USOO9500655B2

(12) United States Patent (10) Patent No.: US 9,500,655 B2 Fant et al. (45) Date of Patent: *Nov. 22, 2016

(54) METHODS FOR DIAGNOSIS, PROGNOSIS (56) References Cited AND METHODS OF TREATMENT U.S. PATENT DOCUMENTS (71) Applicant: Nodality, Inc., South San Francisco, 4,469,863. A 9, 1984 Ts'o et al. CA (US) 4,568,649 A 2/1986 Bertoglio-Matte et al. 4,979,824 A 12/1990 Mathies et al. 5,137,809 A 8, 1992 Loken et al. (72) Inventors: Wendy J. Fantl, San Francisco, CA 5,216,141 A 6/1993 Benner (US); David B. Rosen, Mountain View, 5,234,816 A 8/1993 Terstappen CA (US); Alessandra Cesano, 5,386,023 A 1/1995 Sanghvi et al. Redwood City, CA (US); Santosh K. 5,599,681 A 2/1997 Epstein et al. 5,602,240 A 2f1997 De Mesmaeker et al. Putta, Foster City, CA (US); Garry 5,605,805 A 2f1997 Verwer et al. Nolan, San Francisco, CA (US); Aileen 5,637,684 A 6/1997 Cook et al. Cohen, Palo Alto, CA (US); Erik 5,644,048 A T/1997 Yalu Evensen, Foster City, CA (US) 5,919,646 A 7/1999 Okun et al. 5,968,738 A 10, 1999 Anderson et al. 6,232,299 B1 5, 2001 Jirousek et al. (73) Assignee: NODALITY, INC., South San 6,280,967 B1 8/2001 Ransom et al. Francisco, CA (US) 6,379,917 B1 4/2002 Okun et al. 6,495,333 B1 12/2002 Willmann et al. 6,506,551 B1 1/2003 ChioraZZi et al. (*) Notice: Subject to any disclaimer, the term of this 6,520,108 B1 2/2003 Komura patent is extended or adjusted under 35 6,558,916 B2 5/2003 Veerapandian et al. U.S.C. 154(b) by 0 days. 6,592,822 B1 7/2003 Chandler 6,673,554 B1 1/2004 Kauvar This patent is Subject to a terminal dis 6,733,743 B2 5, 2004 Jordan claimer. (Continued) (21) Appl. No.: 14/279,905 FOREIGN PATENT DOCUMENTS WO WO 99 44067 A1 9, 1999 (22) Filed: May 16, 2014 WO WO 99.54494 A2 10, 1999 WO WO O3,067210 A2 8, 2003 (65) Prior Publication Data WO WO O3,067210 A3 12/2003 WO WO 2006/O12507 A2 2, 2006 US 2015/OO17119 A1 Jan. 15, 2015 WO WO 2006/050333 A2 5, 2006 WO WO 2006/079092 A2 T 2006 WO WO 2006/086111 A2 8, 2006 Related U.S. Application Data (Continued) (60) Continuation of application No. 13/473,829, filed on May 17, 2012, now Pat. No. 8,778,620, which is a OTHER PUBLICATIONS division of application No. 12/460,029, filed on Jul. Halicka, et al. Histone H2AX phosphorylation after cell irradiation 10, 2009, now Pat. No. 8,227,202. with UV-B; relationship to cell cycle phase and induction of (60) Provisional application No. 61/120.320, filed on Dec. apoptosis Cell Cycle. Feb. 2005:4(2):339-45. Epub Feb. 21, 2005. 5, 2008, provisional application No. 61/104,666, filed (Continued) on Oct. 10, 2008, provisional application No. 61/085,789, filed on Aug. 1, 2008, provisional Primary Examiner — Daniel C Gamett application No. 61/079,766, filed on Jul. 10, 2008. (74) Attorney, Agent, or Firm — Wilson Sonsini Goodrich & Rosati (51) Int. Cl. GOIN 33/50 (2006.01) (57) ABSTRACT GOIN 33/574 (2006.01) The present invention provides an approach for the deter GOIN 33/68 (2006.01) mination of the activation States of a plurality of proteins in (52) U.S. Cl. single cells. This approach permits the rapid detection of CPC. G0IN 33/57426 (2013.01); G0IN 33/5041 heterogeneity in a complex cell population based on acti (2013.01); G0IN 33/5044 (2013.01); G0IN Vation states, expression markers and other criteria, and the 33/5055 (2013.01); G0IN 33/5073 (2013.01); identification of cellular subsets that exhibit correlated G0IN 33/6893 (2013.01); G0IN 2333/70589 changes in activation within the cell population. Moreover, (2013.01); G0IN 2800/22 (2013.01); G0IN this approach allows the correlation of cellular activities or 2800/52 (2013.01) properties. In addition, the use of modulators of cellular (58) Field of Classification Search activation allows for characterization of pathways and cell CPC ...... G01N 33/57426; G01N 2800/52: populations. Several exemplary diseases that can be ana G01N 33/5008; G01N 33/5041: G01N lyzed using the invention include AML, MDS, and MPN. 33/5094; C12O 2600/118; C12O 2600/112 See application file for complete search history. 9 Claims, 46 Drawing Sheets US 9,500,655 B2 Page 2

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2 Etopo 3) at Co. : iter F.G. 29 US 9,500,655 B2 1. 2 METHODS FOR DIAGNOSIS, PROGNOSIS esis Although MDS and most MPNs are clinical chronic AND METHODS OF TREATMENT diseases while AML is an acute disease, all three have effects on cellular proliferation and apoptosis of myeloid progeni CROSS-REFERENCE tors and clinically approximately 30% of MDS and 5-10% of MPNS transform into AML. This application is a Continuation Application of U.S. Accordingly, there is a need for a biologically based application Ser. No. 13/473,829, filed May 17, 2012, now clinically relevant re-classification of these disorders that U.S. Pat. No. 8,778,620 which is a Divisional Application of can inform on disease management at the individual level. U.S. application Ser. No. 12/460,029, filed Jul. 10, 2009, This classification, based upon the biologic commonalities now U.S. Pat. No. 8,227,202, which claims the benefit of the 10 of the disorders above, will aid clinicians in both prognosis filing date of U.S. Ser. No. 61/079,766 filed Jul. 10, 2008, and therapeutic selection at the individual patient level thus U.S. Ser. No. 61/085,789 filed Aug. 1, 2008, U.S. Ser. No. improving patient outcomes e.g. Survival and quality of life. 61/104,666 filed Oct. 10, 2008, and U.S. Ser. No. 61/120, There are also needs for a biologically based clinically 320 filed Dec. 5, 2008, all of which are hereby incorporated relevant re-classification of these disorders to aid in new by reference in their entireties. 15 druggable target identification and drug screening for agents that may be active against myeloid malignancies. BACKGROUND OF THE INVENTION SUMMARY OF THE INVENTION Many conditions are characterized by disruptions in cel lular pathways that lead, for example, to aberrant control of In some embodiments, the invention provides methods of cellular processes, with uncontrolled growth and increased diagnosing, prognosing, or determining progression of acute cell Survival. These disruptions are often caused by changes leukemia, myelodysplastic syndrome or myeloproliferative in the activity of molecules participating in cellular path neoplasms in an individual, the method comprising: A ways. For example, alterations in specific signaling path classifying one or more hematopoietic cells associated with ways have been described for many cancers. Despite the 25 acute leukemia, myelodysplastic syndrome or myeloprolif increasing evidence that disruption in cellular pathways erative neoplasms in the individual by a method comprising: mediate the detrimental transformation, the precise molecu a) Subjecting a cell population comprising the one or more lar events underlying these transformations in diseases hematopoietic cells from the individual to a plurality of remain unclear. As a result, therapeutics may not be effective modulators in plurality of cultures, b) characterizing a in treating conditions involving cellular pathways that are 30 plurality of pathways in one or more cells from the plurality not well understood. Thus, the Successful diagnosis of a of cultures by determining an activation level of at least one condition and use of therapies will require knowledge of the activatable element within a plurality of pathways, where i) cellular events that are responsible for the condition pathol at least one of the pathways being characterized in at least Ogy. one of the plurality of cultures is an apoptosis or a DNA Acute myeloid leukemia (AML), myelodysplastic syn 35 damage pathway, ii) the modulators activate or inhibit one or drome (MDS), and myeloproliferative neoplasms (MPN) are more of the plurality of pathways being characterized, and examples of disorders that arise from defects of hematopoi c) classifying one or more hematopoietic cells based on the etic cells of myeloid origin. These hematopoietic disorders pathways characterization; and B making a decision regard are recognized as clonal diseases, which are initiated by ing diagnosis, prognosis or progression of acute leukemia, Somatic and/or inherited mutations that cause dysregulated 40 myelodysplastic syndrome or myeloproliferative neoplasms signaling in a progenitor cell. The wide range of possible in the individual, where the decision is based on the clas mutations and accompanying signaling defects accounts for sification of the cells. In some embodiments, the acute the diversity of disease phenotypes and response to therapy leukemia is acute myeloid leukemia. In some embodiments, observed within this group of disorders. For example, some the pathways are selected from the group consisting of leukemia patients respond well to treatment and survive for 45 apoptosis, cell cycle, signaling, or DNA damage pathways. prolonged periods, while others die rapidly despite aggres In some embodiments, the methods further comprise deter sive treatment. Some patients with myelodysplastic Syn mining whether the apoptosis, cell cycle, signaling, or DNA drome suffer only from anemia while others transform to an damage pathways are functional in the individual based on acute myeloid leukemia that is difficult to treat. Despite the the activation levels of the activatable elements, wherein a emergence of new therapies to treat these disorders the 50 pathway is functional if it is permissive for a response to the percentage of patients who do not benefit from current treatment, wherein if the apoptosis, cell cycle, signaling, and treatment is still high. Patients that are resistant to therapy DNA damage pathways are functional the individual can experience significant toxicity and have very short Survival respond to treatment, and wherein if at least one of the times. While various staging systems have been developed pathways is not functional the individual can not respond to to address this clinical heterogeneity, they cannot accurately 55 treatment. In some embodiments, the methods further com predict at diagnosis the prognosis or predict response to a prise determining whether the apoptosis, cell cycle, signal given therapy or the clinical course that a given patient will ing, or DNA damage pathways are functional in the indi follow. vidual based on the activation levels of the activatable Despite this heterogeneity, it is recognized that these elements, wherein a pathway is functional if it is permissive disorders share both biologic and clinical commonalities. 60 for a response to the treatment, where if the apoptosis and The first commonality is the cell type affected by the DNA damage pathways are functional the individual can disorders i.e. myeloid cell lineage. Second, all three disor respond to treatment. ders share cytogenetic abnormalities and have been shown In some embodiments, the individual has a predefined to have defects in transcription factors common in myeloid clinical parameter. In some embodiments, the predefined cell development. Third, many of the same signaling path 65 clinical parameter is selected from the group consisting of ways, including the RAS, JAK-STAT, and AKT, have been age, de novo acute myeloid leukemia patient, secondary shown to be important for AML, MDS and MPN pathogen acute myeloid leukemia patient, or a biochemical/molecular US 9,500,655 B2 3 4 marker. In some embodiments, a decision is made regarding nine. In some embodiments, the treatment is allogeneic stem diagnosis, prognosis or progression of acute leukemia, cell transplant or autologous stem cell transplant. myelodysplastic syndrome or myeloproliferative neoplasms In some embodiments, where the individual is under 60 in the individual based on the classification of the cells in years old the plurality of distinct modulators and activatable combination with the predefined clinical parameter. elements are selected from the modulators and activatable In some embodiments, the methods of the invention elements listed in table 6. In some embodiments, where the further comprise determining the levels of a cytokine recep individual is over 60 years old the plurality of distinct tor, growth factor receptor and/or a drug transporter in one modulators and activatable elements are selected from the or more hematopoetic cells. In some embodiments, the modulators and activatable elements listed in table 7. In cytokine receptor, growth factor receptor or drug transport 10 Some embodiments, where the individual is a secondary acute myeloid leukemia patient the plurality of distinct ers are selected from the group consisting of MDR1, modulators and activatable elements are selected from the ABCG2, MRP, P-Glycoprotein, CXCR4, FLT3, and c-kit. In modulators and activatable elements listed in table 8 and some embodiments, the levels of the cytokine receptor table 9. In some embodiments, where the individual is a de and/or the drug transporter in combination with the cell 15 novo acute myeloid leukemia patient the plurality of distinct classification and the clinical parameter are indicative of the modulators and the activatable elements are selected from diagnosis, prognosis or progression of acute myeloid leuke the modulators and activatable elements listed in table 10 mia, myelodysplastic syndrome or myeloproliferative neo and table 11. In some embodiments, where the individual plasms. has a wild type FLT3 the plurality of modulators and In some embodiments, the modulators are independently activatable elements are selected from the modulators and selected from the group consisting of growth factor, mito activatable elements listed in table 13. gen, cytokine, chemokine, adhesion molecule modulator, In some embodiments, the invention provides methods of hormone, Small molecule, polynucleotide, antibody, natural predicting a response to a treatment or choosing a treatment compound, lactone, chemotherapeutic agent, immune for acute leukemia, myelodysplastic syndrome or myelopro modulator, carbohydrate, protease, ion, reactive oxygen spe 25 liferative neoplasms in an individual, the method compris cies, and radiation. In some embodiments, the modulators ing: (1) classifying one or more hematopoietic cells associ are independently selected from the group consisting of ated with acute leukemia, myelodysplastic syndrome or FLT3L, GM-CSF, SCF, G-CSF, SDF1a, LPS, PMA, Thapsi myeloproliferative neoplasms in the individual by a method gargin, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, ZVAD, H2O2, comprising: a) Subjecting a cell population comprising the Staurosporine, Etoposide, Mylotarg, Daunorubicin, and 30 one or more hematopoietic cells from the individual to at AraC. least three distinct modulators in separate cultures, wherein: In some embodiments, the activatable element is a protein i) a first modulator is a growth factor or a mitogen, ii) a selected from the group consisting of p-Slp-76, p-Plcg2, second modulator is a cytokine, iii) a third modulator is a p-Stat3, p-Stats, p-Stat1, p-Stat6, p-Creb, cleaved Parp, modulator that slows or stops the growth of cells, and/or p-Chk2, p65/Rel-A, p-Akt. p-S6, p-ERK, Cleaved Caspase 35 induces apoptosis of cells, and/or is an inhibitor of a cellular 8, Cleaved Caspase 3, Cytoplasmic Cytochrome C, and p38. function, b) determining an activation level of at least one In some embodiments, the methods further comprise activatable element in one or more cells from each of the determining the presence or absence of one or more cell separate cultures, wherein: i) a first activatable element is an Surface markers, intracellular markers, or combination activatable element within the PI3K/AKT, or MAPK path thereof. In some embodiments, the cell Surface markers and 40 ways and the activation level is measured in response to the the intracellular markers are independently selected from the growth factor or mitogen, ii) a second activatable element is group consisting of proteins, carbohydrates, lipids, nucleic an activatable element within the STAT pathway and the acids and metabolites. In some embodiments, the presence activation level is measured in response to the cytokine, iii) or absence of one or more cell Surface markers or intracel a third activatable element is an activatable element within lular markers comprises determining the presence or 45 an apoptosis pathway and the activation level is measured in absence of an epitope in both activated and non-activated response to the modulator that slows or stops the growth of forms of the cell surface markers or the intracellular mark cells and/or induces apoptosis of cells, or the third activat ers. In some embodiments, the diagnosing, prognosing or able element is an activatable element within a phospholi determining progression of acute leukemia, myelodysplastic pase C pathway and the activation level is measured in syndrome or myeloproliferative neoplasms in the individual 50 response to the inhibitor, or the third activatable element is is based on both the activation levels of the activatable a phosphatase and the activation level is measured in element and the presence or absence of the one or more cell response to the inhibitor, and c) classifying the one or more Surface markers, intracellular markers, or combination hematopoeitic cells based on the activation levels of the thereof. activatable elements; and (2) making a decision regarding a In some embodiments, the activation level is determined 55 response to a treatment or a selection of treatment for acute by a process comprising the binding of a binding element leukemia, myelodysplastic syndrome or myeloproliferative which is specific to a particular activation state of the neoplasms in the individual based on the classification of the particular activatable element. In some embodiments, the one or more hematopoeitic cells. In some embodiments, the binding element comprises an antibody. acute leukemia is acute myeloid leukemia. In some embodi In some embodiments, the methods further comprise 60 ments, the individual has a predefined clinical parameter. In predicting a response to a treatment or choosing a treatment Some embodiments, the predefined clinical parameter is for acute myeloid leukemia, myelodysplastic syndrome or selected from the group consisting of age, de novo acute myeloproliferative neoplasms in an individual. In some myeloid leukemia patient, secondary acute myeloid leuke embodiments, the treatment is a chemotherapy agent. In mia patient, or a biochemical/molecular marker. Some embodiments, the chemotherapy agent is selected 65 In Some embodiments, activation levels higher than a from the group consisting of cytarabine (ara-C), daunoru threshold level of the activatable element within the STAT bicin, idarubicin, etoposide, mitoxantrone and 6-thiogua pathway in response to the cytokine is indicative that US 9,500,655 B2 5 6 individual can not respond to treatment. In some embodi the cytokine receptor, growth factor receptor or drug trans ments, the activatable element within the STAT pathway is porter are selected from the group consisting of MDR1. selected from the group consisting of p-Stat3, p-Stats. ABCG2, MRP, P-Glycoprotein, CXCR4, FLT3, and c-kit. In p-Stat1, and p-Stat6 and the cytokine is selected from the some embodiments, levels higher than a threshold of the group consisting of IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, drug transporter, growth factor receptor and/or the cytokine and G-CSF. In some embodiments, the activatable element receptor is indicative that the individual can not respond to within the STAT pathway is Stat 1 and the cytokine is IL-27 treatment. or G-CSF. In some embodiments, the methods further comprising In some embodiments, activation levels higher than a determining the activation levels of an activatable element threshold level of the activatable element within the PI3K/ 10 within the Akt pathway in response to an inhibitor, wherein AKT, or MAPK pathway in response to the growth factor or activation levels higher that a threshold of the activatable mitogen is indicative that individual can not respond to element within the Akt pathway in response to the inhibitor treatment. In some embodiments, the activatable element is indicative that the individual can not respond to treatment. within the PI3K/AKT, or MAPK pathway is selected from In Some embodiments, activation levels higher than a the group consisting of p-Akt, p-ERK, p38 and pS6 and the 15 threshold of the activatable element in the PI3K/AKT path growth factor or mitogen is selected from the group con way in response to a growth factor is indicative that the sisting of FLT3L, SCF, G-CSF, SCF, G-CSF, SDF1a, LPS, individual can not respond to treatment. In some embodi PMA, Thapsigargin. ments, the activatable element in the PI3K/Akt pathway is In some embodiments, activation levels higher than a Akt and the growth factor is FLT3L. threshold level of the activatable element within the phos In Some embodiments, activation levels higher than a pholipase C pathway in response to the inhibitor is indica threshold of the activatable element in the apoptosis path tive that individual can respond to treatment. In some way in response to a modulator that slows or stops the embodiments, the activatable element within the phospho growth of cells and/or induces apoptosis of cells is indicative lipase C pathway is selected from the group consisting of that the individual can respond to treatment. In some p-Slp-76, and Plcg2 and the inhibitor is H2O2. 25 embodiments, the activatable element within the apoptosis In some embodiments, activation levels higher than a pathway is Parp-- and the modulator that slows or stops the threshold of an activatable element within the apoptosis growth of cells and/or induces apoptosis of cells is selected pathway in response to a modulator that slows or stops the from the group consisting of Staurosporine, Etoposide, growth of cells and/or induces apoptosis of cells is indicative Mylotarg, Daunorubicin, and AraC. that the individual can respond to treatment. In some 30 In some embodiments, the cytokine is selected from the embodiments, the activatable element within the apoptosis group consisting of G-CSF, IFNg. IFNa, IL-27, IL-3, IL-6, pathway is selected from the group consisting of Parp--, and IL-10. In some embodiments, the growth factor or Cleaved Caspase 3, Cleaved Caspase 8, and Cytochrome C, mitogen is selected from the group consisting of FLT3L, and the modulator that slows or stops the growth of cells SCF, G-CSF, SCF, G-CSF, SDF1a, LPS, PMA, and Thapsi and/or induces apoptosis of cells is selected from the group 35 gargin. In some embodiments, the modulator that slows or consisting of Staurosporine, Etoposide, Mylotarg, Daunoru stops the growth of cells and/or induces apoptosis of cells is bicin, and AraC. selected from the group consisting of Staurosporine, Etopo In some embodiments, the methods further comprise side, Mylotarg, Daunorubicin, and AraC. determining an activation level of an activatable element In some embodiments the inhibitor is selected from the within a DNA damage pathway or a cell cycle pathway in 40 group consisting of AG 490, AG 825, AG 957, AG 1024, response to a modulator that slows or stops the growth of aloisine A, alsterpaullone, aminogenistein, API-2, apigenin, cells and/or induces apoptosis of cells. In some embodi arctigenin, AY-22989, BAY 61-3606, Azacitidine bisindo ments, the activatable element within a DNA damage path lylmaleimide IX, chelerythrine, 10-4'-(N,N-Diethylamino) way is selected from the group consisting of Chikl, Chk2. butyl-2-chlorophenoxazine hydrochloride, dasatinib, 2-Di ATR, ATM, and 14-3-3 and the modulator that slows or stops 45 methylamino-4,5,6,7-tetrabromo-1H-benzimidazole, 5,7- the growth of cells and/or induces apoptosis of cells is Dimethoxy-3-(4-pyridinyl)cquinoline dihydrochloride, selected from the group consisting of Staurosporine, Etopo decitibine, edelfosine, ellagic acid, enzastaurin, ER 27319 side, Mylotarg, Daunorubicin, and AraC. maleate, erlotinib, ET180CH3, fasudil, flavopiridol, gefi In some embodiments, activation levels higher than a tinib, GW 5074, H-7, H-8, H-89, HA-100, HA-1004, threshold of the activatable element within a DNA damage 50 HA-1077, HA-1 100, hydroxyfasudil, indirubin-3'-oxime, pathway and activation levels lower than a threshold of the 5-Iodotubercidin, kenpaullone, KN-62, KY 12420, LFM activatable element within the apoptosis pathway in A13, lavendustin A, luteolin, LY-294.002, LY294.002, mal response to a modulator that slows or stops the growth of lotoxin, ML-9, NSC-154020, NSC-226080, NSC-23 1634, cells and/or induces apoptosis of cells is indicative of a NSC-664704, NSC-680410, NU6102, olomoucine, oxin communication breakdown between the DNA damage 55 dole I, PD-153035, PD-98059, PD 169316, phloretin, phlo response pathway and the apoptotic machinery and that the ridzin, piceatannol, picropodophyllin, PKI, PP1, PP2, pur individual can not respond to treatment. In some embodi Valanol A, quercetin, R406, R788, rapamune, rapamycin, Ro ments, the activatable element within a cell cycle pathway is 31-8220, roscovitine, rottlerin, SB202190, SB203580, selected from the group consisting of Cdc25, p53, CyclinA sirolimus, Sorafenib, SL327, SP600 125, staurosporine, STI Cdk2, CyclinE-Cdk2, CyclinB-Cdk1, p21, p-Histone H3 60 571, SU-11274, SU1498, SU4312, SU6656, 4,5,6,7-Tetra and Gadd45, and the modulator that slows or stops the bromotriazole, TG101348, Triciribine, Tyrphostin AG 490, growth of cells and/or induces apoptosis of cells is selected Tyrphostin AG 825, Tyrphostin AG 957, Tyrphostin AG from the group consisting of Staurosporine, Etoposide, 1024, Tyrphostin SU1498, U0126, VX-509, VX-667, Mylotarg, Daunorubicin, and AraC. VX-680, W-7, wortmannin, XL-019, XL-147, XL-184, In some embodiments, the methods further comprising 65 XL-228, XL-281, XL-518, XL-647, XL-765, XL-820, determining the levels of a drug transporter, growth factor XL-844, XL-880, Y-27632, ZD-1839, ZM-252868, receptor and/or a cytokine receptor. In some embodiments, ZM-447439, H2O2, siRNA, miRNA, Cantharidin, (-)-p- US 9,500,655 B2 7 8 Bromotetramisole, Microcystin LR, Sodium Orthovanadate, further comprising a binding element specific for a cytokine Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo receptor, growth factor receptor or drug transporter are (1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium selected from the group consisting of MDR1, ABCG2, MRP, (IV), Sodium Molybdate, Sodium Perm olybdate, Sodium P-Glycoprotein, CXCR4, FLT3, and c-kit. In some embodi Tartrate, Imidazole, Sodium Fluoride, B-Glycerophosphate, ments, the binding element is an antibody. Sodium Pyrophosphate Decahydrate, Calyculin A, Discod One embodiment of the present invention is a method for ermia calyx, bpV(phen), mpV(pic), DMHV. Cypermethrin, classifying cells of a myeloid disorder based on the biology Dephostatin, Okadaic Acid, NIPP-1, N-(9,10-Dioxo-9,10 of a cell or group of cells derived from a patient with a dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide, myeloid malignancy such as AML, MDS, or MPN. In one C-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br, 10 method of the invention cells are taken and stimulated with C-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br, a modulator, fixed, permeabilized, contacted with a detec C-Bromo-4-(carboxymethoxy)acetophenone, 4-(Car tion element, and analyzed. Fresh or frozen cells may be boxymethoxy)phenacyl Br, and bis(4-Trifluoromethylsulfo used depending on the time between sample acquisition and namidophenyl)-1,4-diisopropylbenzene, phenyarsine oxide, sample analysis. The method of classification can comprise Pyrrolidine Dithiocarbamate, and Aluminum fluoride. 15 correlating the cell with a clinical outcome. Such as the In some embodiments, the activation level of one or more prognosis and/or diagnosis of a condition, or can correlate activatable element is determined by a process comprising with the response to a therapy, such as complete response, the binding of a binding element which is specific to a partial response, remission, no response, progressive dis particular activation state of the particular activatable ele ease, stable disease, hematologic improvement, cytogenetic ment. In some embodiments, the binding element comprises response and adverse reaction. The method can also involve an antibody. In some embodiments, the step of determining staging wherein the staging is selected from the group the activation level comprises the use of flow cytometry, consisting of WHO classification, FAB classification, IPSS immunofluorescence, confocal microscopy, immunohisto score, WPSS score, aggressive, indolent, benign, refractory, chemistry, immunoelectronimicroscopy, nucleic acid ampli limited Stage, extensive stage, including information that fication, gene array, protein array, mass spectrometry, patch 25 may inform on time to progression, progression free Sur clamp, 2-dimensional gel electrophoresis, differential dis vival, overall survival, and event-free survival. Treatments play gel electrophoresis, microsphere-based multiplex pro or therapies may include chemotherapy, biological therapy, tein assays, ELISA, and label-free cellular assays to deter radiation therapy, Small molecules, antibodies, bone marrow mine the activation level of one or more intracellular transplantation, peripheral stem cell transplantation, umbili activatable element in single cells. 30 cal cord blood transplantation, autologous stem cell trans In some embodiments, the invention provides methods of plantation, allogeneic stem cell transplantation, Syngeneic drug screening, the method comprising: A classifying one stem cell transplantation, surgery, induction therapy, main or more hematopoietic cells associated with acute leukemia, tenance therapy, watchful waiting, and other therapy. The myelodysplastic syndrome or myeloproliferative neoplasms classification may comprise correlating the cell with mini in the individual by a method comprising: a) Subjecting a 35 mal residual disease or emerging resistance. cell population comprising the one or more hematopoietic In some embodiments, univariate analysis is performed cells from the individual to a test compound and a plurality on relatively homogeneous clinical groups, such as patents of modulators in plurality of cultures, b) characterizing a over 60 years old, patients under 60 years old, de novo AML plurality of pathways in one or more cells from the plurality patients, and secondary AML patients. In other embodi of cultures by determining an activation level of at least one 40 ments the groups may be molecularly homogeneous groups, activatable element within a plurality of pathways, wherein Such as groups with mutations in the juxtamembrane region i) at least one of the pathways being characterized in at least of the Flt3 receptor, where these mutations can be internal one of the plurality of cultures is an apoptosis or a DNA tandem duplications (ITD) or point mutations. For example, damage pathway, ii) the modulators activate or inhibit one or in patients over 60 years, NRs may have a higher H.O. more of the plurality of pathways being characterized, and 45 response than CRs and/or a higher FLT3L responses than c) classifying the one or more hematopoietic cells based on CRs. In patients under 60 years, NRs may have a higher the pathways characterization; and B making a decision IL-27 response than CRS and/or CRS may undergo apoptosis regarding the test compound and its therapeutic potential for to Etoposide or Ara-C/Daunorubicin more than NRs. In de the treatment of acute leukemia, myelodysplastic syndrome novo AML, CRS may induce apoptosis (cleaved PARP) in or myeloproliferative neoplasms, wherein the decision is 50 response to Etoposide or Ara-C/Daunorubicin, they may based on the classification of the cells. have higher total p-S6 levels than NRS, or NRs may have In some embodiments, the invention provides kits com higher HO responses than CRs. In secondary AML, NRs prising: a) at least two modulators selected from the group may have higher HO responses than CRS, NRS may have consisting of Staurosporine, Etoposide, Mylotarg, Daunoru higher FLT3L, SCF responses than CRS, NRs may have bicin, AraC, G-CSF, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, 55 higher G-CSF, IL-27 responses than CRs, and there may be FLT3L, SCF, G-CSF, SCF, G-CSF, SDF1a, LPS, PMA, overlapping nodes with the over 60 year old patient set. Thapsigargin and H2O2; b) at least three binding elements In some embodiments, the present invention may stratify specific to a particular activation state of the activatable patients with a myeloid disease, monitor the patients for element selected from the group consisting of p-Slp-76, disease recurrence, predict their response to a therapeutic p-Plcg2. p-Stat3, p-Stats, p-Stat1, p-Stat6, p-Creb, Parp--, 60 agent, predict whether they are resistant or refractory to Chk2, p-65/Rel-A, p-Akt. p-S6, p-Erk, Cleaved Caspase 3, drugs, and predict whether they will relapse or have minimal Cleaved Caspase 8, Cytoplasmic Cytochrome C, and p38: residual disease. and c) instructions for diagnosis, prognosis, determining Another embodiment of the invention is a method to acute myeloid leukemia progression, predicting response to stratify patients who have AML, MDS, or MPN by gating a treatment and/or choosing a treatment for acute myeloid 65 the AML, MDS, or MPN cell samples after contacting the leukemia, myelodysplastic syndrome or myeloproliferative cells with the modulator. The method may also comprise the neoplasms in an individual. In some embodiments, the kit steps of: a) providing a population of cells; b) contacting the US 9,500,655 B2 10 cells with a plurality of activation state-specific binding porters, transcription factors/DNA binding factors, regula elements, wherein the plurality of activation state-specific tors of transcription, regulators of translation, HER recep binding elements comprise: i) a first activation state-specific tors, PDGF receptors, Kit receptor, FGF receptors, Eph binding element that binds to a first activable protein; and ii) receptors, Trk receptors, IGF receptors, Insulin receptor, a second activation state-specific binding element that binds Met receptor, Ret, VEGF receptors, TIE1, TIE2, FAK, Jak1, to a second activatable protein; c) using flow cytometry to Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, detect the presence or absence of binding of the first and Btk, ZAP70, Syk, IRAKs, craf, ARaf, BRAF, Mos, Lim second binding elements to determine the activation state of kinase, ILK, Tpl. ALK, TGFB receptors, BMP receptors, the first and second activatable proteins; and d) gating to MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/ separate the cells into discrete Subsets. Also, the method may 10 comprise classifying the cell as a cell that is correlated with 6, MKK4/7, ASK1, Cot, NIK, Bub, Myt 1, Weel, Casein staging of the disease, response to a therapeutic agent, kinases, PDK1, SGK1, SGK2, SGK3, Akt1, Akt2, Akt3, minimal residual disease or emerging resistance and deter p90Rsks, p70S6Kinase, Prks, PKCs, PKAs, ROCK 1, mining method of treatment. ROCK 2, Auroras, CaMKs, MNKS, AMPKs, MELK, One embodiment of the present invention is the use of a 15 MARKs, Chk1, Chk2, LKB-1, MAPKAPKs, Piml, Pim2, modulator that is an activator or an inhibitor, and it may be Pim3, IKKs, Cdks, Jnks, Erks, IKKs. GSK3C, GSK3 B, selected from the group consisting of biological entities, and Cdks, CLKs, PKR, PI3-Kinase class 1, class 2, class 3, physical or environmental stimuli which act extracellularly mTor, SAPK/JNK1,2,3, p38s, PKR, DNA-PK, ATM, ATR, or intracellularly, the chemical and biological modulators Receptor protein tyrosine phosphatases (RPTPs), LAR comprise growth factors, cytokines, mitogens, neurotrans phosphatase, CD45. Non receptor tyrosine phosphatases mitters, adhesion molecules, hormones, Small molecules, (NPRTPs), SHPs, MAP kinase phosphatases (MKPs), Dual inorganic compounds, polynucleotides, antibodies, natural Specificity phosphatases (DUSPs), CDC25 phosphatases, compounds, lectins, lactones, chemotherapeutic agents, bio Low molecular weight tyrosine phosphatase, Eyes absent logical response modifiers, immune modulators, carbohy (EYA) tyrosine phosphatases, Slingshot phosphatases drate, proteases, free radicals, cellular or botanical extracts, 25 (SSH), serine phosphatases, PP2A, PP2B, PP2C, PP1, PP5, cellular or glandular secretions, physiologic fluids Such as inositol phosphatases, PTEN, SHIPs, myotubularins, phos serum, amniotic fluid, or venom; the physical and environ phoinositide kinases, phopsholipases, prostaglandin Syn mental stimuli include electromagnetic, ultraviolet, infrared thases, 5-lipoxygenase, sphingosine kinases, sphingomyeli or particulate radiation, redox potential and pH, the presence nases, adaptor/scaffold proteins, Shc, Grb2, BLNK, LAT, B or absences of nutrients, changes in temperature, changes in 30 cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, oxygen partial pressure, changes in ion concentrations and MyD88, Crk, CrkL, GAD, Nck, Grb2 associated binder the application of oxidative stress. In another embodiment, (GAB), Fas associated death domain (FADD), TRADD, the modulators may be selected from the groups consisting TRAF2, RIP. T-Cell leukemia family, IL-2, IL-4, IL-8, IL-6, of ions, reactive oxygen species, peptides, and protein interferon Y, interferon C. Suppressors of cytokine signaling fragments, either alone or in the context of cells, cells 35 (SOCs), Cbl, SCF ubiquitination ligase complex. APC/C, themselves, viruses, and biological and non-biological com adhesion molecules, integrins, Immunoglobulin-like adhe plexes. sion molecules, selectins, cadherins, catenins, focal adhe In another embodiment, the modulator is a inhibitor sion kinase, p130CAS, fodrin, actin, paxillin, myosin, myo selected from the group consisting of H2O, siRNA, sin binding proteins, tubulin, eg5/KSP. CENPs, B-adrenergic miRNA, Cantharidin, (-)-p-Bromotetramisole, Microcystin 40 receptors, muscarinic receptors, adenylyl cyclase receptors, LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl small molecular weight GTPases, H-Ras, K-Ras, N-Ras, sulfate, Sodium oxodiperoxo (1,10-phenanthroline)vana Ran, Rac, Rho, Cdc42, Arfs, RABS, RHEB, Vav, Tiam, Sos, date, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Dbl, PRK, TSC1.2, Ras-GAP, Arf-GAPs, Rho-GAPs, cas Sodium Permolybdate, Sodium Tartrate, Imidazole, Sodium pases such as, Caspase 2, Caspase 3, Caspase 6. Caspase 7. Fluoride, B-Glycerophosphate, Sodium Pyrophosphate 45 Caspase 8, Caspase 9. Bcl-2. Mcl-1, Bcl-XL, Bcl-w, Bcl-B, Decahydrate, Calyculin A, Discodermia calyx, bpV(phen), A1, Bax, Bak, Bok, Bik, Bad, Bid, Bim, Bmf, Hrk, Noxa, mpV(pic), DMHV. Cypermethrin, Dephostatin, Okadaic Puma, IAPB, XIAP. Smac, Cdk4, Cdk 6, Cdk2, Cdk1, Cdk Acid, NIPP-1, N-(9,10-Dioxo-9,10-dihydro-phenanthren-2- 7, Cyclin D, Cyclin E, Cyclin A, Cyclin B, Rb, p 16, p14Arf, yl)-2,2-dimethyl-propionamide, C-Bromo-4-hydroxyaceto p27KIP p21 CIP. molecular chaperones, Hsp90s, Hsp70, phenone, 4-Hydroxyphenacyl Br, C-Bromo-4-methoxyac 50 Hsp27, metabolic enzymes, Acetyl-CoAa Carboxylase, ATP etophenone, 4-Methoxyphenacyl Br, C-Bromo-4- citrate lyase, nitric oxide synthase, caveolins, endosomal (carboxymethoxy)acetophenone, 4-(Carboxymethoxy) sorting complex required for transport (ESCRT) proteins, phenacyl Br, and bis(4-Trifluoromethylsulfonamidophenyl)- vesicular protein sorting (Vsps), hydroxylases, prolyl-hy 1,4-diisopropylbenzene, phenylarsine oxide, Pyrrolidine droxylases PHD-1, 2 and 3, asparagine hydroxylase FIH Dithiocarbamate, and Aluminium fluoride. 55 transferases, Pin1 prolyl isomerase, topoisomerases, In another embodiment of the invention, the activatable deacetylases. Histone deacetylases, sirtuins, histone acety elements are selected from the group consisting of kinases, lases, CBP/P300 family, MYST family, ATF2, DNA methyl phosphatases, lipid signaling molecules, adaptor/scaffold transferases. Histone H3K4 demethylases, H3K27, proteins, cytokines, cytokine regulators, ubiquitination JHDM2A, UTX, VHL, WT-1, p53, Hdm, PTEN, ubiquitin enzymes, adhesion molecules, cytoskeletal/contractile pro 60 proteases, urokinase-type plasminogen activator (uPA) and teins, heterotrimeric G proteins, Small molecular weight uPA receptor (uPAR) system, cathepsins, metalloprotein GTPases, guanine nucleotide exchange factors, GTPase ases, esterases, hydrolases, separase, potassium channels, activating proteins, caspases, proteins involved in apoptosis, Sodium channels, multi-drug resistance proteins, P-Glyco cell cycle regulators, molecular chaperones, metabolic protein, nucleoside transporters, Ets, Elk, SMADs, Rel-A enzymes, Vesicular transport proteins, hydroxylases, 65 (p65-NFKB), CREB, NFAT, ATF-2, AFT, Myc, Fos, Sp1, isomerases, deacetylases, methylases, demethylases, tumor Egr-1, T-bet, B-catenin, HIFs, FOXOs, E2Fs, SRFs, TCFs, Suppressor genes, proteases, ion channels, molecular trans Egr-1, B-catenin, FOXO STAT1, STAT3, STAT4, STAT US 9,500,655 B2 11 12 5, STAT 6, p53, WT-1, HMGA, pS6, 4-EPB-1, eIF4E transferase and RAF/RAS/ERK inhibitor), bevacizumab binding protein, RNA polymerase, initiation factors, elon (anti-EDGF monoclonal antibody that inhibits angiogen gation factors. esis), ezatiostat (glutathione S1 transferase inhibitor), and One embodiment of the invention is a method for diag clofarabine (nucleoside analog). In M3 AML all-trans ret nosing AML, MDS, or MPN, or predicting the outcome of 5 inoic acid and arsenic trioxide are also used. Therapies patients suffering from AML, MDS, or MPN, or screening traditionally used to treat MDS: supportive care, epo, GCSF, drugs thought to be useful against AML, MDS, or MPN, or Lenalidomide, Decitabine, AZacytidine, cyclosporine A, identifying new druggable targets for these diseases. The Anti-thymocyte globulin, and agents under investigation method comprises classifying a hematopoietic cell, com that include Arsenic trioxide (apoptosis inducer), Sorafenib prising Subjecting a hematopoietic cell to at least one 10 (tyrosine kinase inhibitor), Vorinostat and valproic acid modulator that affects signaling mediated by receptors Sub (histone deacetylase inhibitors), tipifarnib and lonafarnib jecting a hematopoietic cell to at least one modulator that (farnesyl transferase and RAF/RAS/ERK inhibitor), beva affects signaling mediated by receptors selected from the cizumab (anti-EDGF monoclonal antibody that inhibits group comprising SDF-1C, IFN-O, IFN-y, IL-10, IL-6, angiogenesis), FG-2216 (hypoxia-inducible factor stabi IL-27, G-CSF, FLT-3L, IGF-1, M-CSF and SCF; also sub 15 lizer), ezatiostat (glutathione S1 transferase inhibitor), clo jecting the hematopoietic cell to at least one modulator farabine (nucleoside analog). Also included are therapies selected from the group comprising PMA, Thapsigargin, traditionally used to treat MPNs include blood letting, H2O, Etoposide, Mylotarg, AraC, daunorubicin, stauro aspirin, and hydroxyurea. sporine, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluorom One embodiment of the invention involves the use of ethylketone (ZVAD), lenalidomide, EPO, azacitadine, decit multiparametric flow cytometry to examine the biology and abine; determining the expression level at least one protein signalling pathways in AML to determine likelihood of selected from the group comprising ABCG2, C-KIT recep response to agents used in consolidation therapy for AML. tor, and FLT3 LIGAND receptor, determining the activation One embodiment of the invention involves the use of states of a plurality of activatable elements in the cell multiparametric flow cytometry to examine the biology and comprising; and classifying the cell based on the activation 25 signalling pathways in AML to determine likelihood of states and expression levels. Another embodiment of the response to agents in development for the treatment of AML. invention further includes using the modulators IL-3, IL-4, One embodiment of the invention involves the use of GM-CSF, EPO, LPS, TNF-ct, and CD40L. The term “plu multiparameter flow cytometry to examine the biology and rality” as used herein refers to two or more. signalling pathways in myelodysplastic syndrome to classify One embodiment of the invention is a method for diag 30 MDS and inform on likelihood of response to agents such as nosing AML or predicting the outcome of patients Suffering growth factors (e.g. EPO), immunosuppressive agents (e.g. from AML, screening drugs thought to be useful against ATG+/-CSA), epigenetic modulators (e.g. hypomethylators AML, or identifying new druggable targets for these dis Azacytidine and Decitabine and HDAC inhibitors), eases, or predicting the outcome of patients undergoing ara-c immune-modulators (e.g. Lenalidomide). based induction therapy. The method comprises classifying 35 One embodiment of the invention involves the use of a hematopoietic cell, comprising Subjecting a hematopoietic multiparametric flow cytometry to examine the biology and cell to at least one modulator that affects signaling mediated signalling pathways in myelodysplastic syndrome to deter by receptors selected from the group comprising SDF-1C. mine likelihood of progression to AML. IFN-O, IFN-y, IL-10, IL-6, IL-27, G-CSF, FLT-3L, IGF-1, One embodiment of the invention involves the use of M-CSF and SCF; also subjecting the hematopoietic cell to at 40 multiparametric flow cytometry to examine the biology and least one modulator selected from the group comprising signalling pathways in myelodysplastic syndrome to deter PMA, Thapsigargin, H2O, Etoposide, Mylotarg, AraC. mine likelihood of response to agents in development for the daunorubicin, staurosporine, benzyloxycarbonyl-Val-Ala treatment of MDS Asp (OMe) fluoromethylketone (ZVAD), lenalidomide, One embodiment of the invention will look cell signaling EPO, azacitadine, decitabine; determining the expression 45 pathways described above in classifying and diagnosing level at least one protein selected from the group comprising MPN. Modulators can be designed to investigate these ABCG2, C-KIT receptor, and FLT3 LIGAND receptor, pathways and any relevant parallel pathways. determining the activation states of a plurality of activatable One embodiment of the invention involves the use of elements in the cell comprising; and classifying the cell multiparametric flow cytometry to examine the biology and based on the activation states and expression levels. Another 50 signalling pathways in MPN to determine likelihood of embodiment of the invention further includes using the progression to AML. modulators IL-3, IL-4, GM-CSF, EPO, LPS, TNF-C., and One embodiment of the invention involves the use of CD4OL. multiparametric flow cytometry to examine the biology and In another embodiment, for the treatment of myeloid signalling pathways in MPN to determine likelihood of disorders, the method further comprises treatment with a 55 response to agents in development for the treatment of MPN drug selected from the group consisting of therapies tradi Another embodiment of the invention comprises a method tionally used to treat AML: standard induction therapy for drug screening comprising; contacting a population of cytarabine (100-200 mg/m) coupled to an anthracycline AML, MDS, or MPN cells with a test compound and at least Such as daunorubicin or idarubicin, +/-thioguanine, etopo one modulator that affects signaling mediated by receptors side, dexamethasone, consolidation therapy including high 60 selected from the group comprising SDF-1C, IFN-O, IFN-Y, dose (1-3 gram) cytarabine, stem cell transplant or hypom IL-10, IL-6, IL-27, G-CSF, FLT-3L, IGF-1, M-CSF and ethylating drugs such as AZacytidine and Decitabine which SCF; also subjecting the hematopoietic cell to at least one induce differentiation in the affected cells by preventing modulator selected from the group comprising PMA, DNA methylation, Arsenic trioxide (apoptosis inducer), Thapsigargin, H2O, Etoposide, Mylotarg, AraC, daunoru Sorafenib (tyrosine kinase inhibitor), gemtuzumab ozogami 65 bicin, staurosporine, benzyloxycarbonyl-Val-Ala-Asp cin (Mylotarg), Vorinostat and valproic acid (histone (OMe) fluoromethylketone (ZVAD), lenalidomide, EPO, deacetylase inhibitors), tipifarnib and lonafarnib (farnesyl aZacitadine, decitabine; determining the expression level at US 9,500,655 B2 13 14 least one protein selected from the group comprising of a cell that has been stimulated with a modulator ABCG2, C-KIT receptor, and FLT3 LIGAND receptor, and stained with a labeled antibody and then subtracting the determining the activation states of a plurality of activatable value for unstimulated stained cells (log2 elements in the cell comprising; and classifying the cell (MFIstimulated Stained)-log2(MFI Ustimulated Stained). The based on the activation states and expression levels. Another 5 “quadrant frequency metric is the percentage of cells in embodiment of the invention further includes using the each quadrant of the contour plot. FIG. 2B shows that modulators IL-3, IL-4, GM-CSF, EPO, LPS, TNF-C., and additional metrics can also be derived directly from the CD4OL. distribution of cell for a protein in a gated population for In a further embodiment the invention comprises a kit. various condictions. NewlyPos=% of newly positive cells by The subject invention also provides kits for use in deter 10 modulator, based on a positive gate for a stain. AUC mining the physiological status of cells in a sample, the kit unstim=Area under the curve of frequency of un-modulated comprising one or more modulators, inhibitors, specific binding elements for signaling molecules, and may addi cells and modulated cells for a stain. NewlyPos: % Positive tionally comprise one or more therapeutic agents. The kit Cells modulated-% Positive Cellsunmodulated. FIG. 2B may further comprise a software package for data analysis of 15 measures the frequency of cells with a described property the physiological status, which may include reference pro such as cells positive for cleaved PARP (% PARP+), or cells files for comparison with the test profile. The kit may also positive for p-S6 and p-Akt. Similarly, measurements exam include instructions for use for any of the above applica ining the changes in the frequencies of cells may be applied tions. such as the Change in % PARP+ which would measure the In another embodiment, the invention is a method for % PARP+since, Stained-'6 PARP+ Ustimulated Stained The analyzing multiparametric data comprising using the fol AUC, metric also measures changes in population fre lowing measurements: basal, fold change, total phospho, quencies measuring the frequency of cells to become posi quadrant frequency, bimodal, spread, fold over isotype, tive compared to an unstimulated condition. percent over isotype, percent positive above unstimulated FIG. 3 shows a diagram of apoptosis pathways. and unstained, and medium fluorescent intensity of percent 25 FIG. 4A shows that phosphatase inhibitors, such as HO, positive above unstimulated and unstained. can help stratify patient response to induction therapy. FIG. 4B shows H2O target pathways in myeloids cells and INCORPORATION BY REFERENCE monocytes. FIG. 5A show a chemotherapeutic agent, for example All publications, patents, and patent applications men 30 etoposide, can induce DNA damage and apoptosis. Etopo tioned in this specification are herein incorporated by ref side induces DNA damage, which results in the phosphory erence to the same extent as if each individual publication, patent, or patent application was specifically and individu lation and activation of Chk2, a DNA damage checkpoint ally indicated to be incorporated by reference. response protein. If DNA repair is unsuccessful, Subsequent 35 activation of the intrinsic mitochondrial apoptotic pathway BRIEF DESCRIPTION OF THE DRAWINGS results in release of cytochrome c into the cytoplasm, formation of the apoptotsome and cleavage of caspases that The novel features of the invention are set forth with work in a coordinated cascade to cleave crucial Substrates particularity in the appended claims. A better understanding and dismantle the cell and then caspases are activated with of the features and advantages of the present invention will 40 PARP cleavage. FIG. 5B show examples of responses to be obtained by reference to the following detailed descrip Etoposide from NR and CR patients. FIG. 5C show tion that sets forth illustrative embodiments, in which the examples of typical responses to Etoposide from a NR and principles of the invention are utilized, and the accompany CR patients. FIG.5D and FIG.5E show typical CR response ing drawings of which: to p-Chk2 and Cleaved PARP under Unstimulated and FIG. 1 shows some examples of cellular pathways. For 45 Etoposide conditions. FIG.5F and FIG.5G show typical NR example, cytokines such as G-CSF or growth factors such as response to p-Chk2 and Cleaved PARP under Unstimulated FLT-3 Ligand will activate their receptors resulting in acti and Etoposide conditions. Vation of intracellular signaling pathways. Also, chemo FIG. 6 shows how cell populations can be gated to select therapeutics, such as AraC can be transported inside the cell Subpopulations. to cause effects, such as DNA damage, caspase activation, 50 FIG. 7 shows bimodal and spread metrics for analyzing PARP cleavage, etc. cell populations. FIG. 2A shows the use of four metrics used to analyze FIG. 8 shows expression marker metric using fold change data from cells that may be subject to a disease, such as over isotype and percent positive over isotype. AML. For these metrics the median (mean can be used as well) fluorescence intensity (MFI) was computed for the 55 FIG. 9 shows two other methods for analyzing cells cells in one of the gated populations measured under various including the percent of the population that is positive and conditions of staining and stimulation. For example, the has a fluorescence that is greater than the unstimulated/ “basal metric is calculated by subtracting the MFI of cells stained; and the median fluorescent intensity (MFI) of in the absence of a stimulant and stain (autofluorescence) percent positive above unstimulated/stained. from the MFI for cell measured in the absence of a stimulant 60 FIG. 10 shows a p-Stat 1/p-Akt scatter plot, all patient (autofluorescence) (log2(MFIsle, sel)- samples, basal (unstimulated). White=NR, White with log2(MFI, ). The “total phospho' metric is cal box=NR-CR, black=CR, circles scaled by ABCG2 and culated by measuring the fluorescence of a cell that has been MDR-1 expression stimulated with a modulator and stained with a labeled FIG. 11 shows a p-Stat1/p-Akt scatter plot, all patient antibody and then Subtracting the value for autofluorescence 65 samples, H.O. treated. White=NR, white with box=NR-CR, (log2(MFIstimulated stained)-log2(MFIGated Unstained). The black-CR, circles scaled by ABCG2 and MDR-1 expres “fold change' metric is the measurement of the fluorescence S1O. US 9,500,655 B2 15 16 FIG. 12 shows a p-Erk/p-CREB scatter plot, all patient FIGS. 29A and B shows different magnitudes of cytokine samples, basal (unstimulated). White=NR, white with and growth factor induced signaling in JAK/STAT and box=NR-CR, black=CR, circles scaled by ABCG2 and PI3K/S6 pathways within the three apoptotic profiles MDR-1 expression. depicted in FIG. 28. FIG. 29C shows flow cytometry analy FIG. 13 shows a p-Erk/p-CREB scatter plot, all patient sis of p-Stat1 vs. p-Stat3 and p-Stat3 vs. p-Stats in apoptosis samples, IL-3 treated cells. White=NR, white with box=NR competent, etoposide resistant, and staurosporine resistant CR, black=CR, circles scaled by ABCG2 and MDR-1 donor cells under unstimulated, or exposed to IL-27, or expression. G-CSF conditions. FIG. 29D shows apoptotic profiles of FIG. 14 shows a p-Erk/p-CREB scatter plot, all patient staurosporine resistant, etoposide resistant and apoptosis samples, IL-6 treated cells. White=NR, white with box=NR 10 competent after exposure to G-CSF induced p-Stat-3, CR, black=CR, circles scaled by ABCG2 and MDR-1 G-CSF induced p-Stat-5, Total IL-27 induced p-Stat-1, or expression. Total IL-27 induced p-Stat-3 conditions. White=NR, black=CR. FIG. 29E and FIG. 29F shows different magni FIG. 15 shows a p-Erk/p-CREB scatter plot, all patient tudes of induced signaling of apoptosis competent, etopo samples, IL-27 treated cells. White=NR, white with 15 side resistant, and staurosporine resistant donor cells. FIG. box=NR-CR, black=CR, circles scaled by ABCG2 and 29G shows flow cytometry analysis of p-Akt and p-S6 in MDR-1 expression. apoptosis competent, etoposide resistant, and staurosporine FIG.16 shows the use of signaling nodes to select patients resistant donor cells with no stimulation, exposed to FLT3L for specific targeted therapies. or SCF conditions. FIG. 29H shows apoptotic profiles of FIG. 17 shows that grouping of data points increases staurosporine resistant, etoposide resistant and apoptosis predictive value. competent after exposure to G-CSF, GM-CSF induced p-S6, FIG. 18 shows an example of a combination of two Total PMA induced p-S6, Total SCF induced p-S6, or Total independent nodes which correctly classified all but one CR FLT3L induced p-S6 conditions. I shows apoptotic profiles patients and misclassified only 5 NR patients. White=NR, of staurosporine resistant, etoposide resistant, and apoptosis black=CR 25 competent after exposure to Total SDF-1a induced p-Akt, FIG. 19 shows an example of node analysis using an Total SCF induced p-Akt, or Total FLT3L induced p-Akt additive linear model. conditions. White=NR, black=CR. FIG. 20 shows an example of node analysis using an independent combination of nodes model. White=NR, DETAILED DESCRIPTION OF THE black=CR 30 INVENTION FIG. 21 shows an example of analysis using a decision tree model. White=NR, black=CR The present invention incorporates information disclosed FIG.22 shows that analyses using both independent node in other applications and texts. The following patent and combinations and a decision tree model provide node com other publications are hereby incorporated by reference in binations of interest. White=NR, black=CR 35 their entireties: Haskell et al, Cancer Treatment, 5" Ed., FIG. 23 shows an example of an identified node infor W.B. Saunders and Co., 2001: Alberts et al., The Cell, 4" mative for relapse risk in patients who achieved CR and Ed., Garland Science, 2002; Vogelstein and Kinzler. The have FLT3 WT and normal karyotype disease. Genetic Basis of Human Cancer, 2d Ed., McGraw Hill, FIGS. 24A and B show G-CSF-mediated Stat signaling in 2002; Michael, Biochemical Pathways, John Wiley and two patient samples, one taken at diagnosis and the second 40 Sons, 1999; Weinberg, The Biology of Cancer, 2007: Immu at a later timepoint post induction. nobiology, Janeway et al. 7" Ed., Garland, and Leroith and FIG. 25 shows SCF-mediated-p-AKT and p-S6 signaling Bondy, Growth Factors and Cytokines in Health and Dis in two patient samples, one taken at diagnosis and the ease, A Multi Volume Treatise, Volumes 1A and 1B, Growth second at a later timepoint post induction. Factors, 1996. Other conventional techniques and descrip FIG. 26A shows p-AKT and p-S6 levels in CD33", 45 tions can be found in standard laboratory manuals such as CD11b, CD34" cells in an AML patient. FIG. 26B shows Genome Analysis: A Laboratory Manual Series (Vols. I-IV), a double positive gate used to stratify AML patients at Using Antibodies: A Laboratory Manual, Cells: A Labora diagnosis. tory Manual, PCR Primer: A Laboratory Manual, and FIG. 27 shows the frequency of p AKT/pS6 myeloid cells Molecular Cloning: A Laboratory Manual (all from Cold responsive to SCF in different AML patients 50 Spring Harbor Laboratory Press), Stryer, L. (1995) Bio FIG. 28 A shows an example of one embodiment of a chemistry (4th Ed.) Freeman, New York, Gait, “Oligonucle decision tree used for determining various apoptotic signa otide Synthesis: A Practical Approach 1984, IRL Press, tures in AML samples. FIG. 28B shows apoptotic profiles of London, Nelson and Cox (2000), Lehninger, Principles of staurosporine resistant, etoposide resistant, and apoptosis Biochemistry 3rd Ed., W. H. Freeman Pub., New York, N.Y. competent in AML samples. FIG. 28C shows FLT-3-ITD 55 and Berg et al. (2002) Biochemistry, 5th Ed., W. H. Freeman profiles in ITD and WT samples White=NR, black=CR. Pub., New York, N.Y.; and Sambrook, Fritsche and Mania FIG. 28D and FIG. 28E show different apoptotic mecha tis. “Molecular Cloning A laboratory Manual” 3rd Ed. Cold nisms among leukemic samples after in vitro exposure to Spring Harbor Press (2001), all of which are herein incor pan-Caspase inhibitor and Z-VAD in AML samples. FIG. porated in their entirety by reference for all purposes. 28F shows flow cytometry analysis of Cleaved Caspase-3 in 60 Patents and applications that are also incorporated by Major Block, Etoposide Block and Apoptosis Competent reference include U.S. Pat. Nos. 7,381,535 and 7,393,656 donor cells under unstimulated, exposed to staurosporine, or and U.S. Ser. Nos. 10/193,462; 11/655,785; 11/655,789: staurosporine and Z-VAD conditions in AML samples. FIG. 1 1/655,821; 11/338,957, 61/048,886: 61/048,920: 61/048, 28G shows flow cytometry analysis of Cleaved Caspase-3 in 657; and 61/079,766. Some commercial reagents, protocols, Major Block, Etoposide Block and Apoptosis Competent 65 Software and instruments that are useful in some embodi donor cells under unstimulated, exposed to etoposide, or ments of the present invention are available at the Becton etoposide and Z-VAD conditions in AML samples. Dickinson Website http://www.bdbiosciences.com/features/ US 9,500,655 B2 17 18 products/, and the Beckman Coulter website. http://www transduction-based classification of AML, MDS, or MPN ..beckmancoulter.com/Default.asp?bhfv=7. Relevant articles can be performed using clustering of phospho-protein pat include High-content single-cell drug screening with phos terns or biosignatures. See generally FIG. 1. phospecific flow cytometry, Krutzik et al., Nature Chemical In some embodiments, the present invention provides Biology, 23 Dec. 2007: Irish et al., FLt3 ligand Y591 methods for classification, diagnosis, prognosis of disease duplication and Bcl-2 over expression are detected in acute and outcome after administering a therapeutic to treat the myeloid leukemia cells with high levels of phosphorylated disease by characterizing a plurality of pathways in a wild-type p53, Neoplasia, 2007, Irish et al. Mapping normal population of cells. In some embodiments, a treatment is and cancer cell signaling networks: towards single-cell chosen based on the characterization of plurality of path proteomics, Nature, Vol. 6 146-155, 2006; and Irish et al., 10 ways in single cells. In some embodiments, characterizing a Single cell profiling of potentiated phospho-protein net plurality of pathways in single cells comprises determining works in cancer cells, Cell, Vol. 118, 1-20 Jul. 23, 2004; whether apoptosis pathways, cell cycle pathways, signaling Schulz, K. R., et al., Single-cell phospho-protein analysis by pathways, or DNA damage pathways are functional in an flow cytometry, Curr Protoc Immunol, 2007,78:88.17.1-20; individual based on the activation levels of activatable Krutzik, P.O., et al., Coordinate analysis of murine immune 15 elements within the pathways, where a pathway is functional cell Surface markers and intracellular phosphoproteins by if it is permissive for a response to a treatment. For example, flow cytometry, JImmunol. 2005 Aug. 15; 175(4):2357-65; when the apoptosis, cell cycle, signaling, and DNA damage Krutzik, P. O., et al., Characterization of the murine immu pathways are functional the individual can respond to treat nological signaling network with phosphospecific flow ment, and when at least one of the pathways is not functional cytometry, JImmunol. 2005 Aug. 15; 175(4):2366-73; Shulz the individual can not respond to treatment. In some embodi et al., Current Protocols in Immunology 2007, 78:8.17.1-20; ments, if the apoptosis and DNA damage pathways are Stelzer et al. Use of Multiparameter Flow Cytometry and functional the individual can respond to treatment. Immunophenotyping for the Diagnosis and Classification of In some embodiments, the characterization of pathways in Acute Myeloid Leukemia, Immunophenotyping, Wiley, conditions such as AML, MDS and MPN shows disruptions 2000; and Krutzik, P. O. and Nolan, G. P., Intracellular 25 in cellular pathways that are reflective of increased prolif phospho-protein staining techniques for flow cytometry: eration, increased Survival, evasion of apoptosis, insensitiv monitoring single cell signaling events, Cytometry A. 2003 ity to anti-growth signals and other mechanisms. In some October; 55(2):61-70; Hanahan D., Weinberg, The Hall embodiments, the disruption in these pathways can be marks of Cancer, CELL, 2000 Jan. 7: 100(1) 57-70; Krutzik revealed by exposing a cell to one or more modulators that et al. High content single cell drug screening with phophos 30 mimic one or more environmental cue. FIG. 1 shows an phospecific flow cytometry, Nat Chem Biol. 2008 February; example of how biology determines response to therapy. For 4(2): 132-42. Experimental and process protocols and other example, without intending to be limited to any theory, a helpful information can be found at http:/proteomices. Stan responsive cells treated with Ara-C will undergo cell death ford.edu. The articles and other references cited below are through activation of DNA damage and apoptosis pathways. also incorporated by reference in their entireties for all 35 However, a non-responsive cell might escape apoptosis purposes. through disruption in one or more pathways that allows the One embodiment of the present invention involves the cell to Survive. For instance, a non-responsive cell might classification, diagnosis, prognosis of disease and outcome have increased concentration of a drug transporter (e.g., after administering a therapeutic to treat the disease; exem MPR-1), which causes Ara-C to be removed from the cells. plary diseases include AML, MDS and MPN. Another 40 A non-responsive cell might also have disruptions in one or embodiment of the invention involves monitoring and pre more pathways involve in proliferation, cell cycle progres dicting outcome of disease. Another embodiment is drug sion and cell survival that allows the cell to survive. A screening using some of the methods of the invention, to non-responsive cell may have a DNA damage response determine which drugs may be useful in particular diseases. pathway that fails to communicate with apoptosis pathways. In other embodiments, the invention involves the identifi 45 A non-responsive cell might also have disruptions in one or cation of new druggable targets, that can be used alone or in more pathways involve in proliferation, cell cycle progres combination with other treatments. The invention allows the sion and cell survival that allows the cell to survive. The selection of patients for specific target therapies. The inven disruptions in these pathways can be revealed, for example, tion allows for delineation of subpopulations of cells asso by exposing the cell to a growth factor such as FLT3L or ciated with a disease that are differentially susceptible to 50 G-CSF. In addition, the revealed disruptions in these path drugs or drug combinations. In another embodiment, the ways can allow for identification of target therapies that will invention allows to demarkate Subpopulations of cells asso be more effective in a particular patient and can allow the ciated with a disease that have different genetic subclone identification of new druggable targets, which therapies can origins. In another embodiment, the invention provides for be used alone or in combination with other treatments. the identification of a cell type, that in combination other cell 55 Expression levels of proteins. Such as drug transporters and type(s), provide rationnetric or metrics that singly or coor receptors, may not be as informative by themselves for dinately allow for Surrogate identification of Subpopulations disease management as analysis of activatable elements, of cells associated with a disease, diagnosis, prognosis, Such as phosphorylated proteins. However, expression infor disease stage of the individual from which the cells were mation may be useful in combination with the analysis of derived, response to treatment, monitoring and predicting 60 activatable elements, such as phosphorylated proteins. outcome of disease. Another embodiment involves the The discussion below describes some of the preferred analysis of apoptosis, drug transport and/or drug metabo embodiments with respect to particular diseases. However, lism. In performing these processes, one preferred analysis it should be appreciated that the principles may be useful for method involves looking at cell signals and/or expression the analysis of many other diseases as well. markers. One embodiment of cell signal analysis involves 65 Introduction the analysis of phosphorylated proteins and the use of flow Hematopoietic cells are blood-forming cells in the body. cytometers in that analysis. In one embodiment, a signal Hematopoiesis (development of blood cells) begins in the US 9,500,655 B2 19 20 bone marrow and depending on the cell type, further matu with markedly contrasting prognosis. The greatest prognos ration occurs either in the periphery or in secondary lym tic factor is age with children diagnosed with AML faring phoid organs such as the spleen or lymph nodes. Hematopoi much better than adults. Cytogenetics also plays a major etic disorders are recognized as clonal diseases, which are prognostic role in AML. Genetic translocations, such as initiated by Somatic and/or inherited mutations that cause inv(16), tC8:21) and t(15:17) characterize AML with a rela dysregulated signaling in a progenitor cell. The wide range tively favourable prognosis, whereas the cytogenetically of possible mutations and accompanying signaling defects high-risk leukemias include patients with FLt3 ligand muta accounts for the diversity of disease phenotypes observed tions, loss of 5(q) or 7(q), tC6:9) and t(9:22) (Lowenberg et within this group of disorders. Hematopoietic disorders fall al., 1999). More recently, molecular markers have been into three major categories: Myelodysplastic syndromes, 10 recognized as having prognostic value. Nucleophosmin1 myeloproliferative disorders, and acute leukemias. Examples of hematopoietic disorders include non-B lineage (NPM1) predicts good risk AML while the presence internal derived, such as acute myeloid leukemia (AML), Chronic tandem duplications of Flt 3 predicts poor outcome. Myeloid Leukemia (CML), non-B cell acute lymphocytic Causes leukemia (ALL), myelodysplastic disorders, myeloprolif 15 The development of acute myeloid leukemia is currently erative disorders, polycythemias, thrombocythemias, or believed to be a multi-step process. Under the two-hit model non-B atypical immune lymphoproliferations. Examples of proposed by Gilliland et al., a hematopoietic progenitor cell B-Cell or B cell lineage derived disorder include Chronic first acquires a mutation that confers a growth advantage, Lymphocytic Leukemia (CLL), B lymphocyte lineage leu Such as a constitutively activated tyrosine kinase (e.g. Flt kemia, Multiple Myeloma, acute lymphoblastic leukemia 3-ITD). This preleukemic cell thus divides more rapidly. (ALL), B-cell pro-lymphocytic leukemia, precursor B lym Due to the increased proliferation, the odds of acquiring phoblastic leukemia, hairy cell leukemia or plasma cell additional stochastic mutations are increased. When one of disorders, e.g., amyloidosis or Waldenstrom's macroglobu these secondary mutations disrupts normal differentiation of linemia. the progenitor cell (e.g. AML1-ETO), the result is a fully Acute myeloid leukemia (AML), myelodysplastic syn 25 cancerous clone with the regenerative capacity of a progeni drome (MDS), and myeloproliferative neoplasms (MPN) are tor cell, but lacking the developmental checkpoints of dif examples of distinct myeloid hematopoietic disorders. How ferentiation. The unchecked division of this clone produces ever, it is recognized that these disorders share clinical the immature blast population characteristic of AML. (Kelly, overlap in that 30% of patients with MDS and 5-10% of L. M. & Gilliland, D. G. Genetics of myeloid leukemias. patients with MPN will go on to develop AML. Below are 30 Annu Rev Genomics Hum Genet, Epub 2002 Apr. 15). current descriptions of these myeloid disorders. Any endogenous or environmental source of DNA dam Acute Myeloid Leukemia (AML) age has the potential to induce leukemia. As the incidence of AML is characterized by an uncontrolled proliferation of AML increases dramatically with age, the most likely caus immature progenitor cells of myeloid origin including, but ative agent is probably DNA damage from Superoxide not limited to, myeloid progenitor cells, myelomonocytic 35 radicals produced during normal cellular respiration, progenitor cells, and immature megakaryoblasts. It is coupled with imperfect DNA repair. Environmental expo becoming clear that AML is really a heterogeneous collec Sure to high levels of ionizing radiation, Such as nuclear tion of neoplasms with elements of differing pathophysiol industry accidents, increases the risk of developing leuke ogy, genetics and prognosis. Under WHO guidelines, diag mia. Smoking also increases the risk because of concen nosis of AML can be made when blasts (immature cells) are 40 trated levels of benzene in cigarette Smoke. In rare cases, present at 20% or more in peripheral blood or bone marrow AML may occur after long-term exposure to benzene (and sampling. possibly other solvents) used in industry. Some anti-cancer Though rare, AML is one of the most deadly cancers and treatments such as chemotherapy or radiotherapy can result can be very aggressive if untreated. Although AML is a in leukemia being developed years later. The risk is relatively rare disease, accounting for approximately 1.2 45 increased when certain types of chemotherapy drugs are percent of cancer deaths in the US, it is the most common combined with radiotherapy. When leukemia develops form of leukemia accounting for about 50 percent of all because of previous anti-cancer treatment, it is known as leukemia cases. Its incidence is expected to increase as the secondary leukemia or treatment-related leukemia. population ages; up to 85 percent of all acute leukemia cases AML can also arise from genetic causes. For example, involve adults. SEER data predicts that 13,410 people will 50 patients with Down's syndrome, Fanconi anemia, Li Frau be diagnosed with AML in 2008. AML is one of the more meni syndrome, Kostmanns, Kleinfelters, Neurofibromato deadly cancers with an overall survival of 50% in children, sis, Diamond Blackfan anemia and Swachman Diamond 20% in patients<60 years old and 5% in patients 60 years have an increased risk of developing AML. Non-inherited and is uniformly fatal if left untreated. examples include aplastic anemia, paroxysmal nocturnal AML is a quickly progressive malignant disease involv 55 hemoglobinuria and MDS, as well as other blood disorders, ing too many immature blood-forming cells in the blood and Such as the Myeloproliferative neoplasms polycythemia bone marrow, the cells being specifically those that are Vera and essential thrombocythemia. Acute myeloid leuke destined to give rise to granulocytes or monocytes—the two mia is not infectious and cannot be passed on to other types of white blood cells that fight infections. In AML. people. these blasts do not mature and do not die, thus overwhelm 60 Symptoms ing the circulatory system (blasts often represent >90% of The main symptoms of AML are pallor, fatigue and peripheral blood leukocytes), Suppressing normal breathlessness, which are due to anemia caused by the lack hematopoiesis and invading other organs and tissues. It is of red blood cells. Decreased white blood cells lead to an also known as acute myelogenous leukemia or acute non increase in infection and fever. Absence of platelets can lead lymphocytic leukemia (ANLL). 65 to petichiae (rashes of tiny, flat red spots on the legs, chest, AML patients are presently classified into groups or or in the mouth), bleeding of the gums, frequent nosebleeds, Subsets based on age, cytogenetics and molecular analysis, or heavy periods in women. US 9,500,655 B2 21 22 Other symptoms may be caused by an abnormal accumu immunophenotyping techniques can be useful to make a lation of leukemia cells in a particular area of the body. Such diagnosis. Flow cytometry is now routinely used diagnose as bone pain caused by pressure from the accumulation of and classify leukemias, particularly in difficult cases of discriminating between AML and ALL, and is also used to immature cells in the bone marrow, raised bluish-purple determine the tumor burden (e.g. percent blasts). Cytoge areas under the skin (leukemia cutis), caused by leukemia netic techniques help in determining any changes in chro cells in the skin, and hypertrophied (Swollen) gums caused mosome or any translocation, deletions, etc. Another similar by an infiltration of leukemia cells into the gums. Blasts cytogenetic method for diagnosis is fluorescent in situ commonly are found in organs such as the liver, spleen and hybridization (FISH), which can be used to ascertain spe lymph nodes resulting in organomegaly (large organs). cific changes in chromosomal makeup. Soreness or sensitivity in these areas. In addition, headaches 10 One embodiment of the present invention is a method for or seizures may arise when the central nervous system is classifying cells of a myeloid disorder based on the biology infiltrated with contaminating leukemia cells. of a cell or group of cells derived from a patient with a Very rarely, a person does not have any symptoms and the myeloid malignancy such as AML, MDS, or MPN. One leukemia is discovered during a routine blood test. The embodiment of the invention combines one or more of these symptoms of acute myeloid leukemia usually appear over a 15 existing tests with the analysis of signalling mediated by few weeks, and people often fall ill quickly necessitating receptors to diagnose disease, especially AML, MDS, or prompt administration of therapy. MPNs. All tests may be performed in one location and Diagnosis provided as a single service to physicians or other caregiv The duration of signs and symptoms before diagnosis of CS. AML is usually 4 to 6 weeks, and may include fever, pallor, Cell-Signaling Pathways and Differentiating Factors weakness, fatigue, and weight loss. An abnormal result on a Involved complete blood count is the most common and oldest Alterations of kinases and phosphatases lead to inappro method for diagnosing AML. Diagnosis is confirmed by priate signal transduction, whereas alterations of transcrip obtaining a small sample of bone marrow and counting the tion factors give rise to inappropriate gene expression. Both number and percentage of immature blood cells (blasts) in of these mechanisms contribute to the pathogenesis of AML the sample under a microscope, using standard histological 25 by the induction of increased proliferation, reduced apop techniques. Based on the size of the cells, their shape, cell tosis and block of differentiation. The dysregulation of one Surface markers and other traits, one can classify the cells or more of the key signaling pathways (e.g., RAS/MAPK, into specific cell types. PI3K/AKT, and JAK/STAT) is believed to result in growth The percentage of cells in the bone marrow or blood is factor-independent proliferation and clonal expansion of essential for diagnosing an acute leukemia. At least 20 or 30 hematopoietic progenitors (HOX deregulation in acute more percent of blasts in the blood or marrow is generally myeloid leukemia. Journal of Clinical Investigation. 2007, required for the diagnosis of AML. Less than 20 percent vol. 117, no. 4, p. 865-868.) See generally Table 1 below blasts usually indicates a myeloproliferative disease or which depicts pathways relevant for AML Biology. In some myeloproliferative neoplasia. AML can also be diagnosed if embodiments, the pathways depicted in Table 1 are charac the blasts have a chromosome change, which only occurs in 35 terized using the methods described herein by exposing cells a specific type of AML, even though the blast percentage to the modulators listed in the table and measuring the does not reach 20 percent. Sometimes leukemic blasts look readout listed in the table, for each corresponding pathways. similar to normal immature cells in the bone marrow. Disruption in one or more pathways can be revealed by However, under normal circumstances, blasts are generally exposing the cells to the modulators. This can then be used not more than 5 percent of the bone marrow cells. for classification, diagnosis, prognosis of AML, selection of In 90% of cases, morphological and cytochemical studies 40 treatment and/or predict outcome after administering a are sufficient to determine the lineage of the leukemia, but therapeutic. TABLE 1.

Pathway Readout Modulator DNA Damage p-Chk1, p.-Chk2, p-ATM, p-ATR, p Etoposide, Ara-CDaunorubicin, Drug H2AX Pump Inhibitors, Mylotarg Drug transporters MDR-1, ABCG2, MPR Drug Pump Inhibitors Apoptosis Bcl-2, Mcl-1, cytochrome c, Survivin, Staurosporine, Etoposide, Ara XLAP PARP, Casapses 3, 7 and 8 CDaunorubicin, Drug Pump Inhibitors, Mylotarg, Zvad, Caspase Inhibitors, Phosphatases Shp-1, Shp-2, CD45 H2O2 JAK/STAT p-Stat 1, 3, 4, 5, 6 Cytokine and Growth Factors Cell Cycle Myc, Ki-67, Cyclins, DNA stains, p Cytokine and Growth Factors, RB, p16, p21, p27, p15, cyclin D1, Mitogens, Apoptosis inducing agents, cyclin B1, p-Cdk1, p-histoneH3, p CDC25 MAPK Ras, p-Mek, p-Erk, p-S6, p-38 Cytokine and Growth Factors, Mitogens, Cytokines, Growth Factors, Mitogens, chemokines, Receptor Tyrosine Kinase proteins (RTK) ligands FLT3 and other RTKs p-PLCg 1/2, p-CREB, total CREB, Flt3L, Receptor Tyrosine Kinase p-Akt, p-Erk, p-S6 (RTK) ligands Angiogenesis PLCY1, p-Akt, p-Erk VEGF stim Wnt/b-catenin Active B-Catenin, Myc, Cyclin D RTK ligands, growth factors Survival PI3K, PLCg, Stats RKT Growth Factors US 9,500,655 B2 23 24 There are two main classes of receptors which play an receptors (EpoR) to negatively regulate hematopoietic important role in hematopoiesis: Receptors with intrinsic growth (Yi, T. et al. (1995) Blood 85, 87-95). Following tyrosine kinase activity (RTKs) and those that do not contain ligand binding in myeloid cells, SHP-1 associates with their own enzymatic activity and often consist of heterodi IL-3R B chain and down regulates IL-3-induced tyrosine mers of a ligand-binding alpha Subunit and a signal trans phosphorylation and cell proliferation (Yi, T. et al. (1993) ducing beta Subunit, which is frequently shared between a Mol Cell Biol 13, 7577-86). Because SHP-1 downregulates Subset of cytokine receptors. Cytoplasmic tyrosine kinases signaling pathways emanating from receptor tyrosine phosphorylate cytokine receptors thereby creating docking kinases, cytokine receptors, multi-chain recognition recep sites for signaling molecules resulting in activation of a tors and integrins, it is considered a potential tumor Sup specific intracellular signaling pathway. Of the first class, 10 pressor (Wu, C. et al. (2003) Gene 306, 1-12, Bhattacharya, Kit and FLt3 receptor have been shown to play an important R. et al. (2008) J Mol Signal 3, 8). role in the pathogenesis of AML. Extracellular ligand bind SHP-2 (PTPN11) is a ubiquitously expressed, nonrecep ing regulates the intracellular substrate specificity, affinity tor protein tyrosine phosphatase (PTP). It participates in and kinase activity of these proteins. Therefore, the receptor signaling events downstream of receptors for growth factors, transmits its signal through binding and/or phosphorylation 15 cytokines, hormones, antigens and extracellular matrices in of intracellular signaling intermediates. Despite these dif the control of cell growth, differentiation, migration and ferences, the signals transmitted by both classes of receptors death (Qu, C. K. (2000) Cell Res 10, 279-88). Activation of ultimately converge on one or more of the key signaling SHP-2 and its association with Gab1 is critical for sustained pathways, such as the Ras/Raf/MAPK, PI3K/AKT, and Erk activation downstream of several growth factor recep JAK/STAT pathways. tors and cytokines (Maroun, C. R. et al. (2000) Mol Cell Biol The STAT (signal transducer and activator of transcrip 20, 8513-25.). tion) family of proteins, especially STAT3 and STAT5, are FIG. 4 shows the role of phosphaspatases in AML. When emerging as important players in several cancers. (Yu active SHP-1 and SHP-2 dephosphorylates protein kinase 2004 STATs in cancer. (2008) pp. 9). Of particular rel (See Koretzky G A et al. Nat Rev Immunol. 2006 January; evance to AML, the STATs have been shown to be critical 25 6(1):67-78. Review). Treatment of cells with a general for myeloid differentiation and survival, as well as for tyrosine phosphatase inhibitor Such as HO results in an long-term maintenance of normal and leukemic stem cells. increase in phosphorylation of intracellular signalling mol (Schepers et al. STAT5 is required for long-term mainte ecules. In this experiment, AML patients that were complete nance of normal and leukemic human stem/progenitor cells. responders (CR) to one cycle of standard 7+3 induction Blood (2007) vol. 110 (8) pp. 2880-2888) STAT signaling is 30 therapy showed higher levels of phosphorylated PLCy2 and activated by several cytokine receptors, which are differen SLP-76 upon HO treatment when compared with non tially expressed depending on the cell type and the stage of responders (NR). differentiation. Intrinsic or receptor-associated tyrosine FLt3 Ligand Mutations: kinases phosphorylate STAT proteins, causing them to form During normal hematopoietic development, the FLT3 a homodimer. The activated STAT dimer is able to enter the 35 receptor functions in the differentiation and proliferation of cell nucleus and activate the transcription of target genes, multipotent stem cells and their progeny in the myeloid, B many of which are involved in the regulation of apoptosis cell, and T cell lineages. (Gilliland, G. D., and Griffin, J. D. and cell cycle progression. Apart from promoting prolifera The roles of FLT3 in hematopoesis and leukemia. Blood tion and Survival. Some growth factor receptors and signal (2002) 100: 1532-42). FLT3 receptor expression is normally ing intermediates have been shown to play specific and 40 restricted to hematopoietic progenitors, and genetic ablation important roles in myeloid differentiation. For example, experiments have shown that FLT3 is required for the G-CSF- or TPO-induced activation of the Ras-Raf-MAP maturation of these early cells, but is not required in mature Kinase pathway promotes myeloid or megakaryocytic dif cells (Rosnet O., et al. Human FLT3/FLK2 receptor tyrosine ferentiation in the respective progenitor cells by the activa kinase is expressed at the Surface of normal and malignant tion of c/EBPO. (frequently inactivated in myeloid leuke 45 hematopoietic cells. Leukemia (1996) 10: 238–48; Mack mias) and GATA-1, respectively. (B. STEFFEN et al. arehtschian K., et al. Targeted disruption of the flk2/flt3 gene Critical Reviews in Oncology/Hematology. 2005, vol. 56, p. leads to deficiencies in primitive hematopoietic progenitors. 195-221.) Immunity (1995) 3: 147-61). Phosphatases: Mutations in FLT3 are found in 25-45% of all AML One of the earliest events that occurs after engagement of 50 patients (Renneville A., et al. Cooperating gene mutations in myeloid receptors is the phosphorylation of cellular proteins acute myeloid leukemia: a review of the literature. Leukemia on serine, threonine, and tyrosine residues 8, 9, 10. The (2008) 22: 915-31). Of the AML-associated FLT3 muta overall level of phosphorylated tyrosine residues is regulated tions, the most common is the internal tandem duplication by the competing activities of protein tyrosine kinases (ITD), which is found in 25-35% of adult AML patients (Id). (PTKs) and protein tyrosine phosphatases (PTPs). Decreases 55 The ITD is an in-frame duplication of 3-400 nucleotides that in the activity of tyrosine phosphatases may also contribute encodes a lengthened FLT3 juxtamembrane domain (JMD) to an increase in cellular tyrosine phosphorylation following (Schnittger S., et al. FLT3 internal tandem duplication in 234 stimulation. children with acute myeloid leukemia (AML): prognostic SHP-1 (PTPN6) is a non-receptor protein tyrosine phos significance and relation to cellular drug resistance. Blood phatase that is expressed primarily in hematopoietic cells. 60 (2003) 102: 2387-94.). In vitro studies have shown that The enzyme is composed of two SH2 domains, a tyrosine FLT3/ITDs promote ligand-independent receptor dimeriza phosphatase catalytic domain and a carboxy-terminal regu tion, leading to autonomous phosphorylation and constitu latory domain (Yi, T. L. et al. (1992) Mol Cell Biol 12, tive activation of the receptor (Gilliand, G. D., and Griffin, J. 836-46). SHP-1 removes phosphates from target proteins to D. Blood (2002) 100: 1532–42). Structural studies of FLT3 down regulate several tyrosine kinase regulated pathways. 65 suggest that in the wild-type receptor, the JMD produces In hematopoietic cells, the N-terminal SH2 domain of steric hindrance that prevents autodimerization (Griffith, J., SHP-1 binds to tyrosine phosphorylated erythropoietin et al. The Structural Basis for Autoinhibition of FLT3 by the US 9,500,655 B2 25 26 Juxtamembrane Domain. Molecular Cell (2004) 13: 169 siveness to G-CSF stimulation (Gert-Jan, M. et al. G-CSF 78). The ITD-associated lengthening of the JMD appears to receptor truncations found in SCN/AML relieve SOCS3 remove this hindrance, resulting in autodimerization and controlled inhibition of STAT5 but leave suppression of constitutive FLT3 kinase activity. The second class of FLT3 STAT3 intact. Blood (2004) 104: 667-74). Stimulation of mutation, found in 5-10% of AML patients, comprises AML patient blast cells with G-CSF in vitro revealed missense point mutations in exon 20 commonly in codons potentiated Stat3 and Stats phosphorylations that correlated D835, 1836, N841, or Y842 which produce amino acid with poor response to chemotherapy (Irish, J. M., et al. substitutions in the activation loop of the FLT3 tyrosine Single Cell Profiling of Potentiated Phospho-Protein Net kinase domain (TKD) (Yamamoto Y., et al. Activating works in Cancer Cells. Cell (2004) 118: 217-28.). mutation of D835 within the activation loop of FLT3 in 10 The process of angiogenesis may contribute to leukemic human hematologic malignancies. Blood (2001) 97: 2434 cell Survival and a resultant resistance to chemotherapy 39). Investigators have also identified several AML-associ triggered cell death. Vascular endothelial growth factor ated point mutations in the FLT3 JMD (Stirewalt D. L., et al. (VEGF) is a major determinant of angiogenesis. A signifi Novel FLT3 point mutations within exon 14 found in cant proportion of de novo and secondary AML blast popu patients with acute myeloid leukemia. Br. J. Haematol 15 lations produce and secrete VEGF protein. Moreover, blasts (2004) 124: 481-84), and one in the N-terminal portion of from some patients with newly diagnosed AML exhibit the Tyrosine Kinase Domain (Schittenheim M. M., et al. relative overexpression of VEGF Receptor R2 (Padro T. FLT3 K663Q is a novel AML-associated oncogenic kinase: Bieker R, Ruiz S, et al. Overexpression of vascular endothe determination of biochemical properties and sensitivity to lial growth factor (VEGF) and its cellular receptor KDR sunitnib. Leukemia (2006) 20: 2008-14.). (VEGFR-2) in the bone marrow of patients with acute The AML-associated FLT3 mutations generally cause myeloid leukemia. Leukemia 2002; 16:1302). Furthermore, ligand-independent autophosphorylation of the FLT3 recep the incorporation of the anti-VEGF monoclonal antibody tor and Subsequent activation of downstream signaling path bevacizumab (Avastin) into an AML combination therapy ways, such as PI3K, Ras, and JAK/STAT (Renneville, et al. reportedly improved tumor clearance rates. (Karp, J. E., et (2008) 22: 915-31). However, the FLT3-ITD and TKD 25 al. Targeting Vascular Endothelial Growth Factor for mutations are associated with significant biological differ Relapsed and Refractory Adult Acute Myelogenous Leuke ences (Renneville, et al. (2008) 22: 915-31). FLT3-ITD mias. Clinical Cancer Res. (2004) 10:3577-85). mutations constitutively induce STAT5 phosphorylation, In addition to Flt3, a variety of other genes are mutated in while FLT3-TKD mutations only weakly induce STAT5 AML and can be divided into two classes based on whether phosphorylation (Choudry, C. et al. AML-associated Flt3 30 they confer a favorable or non-favorable prognosis. Muta kinase domain mutations show signal transduction differ tions in the chaperone protein-encoding gene NPM1 have ences compared with Flt3-ITD mutations. Blood (2005) been found in 30% of adults with de novo AML, but not in 106: 265-73). Furthermore, FLT3-ITD, but not TKD muta adults with secondary AML (Renneville, et al. (2008) 22: tions Suppress expression of the transcription factors, 915-31). Among patients with cytogenetically normal AML, c/EBPC. and Pu.1, which function in myeloid differentiation. 35 NPM1 mutations are predictive of higher rates of response Additionally, neither class of FLT3 mutation is sufficient to to induction therapy and longer overall Survival, but only in induce AML, Suggesting that additional mechanisms may be the absence of FLT3-ITD mutations. Mutations in the basic involved (Renneville, et al. (2008) 22:915-31). Many inves region leucine Zipper-encoding gene CEBPA are found in tigational new drugs are targeted to FLT3 receptor kinase 15-19% of AML patients, and are predictive of longer activity (Gilliland, G. D., and Griffin, J. D. Blood (2002) 40 overall Survival and longer complete response duration 100: 1532–42). However, the different cell signaling profiles (Baldus, C. D., et al. Clinical outcome of de novo acute of AML-associated mutations suggest that different AML myeloid leukemia patients with normal cytogenetics is patients will exhibit distinct responses to inhibition of FLT3 affected by molecular genetic alterations: a concise review. kinase activity. Pre-screening patient cell samples for a British J. Haematology (2007) 137:387-400). response to a FLT3 kinase inhibitor drug, for example by 45 Mutated genes that confer a non-favorable prognosis examining the effects of drug treatment on pSTAT5 levels, include ERG which encodes a transcription factor activated may predict whether a patient will respond to that drug. by signal transduction pathways that regulates cell differen Clinically, FLT3-TKD mutations correlate with shorter tiation, proliferation, and tissue invasion (Baldus, C. D., et clinical response duration and worse overall Survival. than al. British J. Haematology (2007) 137:387-400.). Overex for patients carrying the FLT3-TKD or wild-type alleles 50 pression of ERG in AML patients is predictive of a higher (Meshinchi, S and Applebaum, F Clin. Can. Res. (2009) 13: rate of relapse and shorter overall survival (Marcucci et al. 4263-4269: Frohling et al. Prognostic significance of acti Overexpression of the ETS-related gene, ERG, predicts a vating FLT3 mutations in younger adults (16 to 60 years) worse outcome in acute myeloid leukemia with normal with acute myeloid leukemia and normal cytogenetics: a karyotype: a Cancer and Leukemia Group B Study. J. study of the AML Study Group Ulm. Blood (2002) 100: 55 Clinical Oncology (2005) 23: 9234–42). High expression of 4372-80.). The presence of the FLT3-ITD mutation, and the BAALC in younger AML patients (under 60 years old) is ratio of the FLT3-ITD mutation to other FLT3 alleles are associated with lower rates of disease-free survival and predictive of clinical response duration, cumulative inci overall survival (Baldus et al. BAALC expression predicts dence of relapse, and patient overall survival (Renneville, et clinical outcome of de novo acute myeloid leukemia patients al. (2008) 22:915-31). 60 with normal cytogenetics: a Cancer and Leukemia Group B In healthy myeloid lineages, G-CSF- promotes cell pro study. Blood (2003) 102: 1613-18). Overexpression of MN1 liferation through activation of JAK/STAT signaling (Touw, in AML patients is associated with a lower rate of response I. P., and Marijke, B., Granulocyte colony-stimulating fac to induction therapy (Baldus, C. D., et al. British J. Hae tor: key factor or innocent bystander in the development of matology (2007) 137: 387-400.). Gain-of-function muta secondary myeloid malignancy? (2007). J. Natl. Cancer. 65 tions in the receptor tyrosine kinase-encoding gene c-KIT Inst. 99: 183-186). A class of AML-associated mutations are predictive of shorter overall complete response duration produces truncated G-CSF receptor, and causes hyperrepon and overall survival in AML patients, and may also be US 9,500,655 B2 27 28 predictive of response to treatment with tyrosine kinase mined clinical parameters include, but are not limited to, inhibitors (Renneville, et al. (2008) 22:915-31). Mutations age, de novo acute myeloid leukemia patient, secondary in the Wlim's Tumor 1 (WT1) gene are found in 10-15% of acute myeloid leukemia patient, or a biochemical/molecular AML cases, and in cytogenetically normal AML patients, marker. In some embodiments, the individual is over 60 are predictive of failure to achieve complete response to years old. In some embodiments, the individual is under 60 chemotherapy (Renneville, et al. (2008) 22:915-31). Point years old. In some embodiments, when the individual is mutations in the RAS oncogenes are found in 10-20% of under 60 years old the activatable elements and modulators AML patients, but prognostic uses of these mutations have are selected from the activatable elements and modulators not yet been identified (Renneville, et al. (2008) 22:915-31). listed in Table 6. In some embodiments, where the individual RAS Mutations: 10 is over 60 years the activatable elements and modulators are Ras proteins normally act as signaling Switches, which selected from the activatable elements and modulators listed alternate between the active (GTP-bound) and inactive in Table 7. In some embodiments, where the individual is a (GDP-bound) states. Somatic point mutations in codons 12, secondary acute myeloid leukemia patient the activatable 13 and 61 of the NRAS and KRAS genes occur in many elements and modulators are selected from the activatable myeloid malignancies, resulting in persistently active forms 15 elements and modulators listed in Table 8 and Table 9. In of the protein. Analyses of patients with MDS revealed a Some embodiments, where the individual is a de novo acute very high risk of transformation to AML in patients with myeloid leukemia patient the activatable elements and N-RAS mutations, providing evidence that these mutations modulators are selected from the activatable elements and might represent an important progression factor in MDS. modulators listed in Table 10 and Table 11. In some embodi Under the two-hit model put forth by Gilliland et al., RAS ments, where the individual has a wild type FLT3 the mutations are likely to provide a growth advantage, which activatable elements and modulators are selected from the when combined with a secondary mutation that blocks activatable elements and modulators listed in Table 13. differentiation, results in AML. Supporting this model, In some embodiments, the activatable elements can N-RAS or K-RAS mutations were found in 22% of cases of demarkate AML cell subpopulations that have different core binding factor AML (CBF-AML), which is defined by 25 genetic Subclone origins. In some embodiments, the activat AML1-ETO or CBFB-MYH11 gene fusions known to dis able elements can demarkate AML Subpopulations that, in rupt differentiation. (Boissel et al. Incidence and prognostic combination with additional Surface molecules, can allow impact of c-Kit, FLT3 LIGAND, and Ras gene mutations in for surrogate identification of AML cell subpopulations. In core binding factor acute myeloid leukemia (CBF-AML). Some embodiments, the activatable elements can demarkate Leukemia (2006) vol. 20 (6) pp. 965-970) 30 AML subpopulations that can be used to determine other One embodiment of the invention will look at any of the protein, epitope-based, RNA, mRNA, siRNA, or metabolic cell signaling pathways described above in classifying dis markers that singly or coordinately allow for surrogate eases, such as AML. Modulators can be designed to inves identification of AML cell Subpopulations, disease stage of tigate these pathways and any relevant parallel pathways. the individual from which the cells were derived, diagnosis, In some embodiments, the invention provides a method 35 prognosis, response to treatment, or new druggable targets. for diagnosis, prognosis, determining progression, predict In some embodiments, the pathways characterization allows ing response to treatment or choosing a treatment for AML. for the delineation of AML cell subpopulations that are the method comprising the steps of (a) Subjecting a cell differentially susceptible to drugs or drug combinations. In population from the individual to a plurality of distinct other embodiments, the cell types or activatable elements modulators in separate cultures, (b) characterizing a plural 40 from a given cell type will, in combination with activatable ity of pathways in one or more cells from the separate elements in other cell types, provide rationnetric or metrics cultures comprising determining an activation level of at that singly or coordinately allow for Surrogate identification least one activatable element in at least three pathways, of AML cell subpopulations, disease stage of the individual where the pathways are selected from the group consisting from which the cells were derived, diagnosis, prognosis, of apoptosis, cell cycle, signaling, or DNA damage path 45 response to treatment, or new druggable targets. ways, and (c) correlating the characterization with diagnosis, Therapeutic Agents Effective Against the Disease prognosis, determining progression, predicting response to Treatment of AML is divided into phases called “induc treatment or choosing a treatment for AML, in an individual, tion' where the goal is to induce the leukemia into “remis where the pathways characterization is indicative of the sion' (defined as no clinically detectable disease, specifi diagnosis, prognosis, determining progression, response to 50 cally <5% marrow blasts with peripheral count recovery) treatment or the appropriate treatment for AML. In some and “post-induction therapy’ or “consolidation' where the embodiments the activatable elements and modulators are goal is to keep the patient in clinical remission. Approxi selected from the activatable elements and modulators listed mately 75% of patients (excluding M3 AML) (<60 years in Tables 1, 2, 3 or 5. In some embodiments, the activatable old) enter into remission after one or two courses of standard elements and modulators are selected from the activatable 55 AML therapy cytarabine (100-200 mg/m) coupled to an elements and modulators listed in Table 12 and are used to anthracycline Such as daunorubicin or idarubicin, +/-thio predict response duration in an individual after treatment. In guanine, etoposide, dexamethasone). Unfortunately only Some embodiments the modulator is selected from the group ~40% of patients (>60 years old) achieve remission despite consisting of FLT3L, GM-CSF, SCF, G-CSF, SDF1a, LPS, significant toxicity. PMA, Thapsigargin, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, 60 The M3 form of AML or Promyelocytic leukemia is ZVAD, H.O., Staurosporine, Etoposide, Mylotarg, Dauno treated differently than the other subtypes. This form arises rubicin, and AraC. In some embodiments, the individual has from the t015:17) translocation involving the RARa gene a predefined clinical parameter and the characterization of located at 17q12. In 99 percent of the cases the translocation multiple pathways in combination with the clinical param is t(15:17), which fuses large parts of RARC. to almost the eter is indicative of the diagnosis, prognosis, determining 65 complete PML coding sequence, generating the PML progression, predicting response to treatment or choosing a RARC. fusion protein. APL is unique among the AML treatment for AML, in an individual. Examples of predeter subtypes in that it can be cured in approximately 90% of US 9,500,655 B2 29 30 cases using a differentiation-inducing therapy of all-trans fying a hematopoietic cell, comprising Subjecting a retinoic acid, which forces the blasts to mature into granu hematopoietic cell to at least one modulator that affects locytes. Arsenic trioxide has also been shown to be particu signaling mediated by receptors subjecting a hematopoietic larly effective in the treatment of M3 AML. cell to at least one modulator that affects signaling mediated If remission induction is Successful, further treatment may by receptors selected from the group comprising SDF-1C. be given to try to destroy any remaining leukemia cells and IFN-O, IFN-y, IL-10, IL-6, IL-27, G-CSF, FLT-3L, IGF-1, help prevent its relapse. The options for AML consolidation M-CSF and SCF; also subjecting the hematopoietic cell to at therapy are several courses of high-dose cytarabine (ara-C) least one modulator selected from the group comprising chemotherapy, Supportive care, experimental therapy, allo PMA, Thapsigargin, H2O, Etoposide, Mylotarg, AraC. geneic (donor) stem cell transplant or autologous stem cell 10 daunorubicin, staurosporine, benzyloxycarbonyl-Val-Ala transplant. Asp (OMe) fluoromethylketone (ZVAD), lenalidomide, Supportive care is important in the treatment of patients EPO, azacitadine, decitabine; determining the expression with AML. One aspect of supportive care is transfusion level at least one protein selected from the group comprising therapy which involves blood transfusion (red blood cells or ABCG2, C-KIT receptor, and FLT3 LIGAND receptor, platelets). Red blood cell transfusions are generally per 15 determining the activation states of a plurality of activatable formed when the patient has symptoms of fatigue in com elements in the cell comprising; and classifying the cell bination with low red cell numbers or low red cell numbers based on said activation states and expression levels. and an inability to make new red blood cells. Platelet Another embodiment of the invention further includes using transfusions are generally performed when the patient is the modulators IL-3, IL-4, GM-CSF, EPO, LPS, TNF-C., and bleeding, has a low platelet count and is not producing CD4OL. adequate platelets to prevent bleeding, or having a procedure One embodiment of the invention involves the use of that may cause bleeding. Patients who receive frequent red multiparametric flow cytometry to examine the biology and blood cell transfusions may suffer from tissue and organ signalling pathways in AML to inform on the choice of damage due to the accumulation of iron. Reactive oxygen consolidation therapy in AML (chemotherapy versus species generated by labile plasma iron are a principal cause 25 hematopoietic cell transplantation) of cellular injury and organ dysfunction in patients with iron One embodiment of the invention involves the use of overload which affects survival and increases the risk of multiparameter flow cytometry to examine the biology and leukemia. Iron chelation therapy is recommended to the signalling pathways in AML to inform on likelihood of patient in these cases. This therapy uses drugs such as response to new agents in development for the treatment of deferasirox, which can chelate extra iron and remove it from 30 AML Such as Mylotarg, tipifarnib, or other agents such as the body through the passage of urine. Decitabine or AZacytidine. Chemotherapy with stem cell transplant is a method for One embodiment of the invention involves the use of giving high dose chemotherapy followed by replacement of multiparametric flow cytometry to examine the biology and blood-forming cells, which have been destroyed by the signalling pathways of myeloid disorders to aid in classifi cancer treatment. The stem cells of healthy donors are used 35 cation and therapeutic selection. for infusion in patients who have undergone chemotherapy. In some embodiments, the invention provides a method These reinfused stem cells grow into (and restore) the blood for predicting a response to a treatment or choosing a cells in the body. Transplantation is most beneficial in high treatment for AML, in an individual, the method comprising risk patients with AML who have achieved a CR (remission) the steps: (a) Subjecting a cell population from the individual in first induction (CR1). There is controversy as to whether 40 to at least two distinct modulators in separate cultures; (b) patients with intermediate risk AML should receive a trans determining an activation level of at least one activatable plant in CR1 if they have a genetically matched sibling. It is element from each of at least three pathways selected from also recommended that patients with intermediate risk AML the group consisting of apoptosis, cell cycle, signaling, and but high risk molecular markers (e.g. Flt 3 ITD) undergo DNA damage pathways in one or more cells from each said allogeneic transplant in CR1. 45 separate cultures, where the activatable elements measured A large number of experimental treatment approaches are in each separate culture are the same or the activatable under the process of development. Agents under investiga elements measured in each separate culture are different; and tion include hypomethylating drugs such as AZacytidine and (c) predicting a response to a treatment or choosing a Decitabine which induce differentiation in the affected cells therapeutic for AML, in the individual based on the activa by preventing DNA methylation, Arsenic trioxide (apoptosis 50 tion level of said activatable elements. In some embodi inducer), Sorafenib (tyrosine kinase inhibitor), gemtuzumab ments, the method further comprises determining whether oZogamicin (Mylotarg), Vorinostat and valproic acid (his the apoptosis, cell cycle, signaling, or DNA damage path tone deacetylase inhibitors), tipifarnib and lonafarnib (farne ways are functional in the individual based on the activation syl transferase), bevacizumab (anti-VEGF monoclonal anti levels of the activatable elements, wherein a pathway is body that inhibits angiogenesis), eZatiostat (glutathione S1 55 functional if it is permissive for a response to a treatment, transferase inhibitor), and clofarabine (nucleoside analog). where if the apoptosis, cell cycle, signaling, and DNA One embodiment of the invention involves the use of damage pathways are functional the individual can respond multiparameter flow cytometry to examine the biology and to treatment, and where if at least one of the pathways is not signalling pathways in AML to inform on likelihood of functional the individual can not respond to treatment. In response to ara-C based induction therapy. 60 Some embodiments, the method further comprises determin One embodiment of the invention involves the use of ing whether the apoptosis, cell cycle, signaling, or DNA multiparametric flow cytometry to examine the biology and damage pathways are functional in the individual based on signalling pathways in AML to inform on the duration of the activation levels of the activatable elements, wherein a response to ara-C based induction therapy. pathway is functional if it is permissive for a response to a One embodiment of the invention is a method for pre 65 treatment, wherein if the apoptosis and DNA damage path dicting the outcome of patients undergoing ara-c based ways are functional the individual can respond to treatment. induction therapy for AML. The method comprises classi In some embodiments, the method further comprises deter US 9,500,655 B2 31 32 mining whether the apoptosis, cell cycle, signaling, or DNA MDS is predominantly a disease of the elderly. Median damage pathways are functional in the individual based on age of diagnosis MDS is 68 years. MDS has an overall the activation levels of the activatable elements, wherein a age-adjusted annual incidence of 3.3 per 100,000, and the pathway is functional if it is permissive for a response to a rate increases with age to 10 per 100,000 among those aged treatment, where a therapeutic is chosen depending of the 70 years or older. Approximately 55% of patients die within functional pathways in the individual. In some embodiments 3 years of diagnosis. (Rollison et al. Epidemiology of the activatable elements and modulators are selected from myelodysplastic syndromes and chronic myeloproliferative the activatable elements and modulators listed in Tables 1, 2, neoplasms in the United States, 2001-2004, using data from 3 or 5. In some embodiments, the activatable elements and the NAACCR and SEER programs. Blood (2008) vol. 112 modulators are selected from the activatable elements and 10 (1) pp. 45-52) Patients with high-risk MDS generally sur modulators listed in Table 12 and are used to predict vive for approximately one year. Morbidity and mortality response duration in an individual after treatment. In some are a result of complications of cytopenias or transformation embodiments the modulator is selected from the group to acute myeloid leukemia. One of the major morbidities of consisting of FLT3L, GM-CSF, SCF, G-CSF, SDF1a, LPS, MDS found in the vast majority of (-60-80%) patients is PMA, Thapsigargin, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, 15 symptomatic anemia, with associated fatigue. Other cytope ZVAD, H.O., Staurosporine, Etoposide, Mylotarg, Dauno nias include neutropenia (-50-60%) and thrombocytopenia rubicin, and AraC. (~40-60%). Dysfunctional neutrophils cause an increased In some embodiments, the invention provides a method of risk of infection. Decreased platelets, as mentioned in the predicting a response to a treatment or choosing a treatment AML section, are associated with bleeding. (PETER L. for AML, in an individual, the method comprising the steps GREENBERG, et. al. Myelodysplastic Syndromes. The of: (a) Subjecting a cell population from said individual to at American Society of Hematology. 2002, p. 136-61.) least three distinct modulators in separate cultures, wherein: Causes (i) a first modulator is a growth factor or a mitogen, (ii) a The initiating event for MDS is DNA injury in a second modulator is a cytokine, (iii) a third modulator is a hematopoietic progenitor cell. The disruption of genes that modulator that slows or stops the growth of cells and/or 25 control the balance of growth and differentiation results in induces apoptosis of cells or, the third modulator is an the clonal proliferation of defective progeny, which are inhibitor; (b) determining the activation level of at least one eliminated by apoptosis before they fully mature. The exces activatable element in one or more cells from each of the sive apoptosis contributes to the peripheral cytopenias char separate cultures, where: (i) a first activatable element is an acteristic of the MDS phenotype. Accumulated genetic dam activatable element within the PI3K/AKT, or MAPK path 30 age, particularly anti-apoptotic mutations, may result in ways and the activation level is measured in response to the neoplastic transformation to acute leukemia. (AUIC, et. al. growth factor or mitogen, (ii) a second activatable element Pathogenesis, etiology and epidemiology of myelodysplas is an activatable element within the JAK/STAT pathways tic syndromes. Haematologica. 1998, vol. 83, p. 71–86: and the activation level is measured in response to the HELLSTROM-LINDBERG E, et. al. Achievements in cytokine, (iii) a third activatable element is an activatable 35 understanding and treatment of myelodysplastic syndromes. element within an apoptosis pathway and the activation level Hematology (American Society of Hematology Education is measured in response to the modulator that slows or stops Program), 2000, p. 110-132: BARRCTTJ, et al. Myelodys the growth of cells and/or induces apoptosis of cells, or the plastic syndrome and aplastic anemia: diagnostic and con third activatable element is activatable element within the ceptual uncertainties. Leukemia Research, 2000, vol. 24, p. phospholipase C pathway and the activation level is mea 40 595-596.) sured in response to the inhibitor, or the third activatable Similar to AML, MDS may develop in individuals who element is a phosphatase and the activation level is mea have been exposed to environmental or occupational toxins Sured in response to the inhibitor; and (c) correlating the that increase the likelihood of Somatic mutations, including, activation levels of said activatable elements with a response but not limited to: Cancer chemotherapy, e.g., alkylating to a treatment or with choosing a treatment for AML in the 45 agents and topoisomerase II inhibitors, excess ionizing individual. Examples of predefined clinical parameters radiation, e.g., atomic bombs and radiotherapy for malignant include age, de novo acute myeloid leukemia patient, sec diseases, and industrial chemicals, e.g., benzene, pesticides, ondary acute myeloid leukemia patient, or a biochemical/ fertilizers, herbicides, heavy metals, Stone and cereal dusts, molecular marker. In some embodiments, the cytokine is nitro-organic explosives, petroleum and diesel derivatives, selected from the group consisting of G-CSF, IFNg. IFNa, 50 and organic solvents (benzene, toluene, Xylene, and IL-27, IL-3, IL-6, and IL-10. In some embodiments, the chloramphenicol). growth factor is selected from the group consisting of Symptoms FLT3L, SCF, G-CSF, and SDF1a. In some embodiments, the MDS is characterized by cytopenias (anemia, neutrope mitogen is selected from the group consisting of LPS, PMA, nia, thrombocytopenia) of any or all of the three hematopoi and Thapsigargin. In some embodiments, the modulator that 55 etic lineages (red blood cells, white blood cells and platelets) slows or stops the growth of cells and/or induces apoptosis with varying degrees of severity. The common symptoms of cells is selected from the group consisting of Stauro include fatigue, bruising, and/or bleeding, pallor, ecchymo sporine, Etoposide, Mylotarg, Daunorubicin, and AraC. sis, epistaxis, gingival bleeding, and bacterial infections. Myelodysplastic Syndromes (MDS) Patients may be asymptomatic at diagnosis. Bleeding (due to Myelodysplastic syndromes (MDS) constitute a hetero 60 lack of platelets) and infection (due to lack of WBCs) are the geneous group of hematologic disorders characterized by two most serious complications in MDS patients. MDS is ineffective hematopoiesis and dysplasia with varying risks Sometimes underdiagnosed, since patients suffering from of transformation to acute myeloid leukemia (AML). In mild to moderate anemia are attributed to a chronic disease addition, evidence of a cellular immunologic response has or a mild renal insufficiency. been implicated in the pathogenesis of a subset of MDS 65 Diagnosis patients (Melchert, et al., Current Opinion in Haematology A combination of cellular morphology (to detect multi 2007 Vol. 14, p 123-129.). lineage dysplasia in the bone) and cytogenetics (to detect US 9,500,655 B2 33 34 characteristic clonal abnormalities) is used for the diagnosis 5q- is associated with favorable prognosis, uniparental of MDS. Basic diagnostic criteria involve microscopic mor disomy in 7q confers substantially lower prognosis (3 phological examination of bone marrow using a variety of months vs. 39 months survival). (Itzykson R, et al. Meeting histological stains. Dysplasia, particularly of megakaryo report: myelodysplastic syndromes at ASH 2007. Leukemia. cytes, evidence of disruption of the normal marrow archi 2008, Vol. 22, pp 893-897) tecture, such as abnormal localization of immature precur A substantial fraction of MDS patients appear cytogeneti sors (ALIP), and an estimate of the blast percentage are cally normal because they harbor Submicroscopic chromo important diagnostic findings in bone marrow examinations. somal lesions. Recently, SNP array-based methods have Bone marrows are also examined for dysgranulopoiesis, been used to detect cryptic genetic lesions in this class of dysmegakaryocytopoiesis, and dyserythropoiesis. Dys 10 patients, although this is not yet standard in clinical practice. granulopoiesis include abnormalities in primary granules (Itzykson R, et al. Meeting report: myelodysplastic Syn Such as decreased or absent secondary granules, large gran dromes at ASH 2007. Leukemia. 2008, Vol. 22, pp 893-897). ules or decreased staining, and nuclear abnormalities or Furthermore, molecular genotyping assays are now being increased blasts. Examples of dysmegakaryocytopoiesis used experimentally to screen for known pathogenic muta include micromegakaryocytes, large mononuclear or 15 tions to help stratify MDS patients. binuclear forms, multiple Small nuclei, and reduced num In the context of MDS, multiparameter flow cytometry is bers. Dyserythropoiesis is characterized by more than 15 used to measure abnormal light scatter properties of dys percent ringed sideroblasts, nuclear fragments, multiple plastic cells, abnormal antigen density, loss of antigens, and nuclei, nuclear lobation, internuclear bridges, megaloblastic asynchronous expression of antigens which are normally erythropoiesis, macronormoblastic erythropoiesis, irregular co-expressed during myeloid maturation, and these param cytoplasmic staining, or less than 5 percent erythroid cells. eters may correlate to the grade of the disease. (STETLER Such morphologic dysplasias are however not specific for STEVENSON M, et al. Diagnostic utility of flow cytometric MDS. Mild megaloblastic changes without dyspoiesis in immunophenotyping in myelodysplastic syndromes. Blood. other cell lines are not considered Sufficient for a diagnosis 2001, vol. 98, p. 979-987.) of MDS. 25 One embodiment of the invention combines one or more In addition to a bone marrow aspirate with biopsy, and a of these existing tests with the analysis of signalling medi CBC with differential, one usually orders a reticulocyte ated by receptors to diagnose disease especially MDS, AML count, serum epo, ferritin, B12, and folate to differentiate or MPN. All tests may be performed in one location and other causes and to optimize treatment of the anemia. Other provided as a single service to physicians or other caregiv helpful tests in MDS include HLA typing (if platelet support 30 CS. and/or potential marrow transplant), HLA-DR15 typing (for Cell-Signaling Pathways and Differentiating Factors possible administration of immunosuppressive therapies), Involved FLAER test (to differentiate MDS from a PNH clone), and Regulation of hematopoiesis in MDS is complex and a JAK2 mutation if the patient has thrombocytosis (to multiple factors are involved. Genetic alterations in signal differentiate essential thrombocythemia). 35 ing molecules have been extensively studied in MDS. These Deletions or amplifications of large chromosomal regions molecules include transcription factors, receptors for growth are more commonly observed in MDS, compared with the factors, RAST signaling molecules, and cell cycle regulators. balanced translocations commonly observed in de novo In the early stages of MDS, there is an increased fre AML. Cytogenetic data help stratify patients in terms of quency of apoptosis resulting in intramedullary apoptotic diagnosis and evaluating prognosis for Survival and risk of 40 bodies. Advanced MDS, which may transform to AML, is transformation to AML (HOFMANN W K, et al. Myelo characterized by increased proliferation and antiapoptotic dysplastic syndrome. Annual Review of Medicine. 2005, vol. factors, such as mutations in p53, RAS, C-MPL or FMS. 56, p. 1-16). Characteristic chromosomal deletions involve (Aulet al. Evaluating the prognosis of patients with myelo chromosome 5 del(5q).-5, chromosome 11 del (11q). dysplastic syndromes. Ann Hematol (2002) vol. 81 (9) pp. chromosome 12 del (12q), chromosome 20 del(20g). 45 485-97) chromosome 7 del(7.q),-7, chromosome 17 del (17p), and Genetic alterations in the RAS signaling pathway are chromosome 13 del (13q). Other frequent structural and/or frequently seen in MDS. The RAS signaling pathway nor numerical chromosomal aberrations include trisomy 8, tri mally promotes cellular proliferation and differentiation. By Somy 21, and inversion 3(q21q26). Rare reciprocal translo contrast, pathogenic RAS pathway mutations generally cations include to 1:7)(q10p10), t(1; 3)(p36:q21), t(3:3)(q21; 50 cause continuous kinase activity and signal transduction. q26), tC6:9)(p23:q34), and t(5; 12)-fusion between The cell Surface receptor for macrophage colony stimulating PDGFR-B and TEL(ETV-6), (q33:p13); t(5:7)(q.33:11.2). factor (M-CSF), encoded by the FMS gene, normally pro Deletion of chromosomal region 5q31 (5q-) is the most motes cellular proliferation and differentiation of monocyte frequent genetic lesion in MDS and is present in more than and macrophages, and is upstream of RAS signaling. Acti 20 percent of MDS patients, garnering its own WHO clas 55 vating mutations in this gene are found in 10% of MDS sification. The pathogenic event associated with this genetic cases, and are associated with poor Survival and increased lesion has been traced to the hemizygous deletion of RPS14, risk of transformation to AML. (PADUA R A, et al. RAS, which encodes a ribosomal Subunit protein, and is also FMS and p53 mutations and poor clinical outcome in implicated in Diamond-Blackfan anemia. (Ebert B L, et al. myelodysplasias: a 10-year follow-up. Leukemia, 1998, vol. Identification of RPS14 as a 5q- syndrome gene by RNA 60 12, p. 887-892; TOBAL K, et al. Mutation of the human interference screen. Nature, 2008, Vol. 451, No. 17, pp FMS gene (M-CSF receptor) in myelodysplastic syndromes 335-340) and acute myeloid leukemia. Leukemia, 1990, vol. 4, p. A chromosomal abnormality commonly implicated in the 486-489.) progression of MDS is monosomy 7q. (STEPHENSONJ, et Activating mutations in FLT3, a receptor-type tyrosine al. Possible co-existence of RAS activation and monosomy 65 kinase also upstream of RAS signaling, have been reported 7 in the leukemic transformation of myelodysplastic Syn in 3-5% of MDS cases. (Georgiou et al. Serial determination dromes. Leukemia Research, 1995, vol. 19, p. 741-8). While of FLT3 mutations in myelodysplastic syndrome patients at US 9,500,655 B2 35 36 diagnosis, follow up or acute myeloid leukaemia transfor of the DNA-damage checkpoint gene CHK2 in myelodys mation: incidence and their prognostic significance. Br J plastic syndromes and acute myeloid leukemias. Leukemia Haematol (2006) vol. 134 (3) pp. 302-6) Inactivation of the Research, 2001, vol. 25, p. 333-338; KIKUKAWA M, et al. neurofibromatosis type 1 (NF1) gene, normally a negative Study of p53 in elderly patients with myelodysplastic syn regulator of RAS signaling, has also been implicated in the dromes by immunohistochemistry and DNA analysis. progression of MDS. (Stephenson J, et al. Possible co American Journal of Pathology: 1999, Vol. 155, p. 717-721: existence of RAS activation and monosomy 7 in the leuke POPPE B, et al. Expression analyses identify MEL as a mic transformation of myelodysplastic syndromes. Leuke prominent target of 11q23 amplification and Support an mia Res 1995; 19:741-8). Gain-of-function mutations have etiologic role for MLL gain of function in myeloid malig also been reported in PTPN11 in patients with MDS. (NEU 10 nancies. Blood. 2004, vol. 103, p. 229-235.) BAUER A, et al. Mutations in the ras proto-oncogenes in Dysregulation of genes that encode angiogenic factors patients with myelodysplastic syndromes. Leukemia. 1994, involved in the growth of hematopoietic cells may play vol. 8, p. 638-641). Among the RAS genes themselves, important role in pathogenesis of MDS. (PRUNERIG, et al. mutations of the N-ras gene are the most frequent and are Angiogenesis in myelodysplastic syndromes. British Jour detected in 20 to 30 percent of human leukemias and 15 nal of Cancer, 1999, Vol. 81, p. 1398-1401.) The immuno approximately 16 percent of MDS cases. K-RAS mutations modulatory cytokine, TNF-C. has been shown to express are found at approximately half that frequency. The majority strong inhibitory activity in hematopoiesis. (BROXMEYER of studies suggest that RAS mutations in MDS are associ H E, et al. The suppressive influences of human tumor ated with poor survival and increased probability of devel necrosis factors on bone marrow hematopoietic progenitor oping AML. (YUNIS JJ, et al. Mechanisms of ras mutation cells from normal donors and patients with leukemia: Syn in myelodysplastic syndrome. Oncogene. 1989, Vol. 4, p. ergism of tumor necrosis factor and interferon-gamma. 609-614; Auletal. Evaluating the prognosis of patients with Journal of Immunology. 1986, vol. 36, p. 4487-4495.) Other myelodysplastic syndromes. Ann Hematol (2002) vol. 81 (9) cytokines reportedly involved in the processes leading to pp. 485-97). ineffective hematopoiesis in MDSs include TGF-B, IL-1B. Although less frequently, AML1, C/EBPO. TEL (ETV6) 25 and TNF-related signaling molecules TRADD/FADD, RIP. and p53 genes are also a target of mutations in MDS. and TNF-related apoptosis inducing ligand (TRAIL) AML 1-binding sites exist upstream of several genes encod (SAWANOBORIM, et al. Expression of TNF receptors and ing factors and receptors that determine the lineage speci related signaling molecules in the bone marrow from ficity of hematopoietic cells. (OKUDAT, et al. AML1, the patients with myelodysplastic syndromes. Leukemia target of multiple chromosomal translocations in human 30 Research, 2003, vol. 27, p. 583-591; PLASILOVA M, et al. leukemia, is essential for normal fetal liver hematopoiesis. TRAIL (Apo2L) Suppresses growth of primary human leu Cell. 1996, vol. 84, p. 321-30.) C/EBPO. is an important kemia and myelodysplasia progenitors. Leukemia, 2002, mediator of granulocyte differentiation and regulates the vol. 16, p. 67-73.) expression of multiple granulocyte-specific genes including One embodiment of the invention will look at any of the the granulocyte colony-stimulating factor (G-CSF) receptor, 35 cell signaling pathways described above in classifying dis neutrophil elastase and myeloperoxidase. C/EBPO. knockout eases, such as MDS. Modulators can be designed to inves mice display a profound block in granulocyte differentiation tigate these pathways and any relevant parallel pathways. (COLLINS SJ, et al. Multipotent hematopoietic cell lines In some embodiments, the invention provides a method derived from C/EBPO. (-/-) knockout mice display granu for diagnosing, prognosing, determining progression, pre locyte macrophage-colony-stimulating factor, granulocyte 40 dicting response to treatment or choosing a treatment for colony-stimulating factor and retinoic acid-induced granu MDS or rationale combinations of drugs, or identification of locytic differentiation. Blood. 2001, vol. 98, p. 2382-8). This new potentially druggable targets the method, the method suggests that any mutation in C/EBPO. will result in defec comprising the steps of (a) Subjecting a cell population from tive hematopoiesis. TEL function is essential for the estab the individual to a plurality of distinct modulators in sepa lishment of hematopoiesis of all lineages in the bone mar 45 rate cultures, (b) characterizing a plurality of pathways in row, Suggesting a critical role for TEL in the normal one or more cells from the separate cultures comprising transition of the hematopoietic activity from fetal liver to determining an activation level of at least one activatable bone marrow. Experiments conducted on the role of TEL element in at least three pathways, where the pathways are genes indicate an ineffective hematopoiesis in the case of an selected from the group consisting of apoptosis, cell cycle, alteration in these genes. (WANG LC, et al. The TEL/ETV6 50 signaling, or DNA damage pathways, and (c) correlating the gene is required specifically for hematopoiesis in the bone characterization with diagnosing, prognosing, determining marrow. Genes and Development. 1998, vol. 12, p. 2392 progression, predicting response to treatment or choosing a 402). Mutations or deletions causing inactivation of the p53 treatment for MDS, in an individual, where the pathways gene in both the alleles have been shown to predispose the characterization is indicative of the diagnosing, prognosing, cells to neoplastic transformation. Inactivation is detected in 55 determining progression, response to treatment or the appro 5 to 10 percent of cases of clinically advanced MDS, priate treatment for MDS. In some embodiments, the indi indicating that p53 mutations may play a role in leukemic vidual has a predefined clinical parameter and the charac progression of MDS. (SUGIMOTO K, et al. Mutations of terization of multiple pathways in combination with the the p53 gene in MDS and MDS-derived leukemia. Blood. clinical parameter is indicative of the diagnosis, prognosis, 1993, vol. 81, p. 3022-6.) 60 determining progression, predicting response to treatment or Apoptotic genes (increased bcl-2 expression) (KURO choosing a treatment for MDS, in an individual. Examples TAKI H. et al. Apoptosis, bcl-2 expression and p53 accu of predetermined clinical parameters include, but are not mulation in MDS, MDS derived acute myeloid leukemia limited to, biochemical/molecular markers. and de novo acute myeloid leukemia. Acta Haematologica, In some embodiments, the activatable elements can 2000, vol. 102, p. 115-123.) and mutations in genes includ 65 demarkate MDS cell subpopulations that have different ing CHK2, p53, MLL have been implicated in the patho genetic Subclone origins. In some embodiments, the activat genesis of MDS (HOFMANNW K, et al. Mutation analysis able elements can demarkate MDS subpopulations that, in US 9,500,655 B2 37 38 combination with additional Surface molecules, can allow pared to combination with G/GM-CSF. Proceeding from the for surrogate identification of MDS cell subpopulations. In American Society of Clinical Oncology Conference. Chi Some embodiments, the activatable elements can demarkate cago, Ill. 2007. Abstract 7082.) MDS subpopulations that can be used to determine other Hematide, a novel synthetic pegylated peptidic com protein, epitope-based, RNA, mRNA, siRNA, or metabolic 5 pound, acts as an erythropoiesis stimulating agent that binds markers that singly or coordinately allow for Surrogate to and activates the erythropoietin receptor. It could restore identification of MDS cell subpopulations, disease stage of hemoglobin to the target range and eliminate the need for red the individual from which the cells were derived, diagnosis, blood cell transfusions, though hematide is immunologically prognosis, response to treatment, or new druggable targets. distinct from EPO (http://www.takeda.com/press/ In some embodiments, the pathways characterization allows 10 article 28646.html). Another growth factor thrombopoeitin for the delineation of MDS cell subpopulations that are (TPO), the ligand for the c-mpl receptor, is a major regulator differentially susceptible to drugs or drug combinations. In of platelet production in vivo. It has been indicated in other embodiments, the cell types or activatable elements several studies that TPO increases platelet counts, platelet from a given cell type will, in combination with activatable size, and increases isotope incorporation into platelets of elements in other cell types, provide rationnetric or metrics 15 recipient animals. Platelet count begins to increase after 3 to that singly or coordinately allow for Surrogate identification 5 days. TPO is thought to affect megakaryocytopoiesis in of MDS cell subpopulations, disease stage of the individual several ways: (1) it increases the size and number of from which the cells were derived, diagnosis, prognosis, megakaryocytes; (2) it produces an increase in DNA con response to treatment, or new druggable targets. tent, in the form of polyploidy, in megakaryocytes; (3) it Therapeutic Agents Effective Against the Disease increases megakaryocyte endomitosis; (4) it produces There are many treatments for MDS. The treatment option increased maturation of megakaryocytes; and (5) it produces typically includes choice of therapy on the basis of risk an increase in the percentage of precursor cells, in the form factors such as patients age, MDS Subtype, and prognostic of small acetylcholinesterase-positive cells, in the bone score. The most commonly used prognostic score for MDS, marrow. RomiploStim, a recombinant Fc-peptide fusion pro the International Prognostic Scoring System (IPSS), is cal 25 tein, is a thrombopoietin receptor agonist which can be used culated based on bone marrow blast percentage, cytogenet for identification of treatments effective in improving throm ics, and the number of cytopenias. Depending on the IPSS bocytopenia. It has recently been used in Phase II trials for score and the patients symptoms, different treatment paths MDS. However, its use is complicated by side effects such are pursued. as disturbances of the gastrointestinal system, and arthralgia. Supportive care is important in the treatment of all 30 Immunosuppressive therapy (IST) has emerged as an patients with MDS. One aspect of supportive care is trans effective therapy for a subset of MDS patients with clonal fusion therapy which involves blood transfusion (red blood amplification of T lymphocytes. T cell clones have been cells or platelets). Red blood cell transfusions are generally identified in 50% of MDS patients and have been implicated performed when the patient has symptoms of fatigue in in Suppression of hematopoiesis through CD8 cytotoxic T combination with low red cell numbers or low red cell 35 lymphocytes. Immunosuppressive agents like anti-thymo numbers and an inability to make new red blood cells. cyte globulin, alone or in combination with cyclosporine, Platelet transfusions are generally performed when the inhibit the effects of T-cell clones. Patients enriched for patient is bleeding, has a low platelet count and is not response to this therapy include the younger age group (sG0 producing adequate platelets to prevent bleeding, or having years), those requiring little to nored blood cell transfusion, a procedure that may cause bleeding. Patients who receive 40 those with marrow hypocellularity, those with the presence frequent red blood cell transfusions may suffer from tissue of paroxysmal nocturnal hemoglobinuria clone, and those and organ damage due to accumulation of iron. Reactive with human leukocyte antigen (HLA)-DR15 phenotype. oxygen species generated by labile plasma iron are a prin using this enrichment criteria, recent data show a 30% cipal cause of cellular injury and organ dysfunction in response rate with improved overall Survival and a decrease patients with iron overload which affects survival and 45 in transformation to AML (Sloand et al JCO 2008 26:2505 increases the risk of leukemia. Iron chelation therapy is 2511) recommended to the patient in these cases. This therapy uses The immunomodulatory drugs are agents that target both drugs such as deferasiroX, which can chelate extra iron and the MDS clone and the bone marrow microenvironment and remove it from the body through the urinary passage. have notable erythropoietic activity in patients with low-risk Low-risk MDS patients are generally empirically treated 50 MDS. Lenalidomide, an amino-derivative of thalidomide with growth factor therapy. Erythropoietin (EPO) therapy is with greater potency and minimal neurotoxicity, has eryth most effective in patients with serum epo-200 IU/L, low ropoietic and cytogenetic remitting activity. The efficacy of int-1 IPSS, and an absence of transfusion requirement. A lenalidomide is greatest in patients with deletions of chro recent study of 403 patients with MDS s?p EPO--/-GCSF mosome 5q. In this Subset, lenalidomide produces and showed a 50% overall response rate to this therapy. (Parket 55 maintains red cell transfusion independence in the majority al Blood 2008 111:574-582) of low-risk patients for about two years. In a study of 148 EPO is thought to overcome reduced sensitivity of eryth patients with MDS RBC dependent anemia and 5q-, 67% of roid precursors to EPO at the initial level of signal trans patients achieved a major erythroid response defined as RBC duction. (Hoefsloot L. H. et al. Erythropoietin-induced acti transfusion independence and an absence of any RBC trans vation of STAT5 is impaired in the myelodysplastic 60 fusion during any consecutive 56 days (8 weeks) and Hgb syndrome. Blood 1997, vol. 89, p. 1690-1700). Reports increase of at least 1 g/dL during the treatment periodt (List show a comparable erythroid response rate when using EPO A. et al. Lenalidomide in the myelodysplastic syndrome alone or EPO plus filgrastim (G-CSF) (response rate 49 with chromosome 5q deletion. New England Journal of percent versus 51 percent), whereas higher EPO dose sched Medicine. 2006, vol. 355, p. 1456-1465.) Among the vast ules were found to have higher response rate than standard 65 majority of MDS patients (over 80 percent) without the 5q EPO dose schedules. (Moyo V M et al. Treating the anemia chromosomal defect, only about 26% respond to lenalido of MDS with erythropoietin: Impact of higher dose com mide. (Blood 2008 111:86-93 (Raza et al) US 9,500,655 B2 39 40 A recent study of gene expression profiling identified a A large number of treatment approaches are under the cohesive set of erythroid-specific genes used as erythroid process of development. Agents under investigation include gene expression signature to predict the response of Arsenic trioxide (apoptosis inducer), Sorafenib (tyrosine lenalidomide. The reduced expression of the erythroid gene kinase inhibitor), Vorinostat and valproic acid (histone signature in responders suggested a defect in erythroid deacetylase inhibitors), tipifarnib and lonafarnib (farnesyl differentiation. This suggests that it might be possible to use transferase), bevacizumab (anti-VEGF monoclonal antibody the response signature to develop a test that can predict the that inhibits angiogenesis), FG-2216 (hypoxia-inducible patients with MDS who will benefit from treatment with factor stabilizer), ezatiostat (glutathione S1 transferase lenalidomide. (Benjamin L. Ebert et al. An Erythroid Dif inhibitor), clofarabine (nucleoside analog). (ALAN F. LIST 10 et al. Insights into the pathogenesis, Classification, and ferentiation Signature Predicts Response to Lenalidomide in treatment of Myelodysplastic Syndromes, Semin. Hematol. Myelodysplastic Syndrome. PLoS Medicine. February 2008. 2008 January; 45(1) 31-8). Pharmacologic differentiators, Vol. 5, no. 2, p. 312-322). such as TLK199, (liposomal glutathione derivative) mediate Hypomethylating drugs such as AZacytidine and Decit proliferation and differentiation of myeloid precursors and abine have been approved for all IPSS scores of MDS. This 15 production of GM-CSF. ATLK-199 trial on MDS patients class of drugs is thought to induce differentiation in the showed hematologic improvement in all three hematopoietic affected cells by preventing DNA methylation. AZacytidine lineages—erythrocytes, neutrophils, platelets. Toxicities is the first FDA-approved drug for the treatment of MDS. It were limited to infusion reactions, nausea, chills and bone is a pyrimidine analog that inhibits DNA methyl transferase. pain. The thrombopoiesis-stimulating agent, IL-11, is an A CALGB study indicates that treatment with azacytidine indirect thrombopoietic cytokine that helps to combat plate produced higher response rate, improved quality of life, let dysfunction and thrombocytopenia in MDS. The major reduced risk of transformation to AML and extended life side effects of this drug include fever, fluid retention, periph expectancy. (Silverman L R et al. Randomized controlled eral edema, pleural effusions and atrial arrhythmias. Pegy trial of azacytidine in patients with the myelodysplastic lated, recombinant human megakaryocyte growth and devel syndrome: a study of the cancer and leukaemia group B. 25 opment factor (PEG-rHuMGDF) stimulates megakaryocyte Journal of Clinical Oncology. 2002. Vol. 20, p. 2429-2440). and platelet production by binding to c-Mpl receptors. Median Survival was significantly prolonged to 24.4 months One embodiment of the invention involves the use of as compared to 15 months with conventional care, with multiparameter flow cytometry to examine the biology and greatest improvement observed in patients with chromo signalling pathways in myelodysplastic syndrome to classify Some 7 abnormalities, including monosomy 7. (Lim Z. Yet 30 MDS identification of possible druggable targets, and al. Outcomes of MDS patients with chromosome abnormali inform on likelihood of response to agents such as growth ties treated with 5-azacytidine. Program and abstracts of factors (e.g. EPO), immunosuppressive agents (e.g. ATG+/- 49" Annual Meeting of the American Society of haematol CSA), epigenetic modulators (e.g. hypomethylators AZacy ogy. December 2007. Atlanta, Ga. Abstract 1449). Further tidine and Decitabine and HDAC inhibitors), immune azacytidine treatment delays the progress of MDS to AML 35 modulators (e.g. Lenalidomide), or a rationale combination to 13 months as compared to 7.6 months in patients given of the above. only conventional care. A relatively higher number of One embodiment of the invention involves the use of patients treated with azacytidine achieved complete remis multiparametric flow cytometry to examine the biology and sion (CR) and hematologic improvements as compared to signalling pathways in myelodysplastic syndrome to deter best supportive care. Decitabine (DNA methyl transferase 40 mine likelihood of progression to AML. inhibitor) is the second FDA-approved drug for treatment of One embodiment of the invention involves the use of patients with MDS. 170 patients were studied with an multiparametric flow cytometry to examine the biology and overall response rate of 17% (9% CR and 8% PR) with a signalling pathways in myelodysplastic syndrome to deter median duration of response of 10.3 months and a tend mine likelihood of response to agents in development for the toward increased time to AML transformation (Cancer 2006 45 treatment of MDS 106:1794-80 (Kantarian et al) One embodiment of the invention involves the use of Combination of hypomethylating agents with histone multiparametric flow cytometry to examine the biology and deacetylase (HDAC) inhibitors (MGCD-0103) is under trial signalling pathways of myeloid disorders to aid in classifi and preliminary data Suggests major responses including cation and therapeutic selection and identification of new CR, partial remission or marrow CR in 35% of patients with 50 potentially druggable targets. refractory MDS and 50% of previously untreated patients. In some embodiments, the invention provides a method (Itzykson et al. Meeting report: myelodysplastic syndromes for predicting a response to a treatment or choosing a at ASH 2007. Leukemia (2008) vol. 22 (5) pp. 893-7). treatment for MDS or designing rationale combinations of Chemotherapy with stem cell transplants is a method for drugs, in an individual, in an individual, the method com giving high dose chemotherapy and replacing blood-form 55 prising the steps: (a) Subjecting a cell population from the ing cells, which have been destroyed by the cancer treat individual to at least two distinct modulators in separate ment. The stem cells of healthy donors are used for infusion cultures; (b) determining an activation level of at least one in patients who have undergone chemotherapy. These rein activatable element from each of at least three pathways fused stem cells grow into (and restore) the blood cells in the selected from the group consisting of apoptosis, cell cycle, body. Although transplant can be curative in MDS, it is often 60 signaling, and DNA damage pathways in one or more cells limited by the patients performance status and the avail from each said separate cultures, where the activatable ability of donors. Transplantation appears to be most ben elements measured in each separate culture are the same or eficial for children with refractory cytopenias and adults the activatable elements measured in each separate culture with chemotherapy-related MDS, which represent only a are different; and (c) predicting a response to a treatment or small fraction of the MDS population. (Itzykson et al. 65 choosing a therapeutic for MDS, in the individual based on Meeting report: myelodysplastic syndromes at ASH 2007. the activation level of said activatable elements. In some Leukemia (2008) vol. 22 (5) pp. 893-7). embodiments, the method further comprises determining US 9,500,655 B2 41 42 whether the apoptosis, cell cycle, signaling, or DNA damage bone marrow. MPN include polycythemia vera (PV), pri pathways are functional in the individual based on the mary or essential thrombocythemia (ET), and primary or activation levels of the activatable elements, wherein a idiopathic myelofibrosis (PMF). The incidence of MPN in pathway is functional if it is permissive for a response to a the USA is 1.3 per 100,000 per year, with a maximum peak treatment, where if the apoptosis, cell cycle, signaling, and at the age of 25-60 years. (PCT/WO 2007085958 A2/3 DNA damage pathways are functional the individual can (CONSORZIO PER GLISTUDI UNI IN) Feb. 8, 2007) respond to treatment, and where if at least one of the MPN predominantly occur in people older than 60 years, pathways is not functional the individual can not respond to though 20 percent of cases occur in individuals of 40 years treatment. In some embodiments, the method further com or less. Men are two times more likely to develop PV than prises determining whether the apoptosis, cell cycle, signal 10 women. Environmental factors, such as exposure to chemi ing, or DNA damage pathways are functional in the indi cals in hair dyes or to electrical wiring increase an individu vidual based on the activation levels of the activatable al’s susceptibility to MPN. elements, wherein a pathway is functional if it is permissive Causes for a response to a treatment, where if the apoptosis and The biology surrounding MPN remained unclear until the DNA damage pathways are functional the individual can 15 discovery of mutations in the JAK2-STAT5 pathway. respond to treatment. In some embodiments, the method JAK2V617F and JAK2 exon 12 mutant kinases can bind further comprises determining whether the apoptosis, cell cytokine receptors and are phosphorylated in the absence of cycle, signaling, or DNA damage pathways are functional in ligand. This leads to ligand independent signal transduction the individual based on the activation levels of the activat of the erythropoietin and other receptors. MPLW515L/K able elements, wherein a pathway is functional if it is mutant thrombopoietin receptors are able to phosphorylate permissive for a response to a treatment, where a therapeutic wild-type JAK2 in the absence of TPO again leading to is chosen depending of the functional pathways in the constitutive activation. (Baxter et al in Lancet April 2005, individual. Skoda etal reported in the NEJM May 2005, Vainchenker et In some embodiments, the invention provides a method of al Nature May 2005, Gilliland etal Ca Cell May 2005, Zhao predicting a response to a treatment or choosing a treatment 25 et al JBC June 2005, PLOS 2006 3(7)e270 Gilliland & for MDS, in an individual, the method comprising the steps Levine labs Blood 2006 108:3472-3476 Tefferi Gilliland of: (a) Subjecting a cell population from said individual to at Although JAK2V617F mutation accounts for >90% of least three distinct modulators in separate cultures, wherein: patients with PV, the mutation is absent in >50% of patients (i) a first modulator is a growth factor or mitogen, (ii) a with ET and PMF suggesting that other as yet undiscovered second modulator is a cytokine, (iii) a third modulator is a 30 genetic aberrations that lead to EPO or TPO receptor path modulator that slows or stops the growth of cells and/or way activation exist and participate in the pathogenesis of induces apoptosis of cells or, the third modulator is an these disorders. inhibitor; (b) determining the activation level of at least one Symptoms activatable element in one or more cells from each of the Many individuals with MPN are asymptomatic at the time separate cultures, where: (i) a first activatable element is an 35 of diagnosis. Depending on the disorder, symptoms may activatable element within the PI3K/AKT, or MAPK path vary from person to person. All patients with MPN have ways and the activation level is measured in response to the increased risk of heart disease, stroke, or other thromboses. growth factor or mitogen, (ii) a second activatable element Similar to leukemia, a common sign for the presence of PV is an activatable element within the STAT pathway and the and PMF is an enlarged spleen. Polycythemia Vera (PV) is activation level is measured in response to the cytokine, (iii) 40 characterized by an increased production of blood cells, a third activatable element is an activatable element within particularly red blood cells, by the bone marrow. This an apoptosis pathway and the activation level is measured in overproduction can lead to an increase in blood Viscosity, response to the modulator that slows or stops the growth of which can impair the functioning of the heart or the brain. cells and/or induces apoptosis of cells, or the third activat Other symptoms may include fatigue, general malaise, dif able element is activatable element within the phospholipase 45 ficulty in breathing, intense itching after bathing in warm C pathway and the activation level is measured in response water, bruising or bleeding Polycythemia Vera has a survival to the inhibitor, or the third activatable element is a phos rate of between 10 and 20 years, with the longest survival phatase and the activation level is measured in response to occurring in young age groups. the inhibitor; and (c) correlating the activation levels of said Primary or essential thrombocythemia is a result of over activatable elements with a response to a treatment or with 50 production of platelet cells. Symptoms include heart attack choosing a treatment for MDS in the individual. Examples or stroke, headache, burning or throbbing pain, redness and of predefined clinical parameters include age, de novo acute Swelling of hands and feet, bruising, gastrointestinal bleed myeloid leukemia patient, secondary acute myeloid leuke ing or blood in the urine. Similar to PV, it occurs primarily mia patient, or a biochemical/molecular marker. In some after 60 years of age, but some cases (20%) occur in persons embodiments, the cytokine is selected from the group con 55 under 40 years of age. Women are 1.5 times more likely to sisting of G-CSF, IFNg. IFNa, IL-27, IL-3, IL-6, and IL-10. develop ET than men. Individuals with ET have normal life In some embodiments, the growth factor is selected from the expectancy with only a low risk of developing cancer. group consisting of FLT3L, SCF, G-CSF, and SDF1a. In Primary or idiopathic myelofibrosis (also known as Some embodiments, the mitogen is selected from the group myelosclerosis) is caused by overproduction of collagen or consisting of LPS, PMA, and Thapsigargin. In some 60 fibrous tissue in the bone marrow. Other symptoms include embodiments, the modulator that slows or stops the growth fatigue, general malaise, difficulty breathing, weight loss, of cells and/or induces apoptosis of cells is selected from the fever and night Sweats, and abnormal bleeding. Individuals group consisting of Staurosporine, Etoposide, Mylotarg, between the 60 and 70 years are most likely to develop the AZacitidine, Dacatabine, Daunorubicin, and AraC. condition. Exposure to petrochemicals (such as benzene and Myeloproliferative Disease/Myeloproliferative Neoplasm 65 toluene) and intense radiation may increase an individuals Myeloproliferative Neoplasms (MPN) are a group of risk of developing the condition. Severe cases of primary disorders that cause an overproduction of blood cells in the myelofibrosis may be fatal within three to six years. US 9,500,655 B2 43 44 Diagnosis Modulators can be designed to investigate these pathways Elevated hematocrit or platelet count suggests PV or ET and any relevant parallel pathways. respectively. Initial evaluation past history and physical In some embodiments, the invention provides a method examination commonly includes a bone marrow aspiration for diagnosing, prognosing, determining progression, pre with FISH for BCR/ABL to rule out CML. Examination of 5 dicting response to treatment or choosing a treatment for either bone marrow or peripheral blood for JAK2V617F, MPN or rationale combination of different drugs, the method MPLW515L/K, or Exon 12 mutations can establish diagno comprising the steps of (a) Subjecting a cell population from S1S. the individual to a plurality of distinct modulators in sepa Primary myelofibrosis is diagnosed in a similar manner as rate cultures, (b) characterizing a plurality of pathways in above and is characterized by fibrotic bone marrow that 10 one or more cells from the separate cultures comprising cannot be explained by another diagnosis such as CML or determining an activation level of at least one activatable MDS. element in at least three pathways, where the pathways are One embodiment of the invention combines one or more selected from the group consisting of apoptosis, cell cycle, of these existing tests with the analysis of signalling medi signaling, or DNA damage pathways, and (c) correlating the ated by receptors to diagnose disease especially MDS, AML 15 characterization with diagnosing, prognosing, determining or MPN. All tests may be performed in one location and progression, predicting response to treatment or choosing a provided as a single service to physicians or other caregiv treatment for MPN, in an individual, where the pathways CS. characterization is indicative of the diagnosing, prognosing, Cell-Signaling Pathways and Differentiating Factors determining progression, response to treatment or the appro Involved priate treatment for MPN. In some embodiments, the indi Dysregulation of the JAK-STAT signaling pathway has vidual has a predefined clinical parameter and the charac been implicated in the development and progression of terization of multiple pathways in combination with the MPN. Alterations in gene expression occur due to the clinical parameter is indicative of the diagnoses, prognoses, activation of the JAK/STAT pathway by exogenous stimuli 25 determining progression, predicting response to treatment or (sepsis or G-CSF treatment), or endogenously through acti choosing a treatment for MPN, in an individual. Examples vating mutations (e.g. JAK2-V617F. (ROBERT KRAL of predetermined clinical parameters include, but are not OVICS, et. al. Altered gene expression in myeloproliferative limited to, biochemical/molecular marker. neoplasms correlates with the activation of signaling by the Therapeutic Agents Effective Against the Disease V617F mutation of JAK2. Blood. November 2005, vol. 106, 30 For the treatment of polycythemia vera, routine phle no. 10, p. 3374-3376.) Several distinct MPN, polycythemia vera, essential thrombocythemia, and myelofibrosis are botomy (removal of one unit of blood) is performed. This found to have JAK2-V617F mutation, supporting the con decreases the viscosity of blood and reduces the risk of cept that hyperactivation of JAK-STAT signaling is involved stroke. Other therapies include hydroxyurea and aspirin. In in the development of MPN. JAK2 mutations are present in 35 severe cases, chemotherapy Such as low dose methotrexate virtually all cases of polycythemia vera, 41 to 72 percent in is preferred to control excess production of red blood cells. essential thrombocythemia, and 39 to 57 percent in primary Interferon has also been used to treat this disease. myelofibrosis. (BAXTER E J, et al. Acquired mutation of Essential thrombocythemia can be treated with drugs the tyrosine kinase JAK2 in human myeloproliferative neo similar to PV such as hydroxyurea and aspirin. plasms. Lancet. 2005, vol. 365, no. 9464, p. 1054-1061.) 40 Treatment of myelofibrosis generally involves blood cell Studies have found 15 gene-expression markers which were transfusion to increase the number of red blood cells and elevated in patients with PV, including polycythemia rubra platelets. Interferon can slow the progression of this disease Vera 1 (PRV1) and nuclear factor erythroid-derived 2 (NF and some patients benefit from splenectomy. In some cases, E2), as well as one marker, ANKRD15, which was down bone marrow transplantation is also performed. regulated. (ROBERT KRALOVICS, et. al. Altered gene 45 There is strong evidence for the efficacy of targeted kinase expression in myeloproliferative neoplasms correlates with inhibitors in CML, and the thought to extend this to other the activation of signaling by the V617F mutation of Jak2. myeloproliferative neoplasms has triggered rampant devel Blood. November 2005, vol. 106, no. 10, p. 3374-3376.) opment of additional therapies in this class. In particular JAK3 important lymphoid development/myeloid differ inhibitors of JAK2 that target the activation of the JAK2 entiation. Loss of function of JAK3 leads to an autosomal 50 STAT5 pathway are underway. However, until new targeted recessive form of severe combined immunodeficiency. Gain drugs become available, most of the MPN must still be of function mutations in JAK3 have been shown to lead to managed with traditional therapies. acute megakaryocytic leukemia. Leukemia and Lymphoma Using multiparametric phospho-protein analysis that March 2008 49 (3):388-397 Phosphatases have been implicated in MPN biology. 55 examine the biology of MPN this invention could: enable These include SHP-1 (Src homology 2 domain containing patient stratification which would provide an improved tyrosine Phosphatase 1), SHP-2 (Src homology 2 domain classification of these diseases; be used for drug screening to containing tyrosine phosphatase 2), TC-PTP (T-cell PTP), produce biologically informed therapeutics choices; and RPTPa (Receptor protein tyrosine phosphatase a). DEP address the potential for responsiveness to new therapies. (Density enhanced phosphatase), PTP-MEG 1 (Protein tyro 60 The benefits of using the present invention for diagnostic sine phosphatase MEG1), PTP-MEG2 (Protein tyrosine tests includes defining the therapeutic possibilities; identi phosphatase MEG2). PTP-MEG2 is thought to be deregu fication of aggressive disease giving potentially improved lated in Normally PTP-MEG2 decreases as cells differenti outcomes; and matching signaling profiles to experimental ate, however PTP-MEG2 displays increased activity in PV. therapeutic outcomes. Additionally, elucidation of disease One embodiment of the invention will look cell signaling 65 mechanisms would identify de novo targets applicable to pathways described above in classifying and diagnosing future drug therapy and cohort selection for drug develop MPN and identification of new potentially druggable targets. ment. US 9,500,655 B2 45 46 One embodiment of the invention involves the use of is an activatable element within the STAT pathway and the multiparametric flow cytometry to examine the biology and activation level is measured in response to the cytokine, (iii) signalling pathways in MPN to determine likelihood of a third activatable element is an activatable element within progression to AML. an apoptosis pathway and the activation level is measured in One embodiment of the invention involves the use of response to the modulator that slows or stops the growth of multiparametric flow cytometry to examine the biology and cells and/or induces apoptosis of cells, or the third activat signalling pathways in MPN to determine likelihood of able element is activatable element within the phospholipase response to agents in development for the treatment of MPN C pathway and the activation level is measured in response One embodiment of the invention involves the use of to the inhibitor, or the third activatable element is a phos multiparametric flow cytometry to examine the biology and 10 phatase and the activation level is measured in response to signalling pathways of myeloid disorders to aid in classifi the inhibitor; and (c) correlating the activation levels of said cation, therapeutic selection and identification of new poten activatable elements with a response to a treatment or with tially druggable targets and design of ratione drug combi choosing a treatment for MPN in the individual. Examples nations. of predefined clinical parameters include age, de novo acute In some embodiments, the invention provides a method 15 myeloid leukemia patient, secondary acute myeloid leuke for predicting a response to a treatment or choosing a mia patient, or a biochemical/molecular marker. In some treatment for MPN, in an individual, the method comprising embodiments, the cytokine is selected from the group con the steps: (a) Subjecting a cell population from the individual sisting of G-CSF, IFNg. IFNa, IL-27, IL-3, IL-6, and IL-10. to at least two distinct modulators in separate cultures; (b) In some embodiments, the growth factor is selected from the determining an activation level of at least one activatable group consisting of FLT3L, SCF, G-CSF, and SDF1a. In element from each of at least three pathways selected from Some embodiments, the mitogen is selected from the group the group consisting of apoptosis, cell cycle, signaling, and consisting of LPS, PMA, and Thapsigargin. In some DNA damage pathways in one or more cells from each said embodiments, the modulator that slows or stops the growth separate cultures, where the activatable elements measured of cells and/or induces apoptosis of cells is selected from the in each separate culture are the same or the activatable 25 group consisting of Staurosporine, Etoposide, Mylotarg, elements measured in each separate culture are different; and Daunorubicin, AraC and Jak2 inhibitors. (c) predicting a response to a treatment or choosing a In some embodiments, the activatable elements can therapeutic for MPN, in the individual based on the activa demarkate MPN cell subpopulations that have different tion level of said activatable elements. In some embodi genetic Subclone origins. In some embodiments, the activat ments, the method further comprises determining whether 30 able elements can demarkate MPN subpopulations that, in the apoptosis, cell cycle, signaling, or DNA damage path combination with additional Surface molecules, can allow ways are functional in the individual based on the activation for surrogate identification of MPN cell subpopulations. In levels of the activatable elements, wherein a pathway is Some embodiments, the activatable elements can demarkate functional if it is permissive for a response to a treatment, MPN subpopulations that can be used to determine other where if the apoptosis, cell cycle, signaling, and DNA 35 protein, epitope-based, RNA, mRNA, siRNA, or metabolo damage pathways are functional the individual can respond mic markers that singly or coordinately allow for Surrogate to treatment, and where if at least one of the pathways is not identification of MPN cell subpopulations, disease stage of functional the individual can not respond to treatment. In the individual from which the cells were derived, diagnosis, Some embodiments, the method further comprises determin prognosis, response to treatment, or new druggable targets. ing whether the apoptosis, cell cycle, signaling, or DNA 40 In some embodiments, the pathways characterization allows damage pathways are functional in the individual based on for the delineation of MPN cell subpopulations that are the activation levels of the activatable elements, wherein a differentially susceptible to drugs or drug combinations. In pathway is functional if it is permissive for a response to a other embodiments, the cell types or activatable elements treatment, where if the apoptosis and DNA damage path from a given cell type will, in combination with activatable ways are functional the individual can respond to treatment. 45 elements in other cell types, provide rationnetric or metrics In some embodiments, the method further comprises deter that singly or coordinately allow for Surrogate identification mining whether the apoptosis, cell cycle, signaling, or DNA of MPN cell subpopulations, disease stage of the individual damage pathways are functional in the individual based on from which the cells were derived, diagnosis, prognosis, the activation levels of the activatable elements, wherein a response to treatment, or new druggable targets. pathway is functional if it is permissive for a response to a 50 General Methods treatment, where a therapeutic is chosen depending of the Embodiments of the invention may be used to diagnose, functional pathways in the individual. predict or to provide therapeutic decisions for disease treat In some embodiments, the invention provides a method of ment, such as MDS, AML, or MPN. In some embodiments, predicting a response to a treatment or choosing a treatment the invention may be used to identify new druggable targets for MPN, in an individual, the method comprising the steps 55 and to design drug combinations. The following will discuss of: (a) Subjecting a cell population from said individual to at instruments, reagents, kits, and the biology involved with least three distinct modulators in separate cultures, wherein: these and other diseases. One aspect of the invention (i) a first modulator is a growth factor or mitogen, (ii) a involves contacting a hematopoietic cell with a modulator; second modulator is a cytokine, (iii) a third modulator is a determining the activation states of a plurality of activatable modulator that slows or stops the growth of cells and/or 60 elements in the cell; and classifying the cell based on said induces apoptosis of cells or, the third modulator is an activation state. inhibitor; (b) determining the activation level of at least one In some embodiments, this invention is directed to meth activatable element in one or more cells from each of the ods and compositions, and kits for analysis, drug screening, separate cultures, where: (i) a first activatable element is an diagnosis, prognosis, for methods of disease treatment and activatable element within the PI3K/AKT, or MAPK path 65 prediction. In some embodiments, the present invention ways and the activation level is measured in response to the involves methods of analyzing experimental data. In some growth factor or mitogen, (ii) a second activatable element embodiments, the physiological status of cells present in a US 9,500,655 B2 47 48 sample (e.g. clinical sample) is used, e.g., in diagnosis or individual, to predict response to a therapy for an individual, prognosis of a condition, patient selection for therapy using to determine the efficacy of a therapy in an individual, and Some of the agents identified above, to monitor treatment, to determine the prognosis for an individual. In some modify therapeutic regimens, and to further optimize the embodiments, the operative pathways can reveal whether selection of therapeutic agents which may be administered 5 apoptosis, cell cycle, signaling, or DNA damage pathways as one or a combination of agents. Hence, therapeutic are functional in an individual, where a pathway is func regimens can be individualized and tailored according to the tional if it is permissive for a response to a treatment. In data obtained prior to, and at different times over the course Some embodiments, when apoptosis, cell cycle, signaling, of treatment, thereby providing a regimen that is individu and DNA damage pathways are functional the individual can ally appropriate. In some embodiments, a compound is 10 respond to treatment, and if at least one of the pathways is contacted with cells to analyze the response to the com not functional the individual can not respond to treatment. In pound. Some embodiments, when the apoptosis and DNA damage In some embodiments, the present invention is directed to pathways are functional the individual can respond to treat methods for classifying a sample derived from an individual ment. In some embodiments, the operative pathways can having or Suspected of having a condition, e.g., a neoplastic 15 reveal new druggable targets. or a hematopoietic condition. The invention allows for In some embodiments, the invention is directed to meth identification of prognostically and therapeutically relevant ods for classifying a cell by contacting the cell with an Subgroups of conditions and prediction of the clinical course inhibitor, determining the presence or absence of an increase of an individual. The methods of the invention provide tools in activation level of an activatable element in the cell, and useful in the treatment of an individual afflicted with a classifying the cell based on the presence or absence of the condition, including but not limited to methods for assigning increase in the activation of the activatable element. In some a risk group, methods of predicting an increased risk of embodiments, the invention is directed to methods of deter relapse, methods of predicting an increased risk of devel mining the presence or absence of a condition in an indi oping secondary complications, methods of choosing a vidual by subjecting a cell from the individual to a modu therapy for an individual, methods of predicting duration of 25 lator and an inhibitor, determining the activation level of an response, response to a therapy for an individual, methods of activatable element in the cell, and determining the presence determining the efficacy of a therapy in an individual, and or absence of the condition based on the activation level methods of determining the prognosis for an individual. The upon treatment with a modulator and an inhibitor. present invention provides methods that can serve as a In some embodiments, the invention is directed to meth prognostic indicator to predict the course of a condition, e.g. 30 ods of determining a phenotypic profile of a population of whether the course of a neoplastic or a hematopoietic cells by exposing the population of cells to a plurality of condition in an individual will be aggressive or indolent, modulators in separate cultures, determining the presence or thereby aiding the clinician in managing the patient and absence of an increase in activation level of an activatable evaluating the modality of treatment to be used. In another element in the cell population from each of the separate embodiment, the present invention provides information to 35 culture and classifying the cell population based on the a physician to aid in the clinical management of a patient so presence or absence of the increase in the activation of the that the information may be translated into action, including activatable element from each of the separate culture. In treatment, prognosis or prediction. Some embodiments at least one of the modulators is an In some embodiments, the invention is directed to meth inhibitor. In some embodiments, the presence or absence of ods of characterizing a plurality of pathways in single cells. 40 an increase in activation level of a plurality of activatable Exemplary pathways include apoptosis, cell cycle, signal elements is determined. In some embodiments, each of the ing, or DNA damage pathways. In some embodiments, the activatable elements belongs to a particular pathway and the characterization of the pathways is correlated with diagnos activation level of the activatable elements is used to char ing, prognosing or determining condition progression in an acterize each of the particular pathways. In some embodi individual. In some embodiments, the characterization of the 45 ments, a plurality of pathways are characterized by exposing pathways is correlated with predicting response to treatment a population of cells to a plurality of modulators in separate or choosing a treatment in an individual. In some embodi cultures, determining the presence or absence of an increase ments, the characterization of the pathways is correlated in activation levels of a plurality of activatable elements in with finding a new druggable target. In some embodiments, the cell population from each of the separate culture, the pathways characterization in combination with a pre 50 wherein the activatable elements are within the pathways determined clinical parameter is indicative of the diagnosis, being characterized and classifying the cell population based prognosis or progression of the condition. In some embodi on the characterizations of said multiple pathways. In some ments, the pathways characterization in combination with a embodiments, the activatable elements and modulators are predetermined clinical parameter is indicative of a response selected from the activatable elements and modulators listed to treatment or of the appropriate treatment for an individual. 55 in Tables 1, 2, 3 or 5. In some embodiments, the activatable In some embodiments, the characterization of the pathways elements and modulators are selected from the activatable in combination with a predetermined clinical parameter is elements and modulators listed in Table 12 and are used to indicative a new druggable target. predict response duration in an individual after treatment. In some embodiments, the invention is directed to meth In some embodiments, the invention is directed to meth ods for determining the activation level of one or more 60 ods for classifying a cell by determining the presence or activatable elements in a cell upon treatment with one or absence of an increase in activation level of an activatable more modulators. The activation of an activatable element in element in the, in combination with additional expression the cell upon treatment with one or more modulators can markers. In some embodiments, expression markers or drug reveal operative pathways in a condition that can then be transporters, such as CD34, CD33, CD45, HLADR, CD11B used, e.g., as an indicator to predict course of the condition, 65 FLT3 Ligand, c-KIT, ABCG2, MDR1, BCRP, MRP1, LRP, to identify risk group, to predict an increased risk of devel and others noted below, can also be used for stratifying oping secondary complications, to choose a therapy for an responders and non-responders. The expression markers US 9,500,655 B2 49 50 may be detected using many different techniques, for ers that singly or coordinately allow for Surrogate identifi example using nodes from flow cytometry data (see the cation of AML, MDS or MPN cell subpopulations, disease articles and patent applications referred to above). Other stage of the individual from which the cells were derived, common techniques employ expression arrays (commer diagnosis, prognosis, response to treatment, or new drug cially available from Affymetrix, Santa Clara Calif.), taqman 5 gable targets. In some embodiments, the pathways charac (commercially available from ABI, Foster City Calif.), terization allows for the delineation of AML, MDS or MPN SAGE (commercially available from Genzyme, Cambridge cell subpopulations that are differentially susceptible to Mass.), sequencing techniques (see the commercial products drugs or drug combinations. In other embodiments, the cell from Helicos, 454, US Genomics, and ABI) and other types or activatable elements from a given cell type will, in commonly know assays. See Golub et al., Science 286: 10 combination with activatable elements in other cell types, 531-537 (1999). Expression markers are measured in provide rationnetric or metrics that singly or coordinately unstimulated cells to know whether they have an impact on allow for surrogate identification of AML, MDS or MPN functional apoptosis. This provides implications for treat cell Subpopulations, disease stage of the individual from ment and prognosis for the disease. Under this hypothesis, which the cells were derived, diagnosis, prognosis, response the amount of drug transporters correlates with the response 15 to treatment, or new druggable targets. of the patient and non-responders may have more levels of The subject invention also provides kits for use in deter drug transporters (to move a drug out of a cell) as compared mining the physiological status of cells in a sample, the kit to responders. In some embodiments, the invention is comprising one or more modulators, inhibitors, specific directed to methods of classifying a cell population by binding elements for signaling molecules, and may addi contacting the cell population with at least one modulator tionally comprise one or more therapeutic agents. The above that affects signaling mediated by receptors selected from reagents for the kit are all recited and listed in the present the group comprising of growth factors, mitogens and cytok application below. The kit may further comprise a software ines. In some embodiments, the invention is directed to package for data analysis of the cellular state and its physi methods of classifying a cell population by contacting the ological status, which may include reference profiles for cell population with at least one modulator that affects 25 comparison with the test profile and comparisons to other signaling mediated by receptors selected from the group analyses as referred to above. The kit may also include comprising SDF-1C, IFN-O, IFN-y, IL-10, IL-6, IL-27, instructions for use for any of the above applications. G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, and Thapsi In some embodiments, the invention provides methods, gargin; determining the activation states of a plurality of including methods to determine the physiological status of a activatable elements in the cell comprising; and classifying 30 cell, e.g., by determining the activation level of an activat the cell based on said activation states and expression levels. able element upon contact with one or more modulators. In In some embodiments, the cell population is also exposed in some embodiments, the invention provides methods, includ a separate culture to at least one modulator that slows or ing methods to classify a cell according to the status of an stops the growth of cells and/or induces apoptosis of cells. activatable element in a cellular pathway. In some embodi In some embodiments, the modulator that slows or stops the 35 ments, the cells are classified by analyzing the response to growth of cells and/or induces apoptosis of cells is selected particular modulators and by comparison of different cell from the group consisting of Etoposide, Mylotarg, AraC. states, with or without modulators. The information can be daunorubicin, staurosporine, benzyloxycarbonyl-Val-Ala used in prognosis and diagnosis, including Susceptibility to Asp (OMe) fluoromethylketone (ZVAD), lenalidomide, disease(s), status of a diseased State and response to changes, EPO, and azacitadine, decitabine. In some embodiments, the 40 in the environment, Such as the passage of time, treatment cell population is also exposed in a separate culture to at with drugs or other modalities. The physiological status of least one modulator that is an inhibitor. In some embodi the cells provided in a sample (e.g. clinical sample) may be ments the inhibitor is HO. In some embodiments, the classified according to the activation of cellular pathways of expression of a growth factor receptor, cytokine receptor interest. The cells can also be classified as to their ability to and/or a drug transporter is also measured. In some embodi 45 respond to therapeutic agents and treatments. The physi ments, the methods comprise determining the expression ological status of the cells can provide new druggable targets level at least one protein selected from the group comprising for the development of treatments. These treatments can be ABCG2, C-KIT receptor, and FLT3 LIGAND receptor. used alone or in combination with other treatments. The Another embodiment of the invention further includes using physiological status of the cells can be used to design the modulators IL-3, IL-4, GM-CSF, EPO, LPS, TNF-C., and 50 combination treatments. CD40L. In some embodiments, the cell population in a One or more cells or cell types, or samples containing one hematopoetic cell population. In some embodiments, the or more cells or cell types, can be isolated from body invention is directed to methods of correlating and/or clas samples. The cells can be separated from body samples by sifying an activation state of an AML, MDS or MPN cell centrifugation, elutriation, density gradient separation, with a clinical outcome in an individual by Subjecting the 55 apheresis, affinity selection, panning, FACS, centrifugation AML, MDS or MPN cell from the individual to a modulator, with Hypaque, Solid Supports (magnetic beads, beads in determining the activation levels of a plurality of activatable columns, or other surfaces) with attached antibodies, etc. By elements, and identifying a pattern of the activation levels of using antibodies specific for markers identified with particu the plurality of activatable elements to determine the pres lar cell types, a relatively homogeneous population of cells ence or absence of an alteration in signaling, where the 60 may be obtained. Alternatively, a heterogeneous cell popu presence of the alteration is indicative of a clinical outcome. lation can be used. Cells can also be separated by using In some embodiments, the activatable elements can demar filters. For example, whole blood can also be applied to kate AML, MDS or MPN cell subpopulations that have filters that are engineered to contain pore sizes that select for different genetic Subclone origins. In some embodiments, the desired cell type or class. Rare pathogenic cells can be the activatable elements can demarkate AML, MDS or MPN 65 filtered out of diluted, whole blood following the lysis of red Subpopulations that can be used to determine other protein, blood cells by using filters with pore sizes between 5 to 10 epitope-based, RNA, mRNA, siRNA, or metabolomic mark um, as disclosed in U.S. patent application Ser. No. 09/790, US 9,500,655 B2 51 52 673. Once a sample is obtained, it can be used directly, may inform on time to progression, progression free Sur frozen, or maintained in appropriate culture medium for vival, overall survival, or event-free survival. short periods of time. Methods to isolate one or more cells The classification of a cell according to the status of an for use according to the methods of this invention are activatable element can comprise classifying a cell as a cell performed according to standard techniques and protocols that is correlated to a patient response to a treatment. In some well-established in the art. See also U.S. Ser. Nos. 61/048, embodiments, the patient response is selected from the 886: 61/048.920; and 61/048,657. See also, the commercial group consisting of complete response, partial response, products from companies such as BD and BCI as identified nodular partial response, no response, progressive disease, above. stable disease and adverse reaction. 10 The classification of a rare cell according to the status of See also U.S. Pat. Nos. 7,381,535 and 7,393,656. All of an activatable element can comprise classifying the cell as a the above patents and applications are incorporated by cell that can be correlated with minimal residual disease or reference as stated above. emerging resistance. See U.S. No. 61/048,886 which is In some embodiments, the cells are cultured post collec incorporated by reference. tion in a media suitable for revealing the activation level of 15 The classification of a cell according to the status of an an activatable element (e.g. RPMI, DMEM) in the presence, activatable element can comprise selecting a method of or absence, of serum such as fetal bovine serum, bovine treatment. Example of methods of treatments include, but serum, human serum, porcine serum, horse serum, or goat are not limited to chemotherapy, biological therapy, radia serum. When serum is present in the media it could be tion therapy, bone marrow transplantation, Peripheral stem present at a level ranging from 0.0001% to 30%. cell transplantation, umbilical cord blood transplantation, In some embodiments, the cells are hematopoietic cells. autologous stem cell transplantation, allogeneic stem cell Examples of hematopoietic cells include but are not limited transplantation, Syngeneic stem cell transplantation, Surgery, to pluripotent hematopoietic stem cells, B-lymphocyte lin induction therapy, maintenance therapy, watchful waiting, eage progenitor or derived cells, T-lymphocyte lineage pro and other therapy. genitor or derived cells, NK cell lineage progenitor or 25 A modulator can be an activator, an inhibitor or a com derived cells, granulocyte lineage progenitor or derived pound capable of impacting cellular signaling networks. cells, monocyte lineage progenitor or derived cells, mega Modulators can take the form of a wide variety of environ karyocyte lineage progenitor or derived cells and erythroid mental cues and inputs. Examples of modulators include but lineage progenitor or derived cells. are not limited to growth factors, mitogens, cytokines, The term “patient’ or “individual” as used herein includes 30 adhesion molecules, drugs, hormones, Small molecules, humans as well as other mammals. The methods generally polynucleotides, antibodies, natural compounds, lactones, involve determining the status of an activatable element. The chemotherapeutic agents, immune modulators, carbohy methods also involve determining the status of a plurality of drates, proteases, ions, reactive oxygen species, radiation, activatable elements. physical parameters such as heat, cold, UV radiation, pep In some embodiments, the invention provides a method of 35 tides, and protein fragments, either alone or in the context of classifying a cell by determining the presence or absence of cells, cells themselves, viruses, and biological and non an increase in activation level of an activatable element in biological complexes (e.g. beads, plates, viral envelopes, the cell upon treatment with one or more modulators, and antigen presentation molecules such as major histocompat classifying the cell based on the presence or absence of the ibility complex). One exemplary set of modulators, include increase in the activation of the activatable element. In some 40 but are not limited to SDF-1C, IFN-C, IFN-y, IL-10, IL-6, embodiments of the invention, the activation level of the IL-27, G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, Thapsi activatable element is determined by contacting the cell with gargin, H.O., Etoposide, Mylotarg, AraC, daunorubicin, a binding element that is specific for an activation state of staurosporine, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluo the activatable element. In some embodiments, a cell is romethylketone (ZVAD), lenalidomide, EPO, azacitadine, classified according to the activation level of a plurality of 45 decitabine, IL-3, IL-4, GM-CSF, EPO, LPS, TNF-C., and activatable elements after the cell have been subjected to a CD4OL. modulator. In some embodiments of the invention, the In some embodiments, the modulator is an activator. In activation levels of a plurality of activatable elements are Some embodiments the modulator is an inhibitor. In some determined by contacting a cell with a plurality of binding embodiments, the invention provides methods for classify elements, where each binding element is specific for an 50 ing a cell by contacting the cell with an inhibitor, determin activation state of an activatable element. ing the presence or absence of an increase in activation level The classification of a cell according to the status of an of an activatable element in the cell, and classifying the cell activatable element can comprise classifying the cell as a based on the presence or absence of the increase in the cell that is correlated with a clinical outcome. In some activation of the activatable element. In some embodiments, embodiments, the clinical outcome is the prognosis and/or 55 a cell is classified according to the activation level of a diagnosis of a condition. In some embodiments, the clinical plurality of activatable elements after the cells have been outcome is the presence or absence of a neoplastic or a subjected to an inhibitor. In some embodiments, the inhibitor hematopoietic condition Such as acute myeloid leukemia is an inhibitor of a cellular factor or a plurality of factors that (AML), myelodysplastic syndrome (MDS) or myeloprolif participates in a signaling cascade in the cell. In some erative neoplasms (MPN). In some embodiments, the clini 60 embodiments, the inhibitor is a phosphatase inhibitor. cal outcome is the staging or grading of a neoplastic or Examples of phosphatase inhibitors include, but are not hematopoietic condition. Examples of staging include, but limited to H2O, siRNA, miRNA, Cantharidin, (-)-p-Bro are not limited to, aggressive, indolent, benign, refractory, motetramisole, Microcystin LR, Sodium Orthovanadate, Roman Numeral staging, TNM Staging, Rai staging, Binet Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo staging, WHO classification, FAB classification, IPSS score, 65 (1,10-phenanthroline)vanadate, bis(maltolato)oxovanadium WPSS score, limited Stage, extensive stage, staging accord (IV), Sodium Molybdate, Sodium Perm olybdate, Sodium ing to cellular markers, occult, including information that Tartrate, Imidazole, Sodium Fluoride, B-Glycerophosphate, US 9,500,655 B2 53 54 Sodium Pyrophosphate Decahydrate, Calyculin A, Discod of a cellular pathway or a signaling pathway are detected ermia calyx, bpV(phen), mpV(pic), DMHV. Cypermethrin, using the methods described herein. For example, patterns Dephostatin, Okadaic Acid, NIPP-1, N-(9,10-Dioxo-9,10 and profiles of one or more phosphorylated polypeptides are dihydro-phenanthren-2-yl)-2,2-dimethyl-propionamide, detected using methods known in art including those C-Bromo-4-hydroxyacetophenone, 4-Hydroxyphenacyl Br, described herein. C-Bromo-4-methoxyacetophenone, 4-Methoxyphenacyl Br, In some embodiments, cells (e.g. normal cells) other than C-Bromo-4-(carboxymethoxy)acetophenone, 4-(Car the cells associated with a condition (e.g. cancer cells) or a boxymethoxy)phenacyl Br, and bis(4-Trifluoromethylsulfo combination of cells are used, e.g., in assigning a risk group, namidophenyl)-1,4-diisopropylbenzene, phenylarsine predicting an increased risk of relapse, predicting an oxide, Pyrrolidine Dithiocarbamate, and Aluminium fluo 10 increased risk of developing secondary complications, ride. In some embodiments, the phosphatase inhibitor is choosing a therapy for an individual, predicting response to H2O. a therapy for an individual, determining the efficacy of a In some embodiments, the methods of the invention therapy in an individual, and/or determining the prognosis provide methods for classifying a cell population or deter for an individual. That is that cells other than cells associated mining the presence or absence of a condition in an indi 15 with a condition (e.g. cancer cells) are in fact reflective of vidual by subjecting a cell from the individual to a modu the condition process. For instance, in the case of cancer, lator and an inhibitor, determining the activation level of an infiltrating immune cells might determine the outcome of the activatable element in the cell, and determining the presence disease. Alternatively, a combination of information from or absence of a condition based on the activation level. In the cancer cell plus the immune cells in the blood that are some embodiments, the activation level of a plurality of responding to the disease, or reacting to the disease can be activatable elements in the cell is determined. The inhibitor used for diagnosis or prognosis of the cancer. can be an inhibitor as described herein. In some embodi In some embodiments, the invention provides methods to ments, the inhibitor is a phosphatase inhibitor. In some carry out multiparameter flow cytometry for monitoring embodiments, the inhibitor is HO. The modulator can be phospho-protein responses to various factors in acute any modulator described herein. In some embodiments, the 25 myeloid leukemia, MDS, or MPN at the single cell level. methods of the invention provides for methods for classi Phospho-protein members of signaling cascades and the fying a cell population by exposing the cell population to a kinases and phosphatases that interact with them are plurality of modulators in separate cultures and determining required to initiate and regulate proliferative signals in cells. the status of an activatable element in the cell population. In Apart from the basal level of protein phosphorylation alone, some embodiments, the status of a plurality of activatable 30 the effect of potential drug molecules on these network elements in the cell population is determined. In some pathways was studied to discern unique cancer network embodiments, at least one of the modulators of the plurality profiles, which correlate with the genetics and disease out of modulators is an inhibitor. The modulator can be at least come. Single cell measurements of phospho-protein one of the modulators described herein. In some embodi responses reveal shifts in the signaling potential of a phos ments, at least one modulator is selected from the group 35 pho-protein network, enabling categorization of cell net consisting of SDF-1C, IFN-O, IFN-y, IL-10, IL-6, IL-27, work phenotypes by multidimensional molecular profiles of G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, Thapsigargin, signaling. See U.S. Pat. No. 7,393,656. See also Irish et. al., H2O, Etoposide, Mylotarg, AraC, daunorubicin, stauro Single cell profiling of potentiated phospho-protein net sporine, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluorom works in cancer cells. Cell. 2004, Vol. 118, p. 1-20. ethylketone (ZVAD), lenalidomide, EPO, azacitadine, decit 40 Flow cytometry is useful in a clinical setting, since abine, IL-3, IL-4, GM-CSF, EPO, LPS, TNF-C., and CD40L relatively small sample sizes, as few as 10,000 cells, can or a combination thereof. In some embodiments of the produce a considerable amount of statistically tractable invention, the status of an activatable element is determined multidimensional signaling data and reveal key cell Subsets by contacting the cell population with a binding element that that are responsible for a phenotype. See U.S. Pat. Nos. is specific for an activation state of the activatable element. 45 7.381,535 and 7,393,656. See also Krutzik et al., 2004). In some embodiments, the status of a plurality of activatable Cytokine response panels have been studied to Survey elements is determined by contacting the cell population altered signal transduction of cancer cells by using a mul with a plurality of binding elements, where each binding tidimensional flow cytometry file which contained at least element is specific for an activation state of an activatable 30,000 cell events. In one embodiment, this panel is element. 50 expanded and the effect of growth factors and cytokines on In some embodiments, the methods of the invention primary AML samples studied. See U.S. Pat. Nos. 7,381,535 provide methods for determining a phenotypic profile of a and 7,393,656. See also Irish et al., CELL July 23; 118(2): population of cells by exposing the population of cells to a 217-28. In some embodiments, the analysis involves work plurality of modulators (recited herein) in separate cultures, ing at multiple characteristics of the cell in parallel after wherein at least one of the modulators is an inhibitor, 55 contact with the compound. For example, the analysis can determining the presence or absence of an increase in examine drug transporter function; drug transporter expres activation level of an activatable element in the cell popu sion; drug metabolism; drug activation; cellular redox poten lation from each of the separate cultures and classifying the tial; signaling pathways; DNA damage repair; and apoptosis. cell population based on the presence or absence of the In Some embodiments, the modulators include growth increase in the activation of the activatable element from 60 factors, cytokines, chemokines, phosphatase inhibitors, and each of the separate culture. In some embodiments, the pharmacological reagents. The response panel is composed phenotypic profile is used to characterize multiple pathways of at least one of: SDF-1C, IFN-O, IFN-y, IL-10, IL-6, IL-27, in the population of cells. G-CSF, FLT-3L, IGF-1, M-CSF, SCF, PMA, Thapsigargin, Patterns and profiles of one or more activatable elements H2O, Etoposide, Mylotarg, AraC, daunorubicin, stauro are detected using the methods known in the art including 65 sporine, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluorom those described herein. In some embodiments, patterns and ethylketone (ZVAD), lenalidomide, EPO, azacitadine, decit profiles of activatable elements that are cellular components abine, IL-3, IL-4, GM-CSF, EPO, LPS, TNF-C., and CD40L. US 9,500,655 B2 55 56 The response of each phospho-protein node is compared dicting response to treatment or choosing a treatment for to the basal state and can be represented by calculating the AML, MDS or MPN in an individual where the individual log fold difference in the Median Fluorescence Intensity has a predefined clinical parameter, the method comprising (MFI) of the stimulated sample divided by the unstimulated the steps of (a) Subjecting a cell population from the indi sample. The data can be analyzed using any of the metrics vidual to a plurality of distinct modulators in separate described in FIGS. 2, 7-9. However, other statistical meth cultures, (b) characterizing a plurality of pathways in one or ods may be used. The growth factor and the cytokine more cells from the separate cultures comprising determin response panel included detection of phosphorylated Stat1. ing an activation level of at least one activatable element in Stat3, Stats, Stat6, PLCY2, S6, Akt, Erk1/2, CREB, p38, and at least three pathways, where (i) the pathways are selected NF-KBp-65. In some embodiments, a diagnosis, prognosis, 10 from the group consisting of apoptosis, cell cycle, signaling, a prediction of outcome Such as response to treatment or or DNA damage pathways (ii) at least one of the pathways relapse is performed by analyzing the two or more phos being characterized in at least one of the separate cultures is phorylation levels of two or more proteins each in response an apoptosis or DNA damage pathway, (iii) the distinct to one or more modulators. The phosphorylation levels of modulators independently activate or inhibit said one or the independent proteins can be measured in response to the 15 more pathways being characterized, and (c) correlating the same or different modulators. See FIG. 17 which shows that characterization with diagnosing, prognosing, determining grouping of data points increases predictive value. progression, predicting response to treatment or choosing a In some embodiments, the AML or other panel of modu treatment for AML, MDS or MPN in an individual, where lators is further expanded to examine the process of DNA the pathways characterization in combination with the clini damage, apoptosis, drug transport, drug metabolism, and the cal parameter is indicative of the diagnosing, prognosing, use of peroxide to evaluate phosphatase activity. Analysis determining progression, response to treatment or the appro can assess the ability of the cell to undergo the process of priate treatment for AML, MDS or MPN. Examples of apoptosis after exposure to the experimental drug in an in predetermined clinical parameters include, but are not lim vitro assay as well as how quickly the drug is exported out ited to, age, de novo acute myeloid leukemia patient, sec of the cell or metabolized. The drug response panel can 25 ondary acute myeloid leukemia patient, or a biochemical/ include but is not limited to detection of phosphorylated molecular marker. In some embodiments, the individual is Chk2, Cleaved Caspase 3, Caspase 8, PARP and mitochon over 60 years old. In some embodiments, the individual is dria-released Cytoplasmic Cytochrome C. Modulators may under 60 years old. In some embodiments the activatable include Stauro, Etoposide, Mylotarg, AraC, daunorubicin. elements and modulators are selected from the activatable Analysis can assess phosphatase activity after exposure of 30 elements and modulators listed in Tables 1, 2, 3 or 5. In some cells to phosphatase inhibitors including but not limited to embodiments, the activatable elements and modulators are hydrogen peroxide (HO), H2O+SCF and H2O+IFNC. selected from the activatable elements and modulators listed The response panel to evaluate phosphatase activity can in Table 12 and are used to predict response duration in an include but is not limited to the detection of phosphorylated individual after treatment. In some embodiments the modu Slp76, PLCg2, Lck, S6, Akt, Erk, Stat1, Sta3, Stats. Later, 35 lator is selected from the group consisting of FLT3L, GM the samples may be analyzed for the expression of drug CSF, SCF, G-CSF, SDF1a, LPS, PMA, Thapsigargin, IFNg, transporters such as MDR1/PGP, MRP1 and BCRP/ IFNa, IL-27, IL-3, IL-6, IL-10, ZVAD. H.O., Staurosporine, ABCG2. Samples may also be examined for XIAP, Sur Etoposide, Mylotarg, Daunorubicin, and AraC. In some vivin, Bcl-2, MCL-1, Bim, Ki-67, Cyclin D1, ID1 and Myc. embodiments, when the individual is under 60 years old the Another method of the present invention is a method for 40 activatable elements and modulators are selected from the determining the prognosis and therapeutic selection for an activatable elements and modulators listed in Table 6. In individual with acute myelogenous leukemia (AML). Using some embodiments, where the individual is over 60 years the signaling nodes and methodology described herein, the activatable elements and modulators are selected from multiparametric flow could separate a patient into “cytara the activatable elements and modulators listed in Table 7. In bine responsive’, meaning that a cytarabine based induction 45 Some embodiments, where the individual is a secondary regimen would yield a complete response or “cytarabine acute myeloid leukemia patient the activatable elements and non-responsive’, meaning that the patient is unlikely to modulators are selected from the activatable elements and yield a complete response to a cytarabine based induction modulators listed in Table 8 and Table 9. In some embodi regimen. Furthermore, for those patients unlikely to benefit ments, where the individual is a de novo acute myeloid from cytarabine based therapy, the individuals blood or 50 leukemia patient the activatable elements and modulators marrow sample could reveal signaling biology that corre are selected from the activatable elements and modulators sponds to either in-vivo or in-vitro sensitivity to a class of listed in Table 10 and Table 11. In some embodiments, where drugs including but not limited to direct drug resistance the individual has a wild type FLT3 the activatable elements modulators, anti-Bcl-2 or pro-apoptotic drugs, proteosome and modulators are selected from the activatable elements inhibitors, DNA methyl transferase inhibitors, histone 55 and modulators listed in Table 13. deacetylase inhibitors, anti-angiogenic drugs, farnesyl trans In some embodiments, the invention provides a method ferase inhibitors, FLt3 ligand inhibitors, or ribonucleotide for predicting a response to a treatment or choosing a reductase inhibitors. An individual with AML with a com treatment for AML, MDS or MPN in an individual, the plete response to induction therapy could further benefit method comprising the steps: (a) Subjecting a cell population from the present invention. The individual’s blood or mar 60 from the individual to at least two distinct modulators in row sample could reveal signaling biology that corresponds separate cultures; (b) determining an activation level of at to likelihood of benefit from further cytarabine based che least one activatable element from each of at least three motherapy versus myeloablative therapy followed by and pathways selected from the group consisting of apoptosis, stem cell transplant versus reduced intensity therapy fol cell cycle, signaling, and DNA damage pathways in one or lowed by stem cell transplantation. 65 more cells from each said separate cultures, where at least In some embodiments, the invention provides a method one of the activatable elements is from an apoptosis or DNA for diagnosing, prognosing, determining progression, pre damage pathway, and where the activatable elements mea US 9,500,655 B2 57 58 Sured in each separate culture are the same or the activatable treatment for AML, MDS or MPN in the individual. elements measured in each separate culture are different; and Examples of predefined clinical parameters include age, de (c) predicting a response to a treatment or choosing a novo acute myeloid leukemia patient, secondary acute therapeutic for AML, MDS or MPN in the individual based myeloid leukemia patient, or a biochemical/molecular on the activation level of said activatable elements. In some 5 marker. In some embodiments, the cytokine is selected from embodiments, the method further comprises determining the group consisting of G-CSF, IFNg. IFNa, IL-27, IL-3, whether the apoptosis, cell cycle, signaling, or DNA damage IL-6, and IL-10. In some embodiments, the growth factor is pathways are functional in the individual based on the selected from the group consisting of FLT3L, SCF, G-CSF, activation levels of the activatable elements, wherein a and SDF1a. In some embodiments, the mitogen is selected pathway is functional if it is permissive for a response to a 10 from the group consisting of LPS, PMA, and Thapsigargin. treatment, where if the apoptosis, cell cycle, signaling, and In some embodiments, the modulator that slows or stops the DNA damage pathways are functional the individual can growth of cells and/or induces apoptosis of cells is selected respond to treatment, and where if at least one of the from the group consisting of Staurosporine, Etoposide, pathways is not functional the individual can not respond to Mylotarg, Daunorubicin, and AraC. treatment. In some embodiments, the method further com 15 In some embodiments, activation levels of an activatable prises determining whether the apoptosis, cell cycle, signal element within the STAT pathway higher than a threshold ing, or DNA damage pathways are functional in the indi level in response to a cytokine are indicative that an indi vidual based on the activation levels of the activatable vidual can not respond to treatment. In some embodiment, a elements, wherein a pathway is functional if it is permissive treatment is chosen based on the ability of the cells to for a response to a treatment, where if the apoptosis and respond to treatment. In some embodiments, the activatable DNA damage pathways are functional the individual can element within the STAT pathway is selected from the group respond to treatment. In some embodiments, the method consisting of p-Stat3, p-Stats, p-Stat1, and p-Stat6 and the further comprises determining whether the apoptosis, cell cytokine is selected from the group consisting of IFNg, cycle, signaling, or DNA damage pathways are functional in IFNa, IL-27, IL-3, IL-6, IL-10, and G-CSF. In some embodi the individual based on the activation levels of the activat 25 ments, the activatable element within the STAT pathway is able elements, wherein a pathway is functional if it is Stat 1 and the cytokine is IL-27 or G-CSF. permissive for a response to a treatment, where a therapeutic In some embodiments, activation levels of an activatable is chosen depending of the functional pathways in the element within the PI3K/AKT, or MAPK pathway higher individual. In some embodiments the activatable elements than a threshold level in response to a growth factor or and modulators are selected from the activatable elements 30 mitogen is indicative that an individual can not respond to and modulators listed in Tables 1, 2, 3 or 5. In some treatment. In some embodiment, a treatment is chosen based embodiments, the activatable elements and modulators are on the ability of the cells to respond to treatment with a selected from the activatable elements and modulators listed modulator. In some embodiments, the activatable element in Table 12 and are used to predict response duration in an within the PI3K/AKT, or MAPK pathway is selected from individual after treatment. In some embodiments the modu 35 the group consisting of p-ERK, p38 and pS6 and the growth lator is selected from the group consisting of FLT3L, GM factor or mitogen is selected from the group consisting of CSF, SCF, G-CSF, SDF1a, LPS, PMA, Thapsigargin, IFNg, FLT3L, SCF, G-CSF, SDF1a, LPS, PMA, and Thapsigargin. IFNa, IL-27, IL-3, IL-6, IL-10, ZVAD. H.O., Staurosporine, In some embodiments, activation levels of an activatable Etoposide, Mylotarg, Daunorubicin, and AraC. element within the phospholipase C pathway higher than a In some embodiments, the invention provides a method of 40 threshold level in response to an inhibitor is indicative that predicting a response to a treatment or choosing a treatment an individual can respond to treatment. In some embodi for AML, MDS or MPN in an individual, the method ment, a treatment is chosen based on the ability of the cells comprising the steps of: (a) Subjecting a cell population from to respond to treatment. In some embodiments, the activat said individual to at least three distinct modulators in able element within the phospholipase C pathway is selected separate cultures, wherein: (i) a first modulator is a growth 45 from the group consisting of p-Slp-76, and Plcg2 and the factor or mitogen, (ii) a second modulator is a cytokine, (iii) inhibitor is H.O. a third modulator is a modulator that slows or stops the In some embodiments, activation levels of an activatable growth of cells and/or induces apoptosis of cells or, the third element within the apoptosis pathway higher than a thresh modulator is an inhibitor; (b) determining the activation old in response to a modulator that slows or stops the growth level of at least one activatable element in one or more cells 50 of cells and/or induces apoptosis of cells is indicative that an from each of the separate cultures, where: (i) a first activat individual can respond to treatment. In some embodiment, a able element is an activatable element within the PI3K/AKT, treatment is chosen based on the ability of the cells to or MAPK pathways and the activation level is measured in respond to treatment. In some embodiments, the activatable response to the growth factor or mitogen, (ii) a second element within the apoptosis pathway is selected from the activatable element is an activatable element within the 55 group consisting of Parp--, Cleaved Caspase 8, and Cyto STAT pathway and the activation level is measured in plasmic Cytochrome C, and the modulator that slows or response to the cytokine, (iii) a third activatable element is stops the growth of cells and/or induces apoptosis of cells is an activatable element within an apoptosis pathway and the selected from the group consisting of Staurosporine, Etopo activation level is measured in response to the modulator side, Mylotarg, Daunorubicin, and AraC. that slows or stops the growth of cells and/or induces 60 In some embodiments, activation levels of an activatable apoptosis of cells, or the third activatable element is acti element within the apoptosis pathway higher than a thresh vatable element within the phospholipase C pathway and the old in response to a modulator that slows or stops the growth activation level is measured in response to the inhibitor, or of cells and/or induces apoptosis of cells and activation the third activatable element is a phosphatase and the levels of an activatable element within the STAT pathway activation level is measured in response to the inhibitor, and 65 higher than a threshold level in response to a cytokine is (c) correlating the activation levels of said activatable ele indicative that an individual can not respond to treatment. In ments with a response to a treatment or with choosing a Some embodiments, the activatable element within the apop US 9,500,655 B2 59 60 tosis pathway is selected from the group consisting of Parp--, In Some embodiments, activation levels higher than a Cleaved Caspase 8, and Cytoplasmic Cytochrome C, and the threshold of an activatable element in the PI3K/AKT path modulator that slows or stops the growth of cells and/or way in response to a growth factor is indicative that the induces apoptosis of cells is selected from the group con individual can not respond to treatment. In some embodi sisting of Staurosporine, Etoposide, Mylotarg, Daunorubi ments, the activatable element in the PI3K/Akt pathway is cin, and AraC. In some embodiments, the activatable ele Akt and the growth factor is FLT3L. ment within the STAT pathway is selected from the group In Some embodiments, activation levels higher than a consisting of p-Stat3, p-Stats, p-Stat1, and p-Stat6 and the threshold of an activatable element in the apoptosis pathway cytokine is selected from the group consisting of IFNg, in response to a modulator that slows or stops the growth of 10 cells and/or induces apoptosis of cells is indicative that the IFNa, IL-27, IL-3, IL-6, IL-10, and G-CSF. In some embodi individual can respond to treatment. In some embodiments, ments, the activatable element within the STAT pathway is the activatable element within the apoptosis pathway is Stat 1 and the cytokine is IL-27 or G-CSF. Parp-- and the modulator that slows or stops the growth of In some embodiments, the methods of the invention cells and/or induces apoptosis of cells is selected from the further comprise determining an activation level of an 15 group consisting of Staurosporine, Etoposide, Mylotarg, activatable element within a DNA damage pathway in Daunorubicin, and AraC. response to a modulator that slows or stops the growth of In some embodiments, the invention provides a method of cells and/or induces apoptosis of cells. In some embodi predicting a response to a treatment or choosing a treatment ments, the activatable element within a DNA damage path for AML in an individual where the individual is a secondary way is selected from the group consisting of Chk2, ATM, acute myeloid leukemia patient, the method comprising the ATR and 14-3-3 and the modulator that slows or stops the steps of: (a) Subjecting a cell population from the individual growth of cells and/or induces apoptosis of cells is selected to IL-27 and G-CSF in separate cultures, (b) determining an from the group consisting of Staurosporine, Etoposide, activation level of pStat1 in one or more cells from each Mylotarg, Daunorubicin, and AraC. separate culture, (c) predicting a response to a treatment or In some embodiments, activation levels higher than a 25 choosing a treatment for AML, in the individual, where if the threshold of an activatable element within a DNA damage activation levels of pStat1 are higher than a threshold level pathway and activation levels lower than a threshold of an in response to both IL-27 and G-CSF the individual can not activatable element within the apoptosis pathway in respond to treatment and if the levels of pStat1 are lower response to a modulator that slows or stops the growth of than a threshold in response to both IL-27 and G-CSF the cells and/or induces apoptosis of cells are indicative of a 30 individual can respond to treatment. In some embodiments, communication breakdown between the DNA damage the treatment is chemotherapy agent. Examples of chemo response pathway and the apoptotic machinery and that an therapy agents include, but are not limited to, cytarabine individual can not respond to treatment. In some embodi (ara-C), daunorubicin (Daunomycin), idarubicin (Idamy ment, a treatment is chosen based on the ability of the cells cin), mitoxantrone and 6-thioguanine. In some embodi to respond to treatment. 35 ments, the treatment is allogeneic stem cell transplant or In some embodiments, the methods of the invention autologous stem cell transplant. further comprise determining an activation level of an In some embodiments, the invention provides a method of activatable element within a cell cycle pathway in response predicting a response to a treatment or choosing a treatment to a modulator that slows or stops the growth of cells and/or for AML, MDS or MPN in an individual, the method induces apoptosis of cells. In some embodiments, the acti 40 comprising the steps of: (a) Subjecting a cell population from vatable element within a DNA damage pathway is selected the individual to FLT3L, (b) determining an activation level from the group consisting of Cdc25, p53, CyclinA-Cdk2, of pAkt in one or more cells from the population (c) CyclinE-Cdk2, CyclinB-Cdk1, p21, and Gadd45 and the predicting a response to a treatment or choosing a treatment modulator that slows or stops the growth of cells and/or for AML, MDS or MPN in the individual, where if the induces apoptosis of cells is selected from the group con 45 activation levels of pAkt are higher than a predetermined sisting of Staurosporine, Etoposide, Mylotarg, Daunorubi threshold in response to FLT3L the individual can not cin, and AraC. respond to treatment. In some embodiments, the method In some embodiments, the methods of the invention further comprises the steps of: (d) Subjecting a cell popula further comprise determining the levels of a drug transporter tion from said individual to IL-27 in a separate culture, (e) and/or a cytokine receptor. In some embodiments, the 50 determining an activation level of Stat1 in one or more cells cytokine receptor or drug transporter are selected from the from the separate culture, (f) predicting a response to a group consisting of MDR1, ABCG2, MRP, P-Glycoprotein, treatment or choosing a treatment for AML, MDS or MPN CXCR4, FLT3, and c-kit. In some embodiments, levels in the individual, where if the activation levels of pStat1 are higher than a threshold of the drug transporter and/or said higher than a predetermined threshold in response to IL-27 cytokine receptor are indicative that an individual can not 55 the individual can not respond to treatment. In some embodi respond to treatment. In some embodiment, a treatment is ments where the individual is over 60 years old the method chosen based on the ability of the cells to respond to further comprises the step of (g) Subjecting a cell population treatment. from the individual to H2O2 in a separate culture, (h) In some embodiments, the methods of the invention determining an activation level of Plcg2 in one or more cells further comprise determining the activation levels of an 60 from the separate culture (i) predicting a response to a activatable element within the Akt pathway in response to an treatment or choosing a treatment for AML, MDS or MPN inhibitor, where activation levels higher that a threshold of in the individual, wherein if the activation levels of Plcg2 are the activatable element within the Akt pathway in response higher than a predetermined threshold in response to H2O2 to the inhibitor are indicative that the individual can not the individual can not respond to treatment. In some embodi respond to treatment. In some embodiment, a treatment is 65 ments where the individual is under 60 years old the method chosen based on the ability of the cells to respond to further comprises the steps of (g) Subjecting a cell popula treatment. tion from said individual to Etoposide in a separate culture, US 9,500,655 B2 61 62 (h) determining an activation level of Parp in one or more In some embodiments, the invention provides methods for cells from the separate culture, and (i) predicting a response predicting risk of relapse at 2 years, wherein the PPV is to a treatment for AML, MDS or MPN in said individual, higher than 60, 70, 80, 90, 95, or 99.9%. In some embodi where if the activation levels of Parp are higher than a ments, the invention provides methods for predicting risk of predetermined threshold in response to Etoposide the indi 5 relapse at 2 years, wherein the PPV is equal or higher than vidual can respond to treatment. In some embodiments, the 95%. In some embodiments, the invention provides methods treatment is chemotherapy agent. Examples of chemo for predicting risk of relapse at 2 years, wherein the NPV is therapy agents include, but are not limited to, cytarabine higher than 60, 70, 80, 90, 95, or 99.9%. In some embodi (ara-C), daunorubicin (Daunomycin), idarubicin (Idamy ments, the invention provides methods for predicting risk of 10 relapse at 2 years, wherein the NPV is higher than 80%. In cin), mitoxantrone and 6-thioguanine. In some embodi some embodiments, the invention provides methods for ments, the treatment is allogeneic stem cell transplant or predicting risk of relapse at 5 years, wherein the PPV is autologous stem cell transplant. higher than 60, 70, 80, 90, 95, or 99.9%. In some embodi In some embodiments, the invention provides methods of ments, the invention provides methods for predicting risk of prediction response to a treatment and/or risk of relapse for 15 relapse at 5 years, wherein the PPV is equal or higher than AML, MDS or MPN in an individual, the method compris 95%. In some embodiments, the invention provides methods ing the steps of: (a) Subjecting a cell population from the for predicting risk of relapse at 5 years, wherein the NPV is individual to SCF. (b) determining an activation level of higher than 60, 70, 80, 90, 95, or 99.9%. In some embodi pAkt and S6 in one or more cells from the population (c) ments, the invention provides methods for predicting risk of predicting a response to a treatment, choosing a treatment or relapse at 5 years, wherein the NPV is higher than 80%. In predicting risk of relapse for AML, MDS or MPN in the some embodiments, the invention provides methods for individual, where if the activation levels of p Akt and S6 are predicting risk of relapse at 10 years, wherein the PPV is higher than a predetermined threshold in response to SCF higher than 60, 70, 80, 90, 95, or 99.9%. In some embodi the individual can not respond to treatment or will have a ments, the invention provides methods for predicting risk of higher probability of relapse. 25 relapse at 10 years, wherein the PPV is equal or higher than In some embodiments, a diagnosis, prognosis, a predic 95%. In some embodiments, the invention provides methods tion of outcome such as response to treatment or relapse is for predicting risk of relapse at 10 years, wherein the NPV performed by analyzing the two or more phosphorylation is higher than 60, 70, 80, 90, 95, or 99.9%. In some levels of two or more proteins each in response to one or embodiments, the invention provides methods for predicting more modulators. The phosphorylation levels of the inde 30 risk of relapse at 10 years, wherein the NPV is higher than pendent proteins can be measured in response to the same or 80%. different modulators. See FIG. 17 which shows that group In some embodiments, the p value in the analysis of the ing of data points increases predictive value. methods described herein is below 0.05, 04, 0.03, 0.02, 0.01, In some embodiments, the invention provides a method of 0.009, 0.005, or 0.001. In some embodiments, the p value is diagnosing, prognosing or predicting a response to a treat 35 below 0.001. Thus in some embodiments, the invention ment or choosing a treatment for AML, MDS or MPN in an provides methods for diagnosing, prognosing, determining individual, the method comprising the steps of: (a) Subject progression or predicting response for treatment of AML. ing a cell population from the individual in separate cultures MDS or MPN wherein the p value is below 0.05, 04, 0.03, to at least two modulators selected from the group consisting 0.02, 0.01, 0.009, 0.005, or 0.001. In some embodiments, the of Staurosporine, Etoposide, Mylotarg, Daunorubicin, AraC. 40 p value is below 0.001. In some embodiments, the invention G-CSF, IFNg, IFNa, IL-27, IL-3, IL-6, IL-10, FLT3L, SCF, provides methods for diagnosing, prognosing, determining G-CSF, SDF1a, LPS, PMA, Thapsigargin and H2O2; b) progression or predicting response for treatment of AML. determining the activation level of at least three activatable MDS or MPN wherein the AUC value is higher than 0.5,0.6, elements selected from the group consisting of p-Slp-76, 07, 0.8 or 0.9. In some embodiments, the invention provides p-Plcg2. p-Stat3, p-Stats, p-Stat1, p-Stat6, p-Creb, Parp--, 45 methods for diagnosing, prognosing, determining progres Chk2, p-65/RelA. p-Akt. p-S6, p-ERK, Cleaved Caspase 8. sion or predicting response for treatment of AML, MDS or Cytoplasmic Cytochrome C, and p38; and (c) diagnosing, MPN wherein the AUC value is higher than 0.7. In some prognosing, or predicting a response to a treatment or embodiments, the invention provides methods for diagnos choosing a treatment for AML, MDS or MPN based on the ing, prognosing, determining progression or predicting activation levels of the activatable elements. In some 50 response for treatment of AML, MDS or MPN wherein the embodiments, the method further comprises determining the AUC value is higher than 0.8. In some embodiments, the expression of a cytokine receptor or drug transporter invention provides methods for diagnosing, prognosing, selected from the group consisting of MDR1, ABCG2, MRP, determining progression or predicting response for treat P-Glycoprotein, CXCR4, FLT3, and c-Kit. ment of AML, MDS or MPN wherein the AUC value is In some embodiments, the invention provides methods for 55 higher than 0.9. predicting response to a treatment for AML, MDS or MPN, Another method of the present invention is a method for wherein the positive predictive value (PPV) is higher than determining the prognosis and therapeutic selection for an 60, 70, 80, 90, 95, or 99.9%. In some embodiments, the individual with myelodysplasia or MDS. Using the signaling invention provides methods for predicting response to a nodes and methodology described herein, multiparametric treatment for AML, MDS or MPN, wherein the PPV is equal 60 flow cytometry could separate a patient into one of five or higher than 95%. In some embodiments, the invention groups consisting of “AML-like', where a patient displays provides methods for predicting response to a treatment for signaling biology that is similar to that seen in acute myel AML, MDS or MPN, wherein the negative predictive value ogenous leukemia (AML) requiring intensive therapy, “Epo (NPV) is higher than 60, 70, 80, 90, 95, or 99.9%. In some Responsive', where a patient’s bone marrow or potentially embodiments, the invention provides methods for predicting 65 peripheral blood, shows signaling biology that corresponds response to a treatment for AML, MDS or MPN, wherein the to either in-vivo or in-vitro sensitivity to erythropoietin, NPV is higher than 85%. “Lenalidomide responsive', where a patient’s bone marrow US 9,500,655 B2 63 64 or potentially peripheral blood, shows signaling biology that (MFIsle, set), also called "fold change in median corresponds to either in-vivo or in-vitro sensitivity to fluorescence intensity'. 4) Measuring the percentage of cells Lenalidomide, “Auto-immune', where a patient’s bone mar in a Quadrant Gate of a contour plot which measures row or potentially peripheral blood, shows signaling biology multiple populations in one or more dimension 5) measuring that corresponds to sensitivity to cyclosporine A (CSA) and 5 MFI of phosphor positive population to obtain percentage anti-thymocyte globulin (ATG). positivity above the background; and 6) use of multimodal In those cases where an individual is classified as “AML ity and spread metrics for large sample population and for like', the individual’s blood or marrow sample could reveal Subpopulation analysis. Other metrics used to analyze data signaling biology that corresponds to either in-vivo or are population frequency metrics measuring the frequency in-vitro sensitivity to cytarabine or to a class of drugs 10 including but not limited to direct drug resistance modula of cells with a described property such as cells positive for tors, anti-Bcl-2 or pro-apoptotic drugs, proteosome inhibi cleaved PARP (% PARP+), or cells positive for p-S6 and tors, DNA methyl transferase inhibitors, histone deacetylase p-Akt (See FIG. 2B). Similarly, measurements examining inhibitors, anti-angiogenic drugs, farnesyltransferase inhibi the changes in the frequencies of cells may be applied Such tors, FLt3 ligand inhibitors, or ribonucleotide reductase 15 as the Change in % PARP+ which would measure the % inhibitors. PARP+since, Stained-'6 PARP+ Ustimulated Stained The In some embodiments of the invention, different gating AUC metric also measures changes in population fre strategies can be used in order to analyze only blasts in the quencies measuring the frequency of cells to become posi sample of mixed population after treatment with the modu tive compared to an unstimulated condition (FIG. 2B). The lator. These gating strategies can be based on the presence of metrics described in FIG. 2B can be use to measure apop one or more specific Surface marker expressed on each cell tosis. For example, these metrics can be applied to cleaved type. In some embodiments, the first gate eliminates cell Caspase-3 and Caspase-8, e.g., Change in % Cleaved Cas doublets so that the user can focus on singlets. The following pase-3 or Cleaved Caspase-8. gate can differentiate between dead cells and live cells and Other possible metrics include third-color analysis (3D Subsequent gating of live cells classifies them into blasts, 25 plots); percentage positive and relative expression of various monocytes and lymphocytes. A clear comparison can be markers; clinical analysis on an individual patient basis for carried out to study the effect of potential modulators, such various parameters, including, but not limited to age, race, as G-SCF on activable elements in: ungated Samples, blasts, cytogenetics, mutational status, blast percentage, CD34+ monocytes, granulocytes and lymphocytes by using two percentage, time of relapse, Survival, etc. See FIG. 2. In dimensional contour plot representations of Stats and Stat3 30 alternative embodiments, there are other ways of analyzing phosphorylation (x and Y axis) of patient samples. The level data, such as third color analysis (3D plots), which can be of basal phosphorylation and the change in phosphylation in similar to Cytobank 2D, plus third D in color. both Stat3 and Stats phosphorylation in response to G-CSF Disease Conditions can be compared. G-CSF increases both STAT3 and STAT5 The methods of the invention are applicable to any phosphorylation and this dual signaling can occur concur 35 condition in an individual involving, indicated by, and/or rently (subpopulations with increases in both pSTAT3 and arising from, in whole or in part, altered physiological status pSTAT5) or individually (subpopulations with either an in a cell. The term "physiological status' includes mechani increase in phospho pSTAT3 or pSTAT5 alone). The advan cal, physical, and biochemical functions in a cell. In some tage of gating is to get a clearer picture and more precise embodiments, the physiological status of a cell is deter results of the effect of various activable elements on blasts. 40 mined by measuring characteristics of cellular components See FIG. 6. of a cellular pathway. Cellular pathways are well known in In some embodiments, a gate is established after learning the art. In some embodiments the cellular pathway is a from a responsive subpopulation. That is, a gate is developed signaling pathway. Signaling pathways are also well known from one data set. This gate can then be applied retrospec in the art (see, e.g., Hunter T., Cell 100(1): 113-27 (2000); tively or prospectively to other data sets (See FIGS. 26 and 45 Cell Signaling Technology, Inc., 2002 Catalogue, Pathway 27). The cells in this gate can be used for the diagnosis or Diagrams pgs. 232-253). A condition involving or charac prognosis of a condition. The cells in this gate can also be terized by altered physiological status may be readily iden used to predict response to a treatment or for treatment tified, for example, by determining the state in a cell of one selection. The mere presence of cells in this gate may be or more activatable elements, as taught herein. indicative of a diagnosis, prognosis, or a response to treat 50 In some embodiments, the present invention is directed to ment. In some embodiments, the presence of cells in this methods for classifying one or more cells in a sample gate at a number higher than a threshold number may be derived from an individual having or Suspected of having a indicative of a diagnosis, prognosis, or a response to treat condition. Example conditions include AML, MDS, or ment. MPN. In some embodiments, the invention allows for iden Some methods of analysis, also called metrics are: 1) 55 tification of prognostically and therapeutically relevant Sub measuring the difference in the log of the median fluores groups of the conditions and prediction of the clinical course cence value between an unstimulated fluorochrome-anti of an individual. In some embodiments, the invention pro body stained sample and a sample that has not been treated vides methods of classifying a cell according to the activa With a stimulant or stained (log (MFIsles)-log tion levels of one or more activatable elements in a cell from (MFIce site)), 2) measuring the difference in the log 60 an individual having or Suspected of having a condition. In of the median fluorescence value between a stimulated Some embodiments, the classification includes classifying fluorochrome-antibody stained sample and a sample that has the cell as a cell that is correlated with a clinical outcome. not been treated with a stimulant or stained (log The clinical outcome can be the prognosis and/or diagnosis (MFIstimulated stained)-log(MFIGated Unstained); 3) Measuring of a condition, and/or staging or grading of a condition. In the change between the stimulated fluorochrome-antibody 65 Some embodiments, the classifying of the cell includes stained sample and the unstimulated fluorochrome-antibody classifying the cell as a cell that is correlated with a patient stained sample log (MFIstimulated Stained)-log response to a treatment. In some embodiments, the classi US 9,500,655 B2 65 66 fying of the cell includes classifying the cell as a cell that is Activation levels for a particular activatable element may correlated with minimal residual disease or emerging resis vary among individual cells so that when a plurality of cells tance. is analyzed, the activation levels follow a distribution. The Activatable Elements distribution may be a normal distribution, also known as a The methods and compositions of the invention may be Gaussian distribution, or it may be of another type. Different employed to examine and profile the status of any activat populations of cells may have different distributions of able element in a cellular pathway, or collections of Such activation levels that can then serve to distinguish between activatable elements. Single or multiple distinct pathways the populations. may be profiled (sequentially or simultaneously), or Subsets In some embodiments, the basis for classifying cells is 10 that the distribution of activation levels for one or more of activatable elements within a single pathway or across specific activatable elements will differ among different multiple pathways may be examined (again, sequentially or phenotypes. A certain activation level, or more typically a simultaneously). In some embodiments, apoptosis, signal range of activation levels for one or more activatable ele ing, cell cycle and/or DNA damage pathways are character ments seen in a cell or a population of cells, is indicative that ized in order to classify one or more cells in an individual. 15 that cell or population of cells belongs to a distinctive The characterization of multiple pathways can reveal opera phenotype. Other measurements, such as cellular levels tive pathways in a condition that can then be used to classify (e.g., expression levels) of biomolecules that may not con one or more cells in an individual. In some embodiments, the tain activatable elements, may also be used to classify cells classification includes classifying the cell as a cell that is in addition to activation levels of activatable elements; it correlated with a clinical outcome. The clinical outcome can will be appreciated that these levels also will follow a be the prognosis and/or diagnosis of a condition, and/or distribution, similar to activatable elements. Thus, the acti staging or grading of a condition. In some embodiments, the vation level or levels of one or more activatable elements, classifying of the cell includes classifying the cell as a cell optionally in conjunction with levels of one or more levels that is correlated with a patient response to a treatment. In of biomolecules that may or may not contain activatable Some embodiments, the classifying of the cell includes 25 elements, of cell or a population of cells may be used to classifying the cell as a cell that is correlated with minimal classify a cell or a population of cells into a class. Once the residual disease or emerging resistance. activation level of intracellular activatable elements of indi As will be appreciated by those in the art, a wide variety vidual single cells is known they can be placed into one or of activation events can find use in the present invention. In more classes, e.g., a class that corresponds to a phenotype. general, the basic requirement is that the activation results in 30 A class encompasses a class of cells wherein every cell has a change in the activatable protein that is detectable by some the same or Substantially the same known activation level, or indication (termed an “activation state indicator”), prefer range of activation levels, of one or more intracellular ably by altered binding of a labeled binding element or by activatable elements. For example, if the activation levels of changes in detectable biological activities (e.g., the activated five intracellular activatable elements are analyzed, pre state has an enzymatic activity which can be measured and 35 defined classes of cells that encompass one or more of the compared to a lack of activity in the non-activated State). intracellular activatable elements can be constructed based What is important is to differentiate, using detectable events on the activation level, or ranges of the activation levels, of or moieties, between two or more activation states (e.g. “off each of these five elements. It is understood that activation and “on levels can exist as a distribution and that an activation level The activation state of an individual activatable element is 40 of a particular element used to classify a cell may be a either in the on or off state. As an illustrative example, and particular point on the distribution but more typically may without intending to be limited to any theory, an individual be a portion of the distribution. phosphorylatable site on a protein can activate or deactivate In some embodiments, the basis for classifying cells may the protein. Additionally, phosphorylation of an adapter use the position of a cell in a contour or density plot. The protein may promote its interaction with other components/ 45 contour or density plot represents the number of cells that proteins of distinct cellular signaling pathways. The terms share a characteristic Such as the activation level of activat “on” and “off” when applied to an activatable element that able proteins in response to a modulator. For example, when is a part of a cellular constituent, are used here to describe referring to activation levels of activatable elements in the state of the activatable element, and not the overall state response to one or more modulators, normal individuals and of the cellular constituent of which it is a part. Typically, a 50 patients with a condition might show populations with cell possesses a plurality of a particular protein or other increased activation levels in response to the one or more constituent with a particular activatable element and this modulators. However, the number of cells that have a plurality of proteins or constituents usually has some pro specific activation level (e.g. specific amount of an activat teins or constituents whose individual activatable element is able element) might be different between normal individuals in the on state and other proteins or constituents whose 55 and patients with a condition. Thus, a cell can be classified individual activatable element is in the off state. Since the according to its location within a given region in the contour activation state of each activatable element is measured or density plot. In other embodiments, the basis for classi through the use of a binding element that recognizes a fying cells may use a series of population clusters whose specific activation state, only those activatable elements in centers, centroids, boundaries, relative positions describe the the specific activation state recognized by the binding ele 60 state of a cell, the diagnosis or prognosis of a patient, ment, representing some fraction of the total number of selection of treatment, or predicting response to treatment or activatable elements, will be bound by the binding element to a combination of treatments, or long term outcome. to generate a measurable signal. The measurable signal In some embodiments, the basis for classifying cells may corresponding to the Summation of individual activatable use an N-dimensional Eigen map that describe the State of a elements of a particular type that are activated in a single cell 65 cell, the diagnosis or prognosis of a patient, selection of is the “activation level for that activatable element in that treatment, or predicting response to treatment or to a com cell. bination of treatments, or long term outcome. US 9,500,655 B2 67 68 In other embodiments, the basis for classifying cells may pH using flow cytometry with carboxy-SNARF-1. Cytom use a Bayesian inference network of activatable elements etry, 1993 November; 14(8):916-21; and Valli, M, et al., interaction capabilities that together, or in part, describe the Intracellular pH Distribution in Saccharomyces cerevisiae state of a cell, the diagnosis or prognosis of a patient, Cell Populations, Analyzed by Flow Cytometry, Applied and selection of treatment, or predicting response to treatment or Environmental Microbiology, March 2005, p. 1515-1521, to a combination of treatments, or long term outcome. See Vol. 71, No. 3. U.S. publication no. 2007/0009923 entitled Use of Bayesian In some embodiments, the activatable element is the Networks for Modeling Signaling Systems, incorporated phosphorylation of immunoreceptor tyrosine-based inhibi herein by reference on its entirety. tory motif (ITIM). An immunoreceptor tyrosine-based inhi In addition to activation levels of intracellular activatable 10 bition motif (ITIM), is a conserved sequence of amino acids elements, levels of intracellular or extracellular biomol (S/I/V/LXYXXI/V/L) that is found in the cytoplasmic tails of ecules, e.g., proteins, may be used alone or in combination many inhibitory receptors of the immune system. After with activation states of activatable elements to classify ITIM-possessing inhibitory receptors interact with their cells. Further, additional cellular elements, e.g., biomol ligand, their ITIM motif becomes phosphorylated by ecules or molecular complexes such as RNA, DNA, carbo 15 enzymes of the Src family of kinases, allowing them to hydrates, metabolites, and the like, may be used in conjunc recruit other enzymes such as the phosphotyrosine phos tion with activatable states or expression levels in the phatases SHP-1 and SHP-2, or the inositol-phosphatase classification of cells encompassed here. called SHIP. These phosphatases decrease the activation of In some embodiments, cellular redox signaling nodes are molecules involved in cell signaling. See Barrow A, Trows analyzed for a change in activation level. Reactive oxygen dale J (2006). “You say ITAM and I say ITIM, let’s call the species (ROS) are involved in a variety of different cellular whole thing off the ambiguity of immunoreceptor signal processes ranging from apoptosis and necrosis to cell pro ling’. Eur J Immunol 36 (7): 1646-53. When phosphory liferation and carcinogenesis. ROS can modify many intra lated, these phospho-tyrosine residues provide docking sites cellular signaling pathways including protein phosphatases, for the Ships which may result in transmission of inhibitory protein kinases, and transcription factors. This activity may 25 signals and effect the signaling of neighboring membrane indicate that the majority of the effects of ROS are through receptor complexes (Paul et al., Blood (2000 96:483). their actions on signaling pathways rather than via non ITIMs can be analyzed by flow cytometry. specific damage of macromolecules. The exact mechanisms Additional elements may also be used to classify a cell, by which redox status induces cells to proliferate or to die, such as the expression level of extracellular or intracellular and how oxidative stress can lead to processes evoking 30 markers, nuclear antigens, enzymatic activity, protein tumor formation are still under investigation. See Mates, JM expression and localization, cell cycle analysis, chromo et al., Arch Toxicol. 2008 May:82(5):271-2; Galaris D., et somal analysis, cell volume, and morphological character al., Cancer Lett. 2008 Jul. 18; 266(1)21-9. istics like granularity and size of nucleus or other distin Reactive oxygen species can be measured. One example guishing characteristics. For example, B cells can be further technique is by flow cytometry. See Chang et al., Lympho 35 subdivided based on the expression of cell surface markers cyte proliferation modulated by glutamine: involved in the such as CD19, CD2O, CD22 or CD23. endogenous redox reaction; Clin Exp Immunol. 1999 Sep Alternatively, predefined classes of cells can be aggre tember: 117(3): 482-488. Redox potential can be evaluated gated or grouped based upon shared characteristics that may by means of an ROS indicator, one example being 2,7- include inclusion in one or more additional predefined class dichlorofluorescein-diacetate (DCFH-DA) which is added to 40 or the presence of extracellular or intracellular markers, the cells at an exemplary time and temperature, Such as 37 similar gene expression profile, nuclear antigens, enzymatic C. for 15 minutes. DCF peroxidation can be measured using activity, protein expression and localization, cell cycle flow cytometry. See Yang K D, Shaio M. F. Hydroxyl analysis, chromosomal analysis, cell Volume, and morpho radicals as an early signal involved in phorbol ester-induced logical characteristics like granularity and size of nucleus or monocyte differentiation of HL60 cells. Biochem Biophys 45 other distinguishing cellular characteristics. Res Commun. 1994; 200: 1650-7 and Wang J. F. Jerrells TR, In some embodiments, the physiological status of one or Spitzer J.J. Decreased production of reactive oxygen inter more cells is determined by examining and profiling the mediates is an early event during in vitro apoptosis of rat activation level of one or more activatable elements in a thymocytes. Free Radic Biol Med. 1996; 20:533-42. cellular pathway. In some embodiments, a cell is classified In some embodiments, other characteristics that affect the 50 according to the activation level of a plurality of activatable status of a cellular constituent may also be used to classify elements. In some embodiments, a hematopoietic cell is a cell. Examples include the translocation of biomolecules classified according to the activation levels of a plurality of or changes in their turnover rates and the formation and activatable elements. In some embodiments, 1, 2, 3, 4, 5, 6, disassociation of complexes of biomolecule. Such com 7, 8, 9, 10 or more activatable elements may be analysed in plexes can include multi-protein complexes, multi-lipid 55 a cell signaling pathway. In some embodiments, the activa complexes, homo- or hetero-dimers or oligomers, and com tion levels of one or more activatable elements of a binations thereof. Other characteristics include proteolytic hematopoietic cell are correlated with a condition. In some cleavage, e.g. from exposure of a cell to an extracellular embodiments, the activation levels of one or more activat protease or from the intracellular proteolytic cleavage of a able elements of a hematopoietic cell are correlated with a biomolecule. 60 neoplastic or hematopoietic condition as described herein. In some embodiments, cellular pH is analyzed. See June, Examples of hematopoietic cells include, but are not limited C H and Moore, and J S. Curr Protoc Immulon, 2004 to, AML, MDS or MPN cells. December: Chapter 5:Unit 5.5; Leyval, D et al., Flow In some embodiments, the activation level of one or more cytometry for the intracellular pH measurement of glutamate activatable elements in single cells in the sample is deter producing Corynebacterium glutamicum, Journal of Micro 65 mined. Cellular constituents that may include activatable biological Methods, Volume 29, Issue 2, 1 May 1997, Pages elements include without limitation proteins, carbohydrates, 121-127; Weider, E D, et al., Measurement of intracellular lipids, nucleic acids and metabolites. The activatable ele US 9,500,655 B2 69 70 ment may be a portion of the cellular constituent, for Another example of a covalent modification of an acti example, an amino acid residue in a protein that may vatable protein is the acetylation of histones. Through the undergo phosphorylation, or it may be the cellular constitu activity of various acetylases and deacetlylases the DNA ent itself, for example, a protein that is activated by trans binding function of histone proteins is tightly regulated. location, change in conformation (due to, e.g., change in pH Furthermore, histone acetylation and histone deactelyation or ion concentration), by proteolytic cleavage, degradation have been linked with malignant progression. See Nature, through ubiquitination and the like. Upon activation, a 2004 May 27; 429(6990): 457-63. change occurs to the activatable element. Such as covalent Another form of activation involves cleavage of the modification of the activatable element (e.g., binding of a activatable element. For example, one form of protein molecule or group to the activatable element, such as 10 regulation involves proteolytic cleavage of a peptide bond. phosphorylation) or a conformational change. Such changes While random or misdirected proteolytic cleavage may be generally contribute to changes in particular biological, detrimental to the activity of a protein, many proteins are biochemical, or physical properties of the cellular constitu activated by the action of proteases that recognize and ent that contains the activatable element. The state of the cleave specific peptide bonds. Many proteins derive from cellular constituent that contains the activatable element is 15 precursor proteins, or pro-proteins, which give rise to a determined to some degree, though not necessarily com mature isoform of the protein following proteolytic cleavage pletely, by the state of a particular activatable element of the of specific peptide bonds. Many growth factors are synthe cellular constituent. For example, a protein may have mul sized and processed in this manner, with a mature isoform of tiple activatable elements, and the particular activation states the protein typically possessing a biological activity not of these elements may overall determine the activation state exhibited by the precursor form. Many enzymes are also of the protein; the state of a single activatable element is not synthesized and processed in this manner, with a mature necessarily determinative. Additional factors, such as the isoform of the protein typically being enzymatically active, binding of other proteins, pH, ion concentration, interaction and the precursor form of the protein being enzymatically with other cellular constituents, and the like, can also affect inactive. This type of regulation is generally not reversible. the state of the cellular constituent. 25 Accordingly, to inhibit the activity of a proteolytically In some embodiments, the activation levels of a plurality activated protein, mechanisms other than “reattachment' of intracellular activatable elements in single cells are deter must be used. For example, many proteolytically activated mined. In some embodiments, at least about 2, 3, 4, 5, 6, 7, proteins are relatively short-lived proteins, and their turn 8, 9, 10 or more than 10 intracellular activatable elements over effectively results in deactivation of the signal. Inhibi are determined. 30 tors may also be used. Among the enzymes that are pro Activation states of activatable elements may result from teolytically activated are serine and cysteine proteases, chemical additions or modifications of biomolecules and including cathepsins and caspases respectively. include biochemical processes such as glycosylation, phos In one embodiment, the activatable enzyme is a caspase. phorylation, acetylation, methylation, biotinylation, gluta The caspases are an important class of proteases that medi mylation, glycylation, hydroxylation, isomerization, pre 35 ate programmed cell death (referred to in the art as "apop nylation, myristoylation, lipoylation, tosis). Caspases are constitutively present in most cells, phosphopantetheinylation, Sulfation, ISGylation, nitrosy residing in the cytosol as a single chain proenzyme. These lation, palmitoylation, SUMOylation, ubiquitination, ned are activated to fully functional proteases by a first pro dylation, citrullination, amidation, and disulfide bond for teolytic cleavage to divide the chain into large and Small mation, disulfide bond reduction. Other possible chemical 40 caspase subunits and a second cleavage to remove the additions or modifications of biomolecules include the for N-terminal domain. The subunits assemble into a tetramer mation of protein carbonyls, direct modifications of protein with two active sites (Green, Cell 94:695-698, 1998). Many side chains, such as o-tyrosine, chloro-, nitrotyrosine, and other proteolytically activated enzymes, known in the art as dityrosine, and protein adducts derived from reactions with “Zymogens, also find use in the instant invention as acti carbohydrate and lipid derivatives. Other modifications may 45 vatable elements. be non-covalent, Such as binding of a ligand or binding of an In an alternative embodiment the activation of the acti allosteric modulator. vatable element involves prenylation of the element. By One example of a covalent modification is the substitution “prenylation', and grammatical equivalents used herein, is of a phosphate group for a hydroxyl group in the side chain meant the addition of any lipid group to the element. of an amino acid (phosphorylation). A wide variety of 50 Common examples of prenylation include the addition of proteins are known that recognize specific protein Substrates farnesyl groups, geranylgeranyl groups, myristoylation and and catalyze the phosphorylation of serine, threonine, or palmitoylation. In general these groups are attached via tyrosine residues on their protein Substrates. Such proteins thioether linkages to the activatable element, although other are generally termed “kinases.” Substrate proteins that are attachments may be used. capable of being phosphorylated are often referred to as 55 In alternative embodiment, activation of the activatable phosphoproteins (after phosphorylation). Once phosphory element is detected as intermolecular clustering of the lated, a Substrate phosphoprotein may have its phosphory activatable element. By “clustering or “multimerization'. lated residue converted back to a hydroxyl one by the action and grammatical equivalents used herein, is meant any of a protein phosphatase that specifically recognizes the reversible or irreversible association of one or more signal Substrate protein. Protein phosphatases catalyze the replace 60 transduction elements. Clusters can be made up of 2, 3, 4, ment of phosphate groups by hydroxyl groups on serine, etc., elements. Clusters of two elements are termed dimers. threonine, or tyrosine residues. Through the action of Clusters of 3 or more elements are generally termed oli kinases and phosphatases a protein may be reversibly phos gomers, with individual numbers of clusters having their phorylated on a multiplicity of residues and its activity may own designation; for example, a cluster of 3 elements is a be regulated thereby. Thus, the presence or absence of one 65 trimer, a cluster of 4 elements is a tetramer, etc. or more phosphate groups in an activatable protein is a Clusters can be made up of identical elements or different preferred readout in the present invention. elements. Clusters of identical elements are termed "homo' US 9,500,655 B2 71 72 dimers, while clusters of different elements are termed tors of cell-to-cell communication that includes interleukins, "hetero' clusters. Accordingly, a cluster can be a homodi interferons, and colony-stimulating factors. The character mer, as is the case for the B-adrenergic receptor. istic features of cytokines lie in their pleiotropy and func Alternatively, a cluster can be a heterodimer, as is the case tional redundancy. Most of the cytokine receptors that for GABA-. In other embodiments, the cluster is a constitute distinct Superfamilies do not possess intrinsic homotrimer, as in the case of TNFO, or a heterotrimer such protein tyrosine kinase domains, yet receptor stimulation the one formed by membrane-bound and soluble CD95 to usually invokes rapid tyrosine phosphorylation of intracel modulate apoptosis. In further embodiments the cluster is a lular proteins, including the receptors themselves. Many homo-oligomer, as in the case of Thyrotropin releasing members of the cytokine receptor superfamily activate the hormone receptor, or a hetero-oligomer, as in the case of 10 Jak protein tyrosine kinase family, with resultant phospho TGFB1. rylation of the STAT family of transcription factors. IL-2, In a preferred embodiment, the activation or signaling IL-4, IL-7 and Interferon Y have all been shown to activate potential of elements is mediated by clustering, irrespective Jak kinases (Frank et al. (1995) Proc. Natl. Acad. Sci. USA of the actual mechanism by which the element’s clustering 92:7779-7783); Scharfeet al. (1995) Blood 86:2077-2085); is induced. For example, elements can be activated to cluster 15 (Bacon et al. (1995) Proc. Natl. Acad. Sci. USA 92.7307 a) as membrane bound receptors by binding to ligands 7311); and (Sakatsume et al. (1995) J. Biol. Chem. 270: (ligands including both naturally occurring or synthetic 17528-17534). Events downstream of Jak phosphorylation ligands), b) as membrane bound receptors by binding to have also been elucidated. For example, exposure of T other surface molecules, or c) as intracellular (non-mem lymphocytes to IL-2 has been shown to lead to the phos brane bound) receptors binding to ligands. phorylation of signal transducers and activators of transcrip In a preferred embodiment the activatable elements are tion (STAT) proteins STAT1C, STAT1 B, and STAT3, as well membrane bound receptor elements that cluster upon ligand as of two STAT-related proteins, p94 and p95. The STAT binding Such as cell Surface receptors. As used herein, 'cell proteins translocate to the nucleus and bind to a specific surface receptor refers to molecules that occur on the DNA sequence, thus suggesting a mechanism by which IL-2 surface of cells, interact with the extracellular environment, 25 may activate specific genes involved in immune cell func and transmit or transduce (through signals) the information tion (Frank et al. Supra). Jak3 is associated with the gamma regarding the environment intracellularly in a manner that chain of the IL-2, IL-4, and IL-7 cytokine receptors (Fujii et may modulate cellular activity directly or indirectly, e.g., via al. (1995) Proc. Natl. Acad. Sci. 92:5482-5486) and (Musso intracellular second messenger activities or transcription of et al. (1995) J. Exp. Med. 181: 1425-1431). The Jak kinases specific promoters, resulting in transcription of specific 30 have been shown to be activated by numerous ligands that genes. One class of receptor elements includes membrane signal via cytokine receptors such as, growth hormone, bound proteins, or complexes of proteins, which are acti erythropoietin and IL-6 (Kishimoto (1994) Stem cells Suppl. vated to cluster upon ligand binding. As is known in the art, 12:37-44). Preferred activatable elements are selected from these receptor elements can have a variety of forms, but in the group p-STAT1, p-STAT3, p-STAT5, p-STAT6. general they comprise at least three domains. First, these 35 p-PLCY2. p-S6, p.Akt, p-Erk, p-CREB, p-38, and NF-KBp receptors have a ligand-binding domain, which can be 65. oriented either extracellularly or intracellularly, usually the In a preferred embodiment the activatable element is a former. Second, these receptors have a membrane-binding member of the nerve growth factor receptor superfamily, domain (usually a transmembrane domain), which can take Such as the Tumor necrosis factor alpha receptor. Tumor the form of a seven pass transmembrane domain (discussed 40 necrosis factor O. (TNF-C. or TNF-alpha) is a pleiotropic below in connection with G-protein-coupled receptors) or a cytokine that is primarily produced by activated macro lipid modification, Such as myristylation, to one of the phages and lymphocytes but is also expressed in endothelial receptor's amino acids which allows for membrane associa cells and other cell types. TNF-alpha is a major mediator of tion when the lipid inserts itself into the lipid bilayer. Finally, inflammatory, immunological, and pathophysiological reac the receptor has an signaling domain, which is responsible 45 tions. (Grell, M., et al., (1995) Cell, 83:793-802). Two for propagating the downstream effects of the receptor. distinct forms of TNF exist, a 26 kDa membrane expressed Examples of Such receptor elements include hormone form and the soluble 17 kDa cytokine which is derived from receptors, Steroid receptors, cytokine receptors, such as proteolytic cleavage of the 26 kDa form. The soluble TNF IL1-C, IL-3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, polypeptide is 157 amino acids long and is the primary IL-10. IL-12, IL-15, IL-18, IL-21, CCR5, CCR7, CCR-1-10, 50 biologically active molecule. CCL20, chemokine receptors, such as CXCR4, adhesion TNF-alpha exerts its biological effects through interaction receptors and growth factor receptors, including, but not with high-affinity cell surface receptors. Two distinct mem limited to, PDGF-R (platelet derived growth factor recep brane TNF-alpha receptors have been cloned and character tor), EGF-R (epidermal growth factor receptor), VEGF-R ized. These are a 55 kDa species, designated p55 TNF-R and (vascular endothelial growth factor), uPAR (urokinase plas 55 a 75 kDa species designated p75 TNF-R (Corcoran. A. E., et minogen activator receptor), ACHR (acetylcholine recep al., (1994) Eur. J. Biochem., 223:831-840). The two TNF tor), IgE-R (immunoglobulin E receptor), estrogen receptor, receptors exhibit 28% similarity at the amino acid level. This thyroid hormone receptor, integrin receptors (B1, B2, B3, B4, is confined to the extracellular domain and consists of four B5, B6, C1, C2, C3, C4, C5, O6), MAC-1 (B2 and cd 11b). repeating cysteine-rich motifs, each of approximately 40 CVB33, opioid receptors (mu and kappa), FC receptors, 60 amino acids. Each motif contains four to six cysteines in serotonin receptors (5-HT, 5-HT6, 5-HT7), B-adrenergic conserved positions. Dayhoff analysis shows the greatest receptors, insulin receptor, leptin receptor, TNF receptor intersubunit similarity among the first three repeats in each (tissue-necrosis factor), statin receptors, FAS receptor, receptor. This characteristic structure is shared with a num BAFF receptor, FLT3 LIGAND receptor, GMCSF receptor, ber of other receptors and cell surface molecules, which and fibronectin receptor. 65 comprise the TNF-R/nerve growth factor receptor superfam In a preferred embodiment the activatable element is a ily (Corcoran. A. E., et al., (1994) Eur. J. Biochem..., 223: cytokine receptor. Cytokines are a family of Soluble media 831-840). US 9,500,655 B2 73 74 TNF signaling is initiated by receptor clustering, either by gene 9:1461-1467; Henkemeyer et al. (1994) Oncogene the trivalent ligand TNF or by cross-linking monoclonal 9:1001-1014; Ruiz et al. (1994) Mech. Dev. 46:87-100; Xu antibodies (Vandevoorde, V., et al., (1997) J. Cell Biol. et al. (1994) Development 120:287-299; Zhou et al. (1994) 137:1627-1638). Crystallographic studies of TNF and the J. Neurosci. Res. 37:129-143; and references in Tuzi and structurally related cytokine, lymphotoxin (LT), have shown Gullick (1994) Br. J. Cancer 69:417-421). Exemplary Eph that both cytokines exist as homotrimers, with subunits receptors include the eph, elk, eck, Sek, mek4, hek, hek2. packed edge to edge in threefold symmetry. Structurally, eek, erk, tyrol, tyro4, tyro5, tyro6, tyrol11, cek4, cekS. cek6. neither TNF nor LT reflect the repeating pattern of the their cek7, cek8, cek9, cek10. bsk, rtk1, rtk2, rtk3, myk1, myk2, receptors. Each monomer is cone shaped and contains two ehkl, ehk2, pagliaccio, htk, erk and nuk receptors. hydrophilic loops on opposite sides of the base of the cone. 10 In another embodiment the receptor element is a member Recent crystal structure determination of a p55 soluble of the hematopoietin receptor Superfamily. Hematopoietin TNF-R/IT complex has confirmed the hypothesis that loops receptor Superfamily is used herein to define single-pass from adjacent monomers join together to form a groove transmembrane receptors, with a three-domain architecture: between monomers and that TNF-R binds in these grooves an extracellular domain that binds the activating ligand, a (Corcoran. A. E., et al., (1994) Eur. J. Biochem..., 223:831 15 short transmembrane segment, and a domain residing in the 840). cytoplasm. The extracellular domains of these receptors In one embodiment, the activatable element is a receptor have low but significant homology within their extracellular tyrosine kinase. The receptor tyrosine kinases can be divided ligand-binding domain comprising about 200-210 amino into Subgroups on the basis of structural similarities in their acids. The homologous region is characterized by four extracellular domains and the organization of the tyrosine cysteine residues located in the N-terminal half of the kinase catalytic region in their cytoplasmic domains. Sub region, and a Trp-Ser-X-Trp-Ser (WSXWS) motif located groups I (epidermal growth factor (EGF) receptor-like), II just outside the membrane-spanning domain. Further struc (insulin receptor-like) and the EPH/ECK family contain tural and functional details of these receptors are provided cysteine-rich sequences (Hirai et al., (1987) Science 238: by Cosman, D. et al., (1990). The receptors of IL-1, IL-2, 1717-1720 and Lindberg and Hunter, (1990) Mol. Cell. Biol. 25 IL-3, IL-4, IL-5. IL-6, IL-7, prolactin, placental lactogen, 10:6316-6324). The functional domains of the kinase region growth hormone GM-CSF, G-CSF, M-CSF and erythropoi of these three classes of receptor tyrosine kinases are etin have, for example, been identified as members of this encoded as a contiguous sequence (Hanks et al. (1988) receptor family. Science 241:42-52). Subgroups III (platelet-derived growth In a further embodiment, the receptor element is an factor (PDGF) receptor-like) and IV (the fibroblast growth 30 integrin other than Leukocyte Function Antigen-1 (LFA-1). factor (FGF) receptors) are characterized as having immu Members of the integrin family of receptors function as noglobulin (Ig)-like folds in their extracellular domains, as heterodimers, composed of various C. and B subunits, and well as having their kinase domains divided in two parts by mediate interactions between a cell's cytoskeleton and the a variable stretch of unrelated amino acids (Yanden and extracellular matrix. (Reviewed in, Giancotti and Ruoslahti, Ullrich (1988) supra and Hanks et al. (1988) supra). 35 Science 285, 13 Aug. 1999). Different combinations of the The family with the largest number of known members is C. and B subunits give rise to a wide range of ligand the Eph family (with the first member of the family origi specificities, which may be increased further by the presence nally isolated from an erythropoietin producing hepatocel of cell-type-specific factors. Integrin clustering is know to lular carcinoma cell line). Since the description of the activate a number of intracellular signals, such as RAS, prototype, the Eph receptor (Hirai et al. (1987) Science 40 MAP kinase, and phosphotidylinosital-3-kinase. In a pre 238:1717-1720), sequences have been reported for at least ferred embodiment the receptor element is a heterodimer ten members of this family, not counting apparently ortholo (other than LFA-1) composed of a B integrin and an O. gous receptors found in more than one species. Additional integrin chosen from the following integrins; B1, B2, B3, 34. partial sequences, and the rate at which new members are B5, B6, C1, C2, C3, C4, C5, and O.6, or is MAC-1 (B2 and still being reported, suggest the family is even larger (Mai 45 cd 11b), or CVB3. sonpierre et al. (1993) Oncogene 8:3277-3288; Andres et al. In a preferred embodiment the element is an intracellular (1994) Oncogene 9:1461-1467; Henkemeyer et al. (1994) adhesion molecule (ICAM). ICAMs-1, -2, and -3 are cellular Oncogene 9:1001-1014; Ruiz et al. (1994) Mech. Dev. adhesion molecules belonging to the immunoglobulin Super 46:87-100; Xu et al. (1994) Development 120:287-299; family. Each of these receptors has a single membrane Zhou et al. (1994) J. Neurosci. Res. 37: 129-143; and refer 50 spanning domain and all bind to B2 integrins via extracel ences in Tuzi and Gullick (1994) Br. J. Cancer 69:417-421). lular binding domains similar in structure to Ig-loops. Remarkably, despite the large number of members in the (Signal Transduction, Gomperts, et al., eds, Academic Press Eph family, all of these molecules were identified as orphan Publishers, 2002, Chapter 14, pp 318-319). receptors without known ligands. In another embodiment the activatable elements cluster As used herein, the terms “Eph receptor or “Eph-type 55 for signaling by contact with other Surface molecules. In receptor refer to a class of receptor tyrosine kinases, contrast to the receptors discussed above, these elements comprising at least eleven paralogous genes, though many cluster for signaling by contact with other Surface mol more orthologs exist within this class, e.g. homologs from ecules, and generally use molecules presented on the Surface different species. Eph receptors, in general, are a discrete of a second cell as ligands. Receptors of this class are group of receptors related by homology and easily recog 60 important in cell-cell interactions, such mediating cell-to nizable, e.g., they are typically characterized by an extra cell adhesion and immunorecognition. cellular domain containing a characteristic spacing of cys Examples of such receptor elements are CD3 (T cell teine residues near the N-terminus and two fibronectin type receptor complex), BCR (B cell receptor complex), CD4. III repeats (Hirai et al. (1987) Science 238:1717-1720; CD28, CD80, CD86, CD54, CD102, CD50 and ICAMs 1, 2 Lindberg et al. (1990) Mol. Cell Biol. 10:6316-6324; Chan 65 and 3. et al. (1991) Oncogene 6:1057-1061; Maisonpierre et al. In a preferred embodiment the receptor element is a T cell (1993) Oncogene 8:3277-3288; Andres et al. (1994) Onco receptor complex (TCR). TCRs occur as either of two US 9,500,655 B2 75 76 distinct heterodimers, C. B. ory both of which are expressed Cell 72, 415: Kouba et al. FEBS Lett. (1993)321, 173; and with the non-polymorphic CD3 polypeptides Y, X, e, S. The Birkenbach et al. (1993) J. Virol. 67, 2209. CD3 polypeptides, especially S and its variants, are critical Known ligands for G protein coupled receptors include: for intracellular signaling. The CfB TCR heterodimer purines and nucleotides, such as adenosine, cAMP, ATP, expressing cells predominate in most lymphoid compart 5 UTP. ADP melatonin and the like; biogenic amines (and ments and are responsible for the classical helper or cyto related natural ligands). Such as 5-hydroxytryptamine, ace toxic T cell responses. In most cases, the CfBTCR ligand is tylcholine, dopamine, adrenaline, histamine, noradrenaline, a peptide antigen bound to a class I or a class II MHC tyraminefoctopamine and other related compounds; peptides molecule (Fundamental Immunology, fourth edition, W. E. Such as adrenocorticotrophic hormone (acth), melanocyte 10 stimulating hormone (msh), melanocortins, neurotensin (nt), Paul, ed., Lippincott-Raven Publishers, 1999, Chapter 10, bombesin and related peptides, endothelins, cholecystoki pp 341-367). nin, gastrin, neurokinin b (nk3), invertebrate tachykinin-like In another embodiment, the activatable element is a peptides, Substance k (nk2), Substance p (ink1), neuropeptide member of the large family of G-protein-coupled receptors. y (npy), thyrotropin releasing-factor (trf), bradykinin, angio It has recently been reported that a G-protein-coupled recep 15 tensin ii, beta-endorphin, c5a anaphalatoxin, calcitonin, tors are capable of clustering. (Kroeger, et al., J Biol Chem chemokines (also called intercrines), corticotrophic releas 276:16, 12736-12743, Apr. 20, 2001; Bai, et al., J Biol Chem ing factor (crif), dynorphin, endorphin, fmlp and other 273:36, 23605-23610, Sep. 4, 1998: Rocheville, et al., J Biol formylated peptides, follitropin (fish), fungal mating phero Chem 275 (11), 7862-7869, Mar. 17, 2000). As used herein mones, galanin, gastric inhibitory polypeptide receptor G-protein-coupled receptor, and grammatical equivalents (gip), glucagon-like peptides (glps), glucagon, gonadotropin thereof, refers to the family of receptors that bind to het releasing hormone (gnrh), growth hormone releasing hor erotrimeric "G proteins.” Many different G proteins are mone(ghrh), insect diuretic hormone, interleukin-8, leutro known to interact with receptors. G protein signaling sys pin (1 h/hcg), met-enkephalin, opioid peptides, oxytocin, tems include three components: the receptor itself, a GTP parathyroid hormone (pth) and pthrp, pituitary adenylyl binding protein (G protein), and an intracellular target 25 cyclase activating peptide (pacap), secretin, Somatostatin, protein. The cell membrane acts as a Switchboard. Messages thrombin, thyrotropin (tsh), vasoactive intestinal peptide arriving through different receptors can produce a single (vip), vasopressin, Vasotocin, eicosanoids such as ip-pros effect if the receptors act on the same type of G protein. On tacyclin, pg-prostaglandins, tX-thromboxanes; retinal based the other hand, signals activating a single receptor can compounds such as vertebrate 11-cis retinal, invertebrate produce more than one effect if the receptor acts on different 30 11-cis retinal and other related compounds; lipids and lipid kinds of G proteins, or if the G proteins can act on different based compounds such as cannabinoids, anandamide, lyso effectors. phosphatidic acid, platelet activating factor, leukotrienes and In their resting state, the G proteins, which consist of the like; excitatory amino acids and ions such as calcium alpha (C.), beta (B) and gamma (Y) subunits, are complexed ions and glutamate. with the nucleotide guanosine diphosphate (GDP) and are in 35 Preferred G protein coupled receptors include, but are not contact with receptors. When a hormone or other first limited to: C1-adrenergic receptor, C.1B-adrenergic receptor, messenger binds to a receptor, the receptor changes confor C2-adrenergic receptor, C2B-adrenergic receptor, B1-adren mation and this alters its interaction with the G protein. This ergic receptor, B2-adrenergic receptor, B3-adrenergic recep spurs a subunit to release GDP, and the more abundant tor, m1 acetylcholine receptor (AChR), m2 AChR, m3 nucleotide guanosine triphosphate (GTP), replaces it, acti 40 AChR, ma AChR, m3 AChR, D1 dopamine receptor, D2 vating the G protein. The G protein then dissociates to dopamine receptor, D3 dopamine receptor, D4 dopamine separate the C. subunit from the still complexed beta and receptor, D5 dopamine receptor, Al adenosine receptor, A2a gamma Subunits. Either the GC. Subunit, or the GBy complex, adenosine receptor, A2b adenosine receptor, A3 adenosine depending on the pathway, interacts with an effector. The receptor, 5-HT1a receptor, 5-HT1b receptor, 5HT1-like effector (which is often an enzyme) in turn converts an 45 receptor, 5-HT1d receptor, 5HT1d-like receptor, 5HT1d beta inactive precursor molecule into an active 'second messen receptor, substance K (neurokinin A) receptor, fMLP recep ger,” which may diffuse through the cytoplasm, triggering a tor (FPR), fMLP-like receptor (FPRL-1), angiotensin II type metabolic cascade. After a few seconds, the GO. converts the 1 receptor, endothelin ETA receptor, endothelin ETB recep GTP to GDP, thereby inactivating itself. The inactivated GO. tor, thrombin receptor, growth hormone-releasing hormone may then reassociate with the GBy complex. 50 (GHRH) receptor, vasoactive intestinal peptide receptor, Hundreds, if not thousands, of receptors convey messages oxytocin receptor, somatostatin SSTR1 and SSTR2. SSTR3, through heterotrimeric G proteins, of which at least 17 cannabinoid receptor, follicle stimulating hormone (FSH) distinct forms have been isolated. Although the greatest receptor, leutropin (LH/HCG) receptor, thyroid stimulating variability has been seen in a subunit, several different Band hormone (TSH) receptor, thromboxane A2 receptor, platelet Y structures have been reported. There are, additionally, 55 activating factor (PAF) receptor, C5a anaphylatoxin recep many different G protein-dependent effectors. tor, CXCR1 (IL-8 receptor A), CXCR2 (IL-8 receptor B), Most G protein-coupled receptors are comprised of a Delta Opioid receptor, Kappa Opioid receptor, mip-1alpha/ single protein chain that passes through the plasma mem RANTES receptor (CRR1), Rhodopsin, Red opsin, Green brane seven times. Such receptors are often referred to as opsin, Blue opsin, metabotropic glutamate mGluR1-6, his seven-transmembrane receptors (STRs). More than a hun 60 tamine H2 receptor, ATP receptor, neuropeptide Y receptor, dred different STRs have been found, including many dis amyloid protein precursor receptor, insulin-like growth fac tinct receptors that bind the same ligand, and there are likely tor II receptor, bradykinin receptor, gonadotropin-releasing many more STRS awaiting discovery. hormone receptor, cholecystokinin receptor, melanocyte In addition, STRs have been identified for which the stimulating hormone receptor, antidiuretic hormone recep natural ligands are unknown; these receptors are termed 65 tor, glucagon receptor, and adrenocorticotropic hormone II “orphan' G protein-coupled receptors, as described above. receptor. In addition, there are at least five receptors (CC and Examples include receptors cloned by Neote et al. (1993) CXC receptors) involved in HIV viral attachment to cells. US 9,500,655 B2 77 78 The two major co-receptors for HIV are CXCR4, (fusin in lymphomas. See Leukemia. See February 2004; 18(2): receptor, LESTR, SDF-1C, receptor) and CCR5 (m-trophic). 356-8. SOCS1 and SHP1 hypermethylation in mantle cell More preferred receptors include the following human lymphoma and follicular lymphoma: implications for epi receptors: melatonin receptor 1a, galanin receptor 1, neuro genetic activation of the Jak/STAT pathway. Chim C S. tensin receptor, adenosine receptor 2a, Somatostatin receptor 5 Wong KY. Loong F. Srivastava G. 2 and corticotropin releasing factor receptor 1. Melatonin In another embodiment the activatable element is a small receptor 1a is particularly preferred. Other G protein coupled molecule, carbohydrate, lipid or other naturally occurring or receptors (GPCRs) are known in the art. synthetic compound capable of having an activated isoform. In one embodiment, Link is a protein to be measured. In addition, as pointed out above, activation of these ele Hematopoietic stem cells (HSCs) give rise to variety of 10 ments need not include Switching from one form to another, hematopoietic cells via pluripotential progenitors. Lineage but can be detected as the presence or absence of the committed progenitors are responsible for blood production compound. For example, activation of cAMP (cyclic throughout adult life. Amplification of HSCs or progenitors adenosine mono-phosphate) can be detected as the presence represents a potentially powerful approach to the treatment of cAMP rather than the conversion from non-cyclic AMP of various blood disorders. Animal model studies demon 15 to cyclic AMP. strated that Link acts as a broad inhibitor of signaling In some embodiments, the activatable element is a pro pathways in hematopoietic lineages. Link is an adaptor tein. Examples of proteins that may include activatable protein which belongs to a family of proteins sharing several elements include, but are not limited to kinases, phos structural motifs, including a Src homology 2 (SH2) domain phatases, lipid signaling molecules, adaptor/scaffold pro which binds phospho-tyrosines in various signal-transduc teins, cytokines, cytokine regulators, ubiquitination ing proteins. The SH2 domain is essential for Link-mediated enzymes, adhesion molecules, cytoskeletal/contractile pro negative regulation of several cytokine receptors (i.e. Mpl. teins, heterotrimeric G proteins, Small molecular weight EpoR, c-Kit, Il-3R and IL7R). Therefore, inhibition of the GTPases, guanine nucleotide exchange factors, GTPase binding of Link to cytokine receptors might lead to enhanced activating proteins, caspases, proteins involved in apoptosis, downstream signaling of the receptor and thereby to 25 cell cycle regulators, molecular chaperones, metabolic improved hematopoiesis in response to exposure to cytok enzymes, Vesicular transport proteins, hydroxylases, ines (i.e. erythropoietin in anemic patients). (Gueller et al. isomerases, deacetylases, methylases, demethylases, tumor Adaptor protein Link associates with Y568 in c-Kit. 1: Suppressor genes, proteases, ion channels, molecular trans Biochem J. 2008 Jun. 30.) It has been shown that overex porters, transcription factors/DNA binding factors, regula pression of Link in Ba/F3-MPLW515L cells inhibits 30 tors of transcription, and regulators of translation. Examples cytokine-independent growth, while Suppression of Link in of activatable elements, activation states and methods of UT7-MPLW515L cells enhances proliferation. Link blocks determining the activation level of activatable elements are the activation of Jak2, Stat3, Erk, and Akt in these cells. described in US Publication Number 20060073474 entitled (Gery et al., Adaptor protein Link negatively regulates the “Methods and compositions for detecting the activation state mutant MPL, MPLW515L associated with myeloprolifera 35 of multiple proteins in single cells' and US Publication tive neoplasms, Blood, 1 Nov. 2007, Vol. 110, No. 9, pp. Number 20050112700 entitled “Methods and compositions 3360-3364.) Thus, Link is an important protein to measure for risk stratification the content of which are incorporate for the evaluation of AML/MDS/MPS. here by reference. See also U.S. Ser. Nos. 61/048,886; In one embodiment, the activatable elements are intrac 61/048.920; and Shulz et al., Current Protocols in Immu ellular receptors capable of clustering. Elements of this class 40 nology 2007, 78:8.17.1-20. are not membrane-bound. Instead, they are free to diffuse In some embodiments, the protein is selected from the through the intracellular matrix where they bind soluble group consisting of HER receptors, PDGF receptors, Kit ligands prior to clustering and signal transduction. In con receptor, FGF receptors, Eph receptors, Trk receptors, IGF trast to the previously described elements, many members of receptors, Insulin receptor, Met receptor, Ret, VEGF recep this class are capable of binding DNA after clustering to 45 tors, TIE1, TIE2, FAK, Jak1, Jak2, Jak3, Tyk2, Src, Lyn, directly effect changes in RNA transcription. Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, In another embodiment the intracellular receptors capable cRaf, ARaf, BRAF. Mos, Lim kinase, ILK, Tpl. ALK, TGFB of clustering are peroxisome proliferator-activated receptors receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, (PPAR). PPARs are soluble receptors responsive to lipo PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, philic compounds, and induce various genes involved in 50 Bub, Myt 1, Weel, Casein kinases, PDK1, SGK1, SGK2, fatty acid metabolism. The three PPAR subtypes, PPARC, SGK3, Akt1, Akt2, Akt3, p90Rsks, p70S6 Kinase, Prks, B, and Y have been shown to bind to DNA after ligand PKCS, PKAs, ROCK1, ROCK 2, Auroras, CaMKs, MNKs, binding and heterodimerization with retinoid X receptor. AMPKs, MELK, MARKs, Chk1, Chk2, LKB-1, MAP (Summanasekera, et al., J Biol Chem, M211261200, Dec. KAPKs, Piml, Pim2, Pim3, IKKs, Cdks, Jnks, Erks, IKKs, 13, 2002.) 55 GSK3C, GSK3 B, Cdks, CLKs, PKR, PI3-Kinase class 1, In another embodiment the activatable element is a class 2, class 3, mTor, SAPK/JNK1,2,3, p38s, PKR, DNA nucleic acid. Activation and deactivation of nucleic acids PK, ATM, ATR, Receptor protein tyrosine phosphatases can occur in numerous ways including, but not limited to, (RPTPs), LAR phosphatase, CD45, Non receptor tyrosine cleavage of an inactivating leader sequence as well as phosphatases (NPRTPs), SHPs, MAP kinase phosphatases covalent or non-covalent modifications that induce struc 60 (MKPs), Dual Specificity phosphatases (DUSPs), CDC25 tural or functional changes. For example, many catalytic phosphatases, Low molecular weight tyrosine phosphatase, RNAS, e.g. hammerhead ribozymes, can be designed to have Eyes absent (EYA) tyrosine phosphatases, Slingshot phos an inactivating leader sequence that deactivates the catalytic phatases (SSH), serine phosphatases, PP2A, PP2B, PP2C, activity of the ribozyme until cleavage occurs. An example PP1, PP5, inositol phosphatases, PTEN, SHIPs, myotubu of a covalent modification is methylation of DNA. Deacti 65 larins, phosphoinositide kinases, phopsholipases, prosta vation by methylation has been shown to be a factor in the glandin synthases, 5-lipoxygenase, Sphingosine kinases, silencing of certain genes, e.g. STAT regulating SOCS genes sphingomyelinases, adaptor/scaffold proteins, Shc, Grb2, US 9,500,655 B2 79 80 BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP. limited Stage, extensive stage, staging according to cellular Dok, KSR, MyD88, Crk, CrkL, GAD, Nck, Grb2 associated markers such as ZAP70 and CD38, occult, including infor binder (GAB), Fas associated death domain (FADD), mation that may inform on time to progression, progression TRADD, TRAF2, RIP. T-Cell leukemia family, IL-2, IL-4, free survival, overall survival, or event-free survival. IL-8, IL-6, interferon Y, interferon C, suppressors of 5 In some embodiments, methods and compositions are cytokine signaling (SOCs), Cbl, SCF ubiquitination ligase provided for the classification of a cell according to the complex. APC/C, adhesion molecules, integrins, Immuno activation level of an activatable element, e.g., in a cellular globulin-like adhesion molecules, selectins, cadherins, pathway wherein the classification comprises classifying a catenins, focal adhesion kinase, p130CAS, fodrin, actin, cell as a cell that is correlated to a patient response to a paxillin, myosin, myosin binding proteins, tubulin, eg5/KSP. 10 treatment. In some embodiments, the patient response is CENPS, B-adrenergic receptors, muscarinic receptors, selected from the group consisting of complete response, adenylyl cyclase receptors, Small molecular weight partial response, nodular partial response, no response, pro GTPases, H-Ras, K-Ras, N-Ras, Ran, Rac, Rho, Cdc42, gressive disease, stable disease and adverse reaction. Arfs, RABS, RHEB, Vav, Tiam, Sos, Dbl, PRK, TSC1.2, In some embodiments, methods and compositions are Ras-GAP Arf-GAPs, Rho-GAPs, caspases, Caspase 2, Cas 15 provided for the classification of a cell according to the pase 3, Caspase 6, Caspase 7, Caspase 8, Caspase 9. Bcl-2. activation level of an activatable element, e.g., in a cellular Mcl-1, Bcl-XL, Bcl-w, Bcl-B, A1, Bax, Bak, Bok, Bik, Bad, pathway wherein the classification comprises classifying the Bid, Bim, Bmf, Hrk, Noxa, Puma, IAPB, XIAP. Smac, cell as a cell that is correlated with minimal residual disease Cdk4, Cdk 6, Cdk 2, Cdk1, Cdk 7, Cyclin D, Cyclin E, or emerging resistance. Cyclin A, Cyclin B, Rb, p16, p14Arf, p27KIP p21CIP. In some embodiments, methods and compositions are molecular chaperones, Hsp90s, Hsp70, Hsp27, metabolic provided for the classification of a cell according to the enzymes, Acetyl-CoAa Carboxylase, ATP citrate lyase, activation level of an activatable element, e.g., in a cellular nitric oxide synthase, caveolins, endosomal sorting complex pathway wherein the classification comprises selecting a required for transport (ESCRT) proteins, vesicular protein method of treatment. Example of methods of treatments sorting (Vsps), hydroxylases, prolyl-hydroxylases PHD-1, 2 25 include, but are not limited to, chemotherapy, biological and 3, asparagine hydroxylase FIH transferases, Pin1 prolyl therapy, radiation therapy, bone marrow transplantation, isomerase, topoisomerases, deacetylases, Histone deacety Peripheral stem cell transplantation, umbilical cord blood lases, sirtuins, histone acetylases, CBP/P300 family, MYST transplantation, autologous stem cell transplantation, allo family, ATF2, DNA methyl transferases. Histone H3K4 geneic stem cell transplantation, Syngeneic stem cell trans demethylases, H3K27, JHDM2A, UTX, VHL, WT-1, p53, 30 plantation, Surgery, induction therapy, maintenance therapy, Hdm. PTEN, ubiquitin proteases, urokinase-type plasmino and watchful waiting. gen activator (uPA) and uPA receptor (uPAR) system, cathe Generally, the methods of the invention involve deter psins, metalloproteinases, esterases, hydrolases, separase, mining the activation levels of an activatable element in a potassium channels, sodium channels, multi-drug resistance plurality of single cells in a sample. proteins, P-Glycoprotein, nucleoside transporters, Ets, Elk, 35 Signaling Pathways SMADs, Rel-A (p65-NFKB), CREB, NFAT, ATF-2, AFT, In some embodiments, the methods of the invention are Myc, Fos, Sp1, Egr-1, T-bet, B-catenin, HIFs, FOXOs, E2Fs, employed to determine the status of an activatable element SRFs, TCFs, Egr-1, B-catenin, FOXO STAT1, STAT3, in a signaling pathway. In some embodiments, a cell is STAT4, STAT5, STAT6, p53, WT-1, HMGA, pS6, 4EPB-1, classified, as described herein, according to the activation eIF4E-binding protein, RNA polymerase, initiation factors, 40 level of one or more activatable elements in one or more elongation factors. signaling pathways. Signaling pathways and their members In some embodiments of the invention, the methods have been described. See (Hunter T. Cell Jan. 7, 2000; described herein are employed to determine the activation 100(1): 13-27). Exemplary signaling pathways include the level of an activatable element, e.g., in a cellular pathway. following pathways and their members: The MAP kinase Methods and compositions are provided for the classifica 45 pathway including Ras, Raf. MEK, ERK and elk; the tion of a cell according to the activation level of an activat PI3K/Akt pathway including PI-3-kinase, PDK1, Akt and able element in a cellular pathway. The cell can be a Bad; the NF-kB pathway including IKKs, IkB and the Wnt hematopoietic cell. Examples of hematopoietic cells include pathway including frizzled receptors, beta-catenin, APC and but are not limited to pluripotent hematopoietic stem cells, other co-factors and TCF (see Cell Signaling Technology, granulocyte lineage progenitor or derived cells, monocyte 50 Inc. 2002 Catalog pages 231-279 and Hunter T. Supra.). In lineage progenitor or derived cells, macrophage lineage some embodiments of the invention, the correlated activat progenitor or derived cells, megakaryocyte lineage progeni able elements being assayed (or the signaling proteins being tor or derived cells and erythroid lineage progenitor or examined) are members of the MAP kinase, Akt, NFkB, derived cells. WNT, RAS/RAF/MEK/ERK, JNK/SAPK, p38 MAPK, Src In some embodiments, the cell is classified according to 55 Family Kinases, JAK/STAT and/or PKC signaling path the activation level of an activatable element, e.g., in a ways. See FIG. 1 generally. cellular pathway comprises classifying the cell as a cell that In some embodiments, the status of an activatable element is correlated with a clinical outcome. In some embodiments, within the PI3K/AKT, or MAPK pathways in response to a the clinical outcome is the prognosis and/or diagnosis of a growth factor or mitogen is determined. In some embodi condition. In some embodiments, the clinical outcome is the 60 ments, the activatable element within the PI3K/AKT or presence or absence of a neoplastic or a hematopoietic MAPK pathway is selected from the group consisting of condition. In some embodiments, the clinical outcome is the Akt, p-Erk, p38 and pS6 and the growth factor or mitogen staging or grading of a neoplastic or hematopoietic condi is selected from the group consisting of FLT3L, SCF, tion. Examples of staging include, but are not limited to, G-CSF, SCF, G-CSF, SDF1a, LPS, PMA and Thapsigargin. aggressive, indolent, benign, refractory, Roman Numeral 65 In some embodiments, the status of an activatable element staging, TNM Staging, Rai staging, Binet staging, WHO within JAk/STAT pathways in response to a cytokine is classification, FAB classification, IPSS score, WPSS score, determined. In some embodiments, the activatable element