Patent Application
20150167062
Recent technological advances have allowed the genome-wide study of gene regulatory networks by localizing proteins to specific regions of DNA or associated chromatin. Deep sequencing of DNA associated with proteins of interest, pulled-down using directed antibodies, provides high-resolution spatial maps of genomic organization. Termed chromatin immunoprecipitation with deep sequencing (ChIP-seq), biologists now possess a new lens through which they can understand cellular and disease biology. However, there are important limitations to this technology. The procedure requires the existence of validated, specific immunoglobulins and only individual protein/DNA complexes may be isolated. The complexity of this experiment has limited the use of ChIP-seq to understand and characterize the effects of chromatin-directed small molecules.
Described herein is a genome-wide assessment of interactions between chemical entities and their target molecules, referred to as Chem-seq technology.
Accordingly, in one aspect, the invention is directed to a method of mapping, across a genome, interaction of a drug or drug candidate with one or more factors that associate with the genome. The method comprises contacting a cell, cell lysate, or nuclear extract that comprises chromatin with the drug. The cell, cell lysate, or nuclear extract that comprises chromatin is maintained under conditions in which the drug or drug candidate can interact with the one or more factors that associate with the genome. The genome of the cell, cell lysate, or nuclear extract that comprises chromatin is fragmented thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with the one or more of the factors which interact with the drug. The sequence of all or a portion of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate is determined, thereby mapping, across the genome, the interaction of the drug or drug candidate with one or more factors that associate with the genome.
In another aspect, the invention is directed to a method of identifying one or more sites on a genome with which a drug or drug candidate interacts. The method comprises contacting a cell, cell lysate, or nuclear extract that comprises chromatin with the drug or drug candidate and maintaining the cell, cell lysate, or nuclear extract that comprises chromatin under conditions in which the drug or drug candidate can interact with one or more factors that associate with the genome. The genome of the cell, cell lysate, or nuclear extract that comprises chromatin is fragmented thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with the one or more of the factors which interact with the drug. The sequence of all or a portion of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate is determined, thereby identifying one or more sites on the genome with which the drug or drug candidate interacts.
In another aspect, the invention is directed to a method of identifying a signature of a condition or disease (e.g., syndrome) across a genome. The method comprises contacting a cell, cell lysate, or nuclear extract that comprises chromatin with a drug or drug candidate that is used or can potentially be used to treat the disease and maintaining the cell, cell lysate, or nuclear extract that comprises chromatin under conditions in which the drug or drug candidate can interact with one or more factors that associate with the genome. In some aspects, the cell or lysate thereof is a cell or lysate thereof obtained from one or more individuals with the disease. The genome of the cell, cell lysate, or nuclear extract that comprises chromatin is fragmented thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with the one or more of the factors which interact with the drug. The sequence of all or a portion of each of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate is determined, thereby identifying the signature of the condition or disease across the genome.
In yet another aspect, the invention is directed to a method of determining whether a drug or drug candidate will effectively treat a condition (e.g., a syndrome; a disease such as cancer, heart disease, etc.) in an individual in need thereof. The method comprises contacting a cell, cell lysate, or nuclear extract that comprises chromatin of the individual with the drug or drug candidate and maintaining the cell, cell lysate, or nuclear extract that comprises chromatin under conditions in which the drug or drug candidate can interact with one or more factors that associate with the genome. The genome of the cell, cell lysate, or nuclear extract that comprises chromatin is fragmented thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with the one or more of the factors which interact with the drug. The sequence of all or a portion of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate is determined, thereby obtaining a signature of the drug's interaction across the individual's genome, wherein if the signature of the drug's interaction across the individual's genome is similar, substantially similar or identical to a signature of a positive control, then the drug or drug candidate will effectively treat the condition in the individual and/or if the signature of the drug's interaction across the individual's genome is similar, substantially similar or identical to a signature of a negative control, then the drug or drug candidate will not effectively treat the condition in the individual.
(a) In one aspect, the disclosure features a kit, the kit comprising one or more of (e.g., each of);
(b) a cell or cell lysate or nuclear extract that comprises chromatin or reagent useful for making of a cell lysate or nuclear extract that comprises chromatin;
(c) a drug or drug candidate, wherein the drug or drug candidate is linked to a detectable label such as biotin or coupled to a solid support (e.g., directly or indirectly);
(d) a factor that interacts with the label (e.g., wherein the factor is attached to a solid support such as a bead) (exemplary factors include an antibody (e.g, attached to a solid support));
(e) optionally, a reagent, for example, a primer or oligo such as a PCR oligo for sequencing one or more sequences identified by the method of the instructions from the kit; and
instructions to maintain the drug or drug candidate and cell or cell lysate or nuclear extract that comprises chromatin under conditions in which the drug or drug candidate can interact with the one or more factors that associate with the genome, and the one or more factors that associate with the genome are covalently linked to the genome of the cell; instructions to fragment the genome of the cell or cell lysate or nuclear extract that comprises chromatin thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with the one or more of the factors which interact with the drug or drug candidate; and instructions to determine all or a portion of the sequence of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate, thereby mapping, across the genome, the interaction of the drug or drug candidate with one or more factors that associate with the genome.
Therapeutic entities elicit their effects by binding specific proteins or other molecules; some of these target molecules are associated with the genome. Protein components of chromatin biology are emerging as pressing therapeutic targets in cancer, inflammation and cardiovascular disease. Indeed, cancer sequencing efforts have identified numerous mutated, rearranged or amplified chromatin-associated factors and transcription factors, prompting coordinated efforts in chromatin drug discovery world-wide. It has not been possible to determine directly how drugs interact with proteins or other molecular entities that are associated with the genome. While it is possible to use mass spectroscopy to identify the proteins that are bound by small molecule chemicals, this approach does not identify the sites throughout the genome that are occupied by such chemical-bound proteins. While it is possible to use ChIP-seq to identify the sites bound by specific proteins throughout the genome, this approach does not reveal whether these proteins are bound by a molecule of interest.
The development of a method that identifies the sites throughout the genome that are bound by molecules (e.g., compounds, drugs), directly and/or indirectly, is described herein. Effectively, these data provide molecular resolution on the sites of compound (e.g., therapeutic) action on the genome, providing spatial target resolution. This information is valuable for e.g., determining the targets of a potential therapeutic chemical, the sites (genes) where a chemical acts throughout the genome, for assessing specificity of the chemical, and/or for identifying biomarkers for disease and therapy.
A broad range of nucleic acids (e.g., DNA; RNA) interacting ligands, which are known to bind to the nucleic acids either directly or indirectly (via their target proteins), can be used as affinity ‘molecules’ to enrich for DNA or RNA derived from cells, tissues or other biological materials.
In some aspects, the method comprises one or more of the following steps shown in
In methods that include a cross linking step, the drug or drug candidate can be added subsequent to the cross-linking, or in the alternative, the method can comprise e.g., using cells (e.g., live cells or lysates thereof) treated with drug or drug candidates or compounds (e.g., using cell permeable compounds or liposome-containing compounds, or other methods of introducing compounds into cells) prior to cross-linking. Another application is to use tissue sections with healthy or diseased cells.
Accordingly, in one aspect, provided herein are methods of mapping, across a genome, interaction of a (one or more) drug or drug candidate with a genome. In some aspects, the drug or drug candidate can interact with the genome directly. In other aspects, the drug or drug candidate can interact with the genome indirectly, e.g., the drug or drug candidate interacts with one or more factors that associates with the genome.
In the methods, the genome is contacted with a drug or drug candidate of interest (a drug or drug candidate to be assessed), thereby producing a combination. In particular aspects, the drug or drug candidate is contacted with a genome that is present in a cell and/or cell lysate. The combination is maintained under conditions in which the drug or drug candidate can interact with the genome directly and/or indirectly e.g., by interacting one or more factors that associate with the genome (e.g., of the cell, cell lysate, or nuclear extract that comprises chromatin). The genome is fragmented, thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with drug or drug candidate directly and/or indirectly. The sequence of all or a portion of the one or more genome fragments that are associated directly or indirectly with the drug or drug candidate is then determined, thereby mapping, across the genome, the interaction of the drug or drug candidate with the genome.
In a particular aspect, the invention is directed to a method of mapping, across a genome, interaction of a drug or drug candidate with one or more factors that associate with the genome. The method comprises contacting a cell, cell lysate, or nuclear extract that comprises chromatin with the drug or drug candidate. The cell, cell lysate, or nuclear extract that comprises chromatin is maintained under conditions in which the drug or drug candidate can interact with the one or more factors that associate with the genome. The genome of the cell, cell lysate, or nuclear extract that comprises chromatin is fragmented thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with the one or more of the factors which interact with the drug or drug candidate. The sequence of all or a portion of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate is determined, thereby mapping, across the genome, the interaction of the drug or drug candidate with one or more factors that associate with the genome.
As used herein “mapping the interaction of a cell, cell lysate, or nuclear extract that comprises chromatin across a genome” refers to the identification of the regions of a genome to which a particular drug or drug candidate interacts either directly or indirectly (e.g., in vivo; in vitro). The regions identified can include a target site of the drug or drug candidate (a site of the genome at which the drug is expected or intended to interact) as well as an off-target site (a site of the genome at which the drug or drug candidate is not expected or intended to interact).
In another aspect, the invention is directed to a method of identifying one or more sites on a genome with which a drug or drug candidate interacts. The method comprises contacting a cell, cell lysate, or nuclear extract that comprises chromatin with the drug or drug candidate and maintaining the cell, cell lysate, or nuclear extract that comprises chromatin under conditions in which the drug or drug candidate can interact with one or more factors that associate with the genome. The genome of the cell, cell lysate, or nuclear extract that comprises chromatin is fragmented thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with the one or more of the factors which interact with the drug or drug candidate. The sequence of all or a portion of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate is determined, thereby identifying one or more sites on the genome with which the drug or drug candidate interacts.
In another aspect, the invention is directed to a method of identifying a signature of a condition or disease (e.g., syndrome) across a genome. The method comprises contacting a cell, cell lysate, or nuclear extract that comprises chromatin with a drug or drug candidate that is used or can potentially be used to treat the disease and maintaining the cell, cell lysate, or nuclear extract that comprises chromatin under conditions in which the drug or drug candidate can interact with one or more factors that associate with the genome. In some aspects, the cell or lysate thereof is a cell or lysate thereof obtained from one or more individuals with the disease. The genome of the cell, cell lysate, or nuclear extract that comprises chromatin is fragmented thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with the one or more of the factors which interact with the drug or drug candidate. The sequence of all or a portion of each of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate is determined, thereby identifying the signature of the condition or disease across the genome.
In yet another aspect, the invention is directed to a method of determining whether a drug or drug candidate will effectively treat a condition (e.g., a syndrome; a disease such as cancer, heart disease, etc.) in an individual in need thereof. The method comprises contacting a cell, cell lysate, or nuclear extract that comprises chromatin of the individual with the drug or drug candidate and maintaining the cell, cell lysate, or nuclear extract that comprises chromatin under conditions in which the drug or drug candidate can interact with one or more factors that associate with the genome. The genome of the cell, cell lysate, or nuclear extract that comprises chromatin is fragmented thereby producing a mixture comprising genome fragments, wherein one or more of the genome fragments are associated with the one or more of the factors which interact with the drug or drug candidate. The sequence of all or a portion of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate is determined, thereby obtaining a signature of the drug or drug candidate's interaction across the individual's genome, wherein if the signature of the drug or drug candidate's interaction across the individual's genome is similar, substantially similar or identical to a signature of a positive control, then the drug or drug candidate will effectively treat the condition in the individual and/or if the signature of the drug or drug candidate's interaction across the individual's genome is similar, substantially similar or identical to a signature of a negative control, then the drug or drug candidate will not effectively treat the condition in the individual. As will be apparent to those of skill in the art, a variety of positive and/or negative controls can be used. An example of a positive control is a signature of the drug or drug candidate's interaction across the genome of a cell of lysate thereof obtained from one or more individuals that are or have been treated successfully with the drug or drug candidate. An example of a negative control is a signature of the drug or drug candidate's interaction across the genome of a cell of lysate thereof obtained from one or more individuals that are not or have not been treated successfully with the drug or drug candidate.
Typically, factors that tightly associate with the genome (e.g., histone) remain bound under the assay conditions described herein; however, factors which do not associate tightly with the genome may not remain bound under the assay conditions described herein. Thus, as described herein, the methods can further comprise covalently linking the one or more factors that associate with the genome to the genome. The one or more factors can be covalently linked to the genome either prior to contacting the genome with the drug or drug candidate or subsequent to contacting the genome with the drug or drug candidate. In one aspect, the cell, cell lysate, or nuclear extract that comprises chromatin can be contacted with the drug or drug candidate after the one or more factors that associate with the genome are covalently linked to the genome. In another aspect, the cell, cell lysate, or nuclear extract that comprises chromatin can be contacted with the drug or drug candidate before the one or more factors are covalently linked to the genome.
Methods for covalently linking one or more factors that associate with the genome of a cell, cell lysate, or nuclear extract that comprises chromatin are known to those of skill in the art. In one aspect, the cell, cell lysate, or nuclear extract that comprises chromatin is contacted with a crosslinking agent such as formaldehyde or paraformaldehyde. In another aspect, the cell, cell lysate, or nuclear extract that comprises chromatin is exposed to UV light (e.g., with a transilluminator).
As described herein, the methods can further comprise reversing the covalent links (e.g., the cross links) between the one or more factors and the genome. In some aspects, the covalent links are reversed prior to determining a sequence of all or a portion of the one or more genome fragments that interact with the drug or drug candidate directly and/or indirectly. In other aspects, the covalent links can be reversed after the genome fragments that are associated with one or more factors which interact with the drug or drug candidate are separated from the other genome fragments in the mixture which are not associated with one or more factors which interact with the drug or drug candidate.
Examples of genomes that can be used in the methods of the invention include a prokaryotic genome (e.g., a bacterial genome) and a eukaryotic genome (e.g., a mammalian genome). In one aspect, the genome is a mammalian (e.g., primate, canine, feline, murine and the like) genome. In a particular aspect, the genome is a human genome.
The regions of the genome that can be identified as interacting with a drug or drug candidate include one or more heterochromatic regions of the genome (heterochromatin) and/or one or more euchromatic regions of a genome (euchromatin). In a particular embodiment, the methods provided herein map the interaction of a drug or drug candidate with euchromatin across a genome.
As described herein, the drug or drug candidate can be contacted with a genome that is present in a cell (in vivo Chem-Seq) or cell lysate (in vitro Chem-Seq). Thus, the methods can further comprise obtaining a cell, cell lysate, or nuclear extract that comprises chromatin. Methods of obtaining cells and/or lysates thereof are well known in the art (e.g., see Marson et al., Cell, 134(3):521-533 (2008) and U.S. Pat. No. 6,410,243 which are incorporated herein by reference). For example, a (one or more) cell can be obtained from an individual (e.g., a patient) or from commercial sources, e.g., a cell and/or tissue bank or storage facility. In particular aspect, cells can be obtained from a sample (e.g., a biological fluid (e.g., blood, urine, lymph, etc.), a tissue sample, a biopsy) from one or more individuals. As will be apparent to those of skill in the art, the cells can be manipulated mechanically and/or chemically (e.g., by suspending cells in any of a variety of lysis buffers as described herein) to obtain cell lysates.
A variety of cells and lysates thereof can be used herein. Examples of cell and lysates thereof include prokaryotic cells, eukaryotic cells and lysates thereof. In a particular aspect, a mammalian (e.g., primate, canine, feline, murine, etc.) cell or lysate thereof is used. In yet another aspect, a human cell or lysate thereof is used. In still another aspect, a live cell is used. Other aspects of the method comprise use of a normal cell, an abnormal cell and/or lysates thereof. Abnormal cells include diseased cells such as cancer cells, heart disease cells, etc. In some embodiments, the cell is fresh, frozen, or preserved (e.g., wherein the cell is a preserved cell from a human sample or sample from a non-human mammal). A sample comprising cells may in some embodiments be obtained from a subject, e.g., a human or a non-human mammal, wherein a drug or drug candidate has been administered to the subject. Cells may be primary cells, immortalized cells, cells of a cell line, genetically modified cells, etc. Cells may be of any cell type or may originate from any organ or tissue in various embodiments. Cells may be, e.g., epithelial cells, hematopoietic cells, immune system cells, nervous system cells, muscle cells, fibroblasts.
The term “drug or drug candidate” refers in some embodiments to a compound that has an activity against (towards) a target of interest (e.g., an IC50 against a target (e.g., a transcription factor) of less than about 1 mM or less than about 100 μM (e.g., less than about 10 μM, less than about 1 μM, or less than about less than about 0.1 μM). As will be appreciated, a compound does not need to be approved by the FDA (or by any government agency responsible for regulating pharmaceuticals in any jurisdiction) to be a drug as used herein. Included herein are compounds (e.g., small molecules) that are active against one or more targets such as a protein (e.g., a protein related to transcription such as a transcription factor). In some embodiments, the drug or drug candidate has been tested and has shown activity in one or more in vitro or in vivo assays. Activity may be, e.g., binding activity (e.g., towards a target), inhibitory or activating activity (e.g., towards a target), activity that alters a cell phenotype or cell state, activity potentially suggesting or indicative that the compound may be of use in treating a disease, etc. Exemplary drug candidates include tool compounds or reference compounds with known activity against a target and compounds identified in an assay such as a screening assay or analogs or derivatives of such compounds, such as analogs or derivatives synthesized, designed, or identified based at least in part on a hit compound in a screen. A drug or drug candidate may modulate, e.g., inhibit or activate, a target of interest. A drug or drug candidate may modulate a target in any of a variety of ways in various embodiments. For example, a drug or drug candidate may bind to a target, may bind to one or more other components that bind to the target, may alter stability or conformation of a target, may affect localization of a target.
In addition, a variety of compounds or drug or drug candidate s can be used in the methods, compositions, kits of the invention. For example, the drug or drug candidate can be a nucleic acid (e.g., antisense RNA, small interfering RNA (siRNA), a short hairpin RNA (shRNA)), a peptide, a small molecule (e.g., a small organic chemical), natural produce (e.g., a macrocycle). In a particular aspect, the drug or drug candidate is a therapeutic drug or drug candidate. In another aspect, the drug or drug candidate is a pharmacologically active small molecule. In yet another aspect, the drug or drug candidate is an anti-cancer drug or drug candidate (e.g., JQ1, tamoxifen). In some embodiments, the drug or drug candidate targets a protein, e.g. inhibits or activates a protein such as a transcription factor or a kinase. In some embodiments, the drug or drug candidate targets DNA or chromatin directly.
The term “small molecule” refers to a compound having a molecular weight of, less than about 7500 amu, less than about 5000 amu, less than about 2500 amu, preferably less than about 2000 amu, even more preferably less than about 1500 amu, still more preferably less than about 1000 amu, or most preferably less than about 750 amu.
As will be appreciated by those of skill in the art, a drug or drug candidate can “interact” with the genome in a variety of ways. For example, a drug or drug candidate can interact with a genome by binding to the genome directly, and/or the drug or drug candidate can interact with a genome indirectly, e.g., by binding to a factor that is associated with (e.g., bound to) the genome.
In still further aspects, the drug or drug candidate can comprise one or more of a variety of detectable labels (e.g., a handle) that allow detection and/or separation of the drug or drug candidate at one or more steps of the methods described herein. Such detectable labels are well known to those of skill in the art. Examples of detectable labels include a (one or more) fluorescent molecule, a peptide (e.g., avidin, streptavidin), a hapten (e.g., dinitrophenyl (DNP), digoxigenin, nitrotyrosine), small molecule (e.g., biotin) or combinations thereof. Methods for introducing a label onto a drug or drug candidate are well known to those of skill in the art. See, for example, Becer, C R., et al., Angew Chem Int Ed, 48:4900-4908 (2009) and Hein, C D, et al., Pharm Res, 25(10:2216-2230 (2008) which are incorporated herein by reference.
In various embodiments, a drug or drug candidate described herein is attached to a label, for example, through a linker. Exemplary drug or drug candidates described herein are provided in the compound of Formula (I) below:
wherein:
Drug or drug or drug candidate is a small molecule
Linker comprise covalent bond-Xn-covalent bond;
each X is independently a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C1-50 hydrocarbon chain wherein one or more methylene units are optionally and independently replaced by —O—, —S—, —Se—, —OP(O)OH—O—,—N(R)P(O)OH—N(R)—, —Si(R)2-, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —(R)NC(O)N(R)—, —(R)C═NN(R)—, —N═N—, —S(O)—, —S(O)2-, —N(R)SO2-, —SO2N(R)—, a haloalkyl group (e.g., —CF2-), a heterocyclic group, an aryl group, or a heteroaryl (e.g., triazole) group;
wherein each occurrence of R is independently hydrogen, a suitable protecting group, or an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety;
n is an integer from 1 to 50; and
Label is a moiety that can be used to isolate and/or identify the drug or drug candidate in a method or kit described herein (e.g., fluorescent molecule, a peptide, a hapten or a halo containing compound).
In some embodiments, the linker comprises a polyethylene glycol moiety (PEG) or a derivative thereof (e.g., a PEG 2, PEG 6, PEG 8, PEG 20, PEG 40, PEG 100, PEG 300, PEG 400, PEG 1000).
In some embodiments, the linker comprises a peptide (e.g., an up to 15 amino acid residue peptide).
In some embodiments, the linker comprises a moiety formed using “click chemistry” (e.g., as described in WO 2006/115547). In one embodiment, the linker comprises an amide bond, an ester bond, a disulfide bond, or a triazole.
As described herein, the drug or drug candidate can interact with the genome indirectly e.g., by interacting with one or more factors that associate with a genome. As used herein, a “factor that associates with the genome” includes a single factor or a combination of factors (a complex of factors such as a transcription complex) that associate with the genome at one or more particular regions. A variety of factors that associate with genomes are well known to those of skill in the art. As is also well known to those of skill in the art, these factors can “associate” with the genome in a variety of ways. For example, a factor can associate with a genome by binding to the genome. Examples of such factors include proteins such as histones, transcription proteins (e.g., a transcription regulatory protein or complexes thereof), a protein or complex thereof involved in maintenance of genome integrity (e.g., a DNA replication and repair protein; a topoisomerase), a chromatin-modifying protein, chromatin associating factor, a receptor (e.g., a steroid receptor; a nuclear hormone receptor), a kinase, a phosphatase, a protesome, a proteosomal component, a structural or scaffolding factor and complexes thereof. Specific examples are provided in Tables 1-4.
Specific examples of biotinylated compounds for analysis by the Chem-Seq methods described herein are shown in
In the methods provided herein, the cell, cell lysate, or nuclear extract that comprises chromatin is contacted with a drug or drug candidate, thereby producing a combination, and the combination is maintained under conditions in which the drug or drug candidate can interact with the one or more factors that associate with the genome. In the methods which further comprise cross linking the one or more factors associated with the genome to the genome (e.g., by contacting the cell, cell lysate, or nuclear extract that comprises chromatin with a cross linking agent), the combination is also maintained under conditions in which the one or more factors that associate with the genome are covalently linked to the genome of the cell. In some embodiments, the target is the genome itself. As will be apparent to one of skill in the art, such conditions include appropriate temperature, pH, incubation periods, buffers, etc. and will depend upon the assay conditions (e.g., the drug or drug candidate being assessed).
The methods provided herein also include a step of fragmenting the genome of the cell, cell lysate, or nuclear extract that comprises chromatin, thereby producing a mixture comprising fragments of the genome (genome fragments), wherein one or more of the fragments are associated with the drug or drug candidate either directly or indirectly. Methods of fragmenting a genome are well known to those of skill in the art. For example, a genome can be fragmented mechanically e.g., using sonication and/or shear-inducing methods, and/or chemically e.g., using enzyme digestion (e.g., micrococcal nuclease).
The methods of the invention further comprise determining a (one or more) sequence of all or a portion (an identifying (e.g., distinct, unique) portion, a portion that allows identification of the sequence within the genome) of the one or more genome fragments that interact with the drug or drug candidate directly and/or indirectly. In a particular aspect, the method comprises determining the sequence of all or a portion of the one or more genome fragments that are associated with the one or more factors which interact with the drug or drug candidate. Methods for determining all or a portion of the sequence that interacts with the drug or drug candidate are well known in the art. Examples of such methods include polymerase chain reaction (PCR), massively parallel sequencing, immobilized nucleic acid polymers, next generation sequencing and the like.
As will be apparent to those of skill in the art, the methods described herein can further comprise isolating one or more (e.g., all) of the genome fragments that are associated with the drug or drug candidate either directly or indirectly from the mixture of genome fragments (e.g., separating one or more genomes fragments that are associated with the drug or drug candidate from the genome fragments that are not associated with the drug or drug candidate).
A variety of methods can be used to isolate or separate the one or more genome fragments that are associated with the drug or drug candidate from the mixture of genome fragments. In a particular aspect, such genome fragments are isolated comprising contacting the mixture of genome fragments with an agent that specifically binds to the drug or drug candidate directly and/or indirectly. Examples of agents that can specifically bind to a label (e.g., a detectable label) present on the drug or drug candidate include a peptide (e.g., biotin, avidin, streptavidin), an enzyme (e.g., alkaline phosphatase, horseradish peroxidase) and an antibody (e.g., a primary antibody, a secondary antibody).
In a particular aspect, the mixture of genome fragments is contacted with a solid support that comprises an agent that specifically binds to the drug or drug candidate directly and/or indirectly. Examples of solid supports for use in the methods include beads, arrays, slides, wells, columns and the like.
The methods described herein can further comprise comparing the results to a suitable control. The controls used in the methods can be a positive and/or negative control. As will be apparent to those of skill in the art, a variety of such controls can be used. An example of a control for use in the methods of mapping, across a genome, interaction of a drug or drug candidate with one or more factors that associate with the genome and/or methods of identifying one or more sites on a genome with which a drug or drug candidate interacts, includes a (one or more) genome map obtained for one or more drug or drug candidates with a known mechanism of action (e.g., genome maps from a plurality of drug or drug candidates (e.g., a class of drug or drug candidates) with a similar mechanism of action; genome maps from a plurality of drug or drug candidates (e.g., multiple classes of drug or drug candidates) with different (e.g., a variety of) mechanisms of action). An example of a control for use in the methods of identifying a signature of a disease across a genome includes a signature (e.g., a known signature) obtained for a similar disease (condition, syndrome) using the same or a similar drug or drug candidate (e.g., a drug or drug candidate that has, or is believed to have, a similar mechanism of action). Examples of a control for use in the methods of determining whether a drug or drug candidate will effectively treat a condition in an individual in need thereof include a signature of the drug or drug candidate's interaction across the genome of a cell of lysate thereof obtained from one or more individuals that are or have been treated successfully with the drug or drug candidate (e.g., a positive control), and/or a signature of the drug or drug candidate's interaction across the genome of a cell of lysate thereof obtained from one or more individuals that are not or have not been treated successfully with the drug or drug candidate (e.g., a negative control).
Biotinylated JQ1 was synthesized from the S enantiomer of JQ-1 referred to herein as (S)-JQ1 or JQ1S. (S)-JQ1 was dissolved in formic acid and stirred at room temperature generated free acid 1. The acid was then coupled with monoprotected PEG-diamine to afford the amide 2. The protecting group (Trt) was removed under acidic condition, and the resulting amine was coupled with biotin to afford final JQ1-PEG-Biotin (e.g., see
Human SK-MEL-5 cells were grown to 80% confluency with a final count of 1-2×108 cells for each location analysis reaction. Cells were chemically crosslinked by the addition of one-tenth volume of fresh 11% formaldehyde solution for 15 minutes at room temperature. Cells were harvested using a silicon scraper, centrifuged and the derived pellets washed three times with 1×PBS. Cell pellets were flash frozen in liquid nitrogen and stored at −80° C. prior to use.
Cells were resuspended, lysed in lysis buffers and sonicated to solubilize and shear crosslinked DNA. Sonication conditions vary depending on cells, culture conditions, crosslinking, equipment, and drug or drug candidate. A Misonix Sonicator 3000 was used and sonication was performed at power 8 for 10×30 second pulses (60 second pause between pulses). Samples were kept on ice at all times.
The resulting whole cell extract was incubated overnight at 4° C. (affinity purification) with 100 μl of Streptavidin Dynal (MyOne Streptavidin T1) magnetic beads that had been pre-incubated with JQ1 as follows: 5 ul of 10 mM JQ1/DMSO solution were mixed with 100 ul of Strepavidin Dynal bead suspension and incubated overnight (o.n.). Drug-bound beads were washed four times with BSA blocking solution to remove unbound drug.
The affinity purification was allowed to proceed overnight. Beads were sequentially washed with the following buffers: (1) lysis buffer 1, (2) lysis buffer 2, (3) Sonication buffer, (4) High-salt sonication buffer, (5) LiCl buffer and (6) TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65° C. with occasional vortexing and crosslinking was reversed by overnight incubation at 65° C. Whole cell extract DNA (reserved from the sonication step) was also treated for crosslink reversal.
Affinity-purified DNA and whole cell extract DNA were then treated with RNAse A, proteinase K and purified with multiple phenol:chloroform:isoamyl alcohol extractions.
Purified DNA was prepared for sequencing according to a modified version of the Illumina/Solexa Genomic DNA protocol. Fragmented DNA was prepared for ligation of Solexa linkers by repairing the ends and adding a single adenine nucleotide overhang to allow for directional ligation. A 1:100 dilution of the Adaptor Oligo Mix (Illumina) was used in the ligation step. A subsequent PCR step with limited (18) amplification cycles added additional linker sequence to the fragments to prepare them for annealing to the Genome Analyzer flow-cell. After amplification, a narrow range of fragment sizes was selected by separation on a 2% agarose gel and excision of a band between 150-300 bp (representing shear fragments between 50 and 200 nt in length and ˜100 bp of primer sequence). The DNA was purified from the agarose and diluted to 10 nM for loading on the flow cell.
The DNA library (2-4 pM) was applied to the flow-cell (8 samples per flow-cell) using the Cluster Station device from Illumina. The concentration of library applied to the flow-cell was calibrated such that polonies generated in the bridge amplification step originated from single strands of DNA. Multiple rounds of amplification reagents were flowed across the cell in the bridge amplification step to generate polonies of approximately 1,000 strands in 1 μm diameter spots. Double stranded polonies were visually checked for density and morphology by staining with a 1:5000 dilution of SYBR Green I (Invitrogen) and visualizing with a microscope under fluorescent illumination. Validated flow-cells were stored at 4° C. until sequencing.
Flow-cells were removed from storage and subjected to linearization and annealing of sequencing primer on the Cluster Station. Primed flow-cells were loaded into the Illumina Genome Analyzer 1G. After the first base was incorporated in the Sequencing-by-Synthesis reaction the process was paused for a key quality control checkpoint. A small section of each lane was imaged and the average intensity value for all four bases was compared to minimum thresholds. Flow-cells with low first base intensities were re-primed and if signal was not recovered the flow-cell was aborted. Flow-cells with signal intensities meeting the minimum thresholds were resumed and sequenced for 26 cycles.
Results:
JQ1 is a BET bromodomain inhibitor with potential applications in multiple human cancers (Delmore, J. E. et al., Cell 146, 904-917; Filippakopoulos, P. et al., Nature 468, 1067-1073; Zuber, J. et al., Nature 478, 524-528). It is a competitive Brd2, Brd3, Brd4 and BrdT inhibitor that competes with acetyl-lysine binding. The JQ1 mechanism of action is largely through targeting BET bromodomains, with consequent loss of Myc gene expression, but how it binds target molecules genome-wide is unknown. Using a biotinylated-JQ1, Chem-seq was used to determine where it associates with the genome of SKMEL5 melanoma cells. DMSO was used to determine the background level of beads alone. As shown in
The method has broad applicability to complement conventional ChIP (which uses antibodies or affinity tags to localize protein:DNA interactions) in cases where ligands for the protein target of interest are available. Importantly, the availability and diversity of these ligands has sharply risen during the past years.
It can be used to reveal the mechanism of drug action as well as the specificity of drugs. Specifically, the method allows a rigorous genome-wide assessment of the specificity and mechanism of small molecule therapeutics, which elicit their effects through binding proteins or nucleic acids where some of the protein targets associate with the genome. The technology described herein allows for the determination of on-target and off-target characterization of drug-genome interactions.
The method is useful for global DNA/RNA-binding studies on entire enzyme and protein families that share a defined mechanism of action. In this case, the function and binding of a whole protein family as such to the genome will be accessible by single analysis using a modified compound targeting the entire protein family. Examples are the genome-wide analyses and thus characterization of the Kinome, HTMome, HDACome etc.
It is useful as a tool in the diagnosis and management of diseases, such as cancer, and thus, can be used in diagnostic laboratories. Drugs have generally a higher stability than antibodies, but, as
The in vivo Chem-Seq was carried with the same protocol for in vitro Chem-Seq described in Example 1, but differed from the in vitro Chem-Seq protocol in the following steps:
1. Human multiple myeloma (MM1.S) cells (final count of 1-2×108) were treated with 5 uM bio-JQ1 for various incubation times (0, 10, 20, 30 min), followed by addition of fresh formaldehyde solution (final formaldehyde concentration in cell medium is 1%) for 20 min at room temperature. Cells were then washed two times with 1×PBS before cell pellets were flash-frozen in liquid nitrogen and stored at −80 degree prior to use.
2. The nuclear cell extracts (derived from cells treated with compound) were incubated with 200 ul of Streptavidin Dynal magnetic beads overnight (o.n.)
Results are shown in
Cell Culture and Treatment with Unbiotinylated Drugs
Multiple Myeloma MM1.S cells (CRL-2974, ATCC) were maintained in RPMI-1640 supplemented with 10% fetal bovine serum and 1% GlutaMAX (Invitrogen, 35050-061). For JQ1 treatment experiments, asynchronous cells were treated with varying concentrations of JQ1 or vehicle (DMSO) for 6. Alternatively, cells were treated with 2 μM AT7519 (Selleck Chemicals) for 6 h.
ChIP coupled with massively parallel DNA sequencing (ChIP-Seq) was performed as previously described (33). The following antibodies were used for Chromatin Immunoprecipitation (ChIP): □nti-BRD4 (Bethyl Labs, A301-985A), □nti-MEDT (Bethyl Labs, A300-793A), □nti-H3K27Ac (Abeam, ab4729), □nti-RNA-Pol II (Santa Cruz, sc-899), □nti-CTCF (Millipore, 07-729) and □nti-CDK9 (sc-484). Illumina sequencing, library construction and ChIP-Seq analysis methods were previously described (33).
The synthesis of active Bio-JQ1(S) started with the (S)-JQ1, the active enantiomer that inhibits the bromodomain extra-terminal (BET) subfamily. As shown below in Scheme S1, removal of the tert-butyl group of the ester on (S)-JQ produced the acid S1. The resulting acid was then coupled with mono-protected (PEG)2 linked diamine to give an amide S2. The Trt protecting group on the terminal amine of compound S2 was removed under acidic condition to generate free amine, which was further coupled with biotin to afford the final active Bio-JQ1(S). The biotinylated inactive enanitomer Bio-JQ1(R) was synthesized in the same synthetic route using inactive enantiomer (R)-JQ 1. See
The first bromodomain of BRD4 was purified as a poly-histidine-tagged recombinant human protein expressed in E. coli, as previously described (18).
Thermal melting experiments were carried out using a 7300 Real Time PCR machine (AB Applied Biosystems). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 1 μM in 20 μL volume. Compounds were added at a final concentration of 10 μM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000. Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 4° C. per minute from 25° C. to 96° C. and fluorescence readings were taken at each interval. The observed temperature shifts, ΔTm obs, were recorded as the difference between the transition midpoints of sample and reference (DMSO) wells containing protein without ligand in the same plate.
Isothermal Titration Calorimetry
ITC was performed using a ITC200 microcalorimeter from MicroCal™ (Northampton, Mass.). All experiments were carried out at 25° C. while stirring at 1000 rpm, in ITC buffer (50 mM HEPES pH 7.4 at 25° C., 150 mM NaCl). The microsyringe was loaded with a solution of the protein sample (190 μM, in ITC buffer). All titrations were conducted using an initial injection of 0.2 μl followed by 19 identical injections of 2 μl with a duration of 5 sec (per injection) and a spacing of 90 sec between injections. The heat of dilution was determined by independent titrations (protein into buffer) and was subtracted from the experimental data. The collected data were implicated in the MicroCal™ Origin software supplied with the instrument to yield enthalpies of binding (ΔH) and binding constants (Ka). A single binding site model was employed. Dissociation constants and thermodynamic parameters are presented in Table 5.
Exponentially growing MM1.S cells (2×108 cells per sample) were treated simultaneously with either 5 uM biotinylated JQ1 (Bio-JQ1) or DMSO (vehicle) and 1% Formaldehyde for 20 min in cell culture medium. Chemical crosslinking was terminated by addition of TRIS buffer, pH 7.5, to a final concentration of 300 mM TRIS. Cells were harvested using a silicon scraper, centrifuged and the derived pellets washed three times with PBS. Cell nuclei were prepared as follows: cells were lysed in 50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 plus protease inhibitor cocktail ‘complete’ (Roche), and cell nuclei were washed once with 10 mM Tris-HCL, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitors. Nuclei were resuspended and sonicated in 50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS (sonication buffer) and protease inhibitor cocktail at 18 W for 10 cycles (30 s each) on ice with 30 s intervals between cycles. Sonicated lysates were cleared by centrifugation and incubated for 16-20 h at 4° C. with magnetic Streptavidin Dynabeads (MyOne Streptavidin T1, Invitrogen) (beads were blocked in PBS containing 0.5% BSA before this incubation step). Following incubation in nuclear sonicated lysate, beads were washed twice in sonication buffer, once in sonication buffer containing 500 mM NaCl, once in LiCl buffer (20 mM Tris-HCL, pH 8.0, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% Na-deoxycholate), and once in 10 mM TRIS, pH 7.5, 0.1 mM EDTA. Bound protein-DNA complexes were subsequently eluted in 50 mM Tris-HCL, pH 8.0, 10 mM EDTA, 10% SDS at 65° C. for 15 min, and crosslinks were reversed by overnight incubation of the eluate at 65° C. Contaminating RNA and protein were digested by addition of RNase and Proteinase K, respectively, and the DNA purified as previously described (34). Finally, purified DNA fragments were massively parallel sequenced and the sequencing data analyzed as described (33).
Exponentially growing, untreated MM1.S cells were fixed with 1% Formaldehyde for 20 min in cell culture medium. Chemical crosslinking was terminated, cell nuclei prepared and sonicated nuclear lysate obtained as described above. Unlike in the in vivo protocol, however, Streptavidin Dynabeads were pre-incubated in PBS containing 0.5% BSA and either 200 □M biotinylated drug or vehicle (DMSO) for 6 h. Drug-bound beads were subsequently washed four times in PBS/0.5% BSA to remove unbound drug, and incubated in nuclear sonicated lysate for 16-20 h at 4° C. All following steps are identical to those described above (in vivo Chem-Seq method).
Exponentially growing, untreated MM1.S cells were fixed with 0.5% Formaldehyde for 5 min in cell culture medium. Chemical crosslinking was terminated by addition of TRIS buffer, pH 7.5, to a final concentration of 300 mM TRIS. Cells were washed 3× in PBS and cell nuclei prepared as follows: Cell nuclei were lysed in 50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 plus protease inhibitor cocktail ‘complete’ (Roche), and cell nuclei were washed once with 10 mM Tris-HCL, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitors. Nuclei were resuspended and sonicated in 50 mM Hepes-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 0.5% Triton-X (sonication buffer). Pellets were sonicated at 9-12 W for 4 cycles (30 s each) in a Misonix sonicator on ice with 1 min rest intervals between cycles. Drug-bound beads were added to the cleared sonicate and the precipitation allowed to proceed for 12-18 hours. Drug-bound beads were subsequently washed four times in sonication buffer, proteins eluted in 1% SDS, and crosslinks were reversed by overnight incubation of the eluate at 65° C. in 1% SDS. Contaminating RNA and protein were digested by sequential incubation with RNase A and Proteinase K, and the DNA purified as previously described (34). Purified DNA fragments were subjected to massively parallel sequencing (Illumina) and the sequencing data analyzed as described (33).
All ChIP-Seq and Chem-seq datasets were aligned using Bowtie (version 0.12.2) (35) to build version NCBI36/HG18 of the human genome. We used the MACS version 1.4.1 (Model based analysis of ChIP-Seq) (36) peak finding algorithm to identify regions of ChIP-Seq enrichment over background. A p-value threshold of enrichment of 1e-9 was used for all datasets. To obtain the normalized read density of ChIP-Seq datasets in any region, ChIP-Seq reads aligning to the region were extended automatically by MACS and the density of reads per basepair (bp) was calculated. The density of reads in each region was normalized to the total number of million mapped reads producing read density in units of reads per million mapped reads per by (rpm/bp).
Aligned and raw data can be found under GEO Accession number GSE43743.
A gene was defined as transcribed if an enriched region for either H3K4me3 or RNA Pol II was located within +/−5 kb of the TSS. H3K4me3 is a histone modification associated with transcription initiation (37).
Active enhancers were defined as regions of enrichment for H3K27Ac outside of promoters (greater than 5kb away from any TSS). H3K27Ac is a histone modification associated with active enhancers (38, 39). Active enhancers form loops with promoters that are facilitated by the Mediator complex (25). Thus, H3K27Ac definitions of enhancers were validated using ChIP-Seq data for the mediator subunit Med1. Enriched regions from Med1 had >90% overlap with H3K27Ac regions in all datasets.
The ratio of RNA Pol II ChIP-Seq levels in initiating to elongating regions a measure known as the traveling ratio (TR), was determined (40). The initiating region was defined as +/−300 bp around the TSS. The elongating region was defined as +300 bp from the TSS to +3,000 bp after the gene end. In order to make higher confidence comparisons, the analysis was limited to genes with detectable signal above noise in the initiating and elongating regions across all samples. The statistical significance of changes in the distribution of traveling ratios was determined using two-tailed t test.
All genes were ranked by increasing density of RNA Pol II ChIP-Seq reads in the promoter region (+/−1 kb of transcription start site (TSS) and binned in increments of 100 genes. The median ChIP-Seq density for promoter regions within each bin was calculated in rpm/bp for both Pol II, BRD4, MED1, H3K27Ac, CDK9.
Active enhancers were aligned at the center in the composite view of ChIP-Seq density profiles. The average ChIP-Seq read density around +/−5kb centered on the active enhancer in 50 bp bin was calculated. Genes were aligned using the position and direction of their transcription start sites and transcription termination sites. Data were expressed as genome-wide ChIP-Seq read density averages in 50 bp bins. Correlation between Chem-seq and ChIP-Seq signals were calculated using the enriched regions from targets of small molecules (BRD4 and CDK9) from ChIP-seq. CTCF enriched regions were used as a comparison. Read density profiles of Chem-seq and ChIP-seq were displayed as scatter plots.
Chem-Seq and ChIP-Seq data was analyzed to identify genomic regions with substantial JQ 1 Chem-Seq signal but no significant BRD4 ChIP-Seq signal, reasoning these sites might represent off-target interactions of JQ1. A generalized linear model (GLM method) was adopted to identify regions with differential signal (41, 42). First identified were the set of genomic regions that were enriched for JQ1 Chem-Seq or BRD4 ChIP-Seq signal in any one of the six datasets being considered (3 replicate JQ1 Chem-Seq datasets and 3 replicate BRD4 ChIP-Seq datasets). Regions from a dataset that overlapped with regions from another data set were merged together to form a representative region that spans the combined genomic region. A total of 25,060 regions were identified. The read density in each region was calculated in units of reads per million mapped reads per by (rpm/bp) for each dataset. The edgeR package was used to model technical variation due to noise among triplicate datasets and the biological variation due to differences in signal between JQ1 Chem-Seq and BRD4 ChIP-Seq datasets (41). Sequencing depth and TMM techniques were used to normalize datasets after common and tagwise dispersions were estimated. The statistical significance of differences between JQ1 Chem-Seq signals and BRD4 ChIP-Seq signals was calculated using an exact test and resulting P values were subjected to Benjamini-Hochberg multiple testing correction. The analysis identified 10 out of the 25,060 genomic regions examined that showed significant differences in JQ1 Chem-Seq and BRD4 ChIP-Seq signals (FDR<=0.25). All 10 regions showed greater BRD4 ChIP-Seq signal relative to JQ1 Chem-Seq signal; we did not detect any regions showing substantial JQ 1 Chem-Seq signal and lacking BRD4 ChIP-Seq signal. These data indicate that, under these experimental conditions, there is no identifiable off-target interaction that results in JQ1 Chem-Seq signal at regions of the genome without BRD4.
To examine the limits of the differential binding analysis described here, simulated datasets that model the presence of JQ1 Chem-Seq signal at regions that were not overlapping with BRD4 ChIP-Seq signals were generated. Then asked was how well the analysis could identify these computationally introduced regions of differential signal. To generate the simulated datasets, each replicate dataset of JQ1 Chem-Seq was spiked with a set of mapped reads from a CTCF ChIP-Seq experiment. The reads mapped specifically to regions of CTCF enrichment, and the selected regions were chosen to avoid overlap with either JQ1 Chem-Seq signal or BRD4 ChIP-Seq signal. Variable numbers of reads were added to simulate the presence of an additional 1%, 2%, 5% or 10% of enriched regions in the JQ 1 Chem-Seq datasets. As the CTCF ChIP-Seq reads were not added to BRD4 ChIP-Seq data, regions of CTCF ChIP-Seq-derived signal in the simulated dataset thus served as true positives when testing for the ability to identify differential signal. The GLM-based analysis was then performed as described above. It was found that the GLM analysis identified essentially all regions of differential signal that were introduced in the simulated dataset. In the simulated dataset where an additional 1% of regions were computationally introduced as true positives, 95% of these regions were identified as having differential signal. In the datasets where an additional 2%, 5% or 10% of regions were computationally introduced as true positives, the identification rate was 98.5%, 99.8% and 99.7%, respectively. Together, these data indicate that the GLM method is capable of accurately identifying instances of differential signal, even when they occur rarely.
The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/659,750, filed on Jun. 14, 2012 and U.S. Provisional Application No. 61/668,718, filed on Jul. 6, 2012 and U.S. Provisional Application No. 61/701,434, filed on Sep. 14, 2012 and U.S. Provisional Application No. 61/800,060, filed on Mar. 15, 2013. The entire teachings of the above application(s) are incorporated herein by reference.
This invention was made with government support under R01-HG002668 from the National Institutes of Health and PF-11-042-01-DMC from the American Cancer Society. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/045929 | 6/14/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61659750 | Jun 2012 | US | |
| 61668718 | Jul 2012 | US | |
| 61701434 | Sep 2012 | US | |
| 61800060 | Mar 2013 | US |
