide sequence is provided at NCBI Reference Sequence NM 014276.3, and reproduced herein below.
By “agent” is meant a small compound, polynucleotide, or polypeptide.
By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression or activity levels, a 25% change, a 40% change, a 50% change, or an even greater change in expression or activity levels (i.e., 75%, 80%, 85%, 90%).
By “analog” is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.
The term “co-administration” or “combined administration” as used herein is defined to encompass the administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “ includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.
By “disease” is meant any condition or disorder that damages, or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include cancer, including but not limited to small B-cell lymphomas, such as mantle cell lymphoma, or chronic lymphocytic leukemia (e.g., small lymphocytic lymphoma), diffuse large B cell lymphoma, splenic marginal zone lymphoma, follicular lymphoma, splenic red pulp lymphoma, MALT lymphoma and leukemias such as chronic lymphocytic leukemia, B cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, and early T cell acute lymphoblastic leukemia).
By “effective amount” is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. In one embodiment, an effective amount of an agent of the invention reduces or stabilizes the growth or proliferation of a neoplastic cell. In other embodiments, an effective amount of an agent of the invention reduces the survival of a neoplastic cell. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By “inhibitory nucleic acid” is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.
The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
The term “jointly therapeutically active” or “joint therapeutic effect” as used herein means that the therapeutic agents may be given separately (in a chronologically staggered manner, especially a sequence-specific manner) in such time intervals as are preferable, in the subject, especially human subject, to be treated, and show an additive or greater effect. In a preferred embodiment, the joint therapeutic effect is an effect greater than the combined effect that each of the compounds would be expected to provide when administered on its own.
By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
By “neoplasia” is meant abnormal cell proliferation. A neoplasm is a collection of cells characterized by increased cell division, poor cellular differentiation, and that is potentially cancerous.
As used herein, “obtaining” as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
By “reference” is meant a standard or controlled condition.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
By “siRNA” is meant a double stranded RNA. Optimally, a siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3′ end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity.
By “specifically binds” is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.
Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).
For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those of ordinary skill in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to a person of ordinary skill in the art.
For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to a person of ordinary skill in the art. Hybridization techniques are well known to a person of ordinary skill in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in
Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.
By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e−3 and e−100 indicating a closely related sequence.
By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
The term “synergistic effect” as used herein refers to action of two therapeutic agents such as, for example, an agent that inhibits Notch signaling and an agent that inhibits B cell receptor signaling producing an effect, for example, slowing the symptomatic progression of a proliferative disease, particularly cancer, or symptoms thereof, which is greater than the simple addition of the effects of each drug administered by themselves. A synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.
Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
Canonical Notch target genes are labeled in grey text (NRARP, HES1, HEY1, NOTCH3, HES4, HEY2, and DTX1).
The invention generally provides therapeutic compositions comprising a combination of an agent that inhibits the activity of or decreases the levels of a Notch protein and an agent that inhibits B-cell receptor (BCR) signalling, and methods of using such combinations to treat cancer (e.g., small B-cell lymphomas, such as mantle cell lymphoma, or chronic lymphocytic leukemia (e.g., small lymphocytic lymphoma), diffuse large B cell lymphoma, splenic marginal zone lymphoma, follicular lymphoma, splenic red pulp lymphoma, MALT lymphoma and leukemias, such as chronic lymphocytic leukemia, B cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, and early T cell acute lymphoblastic leukemia).
Recurrent gain-of-function mutations in genes encoding Notch receptors are associated with poor clinical outcome in two small B-cell lymphoma subtypes, mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL; also known as small lymphocytic lymphoma, SLL), but functional targets of Notch signaling in B cells have not been systematically characterized. As described herein, a gamma-secretase washout strategy was used to rapidly activate Notch signaling in Notch-dependent and -independent MCL lines, and to identify direct Notch regulatory targets through genome-wide expression profiling and chromatin immunoprecipitation (ChIP-Seq) of Notch transcriptional complex (NTC) components.
The invention is based, at least in part, on the discovery that proliferation of Notch-dependent mantle cell lymphoma (MCL) lines was driven by activation of the oncogene MYC via Notch transcriptional complex binding at B-cell-specific 5′ enhancer elements, resulting in secondary activation of MYC target genes and a metabolic program associated with mTORC1 activation. These studies identified novel Notch regulatory targets in B-cell lymphomas associated with NTC binding to proximal and distal regulatory elements, that activate genes encoding cytokine receptors (IL6R, IL 10R, IL21R), as well as SRC-family kinases (FYN, LYN, BLK) and signaling adaptor proteins (BLNK, NEDD9, SH2B2, PIK3AP 1) involved in activation of pathways downstream of B-cell receptor (BCR) signaling. Genome-wide profiling analysis of lymphoma biopsies, plus functional studies of patient-derived lymphoma cells in vitro and in vivo were utilized to validate Notch-dependent regulation of MYC and oncogenic BCR signaling in primary human CLL and MCL.
Genome-wide profiling of mRNA, histone acetylation, and NTC binding in MCL was used to identify differential regulation of enhancers and genes that represent the direct targets of Notch signaling in B cell lymphoma. The findings indicated that Notch signaling drives two distinct oncogenic programs in lymphoma cell lines and primary tumors. First, ICN binds and activates B-cell-specific 5′ MYC enhancers, resulting in activation of a MYC-dependent metabolic program that is shared with other Notch-dependent tumor types. Second, Notch directly activates the expression of cytokine receptors and B cell receptor signaling intermediates, thus potentiating the response of lymphoma cells to activating stimuli. Notably, the data indicated a Notch-dependent increase in B cell-receptor-dependent phosphorylation of PLC2G and downstream activation of NF-KB, a pathway that is known to be central to the proliferation and survival of small B cell lymphomas.
Building on these findings, the invention provides novel therapeutic compositions and methods combining direct B cell receptor inhibition (expected to block B cell receptor signaling and to drive cancerous B cells towards apoptosis and/or disrupts tumor formation) with Notch inhibition (expected to both cease the activation of MYC and to also cease B cell receptor potentiation). In taking both approaches towards B cell inhibition in concert, cancerous B cells are specifically targeted and have increased difficulty escaping the treatment by mutation.
Accordingly, the invention provides therapeutic compositions comprising an agent (e.g., polypeptides, inhibitory nucleic acids, and small molecules) that inhibits a Notch polypeptide (e.g., Notch1, Notch2, Notch3, Notch4) expression or activity and an agent that inhibits B Cell Receptor (BCR) signaling, and methods of using such compositions to inhibit the growth or proliferation of a neoplastic cell. Compositions of the invention are useful for the treatment of cancer (e.g., e.g., small B-cell lymphomas, such as mantle cell lymphoma, or chronic lymphocytic leukemia (e.g., small lymphocytic lymphoma), diffuse large B cell lymphoma, splenic marginal zone lymphoma, follicular lymphoma, splenic red pulp lymphoma, MALT lymphoma and leukemias such as chronic lymphocytic leukemia, B cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, and early T cell acute lymphoblastic leukemia).
Notch proteins are expressed as trans-membrane receptors that undergo sequential proteolytic cleavage upon interaction with Notch ligands expressed on neighboring cells, resulting in gamma secretase-dependent release of the intracellular notch (ICN) fragment. ICN then traffics to the nucleus, where it binds to transcriptional regulatory elements in a Notch transcriptional complex (NTC) with the DNA sequence-specific transcription factor RBPJ, mastermind-like (MAML) proteins, and other co-factors. Nearly all Notch gene mutations reported in CLL and MCL result in frameshift-mediated truncation of the C-terminal PEST domain, which mediates ubiquitination and degradation of ICN. Notch PEST domain truncations have been extensively studied in T-cell acute lymphoblastic leukemia (T-ALL), where they enhance the nuclear accumulation of ICN, but do not confer active signaling in the absence of ligand. This contrasts with Notch gene heterodimerization domain mutations and rearrangements, which do confer ligand-independent signaling, and are common in T-ALL, but are extremely rare in CLL and MCL patients. Immunohistochemistry (IHC) with an antibody that specifically recognizes the gamma-secretase-cleaved NOTCH1 ICN (ICN-1) was previously used to demonstrate NOTCH1 activation in >80% of CLL lymph node biopsies. Strong and diffuse ICN-1 staining was significantly, but not exclusively, associated with cases bearing NOTCH1 PEST mutations. These findings suggested that activation of Notch signaling in lymphoma cells via interaction with ligand-presenting cells in the lymph node microenvironment may be a broadly important feature of this disease.
In vitro models for the study of Notch signaling in B-cell lymphoma have been limited. Two MCL cell lines, Rec-1 and SP-49, were reported to show marked growth inhibition upon treatment with gamma-secretase inhibitors (GSI) or expression of a Notch-inhibiting transgene, suggesting dependency of these lines on ligand-independent Notch signaling (Kridel et al., 2012). Subsequently, ICN-1 activation in Rec-1 was found to be due to a genomic deletion encompassing most of the exons encoding the NOTCH1 extracellular domain, and that this allele confers ligand-independent Notch signaling that is sensitive to GSI inhibition.
The present invention features compositions comprising one or more agents that inhibit Notch signaling and one or more agents that inhibit B cell receptor signaling. Such agents include small molecules, polypeptides, and polynucleotides described herein.
Small molecules capable of inhibiting Notch include gamma-secretase inhibitors (GSI). Exemplary gamma-secretase inhibitors are known in the art, and include, for example, Compound E, MK-0752, PF03084014, RO-4929097, DAPT, N-[N-(3,5-difluorophenacetyl)- L-alanyl]-S-phenylglycine t-butyl ester, tetralin imidazole PF-03084014, LY3039478 and BMS906-024.
Further examples of compounds suitable as Notch inhibitors can include the compounds listed in U.S. Pat. Nos. 8,377,886, 6,756,511, 6,890,956, 6,984,626, 7,049,296, 7,101,895, 7,138,400, 7,144,910, and 7,183,303, incorporated by reference herein in their entirety.
Other Notch inhibitors include antibodies that specifically bind Notch and inhibit or disrupt its activity, or deplete its levels. Exemplary inhibitory Notch antibodies are known in the art, and include, for example, anti-Notch 1 (OMP-52M521) and anti-delta-like-4.
Further examples of antibodies suitable for inhibiting Notch and Notch signaling pathway include the antibodies listed in U.S. Pat. Nos. 9,090,690, 8,945,547, 8,945,873, 7,534,868 and International Patent Application Nos. WO 2008150525, WO 2010059543, WO 2011041336, incorporated by reference herein in their entirety.
Examples of compounds suitable as B cell receptor (BCR) inhibitors can include Bruton tyrosine kinase (BTK) inhibitors, SRC family kinase inhibitors, SYK inhibitors, or protein kinase C inhibitors, and PI3 Kinase inhibitors.
Exemplary B cell receptor inhibitors include, for example, ibrutinib (PC1-32765), acalabrutinib (ACP-ONO-4059 (e.g., GS-4059 or NCT02457598), spebrutinib (e.g., AVL-292, CC-292), and BGB-3111.
Further examples of compounds suitable as BCR inhibitors can include the compounds listed in U.S. Pat. Nos. 8,227,433, 6,306,897, 8,999,999 and International Patent Application Nos. WO2015110923, WO1999054286 (incorporated by reference in their entirety).
Small molecules capable of inhibiting signaling mediated by B cell receptors or Notch can include SRC family kinase inhibitors. Exemplary SRC family kinase inhibitors are known in the art, and include, for example, dasatinib (BMS-354825), KX2-391, bosutinib (SKI-606), and saracatinib (AZD-0530).
Small molecules capable of inhibiting signaling mediated by B cell receptors or Notch can include spleen tyrosine kinase (SYK) inhibitors. Exemplary SYK inhibitors are known in the art, and include, for example, fostamatinib (R788), piceatannol, entospletinib (GS-9973), and GSK2646264.
Small molecules capable of inhibiting signaling mediated by B cell receptors or Notch can include protein kinase C (PKC) inhibitors. Exemplary PKC inhibitors are known in the art, and include, for example, midostaurin (PKC412), enzastaurin (LY317615), sotrastaurin (AEB071), and ruboxistaurin (LY333531).
Small molecules capable of inhibiting signaling mediated by B cell receptors or Notch can include phosphoinositol-3-kinase (PI3K) inhibitors. Exemplary PI3K inhibitors are known in the art, and include, for example, idelalisib (e.g., zydelig, GS-1101, CAL-101), alpelisib (13Y-1-719), AEZS-136, buparlisib (BKM120), copanlisib (BAY 80-6946), CA1,263, CU-DC-907, dactolisib (e.g., NNT-BEZ235, BEZ-235), duvelisib (1PI-145), GNE-477, GSM 059615, 1087114, 1P1-549, INK1117, palomid 529, perifosine (KRX-0401), pictilisib (GDC-0941), ME-401, PI-103, PWT33597, PX-866, RP6503, RP6530, SF-1126, TGR 1202, wortniannin, demethoxyviridin, X1,147 (SAR245408), XL765 (SAR245409), ZSIK474.
Further examples of compounds suitable as PI3K inhibitors can include the compounds listed in U.S. Pat. Nos. 9,403,779, 9,150,579, 9,126,948, 8,940,752, 8,759,359, 8,440,651, U.S. Patent Application Nos. 20140364447, 20100056523, 20100029693, and International Patent Application Nos. WO 2016051374, WO 2015181728, WO 2015160986, WO 2014195888, WO 2011123751 (incorporated by reference herein in their entirety).
In accordance with the present invention, a therapeutically effective amount of each of the combination partners (e.g., an agent that inhibits Notch signaling and an agent that inhibits B cell receptor signaling) may be administered simultaneously or sequentially and in any order, and the components may be administered separately or as a fixed combination. For example, the method of treating a neoplasia according to the invention may comprise (i) administration of the first agent (a) in free or pharmaceutically acceptable salt form and (ii) administration of an agent (b) in free or pharmaceutically acceptable salt form, simultaneously or sequentially in any order, in jointly therapeutically effective amounts, preferably in synergistically effective amounts, e.g. in daily or intermittently dosages corresponding to the amounts described herein. The individual combination partners may be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. Furthermore, the term “administering” also encompasses the use of a pro-drug of a combination partner that converts in vivo to the combination partner as such. The invention is therefore to be understood as embracing all such regimens of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly.
The effective dosage of each of the combination partners employed in the methods of the invention may vary depending on the particular compound or pharmaceutical composition employed, the mode of administration, the condition being treated, and the severity of the condition being treated. Thus, the dosage regimen is selected in accordance with a variety of factors including the route of administration and the renal and hepatic function of the patient. A clinician or physician of ordinary skill in the art can readily determine and prescribe the effective amount of the single therapeutic agents required to alleviate, counter or arrest the progress of the condition.
The optimum ratios, individual and combined dosages, and concentrations of the combination partners that yield efficacy without toxicity are based on the kinetics of the therapeutic agents' availability to target sites, and are determined using methods known to those of skill in the art.
The effective dosage of each of the combination partners may require more frequent administration of one of the agents in the combination. Therefore, to permit appropriate dosing, packaged pharmaceutical products may contain one or more dosage forms that contain the combination of compounds, and one or more dosage forms that contain one of the combination of compounds, but not the other compound(s) of the combination.
When the combination partners are employed or as marketed as single drugs, their dosage and mode of administration can be in accordance with the information provided on the package insert of the respective marketed drug, if not mentioned herein otherwise.
The optimal dosage of each combination partner for treatment of a proliferative disease can be determined empirically for each individual using known methods and will depend upon a variety of factors, including, though not limited to, the degree of advancement of the disease; the age, body weight, general health, gender and diet of the individual; the time and route of administration; and other medications the individual is taking optimal dosages may be established using routine testing and procedures that are well known in the art.
The amount of each combination partner that may be combined with the carrier materials to produce a single dosage form will vary depending upon the individual treated and the particular mode of administration. In some embodiments the unit dosage forms containing the combination of agents as described herein will contain the amounts of each agent of the combination that are typically administered when the agents are administered alone.
Frequency of dosage may vary depending on the compound used and the particular condition to be treated or prevented. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.
The present invention relates to a method of treating a subject having a proliferative disease comprising administering to said subject a combination of an agent that inhibits Notch signaling and an agent that inhibits B cell receptor signaling in a quantity which is jointly therapeutically effective against a neoplastic disease. In particular, the neoplastic disease to be treated is a leukemia or lymphoma.
The present invention further provides a commercial package comprising as therapeutic agents an agent that inhibits Notch signaling and an agent that inhibits B cell receptor signaling, optionally together with instructions for simultaneous, separate or sequential administration thereof for use in the delay of progression or treatment of a proliferative disease in a subject in need thereof.
The invention further provides inhibitory nucleic acids (e.g., antisense molecules, siRNA, shRNA) that inhibit the expression of a Notch polypeptide (e.g., Notch 1, Notch 2, Notch 3, Notch4). In addition, the invention provides inhibitory nucleic acids (e.g., antisense molecules, siRNA, shRNA) that inhibit the expression of a functional component of the B cell receptor. Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes a Notch polypeptide, as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers).
siRNA
Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down-regulating gene expression (Zamore et al., Cell 101: 25-33; Elbashir et al., Nature 411: 494-498, 2001, hereby incorporated by reference). The therapeutic effectiveness of a siRNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38-39.2002).
Given the sequence of a target gene, siRNAs may be designed to inactivate that gene. Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically. The nucleic acid sequence of a gene can be used to design small interfering RNAs (siRNAs). The 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat cancer (e.g., small B-cell lymphomas, such as mantle cell lymphoma, or chronic lymphocytic leukemia (e.g., small lymphocytic lymphoma), diffuse large B cell lymphoma, splenic marginal zone lymphoma, follicular lymphoma, splenic red pulp lymphoma, MALT lymphoma and leukemias such as chronic lymphocytic leukemia, B cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, and early T cell acute lymphoblastic leukemia).
The inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of expression of a Notch polypeptide. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.
In one embodiment of the invention, a double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.
Small hairpin RNAs (shRNAs) comprise an RNA sequence having a stem-loop structure. A “stem-loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). The term “hairpin” is also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, the base-pairing can be exact, i.e. not include any mismatches. The multiple stem-loop structures can be linked to one another through a linker, such as, for example, a nucleic acid linker, a miRNA flanking sequence, other molecule, or some combination thereof.
As used herein, the term “small hairpin RNA” includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). While there may be some variation in range, a conventional stem-loop shRNA can comprise a stem ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. “shRNA” also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. In some instances the precursor miRNA molecule can include more than one stem-loop structure. MicroRNAs are endogenously encoded RNA molecules that are about 22-nucleotides long and generally expressed in a highly tissue- or developmental-stage-specific fashion and that post-transcriptionally regulate target genes. More than 200 distinct miRNAs have been identified in plants and animals. These small regulatory RNAs are believed to serve important biological functions by two prevailing modes of action: (1) by repressing the translation of target mRNAs, and (2) through RNA interference (RNAi), that is, cleavage and degradation of mRNAs. In the latter case, miRNAs function analogously to small interfering RNAs (siRNAs). Thus, one can design and express artificial miRNAs based on the features of existing miRNA genes.
shRNAs can be expressed from DNA vectors to provide sustained silencing and high yield delivery into almost any cell type. In some embodiments, the vector is a viral vector. Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations. Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14x, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells through any means known in the art. A producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein. Such retroviral vector particles then can be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a DNA replication protein.
Examples of delivery methods suitable to deliver siRNA and shRNA molecules of the present invention are disclosed in Nature Materials Vol 12, 2013, pages 967-977, incorporated by reference in its entirety.
Catalytic RNA molecules or ribozymes that include an antisense sequence of the present invention can be used to inhibit expression of a nucleic acid molecule in vivo (e.g., a nucleic acid encoding any component of the Notch signaling pathway (e.g., Notch 1, Notch 2, Notch 3, Notch, 4, canonical Notch signaling modalities) and B Cell receptor (BCR) signaling (e.g. phospholipase C gamma 2, LYN, FYN, PI3K, NF-KB transcription factor pathway). The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 A1, each of which is incorporated by reference.
Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8:183, 1992. Example of hairpin motifs are described by Hampel et al., “RNA Catalyst for Cleaving Specific RNA Sequences,” filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids Research, 18: 299, 1990. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
Essentially any method for introducing a nucleic acid construct into cells can be employed. Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct. A viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA. Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like. Thus the shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.
For expression within cells, DNA vectors, for example plasmid vectors comprising either an RNA polymerase II or RNA polymerase III promoter can be employed. Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters and in some cases, shRNAs are most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al., 2005, Nat. Genet. 39: 914-921). In some embodiments, expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters. Examples of useful promoters in the context of the invention are tetracycline-inducible promoters (including TRE-tight), IPTG-inducible promoters, tetracycline transactivator systems, and reverse tetracycline transactivator (rtTA) systems. Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types. A certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Patent Application PCT/US2003/030901 (Publication No. WO 2004-029219 A2) and Fewell et al., 2006, Drug Discovery Today 11: 975-982, for a description of inducible shRNA.
Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference). Inhibitory nucleic acid molecule can be delivered using a nanoparticle. Nanoparticle compositions suitable for use with inhibitory nucleic acid molecules are known in the art and described for example by Kanasty et al., Nature materials 12: 967-977, 2013, which is incorporated herein by reference. Such nanoparticle delivery compositions include cyclodextrin polymer (CDP)-based nanoparticles, lipid nanoparticles, cationic or ionizable lipid, lipid-anchored PEG, PEGylated nanoparticles, oligonucleotide nanoparticles (ONPs), and siRNA-polymer conjugate delivery systems (e.g., Dynamic PolyConjugate, Triantennary GalNAc-siRNA).
The invention further provides for the use of a combination of the invention (e.g., an agent that inhibits Notch signaling and an agent that inhibits B cell receptor signaling) in combination with another therapeutic agent, such as a conventional chemotherapeutic agent, or agent that mitigates a side effect associated with an agent of the invention. Chemotherapeutic agents can be used with the methods of the present invention including, but are not limited to alkylating agents. Without intending to be limited to any particular theory, alkylating agents directly damage DNA to keep the cell from reproducing. Alkylating agents work in all phases of the cell cycle and are used to treat many different cancers (e.g., small B-cell lymphomas, such as mantle cell lymphoma, or chronic lymphocytic leukemia (e.g., small lymphocytic lymphoma), diffuse large B cell lymphoma, splenic marginal zone lymphoma, follicular lymphoma, splenic red pulp lymphoma, MALT lymphoma and leukemias such as chronic lymphocytic leukemia, B cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, and early T cell acute lymphoblastic leukemia). Alkylating agents are divided into different classes, including, but not limited to: (i) nitrogen mustards, such as, for example mechlorethamine (nitrogen mustard), chlorambucil, cyclophosphamide (Cytoxan®), ifosfamide, and melphalan; (ii) nitrosoureas, such as, for example, streptozocin, carmustine (BCNU), and lomustine; (iii) alkyl sulfonates, such as, for example, busulfan; (iv) riazines, such as, for example, dacarbazine (DTIC) and temozolomide (Temodar®); (v) ethylenimines, such as, for example, thiotepa and altretamine (hexamethylmelamine); and (v) platinum drugs, such as, for example, cisplatin, carboplatin, and oxalaplatin.
Uses of Notch and B Cell Receptor Inhibitors
The invention features methods for inhibiting the proliferation, growth, or viability of a neoplastic cell by contacting the cell with a Notch inhibitor and an agent that inhibits B Cell Receptor signaling. In general, the method includes a step of contacting a neoplastic cell with an effective amount of a compound of the invention. The present method can be performed on cells in culture, e.g., in vitro or ex vivo, or can be performed on cells present in an animal subject, e.g., as part of an in vivo therapeutic protocol. The therapeutic regimen can be carried out on a human or other subject.
The compounds of the invention or otherwise described herein can be tested initially in vitro for their inhibitory effects on the proliferation or survival of neoplastic cells. Examples of cell lines that can be used are any of the MCL cell lines described herein or any other suitable cell line known in the art. Alternatively, the antineoplastic activity of compounds of the invention can be tested in vivo using various animal models known in the art. For example, xenographs of human neoplastic cells or cell lines are injected into immunodeficient mice (e.g., nude or SCID) mice. Compounds of the invention are then administered to the mice and the growth and/or metastasis of the tumor is compared in mice treated with a compound of the invention relative to untreated control mice. Agents that reduce the growth or metastasis of a tumor or increase mice survival are identified as useful in the methods of the invention.
The methods discussed herein can be used to inhibit the proliferation of virtually any neoplastic cell. The invention provides methods for treating a subject having a neoplasia by administering to the subject an effective amount of an agent that inhibits Notch signaling and an agent that inhibits B cell receptor signaling as described herein. In certain embodiments, the subject is a mammal, in particular a human.
Agents which are determined to be effective for the prevention or treatment of neoplasias in animals, e.g., dogs, rodents, may also be useful in treatment of neoplasias in humans. Those skilled in the art of treating neoplasias in humans will know, based upon the data obtained in animal studies, the dosage and route of administration of the compound to humans. In general, the dosage and route of administration in humans is expected to be similar to that in animals.
The identification of those patients who are in need of prophylactic treatment for hyperplastic/neoplastic disease states is well within the ability and knowledge of one skilled in the art. Certain of the methods for identification of patients who are at risk of developing neoplastic disease states which can be treated by the subject method are appreciated in the medical arts, such as family history of the development of a particular disease state and the presence of risk factors associated with the development of that disease state in the subject patient. A clinician skilled in the art can readily identify such candidate patients, by the use of, for example, clinical tests, physical examination and medical/family history.
The invention provides pharmaceutical compositions for the treatment of a neoplasia, comprising an effective amount of an agent that inhibits Notch activity or decreases Notch levels, an agent that inhibits B Cell Receptor signaling and a pharmaceutically acceptable carrier. In particular embodiments, compositions of the invention comprise an agent or combination of agents described herein in combination with a conventional chemotherapeutic agent. In still other embodiments, such compositions are labeled for the treatment of cancer. In a further embodiment, the effective amount is effective to reduce the growth, proliferation, or survival of a neoplastic cell or to otherwise treat or prevent a neoplasia in a subject, as described herein.
In an embodiment, the agent is administered to the subject using a pharmaceutically-acceptable formulation. In certain embodiments, these pharmaceutical compositions are suitable for oral or parenteral administration to a subject. In still other embodiments, as described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human.
The methods of the invention further include administering to a subject a therapeutically effective amount of a compound in combination with a pharmaceutically acceptable excipient. The phrase “pharmaceutically acceptable” refers to those compounds of the invention, compositions containing such compounds, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “pharmaceutically-acceptable excipient” includes pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
Compositions containing a compound(s) include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
Methods of preparing these compositions include the step of bringing into association a agent(s) with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Compositions of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound(s) as an active ingredient. A compound may also be administered as a bolus, electuary or paste.
In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration of the compound(s) include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
In addition to inert diluents, the oral compositions can include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compound(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compound(s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of a compound(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound(s) may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to compound(s) of the present invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to a compound(s), excipients, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
The compound(s) can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically-acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids, such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.
Transdermal patches have the added advantage of providing controlled delivery of a compound(s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the active ingredient across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient in a polymer matrix or gel.
Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of compound(s) in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
When the compound(s) are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically-acceptable carrier.
Regardless of the route of administration selected, the compound(s), which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. An exemplary dose range is from about 0.1 μg to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., 0.1 μg/kg to 10 mg/kg, 0.1-10 μg/kg, 0.1-1 mg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 μg to about 100 μg/kg (e.g., of body weight). Ranges intermediate to the above-recited values are also intended to be part of the invention.
The invention provides kits for the treatment or prevention of cancer. In some embodiments, the kit includes a therapeutic or prophylactic composition containing an effective amount of an agent that inhibits the activity of or decreases the levels of a Notch protein and an effective amount of an agent that inhibits B cell receptor signaling. In one embodiment, the invention provides a commercial package comprising as therapeutic agents a combination comprising a first agent (e.g., an agent that inhibits Notch signaling) or a pharmaceutically acceptable salt thereof, and at least one second agent (e.g., an agent that inhibits B cell receptor signaling) or a pharmaceutically acceptable salt thereof, together with instructions for simultaneous, separate or sequential administration thereof for use in the delay of progression or treatment of a neoplasia.
In particular embodiments, each agent is provided in unit dosage form in a sterile container. Such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
The kit optionally includes instructions for administering the pharmaceutical composition to a subject having or at risk of contracting or developing cancer. The instructions will generally include information about the use of the composition for the treatment or prevention of cancer. In other embodiments, the instructions include at least one of the following: description of the therapeutic/prophylactic agent; dosage schedule and administration for treatment or prevention of cancer or symptoms thereof; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
Rec-1 and SP-49 are the only known MCL cell lines that demonstrate substantial growth inhibition upon treatment with GSI (Kridel et al., 2012) (
To model ligand-dependent Notch activation, MCL cell lines on immobilized recombinant Notch ligand (DLL1ext-IgG) or control protein (IgG) were grown. Analysis by Western blot with an antibody specific for free (gamma secretase-cleaved) ICN-1 demonstrated a time-dependent accumulation of ICN-1 expression in both Mino (
To identify Notch-regulated genes and enhancers genome-wide, a GSI-washout strategy in three MCL cell lines was employed (
Analysis of triplicate RNA-Seq datasets in each state for the three MCL lines revealed primarily gene activation rather than gene repression, consistent with the known role of intracellular Notch proteins as transcriptional activators (
The set of genes up-regulated in all three MCL lines (n=142) included many canonical Notch target genes (HES1, HES4, HEY1, HEY2, NRARP, and NOTCH3), which were also strongly up-regulated in CUTLL1 and HCC-1599. However, a large proportion of genes up-regulated by Notch activation in all MCL lines showed unchanged, or even reduced expression upon Notch activation in CUTLL1 and HCC-1599, indicating that these may represent context-specific Notch targets. A similar pattern was seen in the set of activated genes common to Mino and Rec-1, but not SP-49 (n=56), which included the canonical Notch target gene DTX1 as well as many apparently tissue-specific target genes. Gene set analysis of all genes activated by Notch in at least one GSI-sensitive MCL line and the GSI-insensitive Mino line revealed significant enrichment for gene sets associated with Notch signaling in the mSigDB Hallmark and Reactome collections (
In contrast, a very different pattern was observed in the large set of genes (n=151) that were activated by Notch signaling in both of the GSI-sensitive MCL lines SP-49 and Rec-1, but not in GSI-insensitive Mino. The vast majority of these genes were also Notch-activated in CUTLL1 and HCC-1599 (
Treatment of MCL cell lines with GSI revealed a substantial decrease in c-Myc protein levels for Rec-1 and SP-49 only (
Additional studies to understand the genomic mechanism by which Notch signaling regulates MYC expression in MCL were undertaken. Prior studies across diverse tissues and cancer types have implicated highly tissue-specific distal enhancer elements in MYC activation, including the Notch-dependent 3′ MYC enhancer identified in immature T cells and T-lymphoblastic leukemia (hereafter TNDME). Lymph node biopsies from CLL and MCL showed no evidence of T-NDME acetylation, but do show strong acetylation of enhancer-like elements on the 5′ side of the MYC gene (Ryan et al., 2015). ChIP-Seq was performed for histone H3 Lysine 27 acetylation (H3K27ac) in one CLL and ten MCL cell lines, and noted strong acetylation at the 5′ MYC enhancers in only five lines, including the two Notch gene-rearranged lines Rec-1 and SP-49 (
To directly evaluate enhancer regulation by Notch transcription complex, ChIP-Seq was performed for H3K27ac, RBPJ, and ICN-1 in Notch-rearranged MCL cell lines following GSI-washout and mock-washout experiments. Specific peaks of RBPJ and (in Rec-1) ICN-1 binding at the BNDME sites were noted in the washout (‘notch-on’) samples which were absent or markedly reduced in the mock-washout (notch off) state (
To prove that the BNDME sites are bona fide MYC enhancers, lentiviral guideRNA constructs targeting 15 distinct sites across the MYC locus were designed, including the MYC promoter, RBPJ motifs with the T-NDME and both B-NDME sites, as well as the MYC promoter and other intergenic sites (
Additional studies were undertaken to identify other direct Notch target genes that might play an important role in MCL and CLL biology. Only a small fraction of Notch-activated genes identified in the GSI-washout analysis showed ICN-1 and RBPJ binding, raising the possibility that many of these genes, like MYC, might be activated by Notch-dependent distal elements. To identify such elements, published genome-wide maps were utilized of 3-dimensional genomic interactions associated with RNA Polymerase II via Chromatin Interaction Analysis by Paired-End Tag sequencing (PolII ChIA-PET) in the EBV-immortalized B-lymphoblastoid cell line (LCL) GM12878 (Tang et al., 2015). In support of this approach, strong interactions between both B-NDME sites and the MYC promoter were observed in the GM12878 PolII ChIA-PET data (
GSI-sensitive and -insensitive MCL, and highly significant enrichment over GSI sensitive-only and random gene sets. Notch-activated enhancers identified in these analyses showed properties consistent with Notch target enhancers in other tissues, including dynamic ICN-1 and RBPJ binding in the presence or absence of GSI, and increased H3K27ac signal in the notch-on state.
In total, the combined functional and epigenetic analysis revealed high-confidence direct Notch target genes with linked regulatory elements in the MCL models presented herein. Only a minority of these genes also showed Notch-dependent activation in T-ALL (CUTLL-1) and TNBC (HCC-1599) cell lines, and most have not been previously identified as Notch target genes in any tissue, although all of the canonical Notch target genes identified in the gene expression analysis presented herein was correctly supported as direct ICN-1 targets via promoter binding or ChIA-PET linkage. The positions of ICN-1 peaks with respect to novel target gene promoters were diverse, reflecting a similar diversity seen in canonical Notch target genes (
Next, the set of identified direct Notch target genes for association with pathways identified in the gene set analysis of the RNA-Seq data was examined. Notably, genes involved in cytokine/interleukin signaling (IL6R, IL10RA, IL2 IR) and B cell receptor activation (FYN, LYN, BLK, BLNK, PIK3AP1, SH2B2, NEDD9) were identified as direct Notch targets, indicating that these pathways may be directly modulated by Notch-dependent gene activation. Functional analysis of the set of direct Notch targets with the Ingenuity system predicted a significant activatory effect of Notch-regulated genes on B cell receptor signaling. The large number of transcription factor genes that were predicted to be direct
Notch targets was striking, indicating a broad effect of Notch in activating or reinforcing diverse transcriptional regulatory programs in MCL lines. Interestingly, the NF-KB target gene signature noted in the Notch-activated genes was substantially driven by genes that were not associated with ICN1 peaks, indicating that secondary activation of NF-KB and NF-KB target genes may be an early feature of Notch activation in B-cell lymphoma cells, similar to the phenomenon observed with MYC.
Since rapidly proliferating MCL cell lines show important biological differences from relatively low-grade MCL and CLL cells in vivo, experiments were conducted to validate the activity of Notch target genes and enhancers in primary CLL and MCL cells. RNA-Seq was performed on CLL lymph node biopsies with strong, diffuse ICN-1 staining by IHC and compared it to data from CLL lymph node biopsies with low ICN-1 staining (0 of 4 with NOTCH1 PEST domain mutations). Genome-wide analysis revealed significantly increased expression in the ICN1-high biopsies of many of the strongest Notch target genes identified in the cell line analysis (
Next, ChIP-Seq was performed for ICN1, RBPJ, and H3K27ac in CLL and MCL biopsies. One CLL (CLL-013) and one MCL (MCL-010) biopsy yielded a dramatically higher number of significant RBPJ peaks compared to the others, and both contained NOTCH1 PEST domain mutations (
To functionally demonstrate Notch-dependent activation of Notch target genes in primary CLL and MCL cells, a co-culture model with the immortalized human bone-marrow stromal cell line HS-5 was utilized, which has been widely employed to support the survival of CLL cells in vitro (
Next, the same model was used to evaluate the effect of Notch activation on the activity of signaling pathways linked to lymphoma proliferation and survival. CLL PBMC's were harvested following three days of co-culture with HS5-DLL1 with or without GSI, and then performed an additional brief incubation in the presence of absence of B-cell receptor (BCR)-crosslinking antibodies, followed by flow cytometric analysis of phosphoepitopes associated with BCR signaling and downstream pathways (
Proximal BCR signaling mediators did not show a notch-dependent difference in phosphorylation in the absence of stimulation, but significantly greater phosphorylation of SYK and PLCg2 were noted in Notch-on CLL cells upon BCR crosslinking. These findings indicate that Notch potentiates BCR signaling via up-regulation of proximal pathway regulators, resulting in increased NF-KB activity upon initiation of BCR signaling (
NF-KB is known to be a strong activator of enhancer-mediated gene expression, and in fact, published ChIP-Seq datasets from LCLs show NF-KB protein binding at many ICN-1 bound enhancers, indicating that NF-KB and Notch may act cooperatively to activate many target genes. To test this, additional CLL HS-5 co-culture experiments were performed in the presence of CpG-rich oligodideoxynucleotides, which act as a strong agonist of Toll-like receptor 9 (TLR9) signaling (
Implicit in the present investigation of CLL and MCL lymph node biopsies, as well as co-culture model described herein, is the assumption that Notch activation occurs due to interaction of lymphoma cells with Notch ligand-expressing cells within the lymph node microenvironment. To support this in vivo, a patient-derived xenograft (PDX) model derived from a case of MCL with a NOTCH1 PEST domain mutation was utilized.
Immunohistochemistry showed strong expression of ICN1 in MCL cells within the spleen, but minimal staining in three different, NOTCH1 wild-type MCL PDX models. PDX-XXX mice were treated for five days with either the gamma-secretase inhibitor DBZ or vehicle. Flow cytometry revealed the highest expression of Notch target cell surface proteins in MCL cells within the spleen compared to bone marrow or blood, with substantially decreased expression seen in GSI-treated animals (
Since the initial discovery of recurrent Notch gene mutations in CLL and MCL, it has been clear that aberrant Notch signaling plays a role in the etiology of small B cell lymphomas, but the specific mechanisms by which Notch signaling drives B cell lymphoma growth, and its interaction with other oncogenic signaling pathways have remained largely obscure. The present study reported herein represents a substantial advance by defining a set of direct Notch regulatory targets in B cell lymphoma that is distinct from those identified in other tissue types, indicating unique mechanisms by which small B-cell lymphomas may utilize this pathway to drive malignant biology.
The data presented herein provides the first demonstration of MYC as a critical and direct regulatory target of enhancer activation by ICN/RBPJ in small B cell lymphomas, and the findings reported herein are consistent with other recent data linking Notch signaling to MYC activation in CLL. The BNDME sites are recurrently amplified in a small subset of CLL cases, and an enhancer-like element immediately adjacent to BNDME1 contains a germline polymorphism linked by genome-wide association studies (GWAS) to hereditary risk for CLL, further supporting the central role of these elements in CLL pathogenesis. MYC is a pivotal regulator of cellular growth, directly activating genes responsible for nutrient import, metabolic pathway activation, nucleotide synthesis and core components of the transcriptional and translational machinery. MYC is essential for the proliferation of normal mature B and T cells, as well as most, if not all B-cell lymphomas, and activating genomic rearrangements of the MYC locus are frequently seen in aggressive B cell lymphomas, including blastic transformation of MCL and large-cell transformation of CLL (Richter syndrome), where NOTCH1 mutations and MYC-activating genomic lesions show near-complete mutual exclusivity. Notch-dependent activation of MYC and MYC target genes appears to be a common feature of Notch-dependent cell lines across at least three cancer types (B-cell lymphoma, T-ALL, and TNBC), although the specific distal regulatory elements through which Notch activates MYC in B-cell lymphomas are not utilized in T-ALL. The data presented herein indicates that inhibition of Notch-dependent MYC expression is the primary mechanism by which GSI inhibits growth of Notch-dependent MCL cell lines, since a similar loss of MYC expression and proliferation could be demonstrated via direct CRISPR-Cas9 targeting of the 5′ BNDME sites, while conversely, GSI sensitivity could be largely rescued via expression of a MYC transgene (
CLL and MCL are considered to be low-grade lymphomas, and it is important to note that the growth cycle of these tumors in vivo is different from that of the rapidly proliferating MCL cell lines utilized in the present study (doubling time 24-36 hours). Clinical and biological observations demonstrate that most cases of MCL show slow tumor growth for years after initial presentation, while the majority of CLL cells in most patients are in a quiescent state in both peripheral blood and secondary lymphoid organs, with bursts of proliferation limited to a small subset of cells in proliferation centers. However, the data presented herein, and the findings others, supports an important role for Notch-dependent MYC activation in driving a shift toward anabolic metabolism in primary CLL cells, which may facilitate subsequent cellular growth and proliferation. Co-culture of CLL cells with Notch ligand-expressing stromal cells has been shown to activate expression of hexokinase II and other MYC-activated metabolic regulators, resulting in activation of glycolysis. During activation of normal T cells, MYC is required for initiation of glycolysis and altered amino acid transport and metabolism, resulting in activation of p70-S6 kinase and other mTORC-regulated drivers of protein synthesis. The data presented herein from both proliferating cell lines and non-proliferating primary CLL cells is consistent with an analogous model in which Notch-dependent MYC activation leads to up-regulation of nutrient transporters, as well as HK2 and other metabolic gatekeepers, leading to activation of mTORC1 and S6 phosphorylation. This mechanism could play an important role in the growth of CLL and MCL cells during either proliferation or a pre-proliferative state.
In addition to activating MYC, the data indicated that Notch directly activates genes that encode regulators of B-cell receptor (BCR) signaling, including all three of the SRC family kinases implicated in proximal BCR activation (LYN, BLK, and FYN), as well as signaling adaptor proteins associated with PI3 kinase (PIK3AP; encodes BCAP) and phospholipase C gamma 2 (BLNK). While many details about the oncogenic role of BCR signaling in CLL and MCL are still unclear, phosphorylation of PLCy2 by Bruton tyrosine kinase (BTK) appears to be a critical step, since treatment with the BTK inhibitor ibrutinib drives sustained clinical remission in many CLL and MCL patients, while acquired ibrutinib resistance in lymphoma is often associated with mutations in BTK or PLCG2. A reproducibly stronger increase was observed in PLCy2 phosphorylation upon BCR signaling activation in “notch on” versus GSI-treated CLL cells from HS-5-DLL1 co-cultures, demonstrating that Notch activation potentiates this step of the BCR signaling cascade, likely through increased expression of one or more of the Notch target genes described above.
The validation studies were focused on the MYC and BCR signaling pathways, this work also identified genes encoding a striking array of cell surface signaling receptors as direct Notch targets, including receptors for IL6, IL10, and IL21, interferon gamma, TNF, and others, indicating that Notch may also potentiate signaling through these pathways. IL6R is a particularly strong Notch target, and has been implicated in the pathogenesis of both small B cell lymphomas and several autoimmune disorders. IL6R was among the Notch target genes that showed significantly increased expression in ICN1-high CLL (
The findings presented herein have important implications for the potential use of Notch inhibitors in the treatment of small B cell lymphomas. Notch signaling in lymphomas with wild-type or PEST domain-mutated Notch receptors is predicted to be largely or entirely ligand-dependent, and thus Notch inhibitors might be expected to have little effect on circulating lymphoma cells outside of secondary lymphoid organs, or other microenvironments that support Notch signaling activation. However, there is precedent for selectively targeting lymphoma within a tissue niche, as clinically efficacious agents that inhibit BCR-related signaling, including ibrutinib and the PI3K6 inhibitor idelalisib, show minimal toxicity to circulating CLL cells, and in fact, treatment with these agents is frequently associated with sustained tumor lymphocytosis, despite dramatic shrinkage of lymphadenopathy and eventual clinical remission. BCR signaling-mediated activation of NF-KB, as well as up-regulation of MYC and MYC target genes, are believed to be critical drivers of lymphoma proliferation and survival in the lymph node microenvironment. The potential of Notch inhibitor therapy to target both of these pathways by a single unique mechanism may provide an advantage over existing agents, either alone or in combination therapy. Mutations or rearrangements predicted to yield ligand-independent Notch signaling, as observed in Notch-dependent MCL lines, are essentially absent in low-grade CLL and MCL, although development of a NOTCH1 heterodimerization domain mutation has been observed following large cell (Richter) transformation of CLL. Such patients might represent particularly appealing candidates for Notch-targeting therapy. However, the data presented herein indicates that MYC-activating genomic rearrangements, which are relatively common following high-grade transformation of CLL or MCL, would be likely to show Notch-independent MYC expression and thus reduced susceptibility to Notch inhibitor therapy, indicating that clinical investigators might consider excluding such patients from future trials of Notch-targeting drugs.
The results described herein above, were obtained using the following methods and materials.
MCL-derived cell lines were kindly provided by Dr. Randy Gascoyne, BC Cancer Agency, Canada (Z-138, Maver-1, JVM-2, Granta-519, HBL-2, and UPN-1). The cell lines SP-49, Jeko-1 and Mino were kind gift of Dr. Mariusz Wasik, University of Pennsylvania. Rec-1 and HEK293T cell lines were purchased from the American Type Culture Collection. Mec-1 cells were obtained. All cell lines were authenticated by short tandem repeat (STR) profiling analysis. This study was approved by the Institutional Review Board and MCL and CLL patient samples were collected.
All cell lines were grown in RPMI medium 1640 (Invitrogen) supplemented with 10% FCS, 100 IU per 100 μg per mL penicillin/streptomycin, 1% nonessential amino acids, 1 mM sodium pyruvate and 5μM 2-mercaptoethanol. In GSI washout studies, Rec-1, Mino and SP-49 cells were treated with the GSI compound E (1 μM) (Shelton et al., 2009) for 48-72 hours, washed, and then replated in either 1 μM GSI (washout control) or in DMSO for 4 h (washout) as described in Weng et al., 2006. To activate Notch signaling Mino and Jeko-1 cells were cultured on either immobilized recombinant Notch ligand (DLL1ext-IgG) or control protein (IgG) for 48 hours supplemented with either DMSO or 1 μM GSI, following mock or GSI washout for 4 hours.
Cells were lysed in 50 mM Tris, pH 8.0, containing 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA and supplemented with protease inhibitors. Total protein was determined. Samples were mixed with sample buffer containing 5% f3-mercaptoethanol, separated by 4% to 12% NuPAGE Tris-Acetate gel (Life technologies) and transferred to a nitrocellulose membrane that was blocked for 1 hour in 5% non fat dry milk/BSA in TBST (20 mmol/L Tris-HCl, 0.5 mol/L NaCl, and 0.1% Tween 20). The membrane was probed and incubated with a primary antibody overnight at 4° C. Following washes with TBST, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (Ref) and detected with ECL developing solution (Thermo Scientific). Primary antibodies used are a monoclonal rabbit antibody against the cleaved Notch1 (Val1744, CST; #4147) in 1:1000 dilution, c-MYC and TBP.
RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). cDNA was synthetized with the SuperScript III kit (Invitrogen). qRT-PCR was carried out using 1 μL cDNA, SYBR Green PCR Master Mix (ABI) and gene-specific primers (supplementary table 1) on an ABI ViiA 7 real-time PCR System. cDNA was used as template for each pair of primers in triplicate PCR reactions and resulting qPCR data were analyzed using the ΔΔCt relative quantification protocol.
ChIP-qPCR and ChIP-Seq were performed as previously described (Ref). Briefly, chromatin samples prepared from fixed cells were immunoprecipitated with rabbit IgG (Santa Cruz Biotechnology, sc-3888), rabbit monoclonal anti-Rbpj (CST, #5313), rabbit polyclonal anti-H3K27ac (Active Motif, #39133) and mouse monoclonal anti-EBNA2(PE2) antibody (Abcam, ab90543). Antibody-chromatin complexes were captured with protein G-conjugated agarose beads, washed several times, and eluted. Following reversal of cross-links, RNase and proteinase K treatment, DNA was purified with QIAquick PCR Purification Kit (Qiagen). Input sample was prepared in parallel without immunoprecipitation. Real-time PCR was performed in triplicates for indicated regions using primers listed in supplementary table 2. For ChIP-Seq two replicates were used per experimental condition and libraries were prepared using NEBNext® Ultra™ DNA Library Prep Kit for Illumina according to the manufacturer's instructions. Indexed libraries were validated for quality and size distribution using the Agilent 2100 Bioanalyzer. High-throughput sequencing was performed by using the HiSeq 2500 Illumina Genome Analyzer. ChIP-Seq reads were aligned to the human genome (hg19).
Lentiviral particles were generated with the use of standard procedures (Ref). Briefly, lentivirus was produced in HEK293T cells that were transfected with transfection mix containing 3.9 pg of gRNA expression vectors (Addgene, #57822, #57823, #52963) or pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene, #60954), 1.3 μg of pCMV-VSV-G and 2.6 μg pCMV-delta and FuGENE HD (Promega). Viral supernatant was harvested 48 hours post-transfection. Cell lines were transduced with lentiviral supernatants by spinfection for 90 minutes in the presence of 12 μg/ml of polybrene at 37° C. 3 days after infection, transduced cells were selected either with puromycin (3 days), or were selected by fluorescent marker with cell sorting on a BD FACSAria II SORP. Selected cells were used for RNA extraction and proliferation assay.
RNA-Seq was performed using three replicates per experimental condition. RNA was isolated with RNeasy Plus Mini Kit (Qiagen) from SP-49 cells treated with GSI for 3 days to establish a Notch-off state or cells where Notch was re-activated by GSI washout as described in GSI washout assay or from Mino cells that were cultured with the following modification: supplemented with either immobilized recombinant Notch ligand (DLL1ext-IgG) or control protein (IgG) for 48 hours of purified mRNA was used as template for cDNA synthesis and library construction. Indexed libraries were validated for quality and size distribution using the Agilent 2100 Bioanalyzer and were sequenced on the HiSeq 2500 Illumina Genome Analyzer.
SP-49 cells were stably transduced with pINDUCER-22-MYC (Ref) and single cell clones were isolated by limiting dilution with plating 0.3 cells/well in 96 well plates. Selected clones were treated with DMSO or GSI for 5 days and then MYC expression was induced by increasing concentration of doxycycline for 2 days and cell growth was measured using the CellTiter-Glo Luminescent Cell viability assay (Promega) as recommended by the manufacturer.
SP-49 and Granta-519 were engineered to stably express SFFV-KRAB-dCas9-P2A-mCherry or pLX-304-GFP. GFP+and dCas9-KRAB-mCherry+cells derived from SP-49 or Granta-519 were mixed in 1:1 ratio and transduced with gRNA lentiviruses designed against CD300A and CR2 regulatory regions (gRNA sequences are provided in supplementary table 3), following the puromycin selection for 3 days. Flow antibodies against CR2 and CD300A (Ref) were used to detect the expression in GFP+(negative control) and dCas9-KRAB-mCherry+populations following the epigenetic silencing of CR2 and CD300A.
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/383,111, filed on Sep. 2, 2016. The entire content of this application is hereby incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/049829 | 9/1/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62383111 | Sep 2016 | US |
