Methods for diagnosis, prognosis and methods of treatment
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. A method of drug screening, said method comprising:
classifying one or more hematopoietic cells associated with acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in an 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 at least three pathways in one or more cells from said plurality of cultures by determining an activation level of at least one activatable element within the at least three pathways, wherein
i) at least two of the pathways being characterized are an apoptosis and a DNA damage pathway,
ii) the modulators activate or inhibit one or more of said at least three pathways being characterized, and
c) classifying said one or more hematopoietic cells into groups comprising response or non-response to therapeutic treatment, or risk of relapse based on said pathways characterization, said classification enabling a decision regarding the test compound and its therapeutic potential for the treatment of acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms, wherein said decision is based on said classification of said cells.
2. The method of claim 1 wherein said acute leukemia is acute myeloid leukemia.
3. The method of claim 1 wherein the pathways are selected from the group consisting of apoptosis, cell cycle, signaling, and DNA damage pathways.
4. The method of claim 1 , wherein the individual has a predefined clinical parameter selected from the group consisting of age, de novo acute myeloid leukemia patient, secondary acute myeloid leukemia patient, and a biochemical/molecular marker.
5. The method of claim 4 , wherein said decision is based on said classification of said cells in combination with said predefined clinical parameter.
6. The method of claim 1 , wherein said modulators are independently selected from the group consisting of growth factor, mitogen, cytokine, chemokine, adhesion molecule modulator, hormone, small molecule, polynucleotide, antibody, natural compound, lactone, chemotherapeutic agent, immune modulator, carbohydrate, protease, ion, reactive oxygen species, and radiation.
7. The method of claim 1 wherein said modulators are independently selected from the group consisting of FLT3L, GM-CSF, SCF, G-CSF, SDF1a, LPS, PMA, Thapsigargin, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, ZVAD, H 2 O 2 , Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
8. The method of claim 1 wherein said activation level is based on an 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.
9. The method of claim 8 wherein said activation state is a phosphorylation state.
10. The method of claim 1 , wherein said activatable element is selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids and metabolites.
11. The method of claim 10 wherein said activatable element is a protein capable of being phosphorylated and/or dephosphorylated.
12. The method of claim 11 wherein said 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, p-p65/Rel-A, p-Akt, p-S6, p-ERK, Cleaved Caspase 8, Cleaved Caspase 3, Cytoplasmic Cytochrome C, and p38.
13. The method of claim 1 , wherein said method further comprises determining the presence or absence of one or more cell surface markers, intracellular markers, or a combination thereof.
14. The method of claim 13 wherein said one or more cell surface markers and said intracellular markers are independently selected from the group consisting of proteins, carbohydrates, lipids, nucleic acids and metabolites.
15. The method of claim 13 wherein said determining the presence or absence of one or more cell surface markers or intracellular markers comprises determining the presence or absence of an epitope in both activated and non-activated forms of said one or more cell surface markers or said intracellular markers.
16. The method of claim 13 , wherein diagnosing, prognosing or determining progression of acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual is based on both the activation level of said activatable element and the presence or absence of said one or more cell surface markers, intracellular markers, or combination thereof.
17. The method of claim 1 , wherein said activation level is determined by a process comprising binding of a binding element which is specific to a particular activation state of a particular activatable element.
18. The method of claim 17 , wherein said binding element comprises an antibody.
19. The method of claim 1 , wherein the step of determining the activation level comprises use of flow cytometry, immunofluorescence, confocal microscopy, immunohistochemistry, 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, ELISA, or label-free cellular assays to determine the activation level of one or more intracellular activatable elements in single cells.
20. The method of claim 1 , wherein the at least three pathways are selected from apoptosis, cell cycle, signaling, or DNA damage pathways, further comprising determining whether the apoptosis, cell cycle, signaling, or DNA damage pathways, are functional in said individual based on the activation level of said at least one activatable element, wherein if the apoptosis and DNA damage pathways are functional the individual is predicted to respond to treatment.
21. The method of claim 1 , further comprising determining a level of a cytokine receptor, growth factor receptor and/or a drug transporter in said one or more cells.
22. The method of claim 21 , wherein said cytokine receptor, growth factor receptor or drug transporter are selected from the group consisting of MDR1, ABCG2, MRP, P-Glycoprotein, CXCR4, FLT3L, and c-kit.
23. The method of claim 21 , wherein the level of said cytokine receptor and/or said drug transporter in combination with said cell classification and the clinical parameter are indicative of a diagnosis, prognosis or progression of acute myeloid leukemia, myelodysplastic syndrome or myeloproliferative neoplasms.
24. A method of drug screening, said method comprising:
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 separate cultures, wherein:
i) a first modulator is a growth factor or a mitogen,
ii) a second modulator is a cytokine,
iii) a third modulator is a modulator that slows or stops the growth of cells, and/or induces apoptosis of cells, and/or is an inhibitor of a cellular function,
b) determining an activation level of at least one activatable element in one or more cells from each of said separate cultures, wherein the at least one activatable element is within the PI3K/AKT or MAPK pathways and the activation level is measured in response to said growth factor or mitogen, and
c) classifying said one or more hematopoietic cells into groups comprising response or non-response to therapeutic treatment, or risk or relapse, based on said activation level of said at least one activatable element, said classification enabling a decision regarding the test compound and its therapeutic potential to treat acute leukemia, myelodysplastic syndrome or myeloproliferative neoplasms in said individual based on said classification of said one or more hematopoietic cells.
25. The method of claim 24 wherein said acute leukemia is acute myeloid leukemia.
26. The method of claim 24 wherein said activatable element within the PI3K/AKT or MAPK pathway is selected from the group consisting of p-Akt, p-ERK, p38, p-Creb, and pS6 and said growth factor or mitogen is selected from the group consisting of FLT3L, SCF, G-CSF, SDF1a, LPS, PMA, and Thapsigargin.
27. The method of claim 24 wherein said activatable element is within an apoptosis pathway and said activatable element is selected from the group consisting of cleaved Parp, Cleaved Caspase 3, Cleaved Caspase 8, and Cytochrome C, and said modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
28. The method of claim 24 further comprising determining an activation level of an activatable element within a DNA damage pathway or a cell cycle pathway in response to a modulator that slows or stops the growth of cells and/or induces apoptosis of cells.
29. The method of claim 28 wherein said activatable element within a DNA damage pathway is selected from the group consisting of Chk1, Chk2, ATR, ATM, and 14-3-3 and said modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
30. The method of claim 24 further comprising determining a level of a drug transporter, growth factor receptor and/or a cytokine receptor.
31. The method of claim 28 wherein an said at least one activatable element in the PI3K/Akt pathway is Akt and said growth factor is FLT3L.
32. The method of claim 24 wherein said growth factor or mitogen is selected from the group consisting of FLT3L, SCF, G-CSF, SDFla, LPS, PMA, and Thapsigargin.
33. The method of claim 24 wherein said modulator that slows or stops the growth of cells and/or induces apoptosis of cells is selected from the group consisting of Staurosporine, Etoposide, Mylotarg, Daunorubicin, and AraC.
34. The method of claim 24 wherein said activation level is determined by a process comprising binding of a binding element which is specific to a particular activation state of a particular activatable element.
35. The method of claim 34 wherein said binding element comprises an antibody.
36. The method of claim 24 wherein the step of determining the activation level comprises use of flow cytometry, immunofluorescence, confocal microscopy, immunohistochemistry, 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, ELISA, or label-free cellular assays to determine the activation level of one or more intracellular activatable elements in single cells.