This application is a continuation application which claims the benefit of U.S. application Ser. No. 12/730,170, filed Mar. 23, 2010; which claims the priority of U.S. Provisional App. Ser. No. 61/162,673, filed on Mar. 23, 2009 and U.S. Provisional App. Ser. 61/245,000, filed on Sep. 23, 2009 the disclosures of which are hereby incorporated by reference their entirety for all purposes.
Multiparametric analyses of cells provide an approach for the simultaneous determination of the activation states of a plurality of cellular components. The activation status of the plurality of cellular components can be measured after exposure of cells to extracellular modulators and in so doing allows the signaling capacity of signaling networks to be determined when compared to the activation status of those networks in the absence of such modulators. The induced activation status of a protein rather than the frequently measured basal phosphorylation state of a protein has been shown in several studies to be more informative, as it takes into account (and reveals) signaling deregulation that is the consequence of numerous cytogenetic, epigenetic and molecular changes characteristic of transformed cells. For example, multiparameter flow cytometry at the single cell level can measure the activation status of multiple intracellular signaling proteins and can assign activation states of these molecules to the varied cell sub-sets within complex primary cell populations.
However, usually multiparametric analyses of cells, e.g., multiparametric flow cytometry, require the use of multiple reagents at precise concentrations to produce robust and reproducible results. Since these data can be used as tools to inform clinical decisions, as well as therapeutic development, it would be beneficial to provide kits comprised of components relevant to a particular application with accompanying relevant usage information.
Protein phosphorylation is a critical post translational process in controlling many cell functions such as migration, apoptosis, proliferation and differentiation. Site specific phosphorylation of proteins can be detected, for example, by incubating cells with fluorochrome-conjugated phospho-specific antibodies using flow cytometry. However, only reagents whose parameters (including but not limited to, concentration, kinetics, fluorochrome to protein ratio) have been optimized can be used to generate robust and reproducible data that can be applied to a specific purpose. Kits comprising two or more reagents recognizing intracellular markers and/or extracellular markers along with an appropriate modulator or modulators to evoke a signaling response appropriate for the signal transduction pathway, specific cell type, disease state, or cellular function can save the end user from the tedious and often costly process of selecting, optimizing and standardizing reagents thereby providing the user with a more streamlined and cost-saving approach for profiling cellular networks in single cells.
It is therefore an objective of the present invention to provide kits that meet such demands.
SUMMARY OF THE INVENTION
The present invention involves the preparation of kits to be utilized in multi-parametric analyses (e.g. flow cytometry) on cell populations for the identification of the activation states of cellular signaling molecules (called nodes) in cells. Profiles of node states in cell populations are useful for diagnosis, prognosis, drug discovery, drug development, patient stratification (for example, who will and who will not respond to a drug) and other applications. Methods for determining cell populations and activation states have been disclosed in U.S. Pat. Nos. 7,381,535, 7,393,656, 7,563,584 and U.S. Ser. No. 61/120,320, which are hereby incorporated by reference in their entirety.
One embodiment of the present invention is a kit comprising a combination of binding element cell surface markers and state-specific intracellular markers. The kit can also comprise one or more modulators, therapeutic agents, fixatives, buffers, physical devices and software as described below.
In some embodiments, kits can be directed toward applications such as prediction of a response to a therapeutic agent, diagnosis and prognosis of various diseases or conditions, profiling signaling in specific cell types, analyzing the functional effects of genetic mutations, etc.
In some embodiments, kits can be prepared based on cell types of interest. For example, a kit can have a panel of antibodies that recognize extracellular markers specific to T cells, B cells, myeloid cells, stromal cells, neuronal cells or epithelial cells.
In some embodiments, the cell-type-specific kits can be supplemented with modulators for different signaling pathways. For example, the kit can include one or more cytokines and or growth factors that activate pathways including, but not limited to, JAK/STAT, PI3K/Akt, Ras/Raf/Erk, phosphatase signaling, metabolism, apoptosis, DNA damage response or transcriptional activation pathways. The kits can further comprise control cells, compounds and/or protocols.
In some embodiments, the invention involves kits for analyzing the effect of a compound on a cancer cell, comprising one or more binding elements that recognize particular surface markers expressed by cells in certain disease states. The kits can also comprise a compound used for treating a condition such as cancer. The kits can also comprise binding elements recognizing the activated state of signaling elements that can be activated in response to a compound, including but not limited to phosphorylated, acetylated, methylated or cleaved proteins.
In some other embodiments of the present invention, kits can additionally comprise consumable hardware, such as plates for holding the reagents or performing reactions, pipette tips, and software or files required to carry out the experiment. In some embodiments, the kit can further comprise a software package for data analysis of cell signaling profiles, which can include reference profiles for comparison with a test profile. The kit can also include software to manage or perform the experiment, including the use of the reagents and protocols for conducting appropriate reactions.
In some other embodiments, kits of the present invention enable the detection of activatable elements by sensitive cellular assay methods, such as immunohistochemistry and flow cytometry, which are suitable for clinical applications in detection, prognosis, and screening of cells and tissues from patients who have a disease involving aberrant signaling networks, for example leukemia.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
FIG. 1 illustrates some embodiments of apoptosis pathway kits, comprising various intracellular markers involved in intrinsic and extrinsic apoptosis pathways.
FIG. 2 illustrates some embodiments of apoptosis pathway kits, comprising various intracellular markers involved in intrinsic and extrinsic apoptosis pathways.
INCORPORATION BY REFERENCE
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
DETAILED DESCRIPTION OF THE INVENTION
The present invention incorporates information disclosed in other applications and texts. The following patent and other publications are hereby incorporated by reference in their entireties: Haskell et al., Cancer Treatment, 5th Ed., W.B. Saunders Co. (2001); Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland Science (2002); Vogelstein and Kinzler, The Genetic Basis of Human Cancer, 2d Ed., McGraw Hill (2002); Michael, Biochemical Pathways, John Wiley & Sons (1999); Weinberg, The Biology of Cancer (2007); Janeway et al., Immunobiology, 7th Ed., Garland Science (2008); Leroith & Bondy, Growth Factors and Cytokines in Health and Disease, Vols. 1A and 1B: A Multi Volume Treatise, (JAI Pr, 1996). Patents and applications that are also incorporated by reference in their entirety include U.S. Pat. Nos. 7,381,535; 7,393,656; 7,563,584 and U.S. Pat. Ser. Nos. 10/193,462; 11/655,785; 11/655,789; 11/655,821; 11/338,957, 12/432,720; 12/229,476; 12/432,239; 12/460,029; 12/471,158; 61/216,825; 61/162,673; 61/157,900; 61/151,387; 61/104,666; 61/226,878; 61/218,718; 61/182,518; 61/170,348; 61/144,684; 61/113,823; 61/181,211; 61/162,598; 61/108,803; 61/182,638; 61/177,935; 61/155,373; 12/293,081; 61/186,619; 61/156,754; 61/106,462; 61/176,420; 12/538,643; 12/501,274; 61/079,537; 12/501,295; 61/146,276; and 61/144,955. Some commercial reagents, protocols, software and instruments that are useful in some embodiments of the present invention are available at the Becton Dickinson Website http://www.bdbiosciences.com/features/products/, and the Beckman Coulter website, http://www.beckmancoulter.com/Default.asp?bhfv=7. Method of performing assays on multiparametric flow cytometry are described in e.g., Krutzik et al., High-content single-cell drug screening with phosphospecific flow cytometry, Nature Chemical Biology (2007) 4:132-142; Irish et al., FLt3 ligand Y591 duplication and Bcl-2 over expression are detected in acute myeloid leukemia cells with high levels of phosphorylated wild-type p53, Neoplasia (2007) 109(6):2589-96; Irish et al. Mapping normal and cancer cell signaling networks: towards single-cell proteomics, Nature (2006) 6:146-155; Irish et al., Single cell profiling of potentiated phospho-protein networks in cancer cells, Cell (2004) 118(2):217-18; Schulz, K. R. et al., Single-cell phospho-protein analysis by flow cytometry, Curr Protoc Immunol (2007) 78:8 8.17.1-20; Krutzik, P. O. et al., Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry, J Immunol (2005) 175(4):2357-65; Krutzik, P. O. et al., Characterization of the murine immunological signaling network with phosphospecific flow cytometry, J Immunol (2005) 175(4):2366-73; Stelzer et al., Use of Multiparameter Flow Cytometry and Immunophenotyping for the Diagnosis and Classification of Acute Myeloid Leukemia (John Wiley & Sons, 2000); Krutzik, P. O. et al., Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events, Cytometry A. (2003) 55(2):61-70; Hanahan D. et al., The Hallmarks of Cancer, Cell (2000) 100(1):57-70; Krutzik et al, High content single cell drug screening with phospho-specific flow cytometry, Nature Chemical Biology (2008) 4(2):132-42. Experimental and process protocols and other helpful information can be found at http:/proteomices.stanford.edu. The articles and other references cited below are also incorporated by reference in their entireties for all purposes.
The present invention relates to the processing of cells for analysis. More specifically, the present invention relates to kits comprising binding elements that can be used, e.g., in multi-parametric flow cytometry in order to determine the activation states of a plurality of proteins in single cells.
In one aspect, the present invention provides a kit comprising one or more binding elements for extracellular markers specifically targeted toward certain diseases, cell types and signaling pathways, which can be used, for example, to facilitate research. A kit of the invention can allow for analysis of relevant activatable elements in specific cell types that can provide the information necessary to make a diagnosis, prognosis, drug discovery, predict the response of disease to a therapeutic agent, and provide information relevant to drug development and patient stratification to a specific condition.
In another aspect, the present invention provides a kit comprising one or more binding elements that recognize one or more cell surface markers and one or more intracellular markers to enable rapid screening of the effects of modulators or therapeutic agents on evoked cell signaling.
In yet another aspect, the present invention provides a kit with one or more binding elements and one or more modulators to develop one or more network profiles, such as a network profile that can predict a response to a therapeutic agent or therapeutic regimen.
In some embodiments, the present invention relates to a kit and/or composition to be used in multiparametric flow cytometry on cell populations and activation states for diagnosis, prognosis, drug discovery, drug development, and patient stratification. A kit can comprise one or more binding elements for the cell surface marker of particular cell type or disease state. A kit can also comprise one or more binding elements that recognize intracellular markers of particular cellular pathway or function, modulators, labeling agent, fixatives, permeabilizing agents, etc. The kit generally can be used for determining the status of an activatable element. The kit might also be used for determining the status of a plurality of activatable elements.
A kit of the present invention can include components necessary to determine the activation state of a plurality of activatable elements from single cells, wherein each cell type is selected based on a target disease cellular state or other area of interest. For example, the target diseases can include, but are not limited to hematological diseases such as acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN); the target states and pathways can include, but are not limited to intrinsic apoptosis pathway, extrinsic apoptosis pathway, DNA damage-induced apoptosis pathway, ABL and BCR/ABL function in CML (chronic myelogenous leukemia), phosphatase function, calcium signaling, Protein Kinase C (PKC) function.
In one embodiment, a kit can be used to monitor and predict disease outcome. In another embodiment, a kit can be used in drug screening to determine whether a drug can be useful for treating a particular disease. In some other embodiments, a kit can also be used in the analysis of drug transport and/or drug metabolism, inflammation, autophagy, metabolism, cell proliferation, cell cycle, cell survival, siRNA function, or other functional characteristic.
A kit can also provide a panel of reagents for the analysis of a targeted therapeutic agent. For example, it could be used to study the effect of aJAK2 inhibitor on JAK/STAT pathway activity; PI3K inhibitor on PI3K/Akt or Ras/Raf/Erk pathway activity; Mek inhibitor on Ras/Raf/Erk pathway activity; mTor inhibitor on the TSC/mTor pathway; IK-Kinase inhibitor on NFkB pathway activity; kinase inhibitor acting on a pathway utilizing a tyrosine kinase, including but not limited to, epidermal growth factor receptor, Fibroblast growth factor, and Src family kinase signaling and nucleoside analogues and alkylating agents on the DNA damage response and apoptosis pathways.
A kit can include a composition for the detection of the activation of an element in a cell. A suitable cell includes cell types implicated in a wide variety of disease conditions, even in non-diseased states. Suitable cell types include, but are not limited to, cancer cells of all types including cancer stem cells, cardiomyocytes, dendritic cells, endothelial cells, epithelial cells, lymphocytes (T cell and B cell), mast cells, eosinophils, basophils, neutrophils, natural killer cells, erythrocytes, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as hematopoietic, neural, skin, and monocyte stem cells. Particularly preferred are primary disease state cells, such as primary cancer cells including circulating tumor cells (CTCs).
One embodiment of the present invention is a kit for classifying cells of a myeloid disorder based on the biology of a cell or group of cells derived from a patient with a myeloid malignancy such as AML, MDS, or MPN.
In some embodiments, the kit of the present invention can be directed towards a particular cell type. Specific examples include, but are not limited to, lymphocytes, myeloid cells, such as mature monocytes (CD45+, CD33+, CD11b+), myeloblasts (CD45+, CD34+, CD11b−), lymphoid subsets, such as T cell, B cell, and nucleated red blood cells (nRBCs).
In some embodiments, a kit of the present invention can be geared towards a particular sample, such as peripheral blood and bone marrow. In some preferred embodiments, the cells used in the present invention are populations of leukemic myeloid cells taken from the bone marrow of a leukemic patient or nucleated red blood cells taken from the bone marrow of a leukemic patient.
The terms “patient” or “individual” as used herein includes humans as well as other mammals.
Cell Surface Markers
Cell surface markers are molecules characteristic of the plasma membrane of a cell or in some cases of a specific cell type. The term “extracellular marker” and “cell surface marker”, and “cell surface antigen” and “phenotypic marker” as used herein, include antigens expressed in healthy and/or diseased cells and can be used interchangeably. In some embodiments, a kit of the present invention can comprise a combination of antibodies that recognize cell surface markers including but not limited to CD3, CD4, CD7, CD8, CD11b, CD11c, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD27, CD33, CD34, CD38, CD40, CD45, CD56, CD69, CD71, CD80, CD117, CD138, CD235a, CD235b, Ter119, GP-130, IgM, IgD, IgE, IgG, IgA, CCR5, CCR3, TLR2, TLR4, TLR9. CD3, also known as T3, is a member of the immunoglobulin (Ig) superfamily that plays a role in antigen recognition, signal transduction and T cell activation. It is found on all mature T lymphocytes, NK-T cells, and some thymocytes. CD4 is also a member of the Ig superfamily, which participates in cell-cell interactions, thymic differentiation, and signal transduction. It is primarily expressed on most thymocytes, a subset of T cell and monocytes/macrophages. CD7 is found on T cells, NK cells, thymocytes, hematopoietic progenitors and monocytes. CD7 is also expressed on ALL and some AML cells. CD11b is a member of the integrin family, primarily expressed on granulocytes, monocytes/macrophages, dendritic cells, NK cells, and subsets of T and B cells. CD14 is a GPI-linked membrane glycoprotein, also known as LPS receptor. It is expressed at high levels on macrophages, monocytes and at low level on granulocytes. CD33 is a sialoadhesion Ig superfamily member expressed on myeloid progenitors, monocytes, granulocytes, dendritic cells and mast cells. It is absent on normal platelets, lymphocytes, erythrocytes and hematopoietic stem cells. CD34 is a type I monomeric sialomucin-like glycophosphoprotein. It is selectively expressed on the majority of hematopoietic stem/progenitor cells, bone marrow stromal cells, capillary endothelial cells, embryonic fibroblasts, and some nervous tissues. It is commonly used marker for identifying human hematopoietic stem/progenitor cells. CD45 is commonly known as the leukocyte common antigen. It is a transmembrane tyrosine phosphatase expressed on all hematopoietic cells, except erythrocytes and platelets. It is a signaling molecule that regulates a variety of cellular processes including cell growth, differentiation, cell cycle, and oncogenic transformation. It plays a critical role in T and B cell antigen receptor-mediated activation. CD71 is a type II heterodimeric transmembrane glycoprotein also known as the transferring receptor. It is expressed on proliferating cells, reticulocytes, and erythroid precursors. CD71 plays a role in the control of cellular proliferation by facilitating the uptake of iron via ferrotransferrin binding and the recycling of apotransferrin to the cell surface. CD235a is also known as glycophorin A and CD235b is also known as glycophorin B, major sialoglycoproteins expressed on the red blood cell membrane and erythroid precursors. Mature, non-nucleated red blood cells are characteristically CD235a and/or CD235b positive, but CD45 and CD71 negative.
In some embodiments, a kit to be used for the analysis of myeloid cells in bone marrow can comprise antibodies that recognize 1, 2, 3, 4, 5, 6 or 7 of the following: CD7, CD11b, CD14, CD15, CD33, CD34, and/or CD45.
In some embodiments, a kit to be used for the analysis of nucleated red blood cells can comprise antibodies that recognize 1, 2, 3, 4, 5 or 6 of the following: CD7, CD14, Cd34, CD45, CD71, CD235a and/or CD235b.
State-Specific Binding Elements for Intracellular Markers
In some embodiments, the kits of the invention are employed to monitor the status of an activatable element, such as a signaling protein, in a signaling pathway known in the art including those described herein. Exemplary types of signaling proteins within the scope of the present invention include, but are not limited to, kinases, kinase substrates (e.g. phosphorylated substrates), phosphatases, phosphatase substrates, binding proteins (such as 14-3-3), receptor ligands and receptors (cell surface receptor tyrosine kinases and nuclear receptors)). Kinases and protein binding domains, for example, have been well described. See, for example, Cell Signaling Technology, Inc., 2002 Catalogue “The Human Protein Kinases” and “Protein Interaction Domains” pgs. 254-279).
In some embodiments, a kit can comprise one or more of the state-specific binding elements specific for the activated element(s) of interest. Exemplary binding elements comprise binding elements specific for PI3-Kinase (p85, p110a, p110b, p110d), JAK1, JAK2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nck, Gab, PRK, SHP1, and SHP2, SHIP1, SHIP2, sSHIP, PTEN, Shc, Grb2, PDK1, SGK, Akt1, Akt2, Akt3, TSC1,2, Rheb, mTor, 4E-BP1, p70S6Kinase, S6, LKB-1, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tp12, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK/JNK1,2,3, p38s, Erk1/2, Syk, Btk, BLNK, LAT, ZAP70, Lck, Cbl, SLP-76, PLCγ1, PLCγ2, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, FAK, p130CAS, PAKs, LIMK1/2, Hsp90, Hsp70, Hsp27, SMADs, Rel-A (p65-NFKB), CREB, Histone H2B, Histone H3, HATs, HDACs, PKR, Rb, Cyclin D, Cyclin E, Cyclin A, Cyclin B, p15, p16, p21, p14Arf, p27KIP, p21CIP, Cdk4, Cdk6, Cdk7, Cdk1, Cdk2, Cdk9, Cdc25, A/B/C, Abl, E2F, FADD, TRADD, TRAF2, RIP, Myd88, BAD, Bcl-2, Mcl-1, Bcl-XL, Caspase 2, Caspase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9, XIAPs, Smac, Fodrin, Actin, Src, Lyn, Fyn, Lck, NIK, IκB, p65(RelA), IKKα, PKA, PKCα, PKCβ, PKCθ, PKCδ, CAMK, Elk, AFT, Myc, Egr-1, NFAT, ATF-2, Mdm2, p53, DNA-PK, Chk1, Chk2, ATM, ATR, beta-□catenin, CrkL, GSK3α, GSK3β, FOXO, or glycolytic enzymes including but not limited to M2 pyruvate kinase.
In some preferred embodiments, kits of the present invention comprise one or more of the state-specific binding elements specific for the proteins selected from the group consisting of STAT1, STAT3, STAT5, S6, Erk, Akt, ATM, ATR, Chk1, Chk2, 53BP1, PARP, H2AX, Caspase 3, Caspase 8, CRKL, Histone H3, Cyclin B1, Cyclin D1, Cyclin E, Cyclin A, p15, p16, p21, PLCδ2, p53, SLP-76, and CREB. Other binding elements disclosed in U.S. Pat. No. 7,393,656 are also incorporated here by reference.
In some embodiments of the invention, a kit of the invention can comprise one or more binding elements specific for activation states of activatable elements. The term “binding element” includes any molecule, e.g., peptide, nucleic acid, small organic molecule which is capable of detecting an activation state of an activatable element over another activation state of the activatable element.
In some embodiments, the binding element is a peptide, polypeptide, oligopeptide or a protein. The peptide, polypeptide, oligopeptide or protein can be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, or a combination of both. Thus “amino acid”, or “peptide residue”, as used herein include both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention. The side chains may be in either the (R) or the (S) configuration. In some embodiments, the amino acids are in the D- or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents can be used, for example to prevent or retard in vivo degradation. Proteins including non-naturally occurring amino acids can be synthesized or in some cases, made recombinantly; see van Hest et al., FEBS Lett 428:(1-2) 68-70 May 22, 1998 and Tang et al., Abstr. Pap Am. Chem. S218: U138 Part 2 Aug. 22, 1999, both of which are expressly incorporated by reference herein.
A kit of the present invention can be used to detect any particular activatable element in a sample that is antigenically detectable and antigenically distinguishable from other activatable elements which are present in the sample. For example, activation state-specific antibodies can be used in the present kits to identify distinct signaling cascades within a subset or subpopulation of cells within a complex population, and the ordering of protein activation (e.g., kinase activation) in potential signaling hierarchies. Hence, in some embodiments, the expression and phosphorylation of one or more polypeptides can be detected and quantified using a kit of the present invention. In some embodiments, the expression and phosphorylation of one or more polypeptides that are cellular components of a cellular pathway can be detected and quantified using methods of the present invention. As used herein, the term “activation state-specific antibody” or “activation state antibody” or grammatical equivalents thereof, refer to an antibody that specifically binds to a corresponding and specific antigen. Preferably, the corresponding and specific antigen is a specific form of an activatable element. Also preferably, the binding of the activation state-specific antibody is indicative of a specific activation state of a specific activatable element.
In some embodiments, the binding element is an antibody. In some embodiment, the binding element is an activation state-specific antibody.
The term “antibody” includes full length antibodies and antibody fragments, and may refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below. Examples of antibody fragments, as are known in the art, such as Fab, Fab′, F(ab′)2, Fv, scFv, or other antigen-binding subsequences of antibodies, either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. The term “antibody” comprises monoclonal and polyclonal antibodies. Antibodies can be antagonists, partial antagonists, agonists, partial agonists or neutralizing antibodies.
The antibodies of the present invention may be nonhuman, chimeric, humanized, or fully human. For a description of the concepts of chimeric and humanized antibodies see Clark et al., 2000 and references cited therein (Clark, 2000, Immunol Today 21:397-402). Chimeric antibodies comprise the variable region of a nonhuman antibody, for example VH and VL domains of mouse or rat origin, operably linked to the constant region of a human antibody (see for example U.S. Pat. No. 4,816,567). In some embodiments, the antibodies of the present invention are humanized. By “humanized” antibody as used herein is meant an antibody comprising a human framework region (FR) and one or more complementarity determining regions (CDR\'s) from a non-human (usually mouse or rat) antibody. The non-human antibody providing the CDR\'s is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor”. Humanization relies principally on the grafting of donor CDRs onto acceptor (human) VL and VH frameworks (Winter U.S. Pat. No. 5,225,539). This strategy is referred to as “CDR grafting”. “Backmutation” of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct (U.S. Pat. No. 5,530,101; U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No. 5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213). The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and thus will typically comprise a human Fc region. Methods for humanizing non-human antibodies are well known in the art, and can be essentially performed following the method of Winter and co-workers (Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536). Additional examples of humanized murine monoclonal antibodies are also known in the art, for example antibodies binding human protein C (O\'Connor et al., 1998, Protein Eng 11:321-8), interleukin 2 receptor (Queen et al., 1989, Proc Natl Acad Sci, USA 86:10029-33), and human epidermal growth factor receptor 2 (Carter et al., 1992, Proc Natl. Acad Sci USA 89:4285-9). In an alternate embodiment, the antibodies of the present invention may be fully human, that is the sequences of the antibodies are completely or substantially human. A number of methods are known in the art for generating fully human antibodies, including the use of transgenic mice (Bruggemann et al., 1997, Curr Opin Biotechnol 8:455-458) or human antibody libraries coupled with selection methods (Griffiths et al., 1998, Curr Opin Biotechnol 9:102-108).
Specifically included within the definition of “antibody” are aglycosylated antibodies. By “aglycosylated antibody” as used herein is meant an antibody that lacks carbohydrate attached at position 297 of the Fc region, wherein numbering is according to the EU system as in Kabat. The aglycosylated antibody may be a deglycosylated antibody, which is an antibody for which the Fc carbohydrate has been removed, for example chemically or enzymatically. Alternatively, the aglycosylated antibody may be a nonglycosylated or unglycosylated antibody, that is an antibody that was expressed without Fc carbohydrate, for example by mutation of one or residues that encode the glycosylation pattern or by expression in an organism that does not attach carbohydrates to proteins, for example bacteria.
Activation state specific antibodies can be used to detect kinase activity, however additional means for determining kinase activation are provided by the present invention. For example, substrates that are specifically recognized by protein kinases and phosphorylated thereby are known. Antibodies that specifically bind to such phosphorylated substrates but do not bind to such non-phosphorylated substrates (phospho-substrate antibodies) may be used to determine the presence of activated kinase in a sample.
In a further embodiment, a kit of the invention can include a multiplicity of activation state antibodies that have been immobilized to determine an element activation profile. Antibodies can be non-diffusibly bound to an insoluble support having isolated sample-receiving areas (e.g. a microtiter plate, an array, etc.). The insoluble supports can be made of any composition to which the compositions can be bound, readily separated from soluble material, and otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes, and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, or Teflon™, or other known suitable material. Microtiter plates and arrays are convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like can be included.
The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall kits of the invention, maintains the activity of the composition and is nondiffusable. Methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the antibody on the surface, etc. Following binding of the antibody, excess unbound material is removed by washing. The sample receiving areas can then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
The antigenicity of an activated isoform of an activatable element can be distinguishable from the antigenicity of a non-activated isoform of an activatable element or from the antigenicity of an isoform of a different activation state. In some embodiments, an activated isoform of an element possesses an epitope that is absent in a non-activated isoform of an element, or vice versa. In some embodiments, this difference is due to covalent addition of moieties to an element, such as phosphate moieties, or due to a structural change in an element, as through protein cleavage, or due to an otherwise induced conformational change in an element which causes the element to present the same sequence in an antigenically distinguishable way. In some embodiments, such a conformational change causes an activated isoform of an element to present at least one epitope that is not present in a non-activated isoform, or to not present at least one epitope that is presented by a non-activated isoform of the element. In some embodiments, the epitopes for the distinguishing antibodies are centered around the active site of the element, although as is known in the art, conformational changes in one area of an element can cause alterations in different areas of the element as well.
In some embodiments, the invention is directed to kits to be used for determining the activation level of one or more activatable elements in a cell upon treatment with one or more modulators. The activation of an activatable element in the cell upon treatment with one or more modulators can reveal operative pathways in a condition that can then be used, e.g., as an indicator to predict course of the condition, identify risk group, predict an increased risk of developing secondary complications, choose a therapy for an individual, predict response to a therapy for an individual, determine the efficacy of a therapy in an individual, and determine the clinical outcome for an individual.
A modulator, such as a stimulant or inhibitor, is an element that when added to a biological sample may cause a reaction in the sample, such as altering cellular components such as proteins, lipids, or nucleic acids, which can affect protein signaling networks or gene expression. For a more complete list, see the patents and applications referred to above. Modulators include chemical and biological entities, and physical or environmental stimuli. Modulators can act extracellularly or intracellularly. Chemical and biological modulators include growth factors, mitogens, cytokines, drugs, immune modulators, ions, neurotransmitters, adhesion molecules, hormones, small molecules, inorganic compounds, polynucleotides (e.g., siRNA or RNAi), antibodies, natural compounds, lectins, lactones, chemotherapeutic agents, biological response modifiers, carbohydrate, proteases and free radicals. Modulators include complex and undefined biologic compositions that may comprise cellular or botanical extracts, cellular or glandular secretions, physiologic fluids such as serum, amniotic fluid, or venom. Physical and environmental stimuli include electromagnetic, ultraviolet, infrared or particulate radiation, redox potential and pH, the presence or absences of nutrients, changes in temperature, changes in oxygen partial pressure, changes in ion concentrations and the application of oxidative stress. Modulators can be endogenous or exogenous and may produce different effects depending on the concentration and duration of exposure to the single cells or whether they are used in combination or sequentially with other modulators. Modulators can act directly on the activatable elements or indirectly through the interaction with one or more intermediary biomolecule. Indirect modulation includes alterations of gene expression wherein the expressed gene product is the activatable element or is a modulator of the activatable element.
In some embodiments the modulator is selected from the group consisting of growth factors, mitogens, cytokines, adhesion molecules, drugs, hormones, small molecules, polynucleotides, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulators, carbohydrates, proteases, ions, reactive oxygen species, peptides, and protein fragments, either alone or in the context of cells, cells themselves, viruses, and biological and non-biological complexes (e.g. beads, plates, viral envelopes, antigen presentation molecules such as major histocompatibility complex). In some embodiments, the modulator is a physical stimuli such as heat, cold, UV radiation, and radiation.
In some embodiments, the modulator is an activator. In some embodiments the modulator is an inhibitor. In some embodiments, cells are exposed to one or more modulator.
In some embodiments, the inhibitor is an inhibitor of a cellular factor or a plurality of factors that participates in a cellular pathway (e.g. signaling cascade) in the cell. In some embodiments, the inhibitor is a phosphatase inhibitor. Examples of phosphatase inhibitors include, but are not limited to H2O2, siRNA, miRNA, Cantharidin, (−)-p-Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium Tartrate, Imidazole, Sodium Fluoride, β-Glycerophosphate, Sodium Pyrophosphate Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), mpV(pic), DMHV, Cypermethrin, Dephostatin, Okadaic Acid, NIPP-1, N-(9,10-Dioxo-9,10-dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide, α-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br, α-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br, α-Bromo-4-(carboxymethoxy)acetophenone, 4-(Carboxymethoxy)phenacyl Br, and bis(4-Trifluoromethylsulfonamidophenyl)-1,4-diisopropylbenzene, phenylarsine oxide, Pyrrolidine Dithiocarbamate, and Aluminium fluoride. In some embodiments, the phosphatase inhibitor is H2O2.
Examples of modulators include but are not limited to IL-3, IL-27, IL-6, IL-10, IFN-α, IFN-γ, G-CSF, GM-CSF, EPO, TPO, FLT3L, SCF, SDF-1α, IGF, TRAIL, FASL, TNF, TNFα, Ara-C, Daunorubicin, Etoposide, Staurosporine, Imatinib and salts thereof (marketed as Gleevec), Gemtuzumab (such as Gemtuzumab ozogamicin, marketed as Mylotarg), Azacitidine (marketed as Vidaza), Decitabine (marketed as Dacogen), Vorinostat (marketed as Zolinza), and Thapsigargin, H2O2, and PMA.
In some embodiments, the kits further comprise one or more detection elements, e.g., fluorescent molecules (fluorophores), that can be conjugated to the binding elements that will be used to analyze nodes by technologies including but not limited to flow cytometry. Fluorophores bound to antibody or other binding element can be activated by a laser and re-emit light of a different wavelength. The amount of light detected from the fluorophores is related to the number of binding element targets associated with the cell passing through the beam. Any specific set of detection elements, e.g. fluorescently tagged antibodies, in any embodiment can depend on the types of cells to be studied and the presence of the activatable element within those cells. Several detection elements, e.g. fluorophore-conjugated antibodies, can be used simultaneously, so measurements made as one cell passes through the laser beam consist of scattered light intensities as well as light intensities from each of the fluorophores. Thus, the characterization of a single cell can consist of a set of measured light intensities that may be represented as a coordinate position in a multi-dimensional space. Considering only the light from the fluorophores, there is one coordinate axis corresponding to each of the detection elements, e.g. fluorescently tagged antibodies. The number of coordinate axes (the dimension of the space) is the number of fluorophores used. Modern flow cytometers can measure several colors associated with different fluorophores and thousands of cells per second. Thus, the data from one subject can be described by a collection of measurements related to the number of antigens for each of (typically) many thousands of individual cells. See Krutzik et al., High-content single-cell drug screening with phosphospecific flow cytometry. Nature Chemical Biology, Vol. 4 No. 2, Pgs. 132-42, February 2008. Such methods may optionally include the use of barcoding to increase throughput and reduce consumable consumption. See Krutzik, P. and Nolan, G., Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nature Methods, Vol. 3 No. 5, Pgs. 361-68, May 2006.
Typically detection elements have fluorescent properties either alone or in combination with a secondary element that can be detected. Detection elements can also report through enzymatic activity, such as peroxidase activity, instead of fluorescence.
Suitable fluorescent detection elements include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705 and Oregon green. Suitable optical dyes are described in the 1996 Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference. Suitable fluorescent labels also include, but are not limited to, green fluorescent protein (GFP; Chalfie, et al., Science 263(5148):802-805 (Feb. 11, 1994); and EGFP; Clontech—Genbank Accession Number U55762), blue fluorescent protein (BFP; 1. Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; 2. Stauber, R. H. Biotechniques 24(3):462-471 (1998); 3. Heim, R. and Tsien, R. Y. Curr. Biol. 6:178-182 (1996)), enhanced yellow fluorescent protein (EYFP; 1. Clontech Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, Calif. 94303), luciferase (Ichiki, et al., J. Immunol. 150(12):5408-5417 (1993)), β-galactosidase (Nolan, et al., Proc Natl Acad Sci USA 85(8):2603-2607 (April 1988)) and Renilla WO 92/15673; WO 95/07463; WO 98/14605; WO 98/26277; WO 99/49019; U.S. Pat. No. 5,292,658; U.S. Pat. No. 5,418,155; U.S. Pat. No. 5,683,888; U.S. Pat. No. 5,741,668; U.S. Pat. No. 5,777,079; U.S. Pat. No. 5,804,387; U.S. Pat. No. 5,874,304; U.S. Pat. No. 5,876,995; and U.S. Pat. No. 5,925,558). All of the above-cited references are expressly incorporated herein by reference.
In some embodiments, detection elements for use in the present invention include: Alexa-Fluor dyes (an exemplary list including Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, and Alexa Fluor® 750), Cascade Blue, Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes) (Eugene, Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Tandem conjugate protocols for Cy5PE, Cy5.5PE, Cy7PE, Cy5.5APC, Cy7APC are known in the art. Quantization of fluorescent probe conjugation may be assessed to determine degree of labeling and protocols including dye spectral properties are also well known in the art. In some embodiments the fluorescent label is conjugated to an aminodextran linker which is conjugated to a binding element. Additional labels listed in and are availabel through the on-line and hard copy catalogues of BD Biosciences, Beckman Coulter, AnaSpec, Invitrogen, Cell Signaling Technology, Millipore, eBioscience, Caltag, Santa Cruz Biotech, Abcam and Sigma, the contents of which are incorporated herein by reference.
The kits of the invention can provide binding elements useful for detection protocols that can be carried out by a person, such as a technician in the laboratory. Alternatively, the detection of the binding elements can be carried out using automated systems. In either case, the detection of binding elements for use according to the kits of this invention can be performed according to standard techniques and protocols well-established in the art.
One or more binding elements can be detected and/or quantified by any method that detect and/or quantitates the presence of the activatable element of interest. Such methods can include radioimmunoassay (RIA) or enzyme linked immunoabsorbance assay (ELISA), immunohistochemistry, immunofluorescent histochemistry with or without confocal microscopy, reversed phase assays, homogeneous enzyme immunoassays, and related non-enzymatic techniques, Western blots, whole cell staining, immunoelectronmicroscopy, nucleic acid amplification, gene array, protein array, mass spectrometry, patch clamp, 2-dimensional gel electrophoresis, differential display gel electrophoresis, microsphere-based multiplex protein assays, label-free cellular assays and flow cytometry, etc. U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for modified protein parameters. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Flow cytometry methods are useful for measuring intracellular parameters.
A kit of the present invention can comprise binding elements that can be analyzed by flow cytometry.
When using fluorescent detection elements in a kit of the present invention, different types of fluorescent monitoring systems, e.g., Cytometric measurement device systems, can be used to detect the binding elements. In some embodiments, a kit of the invention can be used in flow cytometric systems dedicated to high throughput screening, e.g. 96 well or greater microtiter plates. Methods of performing assays on fluorescent materials are well known in the art and are described in, e.g., Lakowicz, J. R., Principles of Fluorescence Spectroscopy, New York: Plenum Press (1983); Herman, B., Resonance energy transfer microscopy, in: Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D. L. & Wang, Y.-L., San Diego: Academic Press (1989), pp. 219-243; Turco, N.J., Modern Molecular Photochemistry, Menlo Park: Benjamin/Cummings Publishing Col, Inc. (1978), pp. 296-361.
In some embodiments, fluorescence of the binding elements of a kit can be measured using a fluorimeter. Other methods of detecting fluorescence can also be used, e.g., Quantum dot methods (see, e.g., Goldman et al., J. Am. Chem. Soc. (2002) 124:6378-82; Pathak et al. J. Am. Chem. Soc. (2001) 123:4103-4; and Remade et al., Proc. Natl. Sci. USA (2000) 18:553-8, each expressly incorporated herein by reference) as well as confocal microscopy.
In some embodiments, the binding elements of a kit described herein can be detected using Inductively Coupled Plasma Mass Spectrometer (ICP-MS). (Tanner et al. Spectrochimica Acta Part B: Atomic Spectroscopy, 2007 March; 62(3):188-195.).
In some embodiments, a kit of the instant invention can be used in conjunction with an “In-Cell Western Assay.” In some embodiments, the detecting can be by high pressure liquid chromatography (HPLC), for example, reverse phase HPLC, and in a further aspect, the detecting can be by mass spectrometry.
Iteratively Selecting Binding Elements, Protocols, Detection Elements and Modulators
In some embodiments, the binding elements, protocols, detection elements and modulators in a kit are iteratively evaluated and selected to produce data that distinguishes the different activation states of one or more activation elements. In these embodiments, activation state data can be produced by quantifying the relative amount of the detection element associated with a binding element. The activation state data can then be analyzed to identify performance metrics that characterize the “goodness” of the activation state data. Performance metrics can include but are not limited to: a degree of separation between activation state data associated with activation states, uniformity of the activation state data associated with a same activation state, a degree of association between activation state data and a known characteristic of a cell population. Characteristics of a cell population can include but are not limited to: a cell type/sub-type of the population, a disease state of the cell population, a prognosis of the cell population, a therapeutic response of the cell population and a genotype of the cell population.
In some embodiments, the binding elements, reagents, detection elements and/or modulators are titrated over a set of increasing concentrations to produce different sets of activation state data. The activation state data is then analyzed to determine an optimal concentration of the binding elements, reagents, detection elements and/or modulators. In some embodiments, various combinations of binding elements, reagents, detection elements and modulators used to generate activation state data are evaluated.
According to the specific embodiment, different types of computational analyses can be performed to generate performance metrics based on the activation state data for the binding elements, reagents, detection elements and/or modulators. These computational analyses can be performed using program code executed by a computer comprising a memory and a processor. In most embodiments, the computer can be communicatively coupled with a machine that performs quantification of the detectable element such as a flow cytometer and/or a mass spectrometer. In some embodiments, the activation state data can be pre-processed to generate state metrics that compare the activation state data associated to control data (e.g. data derived from cells that are untreated with a modulator)
In some embodiments, the activation state data can be analyzed to characterize probability density data associated with different activation states. In some embodiments, the activation state data can be gated to identify discrete activation states associated with individual cells and the gated data can be analyzed to identify a degree of separation between the activation states and the degree of uniformity of the activation state data within a same activation states. Suitable methods of gating are outlined in U.S. patent application Ser. No. 12/501,295, the entirety of which is incorporated herein, for all purposes. In some embodiments, histograms can be used to identify the separation between the activation states in the activation state data. Other methods for analyzing probability density data associated with different activation states include binning algorithms. Suitable binning algorithms are outlined in U.S. Publication No. 2009/0307248, the entirety of which is incorporated herein, for all purposes.
In some embodiments, the activation state data can be associated with a characteristic of a cell population and the statistical strength of the association evaluated. Different methods of evaluating the statistical strength of the association include receiver operator curves (ROC curves), correlation analysis, hazard models and classification algorithms. In some instances, the activation state data represents two activation states used to discriminate between two different cell populations. In these instances, the accuracy and sensitivity of the discrimination between the different cell populations can be also evaluated. According to the embodiments, the activation state data that is associated with the characteristic of the cell population can be based on small number of samples (e.g. a proof-of-principle experiment) or a very large number of samples (e.g. hundreds, thousands, millions of samples). In instances where a large number of samples are used, the activation state data can be derived from patient samples and associated with characteristics based on clinical data such as diagnosis, prognosis, genotype and therapeutic response.
In some embodiments, a kit of the invention can be employed to monitor the status of an activatable element in a signaling pathway (activatable element is defined in U.S. Pat. No. 7,393,656 B2, which is hereby incorporated by reference). Signaling pathways and their members have been described extensively. See Hunter, T., Cell (2000) 100: 113-27. The activatable elements monitored include, but are not limited to, elements and regulators of the following signaling pathways: JAK/STAT, PI3K/Akt, PKC, MAP Kinase signaling (Erk, JNK and p38), Ras/Raf, Src, Notch, Hedgehog, WNT signaling pathways. Signaling pathways can be measured in the contest of chemokine signaling (including, for example SDF-1/CXCL12-CXCR4 signaling), DNA damage response, cell cycle regulation, intrinsic apoptosis, and extrinsic apoptosis. Signaling pathways may be measured in response to receptor signaling such as BCR (B cell Receptor), death receptors such as the TNFR family receptors (including, but not limited to, TNFR, TNRF2, CD30, CD40, BAFF-R, TACI, and BCMA), Toll-like Receptor (TLR), and c-KIT/Stem Cell Factor (SCF)/SCF-R (SCF-Receptor).
a. Janus Kinase (JAK)/Signal Transducers and Activators of Transcription (STAT) Pathway:
The JAK/STAT pathway mediates signaling in response to a wide variety of extracellular inputs, including numerous cytokines (See Alberts, et al, IV.15: Signaling through Enzyme-Linked Cell-Surface Receptors). Janus Kinases (JAK) are a membrane-bound receptor tyrosine kinase, and Signal Transducers and Activators of Transcriptions (STATs) are a class of transcription factor that transduce the JAK signal from the cytoplasm directly to the nucleus. Ligand binding to JAK results in receptor dimerization and activation through autophosphorylation of tyrosine residues. Activated JAK subsequently recruits STAT through its SH2 domain and activates it by phosphorylating conserved tyrosine residues, mediating the formation of STAT homodimers. STAT homodimers then translocate to the nucleus, where they act as transcriptional regulators by binding to specific DNA sequences.
There are four known JAK family members in mammals: JAK1, JAK2, JAK3 and TYK2. JAK1, JAK2 and TYK2 are expressed ubiquitously, but JAK3 is only expressed in hematopoietic cells. The JAK2V617F point mutation within the JAK2 JH2 domain, along with several mutations in exon 12 of JAK2 produce constitutive kinase activity, and are associated with myeloid malignancies (Levine, R. L. and Gilliland, D. G., Myeloproliferative disorders, Blood (2008) 112: 2190-98.). Several gain-of-function mutations in JAK3 are associated with acute megakaryoblastic myeloid leukemia (Constantinescu S. N., et al. Mining for JAK-STAT mutations in cancer. Trends Biochem Sci. (2008) 33:122-131.
There are seven members of the STAT family (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and STATE) in mammals. The STAT family of proteins, especially STAT3 and STAT5, are emerging as important players in several cancers. (Yu 2004—STATs in cancer. (2008) pp. 9). Of particular relevance to AML, the STATs have been shown to be critical for myeloid differentiation and survival, as well as for long-term maintenance of normal and leukemic stem cells. (Schepers et al. STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells. Blood (2007) vol. 110 (8) pp. 2880-2888). STAT signaling is activated by several cytokine receptors, which are differentially expressed depending on the cell type and the stage of differentiation. Intrinsic or receptor-associated tyrosine kinases, including but not limited to JAKs, phosphorylate STAT proteins, which induces the formation of STAT homodimers. The activated STAT dimer is able to enter the cell nucleus and activate the transcription of target genes, many of which are involved in the regulation of apoptosis and cell cycle progression. Apart from promoting proliferation and survival, some growth factor receptors and signaling intermediates have been shown to play specific and important roles in myeloid differentiation. For example, G-CSFR- or TPO-induced Ras-activation promotes myeloid or megakaryocytic differentiation in the respective progenitor cells by the activation of c/EBPα (frequently inactivated in myeloid leukemia) and GATA-1, respectively. (Steffen, B. et al. Critical Reviews in Oncology/Hematology. 2005, vol. 56, p. 195-221).
The STAT family of proteins has been implicated in a number of cancers, and STAT3 and STAT5 have been shown to have strong oncogenic potential. (Yu, H. and Jove, R. STATs in cancer. Nat. Rev. Cancer (2008) 4: 97-105). Of particular relevance to AML, the STATs have been shown to be critical for myeloid differentiation and survival, as well as for long-term maintenance of normal and leukemic stem cells. (Schepers et al. STAT5 is required for long-term maintenance of normal and leukemic human stem/progenitor cells. Blood (2007) vol. 110 (8) pp. 2880-2888). In contrast to STAT3 and STAT5, STAT1 negatively regulates cell proliferation and angiogenesis and thereby inhibits tumor formation. Consistent with its function as a tumor suppressor, expression of STAT1 and its downstream targets is reduced in a variety of human tumors (Rawlings, J., The JAK/STAT signaling pathway, J Cell Sci. (2004) 117:1281-83, hereby fully incorporated by reference in its entirety for all purposes). Furthermore, a recent study of Primary mediastinal B-cell lymphoma (PMBL) found that 20 out of 55 (36%) PMBL patients exhibited mutations in the DNA binding domain of STAT6 (Ritz, O., et al. Recurrent mutations of the STAT6 DNA binding domain in primary mediastinal B-cell lymphoma. Blood (2009). doi:10.1182/blood-2009-03-209759).