Methods for diagnosis, prognosis and methods of treatment

Abstract

The present invention provides an approach for the determination of the activation states of a plurality of proteins in single cells. This approach permits the rapid detection of heterogeneity in a complex cell population based on activation states, expression markers and other criteria, and the identification of cellular subsets that exhibit correlated changes in activation within the cell population. Moreover, this approach allows the correlation of cellular activities or properties. In addition, the use of modulators of cellular activation allows for characterization of pathways and cell populations. Several exemplary diseases that can be analyzed using the invention include AML, MDS, and MPN.

1 - 69 . (canceled) 
     
     
         70 . A method of drug screening, said method comprising:
 A] classifying one or more hematopoietic cells associated with acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual by a method comprising:   a) subjecting a cell population comprising said one or more hematopoietic cells from said individual to a test compound and a plurality of modulators in a plurality of cultures,   b) characterizing a plurality of pathways in one or more cells from said plurality of cultures by determining an activation level of at least one activatable element within said plurality of pathways, wherein   i) at least one of said plurality of pathways being characterized in at least one of said plurality of cultures is an apoptosis or a DNA damage pathway,   ii) said plurality of modulators activate or inhibit one or more of said plurality of pathways being characterized, and   c) classifying said one or more hematopoietic cells based on said pathways characterization; and   B] making a decision regarding said test compound and its therapeutic potential for treatment of acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms, wherein said decision is based on said classification of said one or more hematopoietic cells.   
     
     
         71 . The method of  claim 70 , wherein A] further comprises determining levels of a cytokine receptor and/or a drug transporter in said one or more hematopoietic cells. 
     
     
         72 . The method of  claim 70 , wherein said individual has a predefined clinical parameter selected from the group consisting of age, de novo acute myeloid leukemia, secondary acute myeloid leukemia, and a biochemical/molecular marker. 
     
     
         73 . The method of  claim 70 , wherein said plurality of modulators comprise a growth factor, a mitogen, a cytokine, a chemokine, an adhesion molecule modulator, a hormone, a small molecule, a polynucleotide, an antibody, a natural compound, a lactone, a chemotherapeutic agent, an immune modulator, a carbohydrate, a protease, an ion, a reactive oxygen species, or radiation. 
     
     
         74 . The method of  claim 70 , wherein said plurality of modulators comprise SDF-1α, IFN-α, IFN-γ, IL-10, IL-6, IL-27, G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, Thapsigargin, H 2 O 2 , Etoposide, Mylotarg, AraC, daunorubicin, staurosporine, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (ZVAD), lenalidomide, EPO, azacitadine, decitabine, IL-3, IL-4, GM-CSF, EPO, LPS, TNF-α, or CD40L. 
     
     
         75 . The method of  claim 70 , wherein said activation level of said at least one activatable element is determined based on the activation state selected from the group consisting of extracellular protease exposure, novel hetero-oligomer formation, glycosylation state, phosphorylation state, acetylation state, methylation state, biotinylation state, glutamylation state, glycylation state, hydroxylation state, isomerization state, prenylation state, myristoylation state, lipoylation state, phosphopantetheinylation state, sulfation state, ISGylation state, nitrosylation state, palmitoylation state, SUMOylation state, ubiquitination state, neddylation state, citrullination state, deamidation state, disulfide bond formation state, proteolytic cleavage state, translocation state, changes in protein turnover, multi-protein complex state, oxidation state, multi-lipid complex, and biochemical changes in cell membrane. 
     
     
         76 . The method of  claim 70 , wherein said activatable element is selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids, and metabolites. 
     
     
         77 . The method of  claim 76 , wherein said at least one activatable element is a protein capable of being phosphorylated. 
     
     
         78 . The method of  claim 76 , wherein said at least one activatable element is a protein selected from the group consisting of p-Slp-76, p-Plcg2, p-Stat3, p-Stat5, p-Stat1, p-Stat6, p-Creb, cleaved Parp, p-Chk2, p65/Rel-A, p-Akt, p-S6, p-ERK, Cleaved Caspase 8, Cleaved Caspase 3, Cytoplasmic Cytochrome C, and p38. 
     
     
         79 . The method of  claim 70 , wherein said activation level of said at least one activatable element is determined by a process comprising binding of a binding element that is specific to a particular activation state of said at least one activatable element. 
     
     
         80 . The method of  claim 79 , wherein said binding element comprises an antibody. 
     
     
         81 . The method of  claim 70 , wherein A] further comprises determining a presence or absence of one or more cell surface markers, intracellular markers, or a combination thereof. 
     
     
         82 . The method of  claim 70 , wherein said individual is under 60 years old. 
     
     
         83 . The method of  claim 82 , wherein said at least one activatable element are selected from activatable elements listed in table 6. 
     
     
         84 . The method of  claim 70 , wherein said individual is over 60 years old. 
     
     
         85 . The method of  claim 84 , wherein said at least one activatable element are selected from activatable elements listed in table 7. 
     
     
         86 . The method of  claim 70 , wherein said individual has secondary acute myeloid leukemia. 
     
     
         87 . The method of  claim 86 , wherein said at least one activatable element are selected from activatable elements listed in table 8 and table 9. 
     
     
         88 . The method of  claim 70 , wherein said individual has de novo acute myeloid leukemia. 
     
     
         89 . The method of  claim 88 , wherein said at least one activatable element are selected from activatable elements listed in table 10 and table 11.