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 . A method for determining the activation state of a signal transduction pathway signaling protein in a leukocyte-containing sample comprising:
 (a) activating the activatable proteins of at least one signal transduction pathway in the leukocytes of the sample by exposing the leukocyte-containing sample to a pan-kinase activator;   (b) preserving the activated sample with a preservative;   (c) unmasking intracellular epitopes of the preserved leukocytes in the sample;   (d) contacting the unmasked intracellular epitopes of the preserved leukocytes with a plurality of fluorescently labeled capture molecules, said plurality of capture molecules comprising at least two different capture molecules capable of binding to the activated state of at least two different unmasked intracellular epitopes of preserved, activated leukocytes in the sample and at least one control capture molecule, wherein the control capture molecule binds to an epitope on the preserved leukocytes that is unactivated by the pan-kinase activator;   (e) detecting fluorescence of the preserved, activated leukocytes captured by the binding of the capture molecules to the activated state of the unmasked intracellular epitopes;   (f) detecting fluorescence of the preserved leukocytes captured by the binding of the control capture molecule; and   (g) comparing the fluorescence of the detected preserved, activated leukocytes captured by the capture molecules to the fluorescence of the detected preserved leukocytes captured by the control capture molecule.   
     
     
         2 . The method of  claim 1 , further comprising: h) evaluating the compared fluorescence measured in step g) against compared fluorescence measured in an unactivated reference sample. 
     
     
         3 . The method of  claim 2 , wherein the unactivated reference sample is a second aliquot of the sample. 
     
     
         4 . The method of  claim 2 , wherein the unactivated reference sample is a standardized reference sample. 
     
     
         5 . The method of  claim 1 , wherein the activation is performed for about 1 minute to about 10 minutes. 
     
     
         6 . The method of  claim 1 , wherein the activation is performed for at least about 30 minutes. 
     
     
         7 . The method of  claim 2 , wherein the sample is from a patient and the evaluation of the fluorescence indicates that the patient has a signal transduction associated disease or condition when the fluorescence of the activated and unactivated samples are approximately comparable. 
     
     
         8 . The method of  claim 7 , wherein the signal transduction associated disease or condition is inflammation, autoimmune, allergic, fever, sepsis, cancer, diabetes, or heart failure. 
     
     
         9 . The method of  claim 8 , wherein the signal transduction associated disease or condition is sepsis. 
     
     
         10 . The method of  claim 8 , further comprising repeating steps a) to g) with a sample from the patient after the patient has received a therapeutic agent to treat the inflammation, fever, sepsis, cancer, diabetes, or heart failure and monitoring the effectiveness of that therapeutic agent by monitoring for a change in the detected fluorescence between the activated and unactivated samples. 
     
     
         11 . The method of  claim 8 , wherein the sample is from a patient receiving a kinase inhibitor. 
     
     
         12 . The method of  claim 11 , wherein the evaluating of the compared fluorescence indicates that the kinase inhibitor is effective in treating the signal transduction associated disease or condition patient when a change is determined in the detected fluorescence between the activated and unactivated samples. 
     
     
         13 . The method of  claim 2 , wherein the sample has been exposed to a putative kinase inhibitor and the method further comprises ascertaining the effectiveness of the kinase inhibitor when the activated sample does not demonstrate a change in fluorescence of the activatable proteins of the at least one signal transduction pathway. 
     
     
         14 . The method of  claim 13 , wherein the putative kinase inhibitor is a putative inhibitor of ERK or PI3K, further comprising monitoring for the inhibition of ribosomal S6, wherein inhibition of both ERK and PI3K are required for ribosomal S6 inhibition by:
 i) exposing the sample to a known ERK inhibitor and a putative PI3K inhibitor and monitoring for ribosomal S6 inhibition; or   ii) exposing the sample to a known PI3K inhibitor and a putative ERK inhibitor and monitoring for ribosomal S6 inhibition.   
     
     
         15 . The method of  claim 1 , comprising measuring the activity of at least a second signal transduction pathway. 
     
     
         16 . The method of  claim 1 , wherein said intracellular epitopes comprise phosphorylated epitopes. 
     
     
         17 . The method of  claim 1 , wherein said unmasking comprises contacting the fixed cells with an alcohol and a detergent. 
     
     
         18 . The method of  claim 16 , wherein said alcohol is added at a concentration between approximately 25 percent and approximately 90 percent. 
     
     
         19 . The method of  claim 17 , wherein said alcohol is selected from the group consisting of ethanol and methanol. 
     
     
         20 . The method of  claim 1 , wherein said preservative is aldehyde, paraformaldehyde, or formaldehyde. 
     
     
         21 . The method of  claim 16 , wherein said detergent is at a concentration between approximately 0.1 percent and approximately 10 percent. 
     
     
         22 . The method of  claim 21 , wherein said detergent is selected from the group consisting of Triton X-100, Nonidet P-40 (NP-40), and Brij-58. 
     
     
         23 . The method of  claim 1 , wherein said detection is accomplished by cytometry. 
     
     
         24 . The method of  claim 1 , wherein said signal transduction pathway protein is selected from the group consisting of PI3K, ribosomal S6 protein, p44/42 MAP kinase, TYK2, p38 MAP kinase, PKC, PKA, SAPK, ELK, JNK, cJun, RAS, Raf, MEK 1/2, MEK 3/6, MEK 4/7, ZAP-70, LAT, SRC, LCK, ERK 1/2, Rsk 1, PYK2, SYK, PDK1, GSK3, FKHR, AFX, PLCg, PLCγ, FAK, CREB, aIIIβ3, FcsRI, BAD, p70S6K, STAT1, STAT2, STAT3, STATS, STATE, and combinations thereof. 
     
     
         25 . The method of  claim 24 , wherein said signal transduction pathway proteins are p38 and ERK and PI3K or ribosomal S6. 
     
     
         26 . The method of  claim 24 , wherein said signal transduction pathway proteins are p38, ERK, and ribosomal S6. 
     
     
         27 . The method of  claim 24 , wherein the first protein is JNK and the second protein is ribosomal S6. 
     
     
         28 . The method of  claim 1 , wherein said pan-kinase activator is a toll-like receptor 4 (TLR4) activator or lipopolysaccharide (LPS). 
     
     
         29 . The method of  claim 1 , wherein the capture molecule is an antibody or antigen binding fragment thereof. 
     
     
         30 . The method of  claim 29 , wherein said antibody is specific for a phosphorylation state of said signal transduction pathway protein. 
     
     
         31 . The method of  claim 30 , wherein said phosphorylation-state-specific antibody is selected from the group consisting of anti-phospho-p44/42 MAP kinase (Thr202/Tyr204), anti-phospho-TYK2 (Tyr1054/1055), anti-phospho-p38 MAP kinase (Thr180/Tyr182), phospho-PKC-PAN substrate antibody, phospho-PKA-substrate antibody, anti-phospho-SAPK/JNK (Thr183/Tyr185), anti-phospho-tyrosine (P-tyr-100), anti-p44/42 MAPK, anti-phospho-MEK1/2 (Ser217/221), anti-phospho-p90RSK (Ser381), anti-p38 MAPK, anti-JNK/SAPK, anti-phospho-Raf1 (Ser259), anti-phosphoElk-1 (Ser383), anti-phospho-CREB (Ser133), anti-phosphoSEK1/MKK4 (Thr261), anti-phospho-Jun (Ser 63), anti-phosphoMKK3/MKK6 (Ser189/207), anti-AKT, anti-phospho FKHR, anti-FKHR, anti-phospho-Gsk3 alp21, anti-pAFX, anti-PARP, anti-BAD, anti-BADser 112, anti-BADser 136, anti-phospho-BADser 155, anti-p27, anti-p21, anti-cFLIP, antiMYC, anti-p53, anti-NFKB, anti-Ikkα, anti-Ikkβ, anti-phospho-tyrosine, and anti-phospho-threonine. 
     
     
         32 . The method of  claim 1 , wherein said fluorescent label is selected from the group consisting of a chemiluminescent label and FRET label. 
     
     
         33 . The method of  claim 1 , wherein the sample is whole blood. 
     
     
         34 . The method of  claim 1 , wherein the sample comprises the leukocytes isolated from a whole blood sample. 
     
     
         35 . A kit for monitoring the activation state of a signal transduction pathway comprising: a) a pan-kinase activator; and b) at least two different capture molecules that bind at least one signal transduction pathway protein selected from the group consisting of p38, ERK, PI3K, JNK, and ribosomal S6, wherein at least one of the capture molecules binds to either PI3K, JNK, or ribosomal S6. 
     
     
         36 . The kit of  claim 36 , wherein said pan kinase activator is a toll-like receptor 4 activator or LPS. 
     
     
         37 . The kit of  claim 36 , wherein at least one of the capture molecules binds to either PI3K, JNK, or
 p38 and another of the capture molecules binds to ribosomal S6.   
     
     
         38 . A method for determining the activation state of a signal transduction pathway signaling protein in a leukocyte-containing sample, said method comprising:
 (a) activating the activatable proteins of at least one signal transduction pathway in the leukocytes of the sample by exposing the leukocyte-containing sample to a pan-kinase activator;   (b) permeabilizing the activated sample;   (c) contacting the permeabilized sample with a plurality of fluorescently labeled binding elements, said plurality of binding comprising at least two different state-specific binding element capable of binding to the activated state of at least two different intracellular epitopes of permeabilized, activated leukocytes in the sample and at least one control binding element, wherein the control binding element binds to an epitope on the permeabilized leukocytes that is unactivated by the pan-kinase activator;   (d) detecting fluorescence of the permeabilized, activated leukocytes captured by the binding of the binding elements to the activated state of the intracellular epitopes;   (e) detecting fluorescence of the permeabilized leukocytes captured by the binding of the control binding element; and   (f) comparing the fluorescence of the detected permeabilized, activated leukocytes captured by the binding elements to the fluorescence of the detected permeabilized leukocytes captured by the control binding element.